Cytotoxicity Research

**3**

**Chapter 1**

*Miralda I. Blinova*

cornea, ocular surface epithelium

**1. Introduction**

**Abstract**

In Vitro Cytotoxicity Screening

*Olga I. Aleksandrova, Igor N. Okolov, Julia I. Khorolskaya,* 

A large number of artificial tears are currently available in the pharmaceutical market. Selecting the right drug for the patient remains a challenge for both the doctor and the patient. Comparing the cytotoxicity of artificial tears is one of the criteria for the rational selection of a drug that promotes maximum clinical efficacy and a higher safety profile. It is known that cells grown in vitro retain many metabolic features of the parent host tissues and at the same time lack tissue and organ interrelations and regulatory effects of the nervous and endocrine systems and have very limited compensatory capabilities. These features of cell cultures provide an opportunity to investigate the interaction of chemical agents directly with the cell itself, to identify changes in cellular and subcellular structures that can be masked in wholeorganism settings. This study presents the results of assessing the cytotoxicity of tear substitutes, which demonstrate that these drugs can have a cytostatic effect in vitro and differ in their cytotoxic potential. In recent years, the problem of drug therapy of patients with dry eye syndrome has been attracting increasing attention of ophthalmologists, so screening the cytotoxicity of a wide range of tear substitutes using cell

culture-based test systems can promote the rational selection of these drugs.

**Keywords:** cell cultures, cytotoxicity, artificial tear, preservatives, buffers,

Tear substitutes are widely used in ophthalmology today and are the first-line treatment of multifactorial causes that occur in various cases of irritation of the ocular surface, including dry eye syndrome (DES). Artificial tears contain the following substances as active ingredients: sodium hyaluronate, carbomer, hydroxypropyl methylcellulose (HPMC), carmellose sodium, trehalose, as well as a combination of polyvinyl alcohol and povidone and HPMC together with dextran. In addition to the active ingredient, various preservatives are added to maintain the stability of the drops and suppress microorganism growth: benzalkonium chloride (BAC), cetrimide, cetalkonium chloride, Polyquad®, polyhexanide, and also Oxide®, Purite®, and OcuPure®. Substances that increase the viscosity (prolongators) reduce the rate of

as a Criterion for the Rational

Selection of Tear Substitutes

*Natalia A. Mikhailova, Diana M. Darvish and* 

#### **Chapter 1**

## In Vitro Cytotoxicity Screening as a Criterion for the Rational Selection of Tear Substitutes

*Olga I. Aleksandrova, Igor N. Okolov, Julia I. Khorolskaya, Natalia A. Mikhailova, Diana M. Darvish and Miralda I. Blinova*

### **Abstract**

A large number of artificial tears are currently available in the pharmaceutical market. Selecting the right drug for the patient remains a challenge for both the doctor and the patient. Comparing the cytotoxicity of artificial tears is one of the criteria for the rational selection of a drug that promotes maximum clinical efficacy and a higher safety profile. It is known that cells grown in vitro retain many metabolic features of the parent host tissues and at the same time lack tissue and organ interrelations and regulatory effects of the nervous and endocrine systems and have very limited compensatory capabilities. These features of cell cultures provide an opportunity to investigate the interaction of chemical agents directly with the cell itself, to identify changes in cellular and subcellular structures that can be masked in wholeorganism settings. This study presents the results of assessing the cytotoxicity of tear substitutes, which demonstrate that these drugs can have a cytostatic effect in vitro and differ in their cytotoxic potential. In recent years, the problem of drug therapy of patients with dry eye syndrome has been attracting increasing attention of ophthalmologists, so screening the cytotoxicity of a wide range of tear substitutes using cell culture-based test systems can promote the rational selection of these drugs.

**Keywords:** cell cultures, cytotoxicity, artificial tear, preservatives, buffers, cornea, ocular surface epithelium

#### **1. Introduction**

Tear substitutes are widely used in ophthalmology today and are the first-line treatment of multifactorial causes that occur in various cases of irritation of the ocular surface, including dry eye syndrome (DES). Artificial tears contain the following substances as active ingredients: sodium hyaluronate, carbomer, hydroxypropyl methylcellulose (HPMC), carmellose sodium, trehalose, as well as a combination of polyvinyl alcohol and povidone and HPMC together with dextran. In addition to the active ingredient, various preservatives are added to maintain the stability of the drops and suppress microorganism growth: benzalkonium chloride (BAC), cetrimide, cetalkonium chloride, Polyquad®, polyhexanide, and also Oxide®, Purite®, and OcuPure®. Substances that increase the viscosity (prolongators) reduce the rate of

removal of the substance from the ocular surface. These include povidone, polyvinyl alcohol, glycerol, propylene glycol, gelatin, methylcellulose, dextran 70, and carboxymethylcellulose. The next component of eye drops is antioxidants; they prevent the decomposition of the active ingredient by atmospheric oxygen. The most commonly used antioxidants are EDTA, bisulfite, thiosulfate, and metabisulfite. Eye drops may contain buffer substances (systems), which allow maintaining the pH of the drug in the range of 6–8. This is necessary to ensure that the pH of the drops is similar to the normal acidity of a human tear (7.14–7.82). With such similarity, the active substances can easily penetrate the cornea into the anterior chamber of the eye, without causing discomfort during instillation. Examples of buffers are citrate, phosphate, borate, and Tris buffers. Another important component of eye drops is osmotic agents: propylene glycol, glycerol, dextrose, and dextran. These substances provide isotonicity of eye drops in relation to the tear film and maintain osmotic pressure at the level of 305 mOsm/L. Isotonic solutions are better absorbed and well tolerated by the patient.

Thus, in addition to the main pharmaceutical ingredient, eye drops contain a number of excipients, some of which can have an adverse effect on the ocular surface, such as preservatives, buffer system components, and antioxidants.

The inclusion of preservatives in the composition of eye drops is necessary to maintain sterility and prevent their bacterial contamination. According to international standards, the addition of preservatives is mandatory in the manufacture of multidose dosage forms for topical use.

The concentration of the latter in the composition of the eye drops is relatively low; however, the cumulative dose over the entire period of use, especially with their frequent and prolonged use, can be quite high. This is especially important to remember in the context of the development of side effects that can be caused by some of the excipients in eye drops, including artificial tears [1, 2]. The preservatives in the composition of eye drops can be divided into three main types: detergents, oxidizers, and ion buffer systems. Detergent-type preservatives have a broad spectrum of antimicrobial action, which makes them quite toxic for corneal and conjunctival cells [3]. Oxidizing preservatives are less toxic than detergents, while they are effective against bacteria even at low concentrations, which minimizes their adverse effects on conjunctival and corneal epithelial cells [4]. The ion buffer preservative with an antibacterial and antifungal effect is similar to oxidizing agents in its mechanism of action [5]. It is less cytotoxic for the cells of the ocular surface than conventional preservatives but is not yet included in the composition of tear substitutes currently [6].

In the United States, many comparative clinical studies have been conducted to assess the efficacy of tear substitutes. In the published report of the Dry Eye Workshop, it was noted that despite the fact that many tear substitutes improved the ocular surface condition, there was no reliable evidence that any of the drugs was superior to another, while the inflammation of the ocular surface could worsen in the presence of preservatives in their composition [7, 8].

Recently, studies of not only developed but already available drugs have been increasingly using in vitro test systems, among which models with monolayer cell cultures are the simplest and most accessible ones [9–11]. Cells grown in vitro retain many metabolic features of the parent host tissues and at the same time lack tissue and organ interrelations and regulatory effects of the nervous and endocrine systems and have very limited compensatory capabilities. These features of cell cultures provide an opportunity to investigate the interaction of chemical agents directly with the cell itself, to identify changes in cellular and subcellular structures that can be masked in whole-organism settings. It is known that cells affected by various biologically active substances can undergo changes in morphology, cell growth rate, time of death, and degree of disintegration; therefore, it is advisable for each dosage form to assess its effect on cell survival [12]. In toxicology studies,

**5**

**Table 1.**

*In Vitro Cytotoxicity Screening as a Criterion for the Rational Selection of Tear Substitutes*

various cell cultures are used. They differ in origin, belonging to a particular tissue type, sensitivity to various xenobiotics, as well as in their ability to proliferate. The selection of a test system in each case depends on the purposes and objectives of the study. To assess the safety of ophthalmic drugs, the most informative are test

The purpose of this study was to comparatively analyze the cytotoxic effect of 21

*DOI: http://dx.doi.org/10.5772/intechopen.85106*

**2. Materials and methods**

**2.1 Test drugs**

**2.2 Cell cultures used**

**Chemical glass of preservative**

systems based on human eye tissue cells [13–18].

tear substitutes on human corneal epithelial cells in vitro.

The study object was 11 tear substitutes with various systems of

Comod®, Hylosar-Comod®, and Hylomax-Comod® (**Table 2**).

line (Chang conjunctiva, clone 1-5c-4) [17].

**2.3 Study design and methods**

preservatives (**Table 1**) and buffer systems (**Table 2**), Systane® Ultra, Artelac® Balance, Optive®, Cationorm®, Vismed® Light, Blink® contacts, Stillavit®, Ophtolique®, Lacrisifi®, Hypromellose®-P, and Slezin®, and 7 preservative-free tear substitutes, Hylabak®, Thealoz®, Thealoz Duo®, Hylo-Comod®, Hyloparin-

Cells of the immortalized human corneal epithelial cell (HCE) line were used as a test system. This test system has a higher sensitivity to the action of tear substitutes, compared with a test system based on the permanent human conjunctival cell

The effect of artificial tear eye drops on the viability of human corneal epithelial cells was studied in vitro during culturing the cells in Keratinocyte-SFM growth medium (Gibco, USA) containing the test drugs at a concentration of 10% of the

Detergents BAC, EDTA Slezin®

Oxidants Stabilized oxychloro complex

*The main groups of preservatives/oxidants in the tear substitutes.*

**Name of preservative/antioxidant Trade name of tear substitute**

BAC, EDTA Hypromellose®-P BAC, EDTA Lacrisifi® BAC, EDTA Ophtolique® Polyhexanide, EDTA Vismed® Light Polyquad® Systane® Ultra Cetalkonium chloride Cationorm®

Purite® Optive® OcuPure® Blink® contacts

Oxide® Artelac® Balance

Stabilized chlorite complex

Other EDTA Stillavit®

*In Vitro Cytotoxicity Screening as a Criterion for the Rational Selection of Tear Substitutes DOI: http://dx.doi.org/10.5772/intechopen.85106*

various cell cultures are used. They differ in origin, belonging to a particular tissue type, sensitivity to various xenobiotics, as well as in their ability to proliferate. The selection of a test system in each case depends on the purposes and objectives of the study. To assess the safety of ophthalmic drugs, the most informative are test systems based on human eye tissue cells [13–18].

The purpose of this study was to comparatively analyze the cytotoxic effect of 21 tear substitutes on human corneal epithelial cells in vitro.

#### **2. Materials and methods**

#### **2.1 Test drugs**

*Cytotoxicity - Definition, Identification, and Cytotoxic Compounds*

multidose dosage forms for topical use.

in the presence of preservatives in their composition [7, 8].

removal of the substance from the ocular surface. These include povidone, polyvinyl alcohol, glycerol, propylene glycol, gelatin, methylcellulose, dextran 70, and carboxymethylcellulose. The next component of eye drops is antioxidants; they prevent the decomposition of the active ingredient by atmospheric oxygen. The most commonly used antioxidants are EDTA, bisulfite, thiosulfate, and metabisulfite. Eye drops may contain buffer substances (systems), which allow maintaining the pH of the drug in the range of 6–8. This is necessary to ensure that the pH of the drops is similar to the normal acidity of a human tear (7.14–7.82). With such similarity, the active substances can easily penetrate the cornea into the anterior chamber of the eye, without causing discomfort during instillation. Examples of buffers are citrate, phosphate, borate, and Tris buffers. Another important component of eye drops is osmotic agents: propylene glycol, glycerol, dextrose, and dextran. These substances provide isotonicity of eye drops in relation to the tear film and maintain osmotic pressure at the level of 305 mOsm/L. Isotonic solutions are better absorbed and well tolerated by the patient. Thus, in addition to the main pharmaceutical ingredient, eye drops contain a number of excipients, some of which can have an adverse effect on the ocular surface, such as preservatives, buffer system components, and antioxidants.

The inclusion of preservatives in the composition of eye drops is necessary to maintain sterility and prevent their bacterial contamination. According to international standards, the addition of preservatives is mandatory in the manufacture of

The concentration of the latter in the composition of the eye drops is relatively low; however, the cumulative dose over the entire period of use, especially with their frequent and prolonged use, can be quite high. This is especially important to remember in the context of the development of side effects that can be caused by some of the excipients in eye drops, including artificial tears [1, 2]. The preservatives in the composition of eye drops can be divided into three main types: detergents, oxidizers, and ion buffer systems. Detergent-type preservatives have a broad spectrum of antimicrobial action, which makes them quite toxic for corneal and conjunctival cells [3]. Oxidizing preservatives are less toxic than detergents, while they are effective against bacteria even at low concentrations, which minimizes their adverse effects on conjunctival and corneal epithelial cells [4]. The ion buffer preservative with an antibacterial and antifungal effect is similar to oxidizing agents in its mechanism of action [5]. It is less cytotoxic for the cells of the ocular surface than conventional preservatives but is not yet included in the composition of tear substitutes currently [6]. In the United States, many comparative clinical studies have been conducted to assess the efficacy of tear substitutes. In the published report of the Dry Eye Workshop, it was noted that despite the fact that many tear substitutes improved the ocular surface condition, there was no reliable evidence that any of the drugs was superior to another, while the inflammation of the ocular surface could worsen

Recently, studies of not only developed but already available drugs have been increasingly using in vitro test systems, among which models with monolayer cell cultures are the simplest and most accessible ones [9–11]. Cells grown in vitro retain many metabolic features of the parent host tissues and at the same time lack tissue and organ interrelations and regulatory effects of the nervous and endocrine systems and have very limited compensatory capabilities. These features of cell cultures provide an opportunity to investigate the interaction of chemical agents directly with the cell itself, to identify changes in cellular and subcellular structures that can be masked in whole-organism settings. It is known that cells affected by various biologically active substances can undergo changes in morphology, cell growth rate, time of death, and degree of disintegration; therefore, it is advisable for each dosage form to assess its effect on cell survival [12]. In toxicology studies,

**4**

The study object was 11 tear substitutes with various systems of preservatives (**Table 1**) and buffer systems (**Table 2**), Systane® Ultra, Artelac® Balance, Optive®, Cationorm®, Vismed® Light, Blink® contacts, Stillavit®, Ophtolique®, Lacrisifi®, Hypromellose®-P, and Slezin®, and 7 preservative-free tear substitutes, Hylabak®, Thealoz®, Thealoz Duo®, Hylo-Comod®, Hyloparin-Comod®, Hylosar-Comod®, and Hylomax-Comod® (**Table 2**).

#### **2.2 Cell cultures used**

Cells of the immortalized human corneal epithelial cell (HCE) line were used as a test system. This test system has a higher sensitivity to the action of tear substitutes, compared with a test system based on the permanent human conjunctival cell line (Chang conjunctiva, clone 1-5c-4) [17].

#### **2.3 Study design and methods**

The effect of artificial tear eye drops on the viability of human corneal epithelial cells was studied in vitro during culturing the cells in Keratinocyte-SFM growth medium (Gibco, USA) containing the test drugs at a concentration of 10% of the


#### **Table 1.**

*The main groups of preservatives/oxidants in the tear substitutes.*


#### **Table 2.**

*Buffer systems in the study of tear substitutes.*

medium volume at 37°C in a CO2 incubator in an 5% CO2 atmosphere. Cells cultured under standard conditions without the addition of drugs were used as control cells. The concentration of tear substitutes for the experiment was selected on the data of clinical use of the test drugs and our own cytotoxicity studies of artificial tears on cell cultures [17]. Cell viability was assessed by their morphology and functional activity using phase-contrast microscopy (FCM) methods, MTT test, and xCELLigence system.

The morphology of the cells in the course of their culturing with the test drugs was evaluated using an inverted Nikon Eclipse TS100 microscope equipped with a camera. To evaluate the effect of tear substitutes on the metabolic activity of human corneal epithelial cells by MTT method, the cells were inoculated in 96-well plates in 200 μl of the growth medium containing the test drugs and cultured as usual for 2 days. After the culturing period, MTT test was performed. The absorbance of the solutions was measured using Fluorofot Charity analyzer (Russia) at a wavelength of 570 nm and a reference wavelength of 630 nm. Mathematical processing of the data was performed by variation statistics methods using Microsoft Excel 2007. Differences were considered significant at p < 0.05.

To evaluate the effect of tear substitutes on the adhesion and proliferative activity of corneal epithelial cells using xCELLigence system, 1 × 104 HCE cells were inoculated per well of the E-plate in 100 μl of the growth medium containing the test drugs. The plates were placed in the real-time cell analyzer dual purpose (RTCA-DP) (ACEA Biosciences), and adhesion and cell proliferation dynamics was monitored in real time for 24 h. The results were analyzed using RTCA Software 1.2.1 (Roche). The change in impedance at microelectrodes due to cell attachment and spreading was expressed as Cell Index; the value of which is automatically calculated by the program: Cell Index = (RnRb)/t, where Rb is the initial impedance value in the well containing the cell growth medium only (negative control) and Rn is the impedance value at any time t in the well containing the test cells (positive control) in addition to the growth medium. The Cell Index thus reflects changes in the number of cells, the quality of cell attachment, and the morphology of the cells in the well, which may vary over time. The data were presented as the mean value (M) ± standard deviation, the significance of differences was calculated by the Mann-Whitney U-test, and differences were considered significant at p < 0.05.

#### **3. Results and discussion**

#### **3.1 MTT test**

The MTT test, commonly known as a screening method for measuring cell survival and included in most protocols of molecular biology and medicine [19],

**7**

**Figure 1.**

*In Vitro Cytotoxicity Screening as a Criterion for the Rational Selection of Tear Substitutes*

revealed differences in the effect of the test tear substitutes on the metabolic activity of the corneal epithelial cells. The results of the MTT test are presented as a histogram, where the viability of cells cultured in growth media with the addition

of eye drops is expressed as a percentage relative to the control (**Figure 1**).

The MTT test showed that the tear substitutes containing preservatives from the group of detergents, especially BAC at various concentrations, have the greatest toxicity to corneal epithelial cells: Lacrisifi® (BAC 0.1 mg/mL), Slezin® (BAC 0.075 mg/ mL), Ophtolique® (BAC 0.1 mg/mL), Hypromellose®-P (BAC 0.1 mg/mL), and Cationorm® (cetalkonium chloride). Cell viability in the presence of these drugs was close to zero. The exceptions in this group were Vismed® Light (polyhexanide), which did not have a toxic effect at the studied concentration, and Systane® Ultra (Polyquad®), which had a moderate toxic effect. This was probably due to the fact that Vismed® Light contains the preservative polyhexanide, which is rarely included in the composition of tear substitutes. This preservative has a limited antifungal activity and no irritating effect on the human corneal epithelial cells [5]. The eye drops Systane® Ultra contain Polyquad® (polydronium chloride), which is a detergent-type preservative derived from BAC. It is unique in that bacterial cells attract Polyquad®, while corneal epithelial cells tend to repel it. Despite the occurrence of some superficial epithelial lesions, it is better tolerated than other detergent-type preservative agents [20]. Extremely high toxicity to corneal epithelial cells was also shown by the tear substitutes containing oxidants as preservatives: the stabilized oxychloro complex Optive® (Purite®) and the stabilized chlorite complex Artelac® Balance (Oxide®). The least toxic in this group was the Blink® tear substitute with OcuPure® as a preservative. It is thought that oxidative preservatives have a mild cytotoxic effect and are well tolerated and safe [21]; however, it has been found that this group of preservatives can cause superficial punctate keratitis with prolonged use [22]. The group of tear substitutes consisting of only one drug (Stillavit®), which showed moderate toxicity compared to artificial tears with detergent- and oxidative-type preservatives, includes the antioxidant EDTA (sodium edetate). It is a chelating agent, which, while not being a true preservative, can increase the antimicrobial activity of the main disinfectant while reducing its concentration. It chelates the divalent cations of calcium and magnesium, making the microorganisms more vulnerable to the preservative. Since EDTA chelates calcium and magnesium ions, it can also have a slight toxic effect on the corneal

*HCE viability histograms on day 3 of culturing in the medium with a concentration of the test drugs of 10% of the growth medium volume. The drugs were added to the growth medium at the time of inoculating the cells.*

*DOI: http://dx.doi.org/10.5772/intechopen.85106*

*In Vitro Cytotoxicity Screening as a Criterion for the Rational Selection of Tear Substitutes DOI: http://dx.doi.org/10.5772/intechopen.85106*

revealed differences in the effect of the test tear substitutes on the metabolic activity of the corneal epithelial cells. The results of the MTT test are presented as a histogram, where the viability of cells cultured in growth media with the addition of eye drops is expressed as a percentage relative to the control (**Figure 1**).

The MTT test showed that the tear substitutes containing preservatives from the group of detergents, especially BAC at various concentrations, have the greatest toxicity to corneal epithelial cells: Lacrisifi® (BAC 0.1 mg/mL), Slezin® (BAC 0.075 mg/ mL), Ophtolique® (BAC 0.1 mg/mL), Hypromellose®-P (BAC 0.1 mg/mL), and Cationorm® (cetalkonium chloride). Cell viability in the presence of these drugs was close to zero. The exceptions in this group were Vismed® Light (polyhexanide), which did not have a toxic effect at the studied concentration, and Systane® Ultra (Polyquad®), which had a moderate toxic effect. This was probably due to the fact that Vismed® Light contains the preservative polyhexanide, which is rarely included in the composition of tear substitutes. This preservative has a limited antifungal activity and no irritating effect on the human corneal epithelial cells [5]. The eye drops Systane® Ultra contain Polyquad® (polydronium chloride), which is a detergent-type preservative derived from BAC. It is unique in that bacterial cells attract Polyquad®, while corneal epithelial cells tend to repel it. Despite the occurrence of some superficial epithelial lesions, it is better tolerated than other detergent-type preservative agents [20]. Extremely high toxicity to corneal epithelial cells was also shown by the tear substitutes containing oxidants as preservatives: the stabilized oxychloro complex Optive® (Purite®) and the stabilized chlorite complex Artelac® Balance (Oxide®). The least toxic in this group was the Blink® tear substitute with OcuPure® as a preservative. It is thought that oxidative preservatives have a mild cytotoxic effect and are well tolerated and safe [21]; however, it has been found that this group of preservatives can cause superficial punctate keratitis with prolonged use [22]. The group of tear substitutes consisting of only one drug (Stillavit®), which showed moderate toxicity compared to artificial tears with detergent- and oxidative-type preservatives, includes the antioxidant EDTA (sodium edetate). It is a chelating agent, which, while not being a true preservative, can increase the antimicrobial activity of the main disinfectant while reducing its concentration. It chelates the divalent cations of calcium and magnesium, making the microorganisms more vulnerable to the preservative. Since EDTA chelates calcium and magnesium ions, it can also have a slight toxic effect on the corneal

#### **Figure 1.**

*HCE viability histograms on day 3 of culturing in the medium with a concentration of the test drugs of 10% of the growth medium volume. The drugs were added to the growth medium at the time of inoculating the cells.*

*Cytotoxicity - Definition, Identification, and Cytotoxic Compounds*

Borate buffer (B) Systane® Ultra, Blink® contacts, Slezin®

No buffer Artelac® Balance, Ophtolique®

Tris buffer Cationorm®, Hylabak®, Thealoz®, Thealoz Duo®

Combination of buffers Optive® (B + C), Vismed® Light (P + C), Stillavit® (B + P)

**Buffer systems Tear substitutes**

Phosphate buffer (P) Lacrisifi®

*Buffer systems in the study of tear substitutes.*

**Table 2.**

medium volume at 37°C in a CO2 incubator in an 5% CO2 atmosphere. Cells cultured under standard conditions without the addition of drugs were used as control cells. The concentration of tear substitutes for the experiment was selected on the data of clinical use of the test drugs and our own cytotoxicity studies of artificial tears on cell cultures [17]. Cell viability was assessed by their morphology and functional activity using phase-contrast microscopy (FCM) methods, MTT test, and xCELLigence system. The morphology of the cells in the course of their culturing with the test drugs was evaluated using an inverted Nikon Eclipse TS100 microscope equipped with a camera. To evaluate the effect of tear substitutes on the metabolic activity of human corneal epithelial cells by MTT method, the cells were inoculated in 96-well plates in 200 μl of the growth medium containing the test drugs and cultured as usual for 2 days. After the culturing period, MTT test was performed. The absorbance of the solutions was measured using Fluorofot Charity analyzer (Russia) at a wavelength of 570 nm and a reference wavelength of 630 nm.

Citrate buffer (C) Hylo-Comod®, Hyloparin-Comod®, Hylomax-Comod®, Hylosar-Comod®

Mathematical processing of the data was performed by variation statistics methods using Microsoft Excel 2007. Differences were considered significant at p < 0.05. To evaluate the effect of tear substitutes on the adhesion and proliferative

were inoculated per well of the E-plate in 100 μl of the growth medium containing the test drugs. The plates were placed in the real-time cell analyzer dual purpose (RTCA-DP) (ACEA Biosciences), and adhesion and cell proliferation dynamics was monitored in real time for 24 h. The results were analyzed using RTCA Software 1.2.1 (Roche). The change in impedance at microelectrodes due to cell attachment and spreading was expressed as Cell Index; the value of which is automatically calculated by the program: Cell Index = (RnRb)/t, where Rb is the initial impedance value in the well containing the cell growth medium only (negative control) and Rn is the impedance value at any time t in the well containing the test cells (positive control) in addition to the growth medium. The Cell Index thus reflects changes in the number of cells, the quality of cell attachment, and the morphology of the cells in the well, which may vary over time. The data were presented as the mean value (M) ± standard deviation, the significance of differences was calculated by the Mann-Whitney U-test, and differences were considered significant at p < 0.05.

The MTT test, commonly known as a screening method for measuring cell survival and included in most protocols of molecular biology and medicine [19],

HCE cells

activity of corneal epithelial cells using xCELLigence system, 1 × 104

**6**

**3. Results and discussion**

**3.1 MTT test**

cells, which need these ions for metabolism. Although EDTA does not generally have a pronounced toxic effect, there is evidence that patients with severe DES often complain of discomfort after using drugs containing EDTA [23].

The results of the study indicate the cytotoxic effect of tear substitutes with different chemical groups of preservatives on corneal epithelial cells. In scientific literature, this problem is currently covered quite objectively and completely. In addition to the active ingredient, preservatives, and some other excipients, tear substitutes contain various buffer systems (**Table 2**), which can have an adverse effect on corneal and conjunctival epithelial cells. Information on the comparative toxicity of the buffer systems included in the eye drops is almost not available. Nevertheless, separate reports present data on the occurrence of keratopathy and deposition of calcium hydroxyapatite in a transparent layer of the cornea, after using eye drops containing phosphate buffer [24, 25]. Phosphate-containing tear substitutes are widely used in the composition of ophthalmic dosage forms in EU countries, about a third of all buffered drugs contain phosphates as a buffer. The European Committee on Human Medicinal Products (CHMP) gives preference to the use of phosphate-containing drugs, reasoning that the risks do not exceed adverse reactions that occur during their use, since the proportion of complications is less than 1 case per 10,000 sold vials of tear substitutes. Calcification is a multifactorial complication and can occur without the use of phosphate-containing drugs. Preference in selecting phosphate-containing tear substitutes should be given if it is consistent with the low risk of corneal calcification, especially in serious pathology, and on an individual case basis. It is not currently clear what phosphate concentration is critical for the onset of corneal calcification. Quite recently, preference has been given to borate buffers, which possess antimicrobial activity, showed good biocompatibility with the ocular surface both in vivo and in vitro and are considered safer [26, 27]. Tris buffers are also included in some dosage forms and have been found to be effective and low toxic [28].

As our studies have shown, the viability of human corneal epithelial cells depends, among other things, on the composition of the buffer system used in tear substitutes containing no preservatives. The lowest metabolic activity of cells in the study of a line of preservative-free tear substitutes was observed in the presence of Hylosar-Comod® containing citrate buffer together with dexpanthenol. The pronounced cytotoxic effect of Hylosar-Comod® on corneal epithelial cells can be due to the sensitivity of this test system to the combination of the drug ingredients. This question requires further investigation. A higher level of metabolism in cells was detected in the presence of three preservative-free tear substitutes containing citrate buffer (Hylomax-Comod®, Hyloparin-Comod®, and Hylo-Comod®). The average level of cell viability in this case ranged from 73 to 77%. The preservativefree tear substitutes Hylabak®, Thealoz®, and Thealoz Duo® with Tris buffer showed no toxicity to corneal epithelial cells in our studies (**Figure 1**).

#### **3.2 xCELLigence analysis**

Based on the data obtained in the MTT test, eight products with various types of preservatives were selected from a wide range of tear substitutes for xCELLigence analysis: Artelac® and Blink® (oxidants), Ophtolique® and Systane® Ultra (detergents), Stillavit® (EDTA), Hylo-Comod®, Thealoz Duo®, and Hylosar-Comod® (preservative free). These drugs within their groups showed varying degrees of toxicity to the metabolic activity of corneal epithelial cells. The xCELLigence realtime cell analyzer (RTCA) technology is based on the use of microelectronic cell sensors integrated into the bottom of the wells of special culture plates (E-Plate). The resistance measured between the electrodes in a separate well depends on the

**9**

**Figure 2.**

*xCELLigence cell analysis.*

*In Vitro Cytotoxicity Screening as a Criterion for the Rational Selection of Tear Substitutes*

geometry of the electrode, the concentration of ions in the well, and whether the cells are attached to the electrodes. In the absence of cells, the electrode resistance is mainly determined by the ionic environment both at the electrode/solution interface and in the entire volume. The cells attached to the electrode surfaces act as insulators and thus change the local ionic medium at the electrode/solution interface, which will increase the resistance. Thus, the more cells spread on the electrodes, the higher the resistance of the electrodes. The presence of cells on the electrodes in the wells of the E-Plate affects the local state of the ionic environment, which leads to a change in the resistance on the electrodes. The Cell Index value is an indicator of electrical potential, which reflects the cell status. The Cell Index can be used for real-time monitoring of cell viability: their morphology, degree of adhesion, cell growth (proliferation) dynamics, and other important parameters [12]. Continuous monitoring of the effect of tear substitutes on the HCE cell line in real time using the xCELLigence system revealed that the studied drugs at a concentration of 10% of the growth medium volume manifested varying degrees of toxicity to the cultured cells. In the course of monitoring, Cell Index-cell cultivation time plots were obtained, that made it possible to assess the cell viability by the degree of

As can be seen from the plots, of all the drugs tested using the xCELLigence system, the tear substitute Ophtolique® with BAC has the highest toxicity to the corneal epithelial cells (**Figure 2B**). The Cell Index equal to zero throughout the observation period indicates that cell adhesion has never occurred. In the presence of Artelac® Balance (**Figure 2A**) containing the preservative Oxide®, adhesion occurs within 1 h, but after 5 h, the cells begin to detach from the bottom of the wells, and after 10 h, no viable cells were detected. The xCELLigence analysis revealed an adverse effect of preservative-free Hylosar-Comod® containing citrate buffer with dexpanthenol on the cell culture (**Figure 2D**). This drug turned out to be the most toxic drug from the group of preservative-free tear substitutes; its cytotoxic effect was comparable to that of Blink® containing an oxidative-type preservative (**Figure 2A**). In the presence of these drugs, the cells could only adhere and spread. In the presence of Hylo-Comod® (**Figure 2D**) and Systane® Ultra (**Figure 2B**), the corneal epithelial cells adhered and spread, but their proliferation dynamics was low. Cell proliferation in the presence of Stillavit® (**Figure 2C**) was slightly lower than in the control, and the presence of Thealoz Duo® (**Figure 2D**) was comparable to the control, indicating that Stillavit® was moderately toxic and Thealoz Duo® did not

exhibit toxicity at the given concentration to corneal epithelial cells.

*Monitoring of the effect of tear substitutes with various types of preservatives ((A) oxidants, (B) detergents, (C) EDTA, (D) preservative-free drugs) on the viability of HCE cells in real time (proliferation curves).* 

*DOI: http://dx.doi.org/10.5772/intechopen.85106*

their spreading and proliferative activity (**Figure 2**).

#### *In Vitro Cytotoxicity Screening as a Criterion for the Rational Selection of Tear Substitutes DOI: http://dx.doi.org/10.5772/intechopen.85106*

geometry of the electrode, the concentration of ions in the well, and whether the cells are attached to the electrodes. In the absence of cells, the electrode resistance is mainly determined by the ionic environment both at the electrode/solution interface and in the entire volume. The cells attached to the electrode surfaces act as insulators and thus change the local ionic medium at the electrode/solution interface, which will increase the resistance. Thus, the more cells spread on the electrodes, the higher the resistance of the electrodes. The presence of cells on the electrodes in the wells of the E-Plate affects the local state of the ionic environment, which leads to a change in the resistance on the electrodes. The Cell Index value is an indicator of electrical potential, which reflects the cell status. The Cell Index can be used for real-time monitoring of cell viability: their morphology, degree of adhesion, cell growth (proliferation) dynamics, and other important parameters [12]. Continuous monitoring of the effect of tear substitutes on the HCE cell line in real time using the xCELLigence system revealed that the studied drugs at a concentration of 10% of the growth medium volume manifested varying degrees of toxicity to the cultured cells. In the course of monitoring, Cell Index-cell cultivation time plots were obtained, that made it possible to assess the cell viability by the degree of their spreading and proliferative activity (**Figure 2**).

As can be seen from the plots, of all the drugs tested using the xCELLigence system, the tear substitute Ophtolique® with BAC has the highest toxicity to the corneal epithelial cells (**Figure 2B**). The Cell Index equal to zero throughout the observation period indicates that cell adhesion has never occurred. In the presence of Artelac® Balance (**Figure 2A**) containing the preservative Oxide®, adhesion occurs within 1 h, but after 5 h, the cells begin to detach from the bottom of the wells, and after 10 h, no viable cells were detected. The xCELLigence analysis revealed an adverse effect of preservative-free Hylosar-Comod® containing citrate buffer with dexpanthenol on the cell culture (**Figure 2D**). This drug turned out to be the most toxic drug from the group of preservative-free tear substitutes; its cytotoxic effect was comparable to that of Blink® containing an oxidative-type preservative (**Figure 2A**). In the presence of these drugs, the cells could only adhere and spread. In the presence of Hylo-Comod® (**Figure 2D**) and Systane® Ultra (**Figure 2B**), the corneal epithelial cells adhered and spread, but their proliferation dynamics was low. Cell proliferation in the presence of Stillavit® (**Figure 2C**) was slightly lower than in the control, and the presence of Thealoz Duo® (**Figure 2D**) was comparable to the control, indicating that Stillavit® was moderately toxic and Thealoz Duo® did not exhibit toxicity at the given concentration to corneal epithelial cells.

#### **Figure 2.**

*Monitoring of the effect of tear substitutes with various types of preservatives ((A) oxidants, (B) detergents, (C) EDTA, (D) preservative-free drugs) on the viability of HCE cells in real time (proliferation curves). xCELLigence cell analysis.*

*Cytotoxicity - Definition, Identification, and Cytotoxic Compounds*

be effective and low toxic [28].

**3.2 xCELLigence analysis**

cells, which need these ions for metabolism. Although EDTA does not generally have a pronounced toxic effect, there is evidence that patients with severe DES

The results of the study indicate the cytotoxic effect of tear substitutes with different chemical groups of preservatives on corneal epithelial cells. In scientific literature, this problem is currently covered quite objectively and completely. In addition to the active ingredient, preservatives, and some other excipients, tear substitutes contain various buffer systems (**Table 2**), which can have an adverse effect on corneal and conjunctival epithelial cells. Information on the comparative toxicity of the buffer systems included in the eye drops is almost not available. Nevertheless, separate reports present data on the occurrence of keratopathy and deposition of calcium hydroxyapatite in a transparent layer of the cornea, after using eye drops containing phosphate buffer [24, 25]. Phosphate-containing tear substitutes are widely used in the composition of ophthalmic dosage forms in EU countries, about a third of all buffered drugs contain phosphates as a buffer. The European Committee on Human Medicinal Products (CHMP) gives preference to the use of phosphate-containing drugs, reasoning that the risks do not exceed adverse reactions that occur during their use, since the proportion of complications is less than 1 case per 10,000 sold vials of tear substitutes. Calcification is a multifactorial complication and can occur without the use of phosphate-containing drugs. Preference in selecting phosphate-containing tear substitutes should be given if it is consistent with the low risk of corneal calcification, especially in serious pathology, and on an individual case basis. It is not currently clear what phosphate concentration is critical for the onset of corneal calcification. Quite recently, preference has been given to borate buffers, which possess antimicrobial activity, showed good biocompatibility with the ocular surface both in vivo and in vitro and are considered safer [26, 27]. Tris buffers are also included in some dosage forms and have been found to

As our studies have shown, the viability of human corneal epithelial cells depends, among other things, on the composition of the buffer system used in tear substitutes containing no preservatives. The lowest metabolic activity of cells in the study of a line of preservative-free tear substitutes was observed in the presence of Hylosar-Comod® containing citrate buffer together with dexpanthenol. The pronounced cytotoxic effect of Hylosar-Comod® on corneal epithelial cells can be due to the sensitivity of this test system to the combination of the drug ingredients. This question requires further investigation. A higher level of metabolism in cells was detected in the presence of three preservative-free tear substitutes containing citrate buffer (Hylomax-Comod®, Hyloparin-Comod®, and Hylo-Comod®). The average level of cell viability in this case ranged from 73 to 77%. The preservativefree tear substitutes Hylabak®, Thealoz®, and Thealoz Duo® with Tris buffer

showed no toxicity to corneal epithelial cells in our studies (**Figure 1**).

Based on the data obtained in the MTT test, eight products with various types of preservatives were selected from a wide range of tear substitutes for xCELLigence analysis: Artelac® and Blink® (oxidants), Ophtolique® and Systane® Ultra (detergents), Stillavit® (EDTA), Hylo-Comod®, Thealoz Duo®, and Hylosar-Comod® (preservative free). These drugs within their groups showed varying degrees of toxicity to the metabolic activity of corneal epithelial cells. The xCELLigence realtime cell analyzer (RTCA) technology is based on the use of microelectronic cell sensors integrated into the bottom of the wells of special culture plates (E-Plate). The resistance measured between the electrodes in a separate well depends on the

often complain of discomfort after using drugs containing EDTA [23].

**8**

#### **3.3 Observation of the cells morphology**

The results of observation of the HCE cell morphology during their culturing in media containing 10% PCTs with various preservative systems are shown in **Figure 3**. In the pictures presented, the human corneal cells in the control are well spread, have a typical epithelium-like morphology, and have formed a confluent monolayer on cultivation day 3 (**Figure 3E**).

The morphology of the cells in the presence of the tear substitute Thealoz Duo® is comparable to the control. A slightly less dense monolayer than in the control is formed by the cells in the presence of Stillavit® in the growth medium. With Hylo-Comod®, the monolayer of cells is less dense than in the presence of Thealoz Duo® and Stillavit®. Most of the cells in the presence of the former drug are well spread, but their cytoplasm has a granular structure; a lot of unattached cells are observed. In the presence of the tear substitutes Blink® and Systane® Ultra, the monolayer is formed by about 50%; the cells have a vacuolated granular cytoplasm, which indicates their supressed state. With Hylosar-Comod®, a part of the cells have adhered, and intercellular contacts are found between them; in the presence of Artelac®, only single adherent cells are detected, and in the presence of Ophtolique®, no spread cells are found at all; most of the cells have a rounded shape. The cell structure is granular, with vacuoles; invagination of the cytoplasmic membrane is observed. Many cellular fragments are found in the growth medium. These observations suggest a launch of cell death processes. Thus, the results of the MTT test are consistent with the results of the xCELLigence cell analysis and the cell morphology analysis using phase-contrast microscopy methods.

#### **Figure 3.**

*Morphology of HCE cell line on the third day of culturing in a growth medium containing 10% of the test PCTs with various types of preservatives: oxidants (A, D), detergents (C, F), EDTA (B), and preservative-free drugs (G, H, I). Scale ruler 100 nm (20×). Phase-contrast microscopy.*

**11**

*In Vitro Cytotoxicity Screening as a Criterion for the Rational Selection of Tear Substitutes*

A large number of artificial tears are currently available in the pharmaceutical market. Selecting the right drug for the patient remains a challenge for both the doctor and the patient. This study presents the results of assessing the cytotoxicity of tear substitutes, which demonstrate that these drugs can have a cytostatic effect in vitro and differ in their cytotoxic potential. Comparing the cytotoxicity of artificial tears is necessary for the rational selection of a drug that promotes maximum clinical efficacy and a higher safety profile. The tear substitutes Hylabak®, Thealoz®, and Thealoz Duo® that do not contain preservatives in their composition had not cytotoxic effect on the cells. Vismed® Light containing the preservative polyhexanide was not toxic, either. It was found that the so-called mild preservatives can also have an adverse effect on the ocular surface. Among the artificial tears, the greatest toxic effect on corneal epithelial cells was observed in the tear substitutes Ophtolique®, Lacrisifi®, Hypromellose®, Slezin®, and Cationorm® containing BAC at various concentrations as preservative and in the artificial tears Artelac® Balance (Purite®) and Optive® (Oxide®). The study showed the possibility in principle to use in vitro systems for the comparative assessment of the cytotoxic effect of tear substitutes. It should be noted, however, that the test system under consideration has a number of limitations due to the very nature of the method. In particular, studies on cell cultures cannot take into account such aspects that are important in terms of general toxicology as the route of delivery of a chemical agent into the body, its distribution, elimination, and other toxicodynamics issues. Like with the use of other model test systems, extrapolation of the results obtained to the whole body requires great caution, especially when it comes to quantitative indicators. However, in vitro testing provides information on the potential effects of drugs and their specific effects. Given that the problem of drug therapy of patients with DES has been recently attracting increasing attention of ophthalmologists due to both the increasing prevalence of DES and the increasing range of "artificial tear" drugs, screening the cytotoxicity of a wide range of tear substitutes using test systems based on cell cultures can promote the rational selection of these drugs.

*DOI: http://dx.doi.org/10.5772/intechopen.85106*

**4. Conclusion**

**Conflict of interest**

The author declares no competing interests.

*In Vitro Cytotoxicity Screening as a Criterion for the Rational Selection of Tear Substitutes DOI: http://dx.doi.org/10.5772/intechopen.85106*

#### **4. Conclusion**

*Cytotoxicity - Definition, Identification, and Cytotoxic Compounds*

The results of observation of the HCE cell morphology during their culturing in media containing 10% PCTs with various preservative systems are shown in **Figure 3**. In the pictures presented, the human corneal cells in the control are well spread, have a typical epithelium-like morphology, and have formed a confluent monolayer on

The morphology of the cells in the presence of the tear substitute Thealoz Duo® is comparable to the control. A slightly less dense monolayer than in the control is formed by the cells in the presence of Stillavit® in the growth medium. With Hylo-Comod®, the monolayer of cells is less dense than in the presence of Thealoz Duo® and Stillavit®. Most of the cells in the presence of the former drug are well spread, but their cytoplasm has a granular structure; a lot of unattached cells are observed. In the presence of the tear substitutes Blink® and Systane® Ultra, the monolayer is formed by about 50%; the cells have a vacuolated granular cytoplasm, which indicates their supressed state. With Hylosar-Comod®, a part of the cells have adhered, and intercellular contacts are found between them; in the presence of Artelac®, only single adherent cells are detected, and in the presence of Ophtolique®, no spread cells are found at all; most of the cells have a rounded shape. The cell structure is granular, with vacuoles; invagination of the cytoplasmic membrane is observed. Many cellular fragments are found in the growth medium. These observations suggest a launch of cell death processes. Thus, the results of the MTT test are consistent with the results of the xCELLigence cell analysis and the cell morphology analysis using phase-contrast

*Morphology of HCE cell line on the third day of culturing in a growth medium containing 10% of the test PCTs with various types of preservatives: oxidants (A, D), detergents (C, F), EDTA (B), and preservative-free drugs* 

*(G, H, I). Scale ruler 100 nm (20×). Phase-contrast microscopy.*

**3.3 Observation of the cells morphology**

cultivation day 3 (**Figure 3E**).

microscopy methods.

**10**

**Figure 3.**

A large number of artificial tears are currently available in the pharmaceutical market. Selecting the right drug for the patient remains a challenge for both the doctor and the patient. This study presents the results of assessing the cytotoxicity of tear substitutes, which demonstrate that these drugs can have a cytostatic effect in vitro and differ in their cytotoxic potential. Comparing the cytotoxicity of artificial tears is necessary for the rational selection of a drug that promotes maximum clinical efficacy and a higher safety profile. The tear substitutes Hylabak®, Thealoz®, and Thealoz Duo® that do not contain preservatives in their composition had not cytotoxic effect on the cells. Vismed® Light containing the preservative polyhexanide was not toxic, either. It was found that the so-called mild preservatives can also have an adverse effect on the ocular surface. Among the artificial tears, the greatest toxic effect on corneal epithelial cells was observed in the tear substitutes Ophtolique®, Lacrisifi®, Hypromellose®, Slezin®, and Cationorm® containing BAC at various concentrations as preservative and in the artificial tears Artelac® Balance (Purite®) and Optive® (Oxide®). The study showed the possibility in principle to use in vitro systems for the comparative assessment of the cytotoxic effect of tear substitutes. It should be noted, however, that the test system under consideration has a number of limitations due to the very nature of the method. In particular, studies on cell cultures cannot take into account such aspects that are important in terms of general toxicology as the route of delivery of a chemical agent into the body, its distribution, elimination, and other toxicodynamics issues. Like with the use of other model test systems, extrapolation of the results obtained to the whole body requires great caution, especially when it comes to quantitative indicators. However, in vitro testing provides information on the potential effects of drugs and their specific effects. Given that the problem of drug therapy of patients with DES has been recently attracting increasing attention of ophthalmologists due to both the increasing prevalence of DES and the increasing range of "artificial tear" drugs, screening the cytotoxicity of a wide range of tear substitutes using test systems based on cell cultures can promote the rational selection of these drugs.

#### **Conflict of interest**

The author declares no competing interests.

#### **Author details**

Olga I. Aleksandrova1 \*, Igor N. Okolov2 , Julia I. Khorolskaya1 , Natalia A. Mikhailova1 , Diana M. Darvish1 and Miralda I. Blinova1

1 Institute of Cytology of the Russian Academy of Science, Saint Petersburg, Russia

2 Sv. Fyodorov Eye Microsurgery Federal State Institution, Saint-Petersburg, Russia

\*Address all correspondence to: elga.aleks@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**13**

*In Vitro Cytotoxicity Screening as a Criterion for the Rational Selection of Tear Substitutes*

index. Archives of Ophthalmology.

[8] Management and therapy of dry eye disease: Report of the management and therapy Subcommittee of the International Dry Eye WorkShop (2007). The Ocular Surface.

[9] Danchenko EO. Evaluation of cytotoxicity of pharmaceutical substances using cell cultures. Immunopathology, allergology, infectology=Immunopatologija, Allergologija, Infektologija. 2012;**2**:

[10] Eropkin MJ, Eropkina EM. The Cell Cultures as a Model System Toxicity Studies and Screening of Cytoprotective Drugs. SPb.: Morsar AV; 2003 (in Russ.)

[11] Anikina LV, Puhov SA, Dubrovskaja ES, Afanas'eva SV, Klochkov SG. Comparative determination of cell viability using the MTT and Resazurin. Fundamental Research = Fundamental'nye issledovaniya. 2014;**12**:1423-1427. (In Russ.)

[12] Urcan E, Haertel U, Styllou M, Hickel R, Scherthan H, Reichl F. Real-time xCELLigence impedance analysis of the cytotoxicity of dental composite components on human gingival fibroblasts. Dental Materials. 2010;**26**(1):51-58. DOI: 10.1016/j.

[13] Aleksandrova OI, Okolov IN, Takhtaev YV, Khorolskaya YI, Khintuba TS, Blinova MI. Comparative evaluation of the cytotoxicity of antimicrobial eye drops. Ophthalmological Bulletin.

[14] Aleksandrova OI, Khorolskaya YI, Maychuk DY, Blinova MI. A study of the overall cytotoxicity of aminoglycoside and fluoroquinolone antibiotics on

2015;**8**(1):89-97. (In Russ.)

dental.2009.08.007

2000;**118**:615-621

2007;**5**(2):163-178

22-31. (In Russ.)

*DOI: http://dx.doi.org/10.5772/intechopen.85106*

[1] Baudouin C, Labbe A, Liang H, Pauly A, Brignole-Baudouin F. Preservatives in eye drops: The good, the bad and the ugly. Progress in Retinal and Eye Research. 2010;**9**:312-334. DOI: 10.1016/j.preteyeres.2010.03.001

[2] Whitson J, Petroll W. Corneal epithelial cell viability following exposure to ophthalmic solutions containing preservatives and/or antihypertensive agents. Advances in Therapy. 2012;**29**:874-888. DOI:

10.1007/s12325-012-0057-1

efficacy and toxicity of currently available topical ophthalmic preservatives. Saudi Journal of Ophthalmology.

sjopt.2014.06.006

Preservative-System/

2009;**4**(1):59-64

s12325-010-0070-1

[3] Tu E. Balancing antimicrobial

2014;**28**(3):182-187. DOI: 10.1016/j.

[4] Elder D, Crowley P. Antimicrobial preservatives part one: Choosing a preservative system. American Pharmaceutical Review. 2012. Available from: http://www.

americanpharmaceuticalreview.com/ Featured-Articles/38886-Antimicrobial-Preservatives-Part-One-Choosing-a-

[5] Freeman P, Kahook M. Preservatives in topical ophthalmic medications: Historical and clinical perspectives. Expert Review of Ophthalmology.

[6] Ammar D, Noecker R, Kahook M. Effects of benzalkonium chloridepreserved, polyquad-preserved, and sofZia-preserved topical glaucoma medications on human ocular epithelial cells. Advances in Therapy. 2010;**27**(11):837-845. DOI: 10.1007/

[7] Schiffman R, Christianson M, Jacobsen G, Hirsch J, Reis B. Reliability and validity of the ocular surface disease

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*In Vitro Cytotoxicity Screening as a Criterion for the Rational Selection of Tear Substitutes DOI: http://dx.doi.org/10.5772/intechopen.85106*

#### **References**

*Cytotoxicity - Definition, Identification, and Cytotoxic Compounds*

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

1 Institute of Cytology of the Russian Academy of Science, Saint Petersburg, Russia

2 Sv. Fyodorov Eye Microsurgery Federal State Institution, Saint-Petersburg, Russia

, Julia I. Khorolskaya1

and Miralda I. Blinova1

,

\*, Igor N. Okolov2

\*Address all correspondence to: elga.aleks@gmail.com

, Diana M. Darvish1

**12**

**Author details**

Olga I. Aleksandrova1

Natalia A. Mikhailova1

provided the original work is properly cited.

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[2] Whitson J, Petroll W. Corneal epithelial cell viability following exposure to ophthalmic solutions containing preservatives and/or antihypertensive agents. Advances in Therapy. 2012;**29**:874-888. DOI: 10.1007/s12325-012-0057-1

[3] Tu E. Balancing antimicrobial efficacy and toxicity of currently available topical ophthalmic preservatives. Saudi Journal of Ophthalmology. 2014;**28**(3):182-187. DOI: 10.1016/j. sjopt.2014.06.006

[4] Elder D, Crowley P. Antimicrobial preservatives part one: Choosing a preservative system. American Pharmaceutical Review. 2012. Available from: http://www. americanpharmaceuticalreview.com/ Featured-Articles/38886-Antimicrobial-Preservatives-Part-One-Choosing-a-Preservative-System/

[5] Freeman P, Kahook M. Preservatives in topical ophthalmic medications: Historical and clinical perspectives. Expert Review of Ophthalmology. 2009;**4**(1):59-64

[6] Ammar D, Noecker R, Kahook M. Effects of benzalkonium chloridepreserved, polyquad-preserved, and sofZia-preserved topical glaucoma medications on human ocular epithelial cells. Advances in Therapy. 2010;**27**(11):837-845. DOI: 10.1007/ s12325-010-0070-1

[7] Schiffman R, Christianson M, Jacobsen G, Hirsch J, Reis B. Reliability and validity of the ocular surface disease index. Archives of Ophthalmology. 2000;**118**:615-621

[8] Management and therapy of dry eye disease: Report of the management and therapy Subcommittee of the International Dry Eye WorkShop (2007). The Ocular Surface. 2007;**5**(2):163-178

[9] Danchenko EO. Evaluation of cytotoxicity of pharmaceutical substances using cell cultures. Immunopathology, allergology, infectology=Immunopatologija, Allergologija, Infektologija. 2012;**2**: 22-31. (In Russ.)

[10] Eropkin MJ, Eropkina EM. The Cell Cultures as a Model System Toxicity Studies and Screening of Cytoprotective Drugs. SPb.: Morsar AV; 2003 (in Russ.)

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[12] Urcan E, Haertel U, Styllou M, Hickel R, Scherthan H, Reichl F. Real-time xCELLigence impedance analysis of the cytotoxicity of dental composite components on human gingival fibroblasts. Dental Materials. 2010;**26**(1):51-58. DOI: 10.1016/j. dental.2009.08.007

[13] Aleksandrova OI, Okolov IN, Takhtaev YV, Khorolskaya YI, Khintuba TS, Blinova MI. Comparative evaluation of the cytotoxicity of antimicrobial eye drops. Ophthalmological Bulletin. 2015;**8**(1):89-97. (In Russ.)

[14] Aleksandrova OI, Khorolskaya YI, Maychuk DY, Blinova MI. A study of the overall cytotoxicity of aminoglycoside and fluoroquinolone antibiotics on

cell cultures. Ophthalmology Bulletin. 2015;**5**:39-48. (In Russ.)

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[18] Aleksandrova OI, Okolov IN, Khorolskaya YI, Panova IE, Blinova MI. The effect of non-steroidal antiinflammatory eye drops on corneal and conjunctival epithelial cells in vitro. Ophthalmology. 2017;**15**(3):251-259. DOI: 10.18008/1816-5095-2017-3- 251-259. (In Russ.)

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[21] Noecker R. Effects of common ophthalmic preservatives on ocular health. Advances in Therapy. 2001;**18**(5):205-215

[22] Schrage N, Frentz M, Spoeler F. The ex vivo eye irritation test (EVEIT) in evaluation of artificial tears: Puritepreserved versus unpreserved eye drops. Graefe's Archive for Clinical and Experimental Ophthalmology. 2012;**250**(9):1333-1340. DOI: 10.1007/ s00417-012-1999-3

Chapter 2

Abstract

Steinernema feltiae.

Steinernema feltiae

1. Introduction

15

Study of the Cytotoxic Activity of

Haarlem Oil on Different Cell

Lines and a Higher Organism,

Khairan Khairan,Torsten Burkholz, Mareike Kelkel,

Vincent Jamier, Karl-Herbert Schäfer and Claus Jacob

Keywords: Haarlem oil, cytotoxicity activity, organosulfur compounds,

Haarlem oil (HO) was first introduced in the Netherlands by Thomas Monsieur, a French scientist, as a dietary supplement and stamina aid. In the sixteenth century, a Dutch researcher named Claas Tilly used HO for the first time for the treatment of kidney and urinary stones. HO became increasingly popular in the Netherlands as a health product (Figure 1). HO production at that time continued to increase, eventually attracting the attention of scientists because of its efficacy and usefulness in the fields of health and beauty. In the nineteenth century, HO's effects and uses started to be studied pharmacologically. In 1924, HO was widely traded in France as a food supplement. In the 1980s and 1990s, HO again received

Haarlem oil (HO) is a semisynthetic product made by combining terpene oil and sulfur atoms at high temperatures. HO contains organosulfur compounds; these compounds are known to have strong antioxidant properties, such as superoxide dismutase (SOD). This study provides a brief overview of the effects of HO cytotoxicity on several cell lines using several cytotoxicity test methods. The crystal violet (CV) staining assay showed that HO had a strong toxic effect on the A549 cell line. The test results of the trypan blue and celltiter-Glo assay methods showed that HO has a strong cytotoxic effect on HL-60 cells. The results of the MTT and XTT assays indicated that HO produced a fairly strong toxic effect on HL-60 cells and U937 cells. A hoechst staining assay showed that HO was able to increase (induce) apoptotic cell levels and reduce mitotic cell levels after 24 hours of incubation. However, in this study, we were not able to detect any effect of HO on activation and inhibition of the K562 cell line through the NF-κB pathway. Meanwhile, the live and dead assay showed that HO tends to cause apoptosis. The nematicidal assay showed that HO showed moderate activity against

Steinernema feltiae

[23] Samar K. Basak preservatives and ocular surface diseases. Kerala Journal of Ophthalmology. 2016;**18**(4):311-316

[24] Schrage N, Frentz M, Reim M. Changing the composition of buffered eye-drops prevents undesired side effects. The British Journal of Ophthalmology. 2010;**94**:1519-1522. DOI: 10.1136/bjo.2009.177386

[25] Mueller-Lierheim W. Traenenersatz- und Kontaktlinsenbenetzungsloesungen. In: Köln Biermann, editor. Aktuelle Kontaktologie; 2015. 8-15

[26] Houlsby R, Ghajar M, Chavez G. Antimicrobial activity of boratebuffered solutions. Antimicrobial Agents and Chemotherapy. 1986;**29**:803-806

[27] Lehmann D, Cavet M, Richardson M. Nonclinical safety evaluation of boric acid and a novel borate-buffered contact lens multi-purpose solution, Biotrue™ multi-purpose solution. Contact Lens & Anterior Eye. 2010;**33**(Suppl 1):S24-S32. DOI: 10.1016/j.clae.2010.06.010

[28] Graupner O, Hausmann C. The alternation of the pH in the anterior chamber of the rabbits eye burned with smallest volumes of high concentrated acid and base [in German]. Albrecht von Graefes Archiv für Klinische und Experimentelle Ophthalmologie. 1968;**176**:48-53

#### Chapter 2

*Cytotoxicity - Definition, Identification, and Cytotoxic Compounds*

[22] Schrage N, Frentz M, Spoeler F. The ex vivo eye irritation test (EVEIT) in evaluation of artificial tears: Puritepreserved versus unpreserved eye drops. Graefe's Archive for Clinical and Experimental Ophthalmology. 2012;**250**(9):1333-1340. DOI: 10.1007/

[23] Samar K. Basak preservatives and ocular surface diseases. Kerala Journal of Ophthalmology. 2016;**18**(4):311-316

[24] Schrage N, Frentz M, Reim M. Changing the composition of buffered eye-drops prevents undesired side effects. The British Journal of Ophthalmology. 2010;**94**:1519-1522. DOI: 10.1136/bjo.2009.177386

[25] Mueller-Lierheim W. Traenenersatz- und

Kontaktologie; 2015. 8-15

1986;**29**:803-806

1968;**176**:48-53

Kontaktlinsenbenetzungsloesungen. In: Köln Biermann, editor. Aktuelle

[26] Houlsby R, Ghajar M, Chavez G. Antimicrobial activity of boratebuffered solutions. Antimicrobial Agents and Chemotherapy.

[27] Lehmann D, Cavet M, Richardson M. Nonclinical safety evaluation of boric acid and a novel borate-buffered contact lens multi-purpose solution, Biotrue™ multi-purpose solution. Contact Lens & Anterior Eye. 2010;**33**(Suppl 1):S24-S32.

DOI: 10.1016/j.clae.2010.06.010

[28] Graupner O, Hausmann C. The alternation of the pH in the anterior chamber of the rabbits eye burned with smallest volumes of high concentrated acid and base [in German]. Albrecht von Graefes Archiv für Klinische und Experimentelle Ophthalmologie.

s00417-012-1999-3

cell cultures. Ophthalmology Bulletin.

2015;**5**:39-48. (In Russ.)

(In Russ.)

(In Russ.)

251-259. (In Russ.)

p. 165-169

[15] Aleksandrova OI, Okolov IN, Khorolskaya YI, Blinova MI, Churakov TK. Evaluation of the effect of benzalkonium chloride on the cytotoxicity of Nettacin and Tobrex eye drops in vitro. Modern Technologies in Ophthalmology. 2016;**3**:163-166.

[16] Aleksandrova OI, Okolov IN, Khorolskaya YI, Panova IE, Blinova MI. Possibilities of cellular technologies for rational pharmacotherapy of ocular pathologies. Modern Technologies in Ophthalmology. 2017;**7**:5-7. (In Russ.)

[17] Aleksandrova OI, Okolov IN, Khorolskaya YI, Panova IE, Blinova MI. Evaluation of cytotoxicity of tear substitutes using an in vitro system. Ophthalmology. 2017;**14**(1):59-64. DOI: 10.18008/1816-5095-2017-1-59-66.

[18] Aleksandrova OI, Okolov IN, Khorolskaya YI, Panova IE, Blinova MI. The effect of non-steroidal antiinflammatory eye drops on corneal and conjunctival epithelial cells in vitro. Ophthalmology. 2017;**15**(3):251-259. DOI: 10.18008/1816-5095-2017-3-

[19] Langdon SP, editor. Cancer Cell Culture: Methods and Protocols. Ser. Methods in Molecular Medicine. Vol. 88. Totowa, NJ: Humana Press; 2003.

[20] Epstein S, Ahdoot M, Marcus E, Asbell P. Comparative toxicity of preservatives on immortalized corneal and conjunctival epithelial cells. Journal of Ocular Pharmacology and Therapeutics. 2009;**25**(2):113-119

[21] Noecker R. Effects of common ophthalmic preservatives on ocular health. Advances in Therapy.

2001;**18**(5):205-215

**14**

## Study of the Cytotoxic Activity of Haarlem Oil on Different Cell Lines and a Higher Organism, Steinernema feltiae

Khairan Khairan,Torsten Burkholz, Mareike Kelkel, Vincent Jamier, Karl-Herbert Schäfer and Claus Jacob

### Abstract

Haarlem oil (HO) is a semisynthetic product made by combining terpene oil and sulfur atoms at high temperatures. HO contains organosulfur compounds; these compounds are known to have strong antioxidant properties, such as superoxide dismutase (SOD). This study provides a brief overview of the effects of HO cytotoxicity on several cell lines using several cytotoxicity test methods. The crystal violet (CV) staining assay showed that HO had a strong toxic effect on the A549 cell line. The test results of the trypan blue and celltiter-Glo assay methods showed that HO has a strong cytotoxic effect on HL-60 cells. The results of the MTT and XTT assays indicated that HO produced a fairly strong toxic effect on HL-60 cells and U937 cells. A hoechst staining assay showed that HO was able to increase (induce) apoptotic cell levels and reduce mitotic cell levels after 24 hours of incubation. However, in this study, we were not able to detect any effect of HO on activation and inhibition of the K562 cell line through the NF-κB pathway. Meanwhile, the live and dead assay showed that HO tends to cause apoptosis. The nematicidal assay showed that HO showed moderate activity against Steinernema feltiae.

Keywords: Haarlem oil, cytotoxicity activity, organosulfur compounds, Steinernema feltiae

#### 1. Introduction

Haarlem oil (HO) was first introduced in the Netherlands by Thomas Monsieur, a French scientist, as a dietary supplement and stamina aid. In the sixteenth century, a Dutch researcher named Claas Tilly used HO for the first time for the treatment of kidney and urinary stones. HO became increasingly popular in the Netherlands as a health product (Figure 1). HO production at that time continued to increase, eventually attracting the attention of scientists because of its efficacy and usefulness in the fields of health and beauty. In the nineteenth century, HO's effects and uses started to be studied pharmacologically. In 1924, HO was widely traded in France as a food supplement. In the 1980s and 1990s, HO again received

the attention of scientists because of its sulfur content. At present, HO is produced semi-synthetically from natural products, including terpene oil and sulfur elements, which are combined at high temperatures. Research shows that, in addition to sulfur compounds, HO also contains iminosugars. Iminosugars are compounds containing nitrogen atom bases in their endocyclic structure. It is known that this nitrogen atom is partially responsible for HO's activity. A study of the literature also shows that HO contains polyunsaturated essential oils; polyunsaturated essential oils are known to have strong antioxidant effects, such as in superoxide dismutase (SOD) [1–3].

it is itself a parasite in other parasites, especially insects and their eggs [8]. S. feltiae was selected as a test organism is because this organism is a complex organism and

Study of the Cytotoxic Activity of Haarlem Oil on Different Cell Lines and a Higher…

2. Cytotoxicity studies of Haarlem oil study cytotoxicity by crystal

Crystal violet (CV), or Tris(4-(dimethylamino)phenyl)methylium chloride, is a triarylmethane dye which is used to investigate cell viability responses. CV staining is used mainly in living cell membranes, because this dye is able to bind to proteins and DNA in cells. Cells that die will lose adherence and then disappear from the cell population. Losses from the population of cells reduce the amount of dye staining in a culture, which enables the researcher to count the number of cells in a monolayer culture via absorption of dye by the cells. This test is a simple, fast method of cell viability screening and is useful for acquiring information about relative cell density. CV can also be used to measure the cytotoxicity of a compound [11, 12]. The CV test has major advantages over other cytotoxic tests: in a CV test, after staining, changes in cell morphology can be observed and stored for a long time. At present, research on the effects of HO cytotoxicity is still limited. Therefore, this article provides a brief overview of the effects of HO cytotoxicity on several cell lines such as 3T3, CT26, HT29, A549, HUVEC, MCF7, HepG2, and OVCAR cell lines. At present, HO is sold as a supplement for maintaining stamina and as an antioxidant. HO has been studied previously because of its organosulfur compound content, which may have potential for medical treatments. The mechanism of HO activity against cells is not yet fully understood. Knowledge of the biological activity of HO, especially as related to the behavior of redox-modulators, is also still very

In this study, the results of HO cytotoxicity on several cells are expressed in percentages of cell viability, by measuring the Optical Density (OD) of cells at a wavelength of 590 nm (OD590). OD indicates the absorbance of a sample measured at a specific wavelength, and is a common method used to measure concentration,

To determine the cytotoxicity of HO, we used several levels of HO concentration, from 13.125 to 50 ppm. In this test, we used a 0.1% cytotoxic agent solution of detergent Nonylphenoxypolyethoxy ethanol (NP40). This detergent acts to break down and open membranes in cells, including the membrane's core. Figure 1 shows HO's effect on 3T3, CT26, HT29, A549, HUVEC, MCF7, HepG2, and OVCAR cell

The results show that HO has almost the same toxic effect as NP40. In the OVCAR cell line, HO had the highest toxic effect after 72 hours of incubation, while for the HT29 cell line, HO had a very small effect. HO had relatively similar cytotoxic effects

EC50 (half maximal effective concentration) is the concentration of a drug, compound, antibody, or toxicant capable of inhibiting growth by 50% after exposure for a certain amount of time. The EC50 value of HO on several cell lines was determined using OriginPro 7.5 software. The EC50 values of HO obtained from the CV staining assay method can be seen in Table 1. Table 1 shows that HO had the most active toxic effect on A549 cell lines (adenocarcinomic human alveolar basal epithelial cells), with an EC50 value of 6.30 ppm. In addition, HO also showed a strong cytotoxic effect on CT26 and HepG2 cell lines, with EC50 values of 6.49 and 6.50, respectively. Meanwhile, HO showed EC50 values of 15.19 and 15.20 ppm,

on the 3T3, CT26, A549, HUVEC, MCF7, and HepG2 cell lines (Figure 2).

can be used as a model for whole organisms [9, 10].

DOI: http://dx.doi.org/10.5772/intechopen.85467

growth conditions, and cell reproduction abilities.

respectively, on the HUVEC and 3T3 cell lines [13].

lines at various concentrations.

violet staining assay

limited [1, 12].

17

HO has been reported to be effective in treating rheumatic diseases, bronchitis, and diseases of the liver and kidneys [2, 3]. Histological and bio-clinical tests on mice showed that the sulfo-terpene in HO stimulated the adrenal cortex by causing mice to secrete adrenocorticotropic hormone (ACTH) and eliminating corticosteroids in urine [4]. HO also has strong antiseptic properties, which are thought to originate from its terpene content. Other studies have shown that, when applied to bronchial-pulmonary tissue for 15–60 minutes, HO anti-inflammatory action and increased the action of SOD by increasing the levels of thiol (–SH) group in plasma. Antioxidant activity tests have shown that HO is able to increase SOD enzyme activity. Toxicology and posology tests showed no cases of intoxication in patients given HO at a dose of 2500 mg/kg [5]. X-ray analysis of bronchitis patients treated with HO at a dose of 10 mg/kg for 10 days showed improvements [6].

In the body, sulfur is essential because it functions as a regulatory agent in bile glands, as a stimulator in the respiratory system, and as a toxin neutralizer, and also plays a role in the allergic response. In cells, sulfur plays a role in the synthesis of proteins and also contributes to the synthesis of essential amino acids (such as cysteine and methionine), vitamins (thiamine or vitamin B1, Biotin, and B6), and coenzyme A (CoA), which plays a role in many metabolic processes. Sulfur compounds also play a major role in the prevention of various cancers and infection by microorganisms such as bacteria and fungi.

HO is a natural product containing unusual sulfur compounds, such as thiosulfinate, polysulphane, 1,2-dithiin, and 1,2-dithiole-3-thione. HO also contains several other isothiocyanates. These compounds are also found in many other natural ingredients such as onions (Allium sp.), mustard, and asparagus. Unusual sulfur compounds generally contain reactive sulfur species (RSS), which function as antioxidants and have cytotoxic activities, which give them their anticancer, antibacterial, antifungal, and antisclerodermal effects [7]. This study presents a brief overview of the effects of HO cytotoxicity on several cell lines, using several cytotoxicity test methods such as Crystal Violet (CV), CellTiter-Glo, Trypan Blue, MTT, XTT, NF-κB pathway, Hoechst staining, and Live and Dead assays. This article also provides brief information about HO activity against Steinernema feltiae (S. feltiae). S. feltiae is a microscopic entomopathogenic nematode. This means that

Figure 1. Products of Haarlem oil.

Study of the Cytotoxic Activity of Haarlem Oil on Different Cell Lines and a Higher… DOI: http://dx.doi.org/10.5772/intechopen.85467

it is itself a parasite in other parasites, especially insects and their eggs [8]. S. feltiae was selected as a test organism is because this organism is a complex organism and can be used as a model for whole organisms [9, 10].

#### 2. Cytotoxicity studies of Haarlem oil study cytotoxicity by crystal violet staining assay

Crystal violet (CV), or Tris(4-(dimethylamino)phenyl)methylium chloride, is a triarylmethane dye which is used to investigate cell viability responses. CV staining is used mainly in living cell membranes, because this dye is able to bind to proteins and DNA in cells. Cells that die will lose adherence and then disappear from the cell population. Losses from the population of cells reduce the amount of dye staining in a culture, which enables the researcher to count the number of cells in a monolayer culture via absorption of dye by the cells. This test is a simple, fast method of cell viability screening and is useful for acquiring information about relative cell density. CV can also be used to measure the cytotoxicity of a compound [11, 12].

The CV test has major advantages over other cytotoxic tests: in a CV test, after staining, changes in cell morphology can be observed and stored for a long time. At present, research on the effects of HO cytotoxicity is still limited. Therefore, this article provides a brief overview of the effects of HO cytotoxicity on several cell lines such as 3T3, CT26, HT29, A549, HUVEC, MCF7, HepG2, and OVCAR cell lines.

At present, HO is sold as a supplement for maintaining stamina and as an antioxidant. HO has been studied previously because of its organosulfur compound content, which may have potential for medical treatments. The mechanism of HO activity against cells is not yet fully understood. Knowledge of the biological activity of HO, especially as related to the behavior of redox-modulators, is also still very limited [1, 12].

In this study, the results of HO cytotoxicity on several cells are expressed in percentages of cell viability, by measuring the Optical Density (OD) of cells at a wavelength of 590 nm (OD590). OD indicates the absorbance of a sample measured at a specific wavelength, and is a common method used to measure concentration, growth conditions, and cell reproduction abilities.

To determine the cytotoxicity of HO, we used several levels of HO concentration, from 13.125 to 50 ppm. In this test, we used a 0.1% cytotoxic agent solution of detergent Nonylphenoxypolyethoxy ethanol (NP40). This detergent acts to break down and open membranes in cells, including the membrane's core. Figure 1 shows HO's effect on 3T3, CT26, HT29, A549, HUVEC, MCF7, HepG2, and OVCAR cell lines at various concentrations.

The results show that HO has almost the same toxic effect as NP40. In the OVCAR cell line, HO had the highest toxic effect after 72 hours of incubation, while for the HT29 cell line, HO had a very small effect. HO had relatively similar cytotoxic effects on the 3T3, CT26, A549, HUVEC, MCF7, and HepG2 cell lines (Figure 2).

EC50 (half maximal effective concentration) is the concentration of a drug, compound, antibody, or toxicant capable of inhibiting growth by 50% after exposure for a certain amount of time. The EC50 value of HO on several cell lines was determined using OriginPro 7.5 software. The EC50 values of HO obtained from the CV staining assay method can be seen in Table 1. Table 1 shows that HO had the most active toxic effect on A549 cell lines (adenocarcinomic human alveolar basal epithelial cells), with an EC50 value of 6.30 ppm. In addition, HO also showed a strong cytotoxic effect on CT26 and HepG2 cell lines, with EC50 values of 6.49 and 6.50, respectively. Meanwhile, HO showed EC50 values of 15.19 and 15.20 ppm, respectively, on the HUVEC and 3T3 cell lines [13].

the attention of scientists because of its sulfur content. At present, HO is produced semi-synthetically from natural products, including terpene oil and sulfur elements, which are combined at high temperatures. Research shows that, in addition to sulfur compounds, HO also contains iminosugars. Iminosugars are compounds containing nitrogen atom bases in their endocyclic structure. It is known that this nitrogen atom is partially responsible for HO's activity. A study of the literature also shows that HO contains polyunsaturated essential oils; polyunsaturated essential oils are known to have strong antioxidant effects, such as in superoxide dismutase

Cytotoxicity - Definition, Identification, and Cytotoxic Compounds

HO has been reported to be effective in treating rheumatic diseases, bronchitis, and diseases of the liver and kidneys [2, 3]. Histological and bio-clinical tests on mice showed that the sulfo-terpene in HO stimulated the adrenal cortex by causing mice to secrete adrenocorticotropic hormone (ACTH) and eliminating corticosteroids in urine [4]. HO also has strong antiseptic properties, which are thought to originate from its terpene content. Other studies have shown that, when applied to bronchial-pulmonary tissue for 15–60 minutes, HO anti-inflammatory action and increased the action of SOD by increasing the levels of thiol (–SH) group in plasma. Antioxidant activity tests have shown that HO is able to increase SOD enzyme activity. Toxicology and posology tests showed no cases of intoxication in patients given HO at a dose of 2500 mg/kg [5]. X-ray analysis of bronchitis patients treated

In the body, sulfur is essential because it functions as a regulatory agent in bile glands, as a stimulator in the respiratory system, and as a toxin neutralizer, and also plays a role in the allergic response. In cells, sulfur plays a role in the synthesis of proteins and also contributes to the synthesis of essential amino acids (such as cysteine and methionine), vitamins (thiamine or vitamin B1, Biotin, and B6), and coenzyme A (CoA), which plays a role in many metabolic processes. Sulfur compounds also play a major role in the prevention of various cancers and infection by

with HO at a dose of 10 mg/kg for 10 days showed improvements [6].

HO is a natural product containing unusual sulfur compounds, such as thiosulfinate, polysulphane, 1,2-dithiin, and 1,2-dithiole-3-thione. HO also contains several other isothiocyanates. These compounds are also found in many other natural ingredients such as onions (Allium sp.), mustard, and asparagus. Unusual sulfur compounds generally contain reactive sulfur species (RSS), which function as antioxidants and have cytotoxic activities, which give them their anticancer, antibacterial, antifungal, and antisclerodermal effects [7]. This study presents a brief overview of the effects of HO cytotoxicity on several cell lines, using several cytotoxicity test methods such as Crystal Violet (CV), CellTiter-Glo, Trypan Blue, MTT, XTT, NF-κB pathway, Hoechst staining, and Live and Dead assays. This article also provides brief information about HO activity against Steinernema feltiae (S. feltiae). S. feltiae is a microscopic entomopathogenic nematode. This means that

microorganisms such as bacteria and fungi.

(SOD) [1–3].

Figure 1.

16

Products of Haarlem oil.

#### Cytotoxicity - Definition, Identification, and Cytotoxic Compounds

results also showed that HO was able to reduce the ATP in HL-60 cells at all incubation times [15]. This shows that the reduction in ATP by HO occurs very

Study of the Cytotoxic Activity of Haarlem Oil on Different Cell Lines and a Higher…

Trypan Blue assay is a method used to determine the viability of a cell. The basic principle of this method is that normal cells have intact cell membranes that are able to bind to foreign substances, such as the Trypan Blue dye. In abnormal cells, however, the cell membrane does not have the ability to bind foreign substances onto the cell [16]. In this test, a cell suspension is mixed with Trypan Blue dye, then the cell is observed visually and the viability is calculated using a microscope. Observations were made by examining whether these cells would repel or uptake the dye. In this test, viable cells exhibit clear cytoplasm because their cell membranes cannot be penetrated by the dye, while nonviable cells show blue cytoplasm, because damage to the cell membranes allow them to be easily penetrated by foreign substances such as Trypan Blue. The results showed that HO had a significant cytotoxic effect on cells at all treatment durations and concentrations used. These results also showed that HO at high concentrations (i.e., HO: MilliQ water (1:5) and (1:1)) had a strong effect on the percentage of viability of HL-60 cells, with a percent viability of <10% after 8 hours of incubation. If the incubation time is further extended to 24 and 48 hours, the viability decreases to nearly 0% [15]. These results indicate that, out of all the cells tested, HO had the strongest cytotoxic

The MTT test is a colorimetric, enzyme-based method commonly used to test mitochondrial dehydrogenase activity in cells. This method is frequently used because it is easy, safe, and has a high sensitivity. It is the most commonly used method for testing cell toxicity and viability [17]. The MTT test is used to evaluate the ability of cells to reduce tetrazolium salt or 3-(4, 5-dimethylthiazole-2-yl) 2, 5 diphenyltetrazolium bromide to form insoluble formazan violet crystals. Colored tetrazolium salt, when interacting with cells, will turn purple (formazan). This

color is caused by cells undergoing metabolic reduction by the enzyme

The cytotoxicity effect of HO on HL-60 cell line using several cytotoxicity assays. Data are expressed as the

quickly (Figure 3).

DOI: http://dx.doi.org/10.5772/intechopen.85467

effect on HL-60 cells (Figure 3).

Figure 3.

19

percentage of viability % SD.

#### Figure 2.

The percentage of cell viabilities of 3T3, CT26, HT29, A549, HUVEC, MCF7, HepG2 and OVCAR cell lines after been exposed to HO for 72 hours by CV staining assay. Data are expressed as the percentage of viability % SD.


#### Table 1.

The characteristics and EC50 values of HO on several cell lines were tested with a CV staining assay.

#### 2.1 Study cytotoxicity on HL-60 cell line

The CellTiter-Glo luminescent cell viability assay is a homogeneous method used to determine the number of active cells in cell cultures. This method quantifies cells based on the presence of adenosine triphosphate (ATP) nucleotides in cells. In this method, the presence of ATP is interpreted as an indicator of cell proliferation and an indicator of energy changes in the cell's biological system. ATP is commonly found in living cells because it plays a role in catabolic and anabolic cell processes. The measurement of ATP is fundamental in the study of cells, as the quantities of ATP directly correlate with cell populations [14]. This method uses the enzyme luciferase, which uses ATP to produce luminescence. This luminescence is then measured by the amount of light, or signal, produced, which is strongly correlated to the amount of ATP in the cell population. ATP quantities are highly correlated with the number of living cells [14].

In this method, the test compounds are diluted at certain concentrations and incubated for 8, 24, and 48 hours. The amount of ATP is measured at the end of the incubation time. The results showed that, at a concentration of 1:10, HO was able to reduce ATP in HL-60 cells by 50%, while at a concentration of 1:5, HO was unable to reduce ATP, because the cells were lysed or killed due to a decrease in cell glycogenesis. Glycogenesis is crucial for the formation of ATP; and the decrease in glycogenesis results in the halt of glycogen formation, leading to cell death. The

Study of the Cytotoxic Activity of Haarlem Oil on Different Cell Lines and a Higher… DOI: http://dx.doi.org/10.5772/intechopen.85467

results also showed that HO was able to reduce the ATP in HL-60 cells at all incubation times [15]. This shows that the reduction in ATP by HO occurs very quickly (Figure 3).

Trypan Blue assay is a method used to determine the viability of a cell. The basic principle of this method is that normal cells have intact cell membranes that are able to bind to foreign substances, such as the Trypan Blue dye. In abnormal cells, however, the cell membrane does not have the ability to bind foreign substances onto the cell [16]. In this test, a cell suspension is mixed with Trypan Blue dye, then the cell is observed visually and the viability is calculated using a microscope. Observations were made by examining whether these cells would repel or uptake the dye. In this test, viable cells exhibit clear cytoplasm because their cell membranes cannot be penetrated by the dye, while nonviable cells show blue cytoplasm, because damage to the cell membranes allow them to be easily penetrated by foreign substances such as Trypan Blue. The results showed that HO had a significant cytotoxic effect on cells at all treatment durations and concentrations used. These results also showed that HO at high concentrations (i.e., HO: MilliQ water (1:5) and (1:1)) had a strong effect on the percentage of viability of HL-60 cells, with a percent viability of <10% after 8 hours of incubation. If the incubation time is further extended to 24 and 48 hours, the viability decreases to nearly 0% [15]. These results indicate that, out of all the cells tested, HO had the strongest cytotoxic effect on HL-60 cells (Figure 3).

The MTT test is a colorimetric, enzyme-based method commonly used to test mitochondrial dehydrogenase activity in cells. This method is frequently used because it is easy, safe, and has a high sensitivity. It is the most commonly used method for testing cell toxicity and viability [17]. The MTT test is used to evaluate the ability of cells to reduce tetrazolium salt or 3-(4, 5-dimethylthiazole-2-yl) 2, 5 diphenyltetrazolium bromide to form insoluble formazan violet crystals. Colored tetrazolium salt, when interacting with cells, will turn purple (formazan). This color is caused by cells undergoing metabolic reduction by the enzyme

Figure 3.

The cytotoxicity effect of HO on HL-60 cell line using several cytotoxicity assays. Data are expressed as the percentage of viability % SD.

2.1 Study cytotoxicity on HL-60 cell line

Figure 2.

% SD.

Table 1.

18

with the number of living cells [14].

The CellTiter-Glo luminescent cell viability assay is a homogeneous method used to determine the number of active cells in cell cultures. This method quantifies cells based on the presence of adenosine triphosphate (ATP) nucleotides in cells. In this method, the presence of ATP is interpreted as an indicator of cell proliferation and an indicator of energy changes in the cell's biological system. ATP is commonly found in living cells because it plays a role in catabolic and anabolic cell processes. The measurement of ATP is fundamental in the study of cells, as the quantities of ATP directly correlate with cell populations [14]. This method uses the enzyme luciferase, which uses ATP to produce luminescence. This luminescence is then measured by the amount of light, or signal, produced, which is strongly correlated to the amount of ATP in the cell population. ATP quantities are highly correlated

The characteristics and EC50 values of HO on several cell lines were tested with a CV staining assay.

The percentage of cell viabilities of 3T3, CT26, HT29, A549, HUVEC, MCF7, HepG2 and OVCAR cell lines after been exposed to HO for 72 hours by CV staining assay. Data are expressed as the percentage of viability

Cytotoxicity - Definition, Identification, and Cytotoxic Compounds

Cell line Characteristics EC50 value [ppm] 3T3 Fibroblast cell line 15.19 HUVEC Human umbilical vein endothelial cells line 15.20 CT26 Colon carcinoma cell line 6.49 HT29 Human colon carcinoma cells line >50.0 MCF7 Breast cancer cell line 6.50 A549 Adeno carcinomic human alveolar basal epithelial cells line 6.30 HepG2 Perpetual liver tissue cell line 6.50 OVCAR Human epithelial carcinoma of the ovary cell line 9.07

In this method, the test compounds are diluted at certain concentrations and incubated for 8, 24, and 48 hours. The amount of ATP is measured at the end of the incubation time. The results showed that, at a concentration of 1:10, HO was able to reduce ATP in HL-60 cells by 50%, while at a concentration of 1:5, HO was unable to reduce ATP, because the cells were lysed or killed due to a decrease in cell glycogenesis. Glycogenesis is crucial for the formation of ATP; and the decrease in glycogenesis results in the halt of glycogen formation, leading to cell death. The

dehydrogenase to form NADH or NADPH. It is the absorbance value of this purple color that is measured. This absorbance value is used to determine the cell viability. If the absorbance value observed is smaller than the absorbance value of the control, the cell is undergoing reduction; in other words the cell's ability to proliferate is low. However, on the contrary, if the absorbance produced is higher than the control, the cell's ability to proliferate is very high. If the level of proliferation is too high, however, this can result in cell death, due to potential changes in cell morphology.

wavelength of 490 nm. The results showed that at a concentration of 1:5, HO had a

Study of the Cytotoxic Activity of Haarlem Oil on Different Cell Lines and a Higher…

mitotic cells was quantified as a fraction. The nuclei of apoptotic cells were

treatment with HO at a concentration of 1:10. After each treatment, Hoechst staining was performed for 15 minutes and apoptotic and mitotic cells were counted with the fluorescent microscope. The results showed that HO increased the number of apoptotic cells and reduced the number of mitotic cells after 24 hours of incubation (Figure 5). The Hoechst staining assay is one method to identify cell apoptosis

The U-937 cell line was treated for 4, 8, 16, and 24 hours with HO at a concentration of 1:10. After each treatment, Hoechst staining was performed for 15 minutes. Apoptotic and mitotic cells were counted with the fluorescent microscope, n = 4. Significances are expressed against the control. Data are presented as viability

% SD. Significances: ns = p ≥ 0.05, \* = p < 0.05, \*\* = p < 0.01 and \*\*\* = p < 0.001.

observed under a fluorescent microscope using a specific dye, in this case, Hoechst stain 33,342 (Sigma, Bornem, Belgium). The fraction of cells undergoing apoptosis

Figure 5 shows the ratio of apoptotic and mitotic cells in the U-973 cell line after

Apoptosis is a biological mechanism characterized as "programmed cell death." Apoptosis is used by multicellular organisms to remove cells that are not needed by the body. Apoptosis shows distinctive morphological features, such as plasma membrane blebbing, cell shrinkage, chromatin condensation, and DNA fragmentation. Mitosis is the process of cell division that identically divides genomes into two daughter cells. Mitosis is generally followed by cytokinesis, which divides the cytoplasm and cell membrane. This process produces two identical daughter cells, which have almost the same distribution of organelles and cell components. Mitosis and cytokinesis make up the mitotic phase (M phase) in the cell cycle, where the initial cell is divided into two daughter cells that have the same genetic origin as the initial cell [21]. In testing HO's effect on U-973 cells, the percentage of apoptotic and

cytotoxic effect on the U937 cell line (Figure 4).

DOI: http://dx.doi.org/10.5772/intechopen.85467

were calculated (at least 300 cells).

Figure 5.

21

through cell cycle analysis, mainly sub G1 phase cells.

This test uses HL-60 cells, which are commonly used as models to study the differentiation of myeloid cells in humans [18, 19]. HL-60 cells exposed to HO at concentrations of 1:100, 1:10, and 1:5 with incubation times of 8, 24, and 48 hours are presented in Figure 3. The results indicate that, at a concentration of 1:5, HO was effective at all incubation times, with the percentage of cell viability between 40 and 65%. This means that only 40–65% of cells were able to proliferate. However, at HO concentrations of 1:100 and 1:5, the results were effectively identical, with the percentage of cell viability ranging from 70 to 85% [15] (Figure 3).

#### 2.2 Study cytotoxicity on U-973 cell line

In this section, we tried to determine HO's cytotoxicity on the U-937 cell line using the XTT and Hoechst staining assay methods, as well as testing HO's activation and inhibition of U-937 cells through the NF-κB pathway. U937 cells are human histiocytic lymphoma cells. The principle of the XTT test is the breakdown of the tetrazolium salt into formazan by succinate-tetrazolium reductase in the mitochondria, involving electron transfer by mitochondrial and non-mitochondrial enzymes. Compared to MTT, the XTT test is faster, more reproducible, and gives more sensitive results. The viable cells in the XTT test were measured based on the activity of mitochondrial enzymes in reducing tetrazolium salt [20]. In this test, the U-937 cell line was treated with HO for 6, 24, 48, and 72 hours. 24 hours after the HO treatment, the cells were then treated with XTT for 3 hours (6 hours treatment) or 4 hours (24, 48, and 72 hours treatment) and measured with a plate reader at a

Figure 4.

U937 cell lines were treated with HO using XTT assays. Data are expressed as the percentage of viability % SD.

#### Study of the Cytotoxic Activity of Haarlem Oil on Different Cell Lines and a Higher… DOI: http://dx.doi.org/10.5772/intechopen.85467

wavelength of 490 nm. The results showed that at a concentration of 1:5, HO had a cytotoxic effect on the U937 cell line (Figure 4).

Apoptosis is a biological mechanism characterized as "programmed cell death." Apoptosis is used by multicellular organisms to remove cells that are not needed by the body. Apoptosis shows distinctive morphological features, such as plasma membrane blebbing, cell shrinkage, chromatin condensation, and DNA fragmentation. Mitosis is the process of cell division that identically divides genomes into two daughter cells. Mitosis is generally followed by cytokinesis, which divides the cytoplasm and cell membrane. This process produces two identical daughter cells, which have almost the same distribution of organelles and cell components. Mitosis and cytokinesis make up the mitotic phase (M phase) in the cell cycle, where the initial cell is divided into two daughter cells that have the same genetic origin as the initial cell [21]. In testing HO's effect on U-973 cells, the percentage of apoptotic and mitotic cells was quantified as a fraction. The nuclei of apoptotic cells were observed under a fluorescent microscope using a specific dye, in this case, Hoechst stain 33,342 (Sigma, Bornem, Belgium). The fraction of cells undergoing apoptosis were calculated (at least 300 cells).

Figure 5 shows the ratio of apoptotic and mitotic cells in the U-973 cell line after treatment with HO at a concentration of 1:10. After each treatment, Hoechst staining was performed for 15 minutes and apoptotic and mitotic cells were counted with the fluorescent microscope. The results showed that HO increased the number of apoptotic cells and reduced the number of mitotic cells after 24 hours of incubation (Figure 5). The Hoechst staining assay is one method to identify cell apoptosis through cell cycle analysis, mainly sub G1 phase cells.

#### Figure 5.

dehydrogenase to form NADH or NADPH. It is the absorbance value of this purple color that is measured. This absorbance value is used to determine the cell viability. If the absorbance value observed is smaller than the absorbance value of the control, the cell is undergoing reduction; in other words the cell's ability to proliferate is low. However, on the contrary, if the absorbance produced is higher than the control, the cell's ability to proliferate is very high. If the level of proliferation is too high, however, this can result in cell death, due to potential changes in cell morphology. This test uses HL-60 cells, which are commonly used as models to study the differentiation of myeloid cells in humans [18, 19]. HL-60 cells exposed to HO at concentrations of 1:100, 1:10, and 1:5 with incubation times of 8, 24, and 48 hours are presented in Figure 3. The results indicate that, at a concentration of 1:5, HO was effective at all incubation times, with the percentage of cell viability between 40 and 65%. This means that only 40–65% of cells were able to proliferate. However, at HO concentrations of 1:100 and 1:5, the results were effectively identical, with the percentage of cell viability ranging from 70 to 85% [15] (Figure 3).

Cytotoxicity - Definition, Identification, and Cytotoxic Compounds

In this section, we tried to determine HO's cytotoxicity on the U-937 cell line using the XTT and Hoechst staining assay methods, as well as testing HO's activation and inhibition of U-937 cells through the NF-κB pathway. U937 cells are human histiocytic lymphoma cells. The principle of the XTT test is the breakdown of the tetrazolium salt into formazan by succinate-tetrazolium reductase in the mitochondria, involving electron transfer by mitochondrial and non-mitochondrial enzymes. Compared to MTT, the XTT test is faster, more reproducible, and gives more sensitive results. The viable cells in the XTT test were measured based on the activity of mitochondrial enzymes in reducing tetrazolium salt [20]. In this test, the U-937 cell line was treated with HO for 6, 24, 48, and 72 hours. 24 hours after the HO treatment, the cells were then treated with XTT for 3 hours (6 hours treatment) or 4 hours (24, 48, and 72 hours treatment) and measured with a plate reader at a

U937 cell lines were treated with HO using XTT assays. Data are expressed as the percentage of viability

2.2 Study cytotoxicity on U-973 cell line

Figure 4.

% SD.

20

The U-937 cell line was treated for 4, 8, 16, and 24 hours with HO at a concentration of 1:10. After each treatment, Hoechst staining was performed for 15 minutes. Apoptotic and mitotic cells were counted with the fluorescent microscope, n = 4. Significances are expressed against the control. Data are presented as viability % SD. Significances: ns = p ≥ 0.05, \* = p < 0.05, \*\* = p < 0.01 and \*\*\* = p < 0.001.

#### 2.3 Study cytotoxicity on K562 cell lines

NF-κB, or nuclear factor kappa-light-chain enhancer of activated B cells, is a protein complex that controls the DNA transcription process. NF-κB is an important regulator in determining the fate of a cell, such as apoptosis (programmed cell death), control of cell proliferation, and tumorigenesis. The NF-κB pathway is activated by cell exposure to lipopolysaccharide (LPS), inflammatory cytokines such as TNF (Tumor Necrosis Factor) or IL-1 (Interleukin-1), growth factors, lymphokines, oxidant-free radicals, inhaled particles, viral infection, or expression of certain viral or bacterial gene products, UV irradiation, B or T cell activation, and other physiological and non- physiological stimuli. The best NF-κB activators are proinflammatory cytokines IL-1 and TNF, because they cause phosphorylation of κB on the N-terminus domain side. TNF is an excellent activator for binding to TRADD (TNF-Associated Receptor DEATH Domain Protein) receptors and proteins. TRADD binds TRAF2 (TNF Receptor-Associated Factor-2), which recruits NIK (NF-κB-Inducible Kinase) [22, 23]. In this test, we used TNF to activate NF-κB. The activated NF-κB would then cause the expression of genes that keep cells undergoing proliferation and protect cells from conditions that cause death through apoptosis.

a decrease in the synthesis of IL-6 is regulated by the NF-κB pathway; thus, we suspect that this pathway may be involved, but our results were unable to detect any effect of HO on activation and inhibition of the K562 cell line (Figure 6).

Study of the Cytotoxic Activity of Haarlem Oil on Different Cell Lines and a Higher…

is used to analyze the oxidative effects of stress on compounds [24].

lowing HO exposure at a concentration of 1:5 after 24 hours incubation.

Steinernema feltiae (S. feltiae) is an entomopathogenic nematode used as a 'phytoprotectant' due to its consumption of fly eggs and larvae. Because S. feltiae is

Schematic representation of a live and dead assay using Calcein-AM and PI staining to determine viable and

In this study, testing on the Neuro 2a cell line was carried out using the Live and Dead assay method. In this method, calcein-AM and propidium iodide are used to determine the viability of Neuro 2a cells, a neuroblastoma-derived cell line (Figure 7). There were three reasons for using the Neuro 2a cell line in this study. First, Neuro 2a cells contain GSH at concentrations five times higher than other cell lines, such as PC12 cell line (derived from a transplantable rat pheochromocytoma) [24]. Second, this cell line has expanded to be used as a model to determine the neuronal function of the system. Third, cells from the Neuro 2a cell line are very easy to differentiate using retinoic acid, so that it is structurally close to "real neurons". In this study, the Neuro 2a cell line was used to determine the effect of HO with the presence or absence of Hydrogen peroxide (H2O2). Hydrogen peroxide

HO activity shows that HO at a concentration of 1:1 with or without H2O2 had a moderate toxic effect on Neuro 2a cells, causing a percent viability of about 6%. The morphology of Neuro 2a cell structure after exposure to HO can be seen in Figure 8. The results show that Neuro 2a cells tend to experience apoptosis (bubbling) fol-

2.4 Study cytotoxicity on Neuro 2a cell line

DOI: http://dx.doi.org/10.5772/intechopen.85467

2.5 Activity on Steinernema feltiae

Figure 7.

23

dead cells [25].

The cell type used in this test was a K562 cell line. K562 cells are human chronic myelogenous leukemia lymphoblastoid cells. The K562 cell line was treated with HO at various concentrations for 2 hours, followed by the addition of 20 ng/ml TNF-α for 6 hours. The experiment was repeated five times. The inhibition of the NF-κB pathway in the K562 cell line was analyzed based on the percentage of luciferase enzyme activity.

The results show no detectable effect of HO on the activation and inhibition of the K562 cell line through the NF-κB pathway. According to Keophiphath,

#### Figure 6.

The transfected K562 cells were treated with HO with concentrations of: 1:100, 1:10, and 1:5 for 2 hours, followed by a TNF-α-treatment for 6 hours, n = 5. The vehicle was normalized to a cell viability of 100%. Significances are expressed against the control. Data are presented as viability % SD. Significances: ns = p ≥ 0.05, \* = p < 0.05, \*\* = p < 0.01 and \*\*\* = p < 0.001.

a decrease in the synthesis of IL-6 is regulated by the NF-κB pathway; thus, we suspect that this pathway may be involved, but our results were unable to detect any effect of HO on activation and inhibition of the K562 cell line (Figure 6).

#### 2.4 Study cytotoxicity on Neuro 2a cell line

2.3 Study cytotoxicity on K562 cell lines

Cytotoxicity - Definition, Identification, and Cytotoxic Compounds

apoptosis.

enzyme activity.

Figure 6.

22

NF-κB, or nuclear factor kappa-light-chain enhancer of activated B cells, is a protein complex that controls the DNA transcription process. NF-κB is an important regulator in determining the fate of a cell, such as apoptosis (programmed cell death), control of cell proliferation, and tumorigenesis. The NF-κB pathway is activated by cell exposure to lipopolysaccharide (LPS), inflammatory cytokines such as TNF (Tumor Necrosis Factor) or IL-1 (Interleukin-1), growth factors, lymphokines, oxidant-free radicals, inhaled particles, viral infection, or expression of certain viral or bacterial gene products, UV irradiation, B or T cell activation, and other physiological and non- physiological stimuli. The best NF-κB activators are proinflammatory cytokines IL-1 and TNF, because they cause phosphorylation of κB on the N-terminus domain side. TNF is an excellent activator for binding to TRADD (TNF-Associated Receptor DEATH Domain Protein) receptors and proteins. TRADD binds TRAF2 (TNF Receptor-Associated Factor-2), which recruits NIK (NF-κB-Inducible Kinase) [22, 23]. In this test, we used TNF to activate NF-κB. The activated NF-κB would then cause the expression of genes that keep cells undergoing proliferation and protect cells from conditions that cause death through

The cell type used in this test was a K562 cell line. K562 cells are human chronic myelogenous leukemia lymphoblastoid cells. The K562 cell line was treated with HO at various concentrations for 2 hours, followed by the addition of 20 ng/ml TNF-α for 6 hours. The experiment was repeated five times. The inhibition of the NF-κB pathway in the K562 cell line was analyzed based on the percentage of luciferase

The results show no detectable effect of HO on the activation and inhibition of

the K562 cell line through the NF-κB pathway. According to Keophiphath,

The transfected K562 cells were treated with HO with concentrations of: 1:100, 1:10, and 1:5 for 2 hours, followed by a TNF-α-treatment for 6 hours, n = 5. The vehicle was normalized to a cell viability of 100%. Significances are expressed against the control. Data are presented as viability % SD. Significances:

ns = p ≥ 0.05, \* = p < 0.05, \*\* = p < 0.01 and \*\*\* = p < 0.001.

In this study, testing on the Neuro 2a cell line was carried out using the Live and Dead assay method. In this method, calcein-AM and propidium iodide are used to determine the viability of Neuro 2a cells, a neuroblastoma-derived cell line (Figure 7). There were three reasons for using the Neuro 2a cell line in this study. First, Neuro 2a cells contain GSH at concentrations five times higher than other cell lines, such as PC12 cell line (derived from a transplantable rat pheochromocytoma) [24]. Second, this cell line has expanded to be used as a model to determine the neuronal function of the system. Third, cells from the Neuro 2a cell line are very easy to differentiate using retinoic acid, so that it is structurally close to "real neurons". In this study, the Neuro 2a cell line was used to determine the effect of HO with the presence or absence of Hydrogen peroxide (H2O2). Hydrogen peroxide is used to analyze the oxidative effects of stress on compounds [24].

HO activity shows that HO at a concentration of 1:1 with or without H2O2 had a moderate toxic effect on Neuro 2a cells, causing a percent viability of about 6%. The morphology of Neuro 2a cell structure after exposure to HO can be seen in Figure 8. The results show that Neuro 2a cells tend to experience apoptosis (bubbling) following HO exposure at a concentration of 1:5 after 24 hours incubation.

#### 2.5 Activity on Steinernema feltiae

Steinernema feltiae (S. feltiae) is an entomopathogenic nematode used as a 'phytoprotectant' due to its consumption of fly eggs and larvae. Because S. feltiae is

Figure 7.

Schematic representation of a live and dead assay using Calcein-AM and PI staining to determine viable and dead cells [25].

percentage of 65%. HO concentrations of 1:100 and 1:10 had no significant effect. This shows that increasing concentration of HO will increase the toxicity towards

Effects of HO treatment on S. feltiae after incubation for 24 hours. A) A powder cake of S. feltiae. B) Live

Study of the Cytotoxic Activity of Haarlem Oil on Different Cell Lines and a Higher…

DOI: http://dx.doi.org/10.5772/intechopen.85467

We found that HO has a strong toxic effect on the A549 cell line, with an EC50 value of 6.30 ppm in a CV staining assay. The Trypan Blue and CellTiter-Glo assay showed that HO also has a strong cytotoxic effect on HL-60 cells, especially at concentrations of 1:10 and 1:5. However, the results of the MTT assay showed that HO at a concentration of 1:5 had greater effectiveness than a concentration of 1:10, with a percentage of cell viability between 40 and 65%. The XTT results showed that HO at a concentration of 1:5 had a cytotoxic effect on the U937 cell line. The Hoechst staining assay showed that HO was able to increase (induce) apoptotic cells and reduce mitotic cells after 24 hours of incubation. The results also showed no detectable effect of HO on the activation and inhibition of the K562 cell line through the NF-κB pathway. Meanwhile, after HO exposure at a concentration of 1:5 and 24 hours of incubation, the Neuro 2a cell line tends to activate the apoptotic pathway. The nematicidal test showed that only at a concentration of 1:5 did HO showed

significant activity, with a percentage of S. feltiae viability of 70%.

S. feltiae, which increases the organism's motility.

sample of S. feltiae. C) Dead sample of S. feltiae (red arrow).

3. Conclusion

25

Figure 9.

#### Figure 8.

A) Survival assay of HO on Neuro 2A cells for 24 h on 96-well plate. White bars, the HO was tested in the absence H2O2 and gray bars the HO was tested in the presence H2O2. The control containing water was normalized to 100% viability, significances are expressed to the control. Data presented as viability % SD. Significances: ns p≥0.05, \* p< 0.05, \*\* p < 0.01 and \*\*\* p < 0.001. B) Survival assay of Neuro 2A cells upon treatment with HO. Neuronal cells were plated at a density of 10,000 cells/well in 96-well tissue culture plate. After 24 h, the culture medium containing the HO was replaced with 0.2 ml of dye solution (calcein-AM and PI) and cells were observed under the microscope (Magnification 40 x). Cells treated with HO 1:5.

not a pest but a helpful organism for gardening, these nematodes can be used in normal laboratories without any special security protocols. This nematode is easy to culture, and simple toxicity screens using a normal light microscope are possible [26]. S. feltiae is a standard model for related environmental and agricultural vermin and pests. S. feltiae is normally used by gardeners as a biological defense against garden pests like flies, bugs, snails, and various other organisms. In the past few years, nematodes have grown in popularity as subjects in laboratory experiments. These small organisms are cheap, easy to handle, and they do not require any specific handling. The fact that they are useful animals in the environment with very short lifespans makes them attractive experimental subjects [27].

The effect of HO on Steinernema feltiae (S. feltiae) worms was assessed based on the nematode movement test after 24 hours of incubation. In this study, dimethyl sulfoxide (DMSO) was used as a solvent. In the activity test, this solvent was used as a control. The movement generated in the movement test was compared to the control. The HO concentrations used in this method ranged from 1:100 to 1:5. Due to the low solubility in water, the HO solution was sonicated or, alternatively, centrifuged, before it was applied to the nematodes. The two different preparations showed no toxic effect on S. feltiae (Figure 9). Figure 9 shows that S. feltiae showed the most activity when treated with HO at a concentration of 1:5, with a viability

Study of the Cytotoxic Activity of Haarlem Oil on Different Cell Lines and a Higher… DOI: http://dx.doi.org/10.5772/intechopen.85467

#### Figure 9.

not a pest but a helpful organism for gardening, these nematodes can be used in normal laboratories without any special security protocols. This nematode is easy to culture, and simple toxicity screens using a normal light microscope are possible [26]. S. feltiae is a standard model for related environmental and agricultural vermin and pests. S. feltiae is normally used by gardeners as a biological defense against garden pests like flies, bugs, snails, and various other organisms. In the past few years, nematodes have grown in popularity as subjects in laboratory experiments. These small organisms are cheap, easy to handle, and they do not require any specific handling. The fact that they are useful animals in the environment with

A) Survival assay of HO on Neuro 2A cells for 24 h on 96-well plate. White bars, the HO was tested in the absence H2O2 and gray bars the HO was tested in the presence H2O2. The control containing water was normalized to 100% viability, significances are expressed to the control. Data presented as viability % SD. Significances: ns p≥0.05, \* p< 0.05, \*\* p < 0.01 and \*\*\* p < 0.001. B) Survival assay of Neuro 2A cells upon treatment with HO. Neuronal cells were plated at a density of 10,000 cells/well in 96-well tissue culture plate. After 24 h, the culture medium containing the HO was replaced with 0.2 ml of dye solution (calcein-AM and PI) and cells were observed under the microscope (Magnification 40 x). Cells treated with HO 1:5.

Cytotoxicity - Definition, Identification, and Cytotoxic Compounds

Figure 8.

24

very short lifespans makes them attractive experimental subjects [27].

The effect of HO on Steinernema feltiae (S. feltiae) worms was assessed based on the nematode movement test after 24 hours of incubation. In this study, dimethyl sulfoxide (DMSO) was used as a solvent. In the activity test, this solvent was used as a control. The movement generated in the movement test was compared to the control. The HO concentrations used in this method ranged from 1:100 to 1:5. Due to the low solubility in water, the HO solution was sonicated or, alternatively, centrifuged, before it was applied to the nematodes. The two different preparations showed no toxic effect on S. feltiae (Figure 9). Figure 9 shows that S. feltiae showed the most activity when treated with HO at a concentration of 1:5, with a viability

Effects of HO treatment on S. feltiae after incubation for 24 hours. A) A powder cake of S. feltiae. B) Live sample of S. feltiae. C) Dead sample of S. feltiae (red arrow).

percentage of 65%. HO concentrations of 1:100 and 1:10 had no significant effect. This shows that increasing concentration of HO will increase the toxicity towards S. feltiae, which increases the organism's motility.

#### 3. Conclusion

We found that HO has a strong toxic effect on the A549 cell line, with an EC50 value of 6.30 ppm in a CV staining assay. The Trypan Blue and CellTiter-Glo assay showed that HO also has a strong cytotoxic effect on HL-60 cells, especially at concentrations of 1:10 and 1:5. However, the results of the MTT assay showed that HO at a concentration of 1:5 had greater effectiveness than a concentration of 1:10, with a percentage of cell viability between 40 and 65%. The XTT results showed that HO at a concentration of 1:5 had a cytotoxic effect on the U937 cell line. The Hoechst staining assay showed that HO was able to increase (induce) apoptotic cells and reduce mitotic cells after 24 hours of incubation. The results also showed no detectable effect of HO on the activation and inhibition of the K562 cell line through the NF-κB pathway. Meanwhile, after HO exposure at a concentration of 1:5 and 24 hours of incubation, the Neuro 2a cell line tends to activate the apoptotic pathway. The nematicidal test showed that only at a concentration of 1:5 did HO showed significant activity, with a percentage of S. feltiae viability of 70%.

### Acknowledgements

We would like to thank Dr. J. Lefevre for kindly providing HO for our research.

References

9780470517437

1972;21(5):199-200

[1] Compain P, Martin OR. Iminosugars:

DOI: http://dx.doi.org/10.5772/intechopen.85467

Study of the Cytotoxic Activity of Haarlem Oil on Different Cell Lines and a Higher…

Boca Raton: CRC Press; 1990. 381 p.

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[13] Khairan K, Jamier V, Jacob C. Study cytotoxicity of Haarlem oil by crystal violet staining assay. Research Journal of Chemistry and Environment. 2018;22 (Special Issue II):198-202. E-ISSN:

Brimacombe K, editors. Assay Guidance

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[14] Terry G, Riss L, Richard A, Moravec BS, Andrew LN, Sarah Duellman MS. Cell viability assay. In: Sittampalam GS, Coussens NP,

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[6] Chan BA, Coward JIG.

10.1007/978-3-319-19096-9

[8] Gaugler R, Lewis E, Stuart RJ. Ecology in the service of biological control: The case of entomopathogenic nematodes. Oecologia. 1997;109: 483-489. DOI: 10.1007/s004420050108

[9] Jorgensen LV, Cornett C, Justesen U, Skibsted LH, Dragsted DO. Twoelectron electrochemical oxidation of quercetin and kaempferol changes only the flavonoid C-ring. Free Radical Research. 1998;29:339-350. DOI: 10.1080/10715769800300381

[10] Gaugler R. Entomopathogenic nematodes in biological control. 1st ed.

27

2013;5:565-578

[4] Lefevre JR. Du Boistesselin. Theraphy Journal. 1959;14:1044-1052

[2] Wittop-Koning DA. History of Haarlem oil. Ceskoslovenská Farmacie.

[3] Wittop-Koning DA. Contribution to the history of Haarlem oil. Pharmaceutisch

[5] Jae J, An YP, Lee SY, Kim SH, Lee MJ, Lee MS, et al. Transduced human PEP-1–heat shock protein 27 efficiently protects against brain ischemic insult. FEBS Journal. 2008;275:1296-1308. DOI: 10.1111/j.1742-4658.2008.06291.x

Chemotherapy advances in small-cell lung cancer. Journal of thoracic disease.

[7] Stephen MR, Kehrer JP, Klotz LO. Studies on Experimental Toxicology and Pharmacology. Switzerland: Springer International Publishing; 2015. DOI:

### Conflict of interest

None declared.

### Author details

Khairan Khairan1,2\*, Torsten Burkholz3 , Mareike Kelkel<sup>4</sup> , Vincent Jamier<sup>5</sup> , Karl-Herbert Schäfer<sup>6</sup> and Claus Jacob<sup>7</sup>

1 Department of Pharmacy, Universitas Syiah Kuala, Banda Aceh, Indonesia

2 Pusat Riset Obat Herbal, Universitas Syiah Kuala, Banda Aceh, Indonesia

3 Department of Applied Materials Engineering, Institute of Air Handling and Refrigeration (ILK), Dresden, Germany

4 Laboratoire de Biologie Moléculaireet Cellulaire du Cancer (LBMCC), Hôpital Kirchberg, Luxembourg

5 Leitat Technological Center, Terrassa, Spain

6 Department of Microsystems Technique, University of Applied Sciences, Zweibruecken, Germany

7 School of Pharmacy, Universitaet des Saarlandes, Saarbruecken, Germany

\*Address all correspondence to: khairankhairan@unsyiah.ac.id

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Study of the Cytotoxic Activity of Haarlem Oil on Different Cell Lines and a Higher… DOI: http://dx.doi.org/10.5772/intechopen.85467

#### References

Acknowledgements

Conflict of interest

None declared.

Author details

Khairan Khairan1,2\*, Torsten Burkholz3

Karl-Herbert Schäfer<sup>6</sup> and Claus Jacob<sup>7</sup>

Refrigeration (ILK), Dresden, Germany

5 Leitat Technological Center, Terrassa, Spain

provided the original work is properly cited.

Kirchberg, Luxembourg

Zweibruecken, Germany

26

We would like to thank Dr. J. Lefevre for kindly providing HO for our research.

Cytotoxicity - Definition, Identification, and Cytotoxic Compounds

, Mareike Kelkel<sup>4</sup>

1 Department of Pharmacy, Universitas Syiah Kuala, Banda Aceh, Indonesia

2 Pusat Riset Obat Herbal, Universitas Syiah Kuala, Banda Aceh, Indonesia

3 Department of Applied Materials Engineering, Institute of Air Handling and

4 Laboratoire de Biologie Moléculaireet Cellulaire du Cancer (LBMCC), Hôpital

6 Department of Microsystems Technique, University of Applied Sciences,

7 School of Pharmacy, Universitaet des Saarlandes, Saarbruecken, Germany

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: khairankhairan@unsyiah.ac.id

, Vincent Jamier<sup>5</sup>

,

[1] Compain P, Martin OR. Iminosugars: from synthesis to therapeutic applications. CNRS, University of Orléans, France: John Wiley & Sons; 2007. 457 p. DOI: 10.1002/ 9780470517437

[2] Wittop-Koning DA. History of Haarlem oil. Ceskoslovenská Farmacie. 1972;21(5):199-200

[3] Wittop-Koning DA. Contribution to the history of Haarlem oil. Pharmaceutisch Weekblad. 1972;107:165-172

[4] Lefevre JR. Du Boistesselin. Theraphy Journal. 1959;14:1044-1052

[5] Jae J, An YP, Lee SY, Kim SH, Lee MJ, Lee MS, et al. Transduced human PEP-1–heat shock protein 27 efficiently protects against brain ischemic insult. FEBS Journal. 2008;275:1296-1308. DOI: 10.1111/j.1742-4658.2008.06291.x

[6] Chan BA, Coward JIG. Chemotherapy advances in small-cell lung cancer. Journal of thoracic disease. 2013;5:565-578

[7] Stephen MR, Kehrer JP, Klotz LO. Studies on Experimental Toxicology and Pharmacology. Switzerland: Springer International Publishing; 2015. DOI: 10.1007/978-3-319-19096-9

[8] Gaugler R, Lewis E, Stuart RJ. Ecology in the service of biological control: The case of entomopathogenic nematodes. Oecologia. 1997;109: 483-489. DOI: 10.1007/s004420050108

[9] Jorgensen LV, Cornett C, Justesen U, Skibsted LH, Dragsted DO. Twoelectron electrochemical oxidation of quercetin and kaempferol changes only the flavonoid C-ring. Free Radical Research. 1998;29:339-350. DOI: 10.1080/10715769800300381

[10] Gaugler R. Entomopathogenic nematodes in biological control. 1st ed. Boca Raton: CRC Press; 1990. 381 p. DOI: 10.1201/9781351071741

[11] Feoktistova M, Geserick P, Leverkus M. Crystal violet assay for determining viability of cultured cells. Cold Spring Harbor Protocols. 2016;4:343-346. DOI: 10.1101/pdb.prot087379

[12] Castro-Garza J, Barrios-Garcia HB, Cruz-Vega DE, Said Fernandez S, Carranza-Rosales P, Molina-Torres CA, et al. Use of a colorimetric assay to measure differences in cytotoxicity of Mycobacterium tuberculosis strains. Journal of Medical Microbiology. 2007; 56(6):733-737. DOI: 10.1099/ jmm.0.46915-0

[13] Khairan K, Jamier V, Jacob C. Study cytotoxicity of Haarlem oil by crystal violet staining assay. Research Journal of Chemistry and Environment. 2018;22 (Special Issue II):198-202. E-ISSN: 2278-4527

[14] Terry G, Riss L, Richard A, Moravec BS, Andrew LN, Sarah Duellman MS. Cell viability assay. In: Sittampalam GS, Coussens NP, Brimacombe K, editors. Assay Guidance Manual [Internet]. Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2016. p. 1-31

[15] Bahi M, Jacob C, Khairan K. Cytotoxic effect of Haarlem oil on HL-60 cell line and Steinernema feltiae. Jurnal Kedokteran Hewan. 2016;10(2):109-114. P-ISSN : 1978-225X; E-ISSN : 2502-5600

[16] Shapiro HM. Practical Flow Cytometry. 2nd ed. New York: John Wiley & Sons; 1988. p. 129

[17] Berridge MV, Herst PM, Tan AS. Tetrazolium dyes as tools in cell biology: New insights into their cellular reduction. Biotechnology Annual Review. 2005;11:127-152. DOI: 10.1016/ S1387-2656(05)11004-7

[18] Gallagher R, Collins S, Trujillo J. Characterization of the continuous, differentiating myeloid cell line (HL-60) from a patient with acute promyelocytic leukemia. Blood. 1979; 54(3):713-733

[19] Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods. 1983; 65(1–2):55-63

[20] Roehm NW. An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT. Journal of Immunological Methods. 1991;142:257

[21] Susan E. Apoptosis: A review of programmed cell death. Toxicologic Pathology. 2007;35(4):495-516

[22] Santoro MG, Crisari A, Benedetto A, Amici C. Modulation of the growth of a human Erythroleukemic cell line (K562) by prostaglandins: Antiproliferative action of prostaglandin A1. Cancer Research. 1986;46:6073-6077

[23] Pobezinskaya YL, Liu Z. The role of TRADD in death receptor signaling. Cell Cycle. 2012;11(5):871-876. DOI: 10.4161/cc.11.5.19300

[24] Kaneshiro ES, Wyder MA, Wu YP, Cushion MT. Reliability of calcein acetoxy methyl-ester and ethidium homodimer or propidium iodide for viability assessment of microbes. Journal of Microbiological Methods. 1993;17:1-16. DOI: 10.1016/S0167-7012 (93)80010-4

[25] Calderon FH, Bonnefont A, Munoz FJ, Fernandez V, Videla LA, Inestrosa NC. PC12 and neuro 2a cells have different susceptibilities to acetylcholinesterase-amyloid complexes, amyloid 25-35 fragment, glutamate, and hydrogen peroxide. Journal of Neuroscience Research. 1999; 56:620-631. DOI: 10.1002/(SICI) 1097-4547(19990615)56:6<620::AID-JNR8>3.0.CO;2-F

[26] Clennan EL, Liao C. The hydroperoxysulfoniumylide. An aberration or a ubiquitous intermediate? Tetrahedron. 2006;62:10724-10728. DOI: 10.1016/j.tet.2006.07.111

[27] Buecher EJ, Popiel I. Liquid culture of the Entomogenous nematode Steinernemafeltiae with its bacterial Symbiont. Journal of Nematology. 1989; 21:500-504

**29**

**Chapter 3**

Family

**Abstract**

against different cancer cell lines.

**1. Introduction**

**Keywords:** essential oils, Lamiaceae family, cytotoxic activity

sis. Also, these cells frequently evade the immune system.

vessels also remove waste products from tumors [1].

Cytotoxic Activity of Essential Oils

Cancer is considered one of the most lethal diseases in the world, with a prevalence of 439.2 cases and 163.5 deaths per 100,000 inhabitants, in the period from 2011 to 2015; this disease has a greater impact in underdeveloped countries. For the treatment of this disease, a combination of chemotherapy with surgery or radiation is generally used, however, it is not exempt from adverse effects or resistance of the tumor to this type of treatment, for this reason the search for new treatments is constant. The plants are a possible source to achieve this; Lamiaceae is a family of plants widely distributed on the planet and has been used traditionally for the treatment of different diseases, and various essential oils with the potential for cancer treatment have been isolated from this species. The scope of this review is to present 46 essential oils isolated from different species of Lamiaceae which have been tested

Cancer is a complex disease due to its multiple etiologies, and cancer cells are different to normal cells in many ways. The main characteristics of cancer cells are the cell growth out-of-control in a part of the body that spreads to surrounding tissue, and cancer cells are less specialized than the normal cells. Cancer cells ignore signals that normally tell cells to stop dividing or that begin the process of apopto-

These cells influence the normal cells, molecules, and blood vessels which feed tumors supplying with oxygen and nutrients, which they need to grow. These blood

Cancer has a large global impact; between 2011 and 2015, the number of new cases of cancer was 439.2 per 100,000 habitants, the cancer mortality rate was 163.5

In 2012, approximately 57% of new cancer cases were detected in less developed

per 100,000, and cancer mortality was higher for men than women [2].

countries such as those in Central America and some parts of Africa and Asia,

of Some Species from Lamiaceae

*Cuauhtémoc Pérez-González, Julia Pérez-Ramos,* 

*Carlos Alberto Méndez-Cuesta, Roberto Serrano-Vega,* 

*Miguel Martell-Mendoza and Salud Pérez-Gutiérrez*

#### **Chapter 3**

[18] Gallagher R, Collins S, Trujillo J. Characterization of the continuous, differentiating myeloid cell line (HL-60) from a patient with acute promyelocytic leukemia. Blood. 1979;

Cytotoxicity - Definition, Identification, and Cytotoxic Compounds

56:620-631. DOI: 10.1002/(SICI) 1097-4547(19990615)56:6<620::AID-

[26] Clennan EL, Liao C. The hydroperoxysulfoniumylide. An

aberration or a ubiquitous intermediate? Tetrahedron. 2006;62:10724-10728. DOI: 10.1016/j.tet.2006.07.111

[27] Buecher EJ, Popiel I. Liquid culture of the Entomogenous nematode Steinernemafeltiae with its bacterial Symbiont. Journal of Nematology. 1989;

JNR8>3.0.CO;2-F

21:500-504

[19] Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. Journal of Immunological Methods. 1983;

[20] Roehm NW. An improved

Methods. 1991;142:257

colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT. Journal of Immunological

[21] Susan E. Apoptosis: A review of programmed cell death. Toxicologic Pathology. 2007;35(4):495-516

[22] Santoro MG, Crisari A, Benedetto A, Amici C. Modulation of the growth of a human Erythroleukemic cell line (K562) by prostaglandins: Antiproliferative action of prostaglandin A1. Cancer Research. 1986;46:6073-6077

[23] Pobezinskaya YL, Liu Z. The role of TRADD in death receptor signaling. Cell

[24] Kaneshiro ES, Wyder MA, Wu YP, Cushion MT. Reliability of calcein acetoxy methyl-ester and ethidium homodimer or propidium iodide for viability assessment of microbes. Journal of Microbiological Methods. 1993;17:1-16. DOI: 10.1016/S0167-7012

[25] Calderon FH, Bonnefont A, Munoz FJ, Fernandez V, Videla LA, Inestrosa NC. PC12 and neuro 2a cells have different susceptibilities to acetylcholinesterase-amyloid complexes, amyloid 25-35 fragment, glutamate, and hydrogen peroxide. Journal of Neuroscience Research. 1999;

Cycle. 2012;11(5):871-876. DOI:

10.4161/cc.11.5.19300

(93)80010-4

28

54(3):713-733

65(1–2):55-63

## Cytotoxic Activity of Essential Oils of Some Species from Lamiaceae Family

*Cuauhtémoc Pérez-González, Julia Pérez-Ramos, Carlos Alberto Méndez-Cuesta, Roberto Serrano-Vega, Miguel Martell-Mendoza and Salud Pérez-Gutiérrez*

#### **Abstract**

Cancer is considered one of the most lethal diseases in the world, with a prevalence of 439.2 cases and 163.5 deaths per 100,000 inhabitants, in the period from 2011 to 2015; this disease has a greater impact in underdeveloped countries. For the treatment of this disease, a combination of chemotherapy with surgery or radiation is generally used, however, it is not exempt from adverse effects or resistance of the tumor to this type of treatment, for this reason the search for new treatments is constant. The plants are a possible source to achieve this; Lamiaceae is a family of plants widely distributed on the planet and has been used traditionally for the treatment of different diseases, and various essential oils with the potential for cancer treatment have been isolated from this species. The scope of this review is to present 46 essential oils isolated from different species of Lamiaceae which have been tested against different cancer cell lines.

**Keywords:** essential oils, Lamiaceae family, cytotoxic activity

#### **1. Introduction**

Cancer is a complex disease due to its multiple etiologies, and cancer cells are different to normal cells in many ways. The main characteristics of cancer cells are the cell growth out-of-control in a part of the body that spreads to surrounding tissue, and cancer cells are less specialized than the normal cells. Cancer cells ignore signals that normally tell cells to stop dividing or that begin the process of apoptosis. Also, these cells frequently evade the immune system.

These cells influence the normal cells, molecules, and blood vessels which feed tumors supplying with oxygen and nutrients, which they need to grow. These blood vessels also remove waste products from tumors [1].

Cancer has a large global impact; between 2011 and 2015, the number of new cases of cancer was 439.2 per 100,000 habitants, the cancer mortality rate was 163.5 per 100,000, and cancer mortality was higher for men than women [2].

In 2012, approximately 57% of new cancer cases were detected in less developed countries such as those in Central America and some parts of Africa and Asia,

where 65% of cancer deaths occurred. In 2030, it is expected that the number of new cancer cases will rise to 23.6 million [2].

In 2017, it was estimated that in the USA, national expenditures for cancer care were \$147.3 billion dollars, and the cost will rise with the increase in cancer prevalence and population age.

Currently, many types of cancer treatment are used. Most patients with cancer undergo a combination of treatments, such as surgery with chemotherapy, radiation, immunotherapy, targeted therapy, or hormone therapy. Chemotherapy is one of the most common cancer treatments, but the drugs used produce severe side effects, such as nausea, vomiting, and alopecia, among others, which diminish the quality of life of the patients.

The use of plants in the treatment of many diseases is an ancient practice that has an increased use in recent years. Medicinal plants are a source of compounds with biological activities as anticancer agents, and over 50% of the drugs used in the clinical treatment of cancer, such as Taxol, camptothecin, vincristine, and vinblastine, were obtained from natural sources.

Essential oils (EOs) are a highly complex, volatile, and odorous mixture. The main components are monoterpenes, sesquiterpenes, and aromatic compounds. EOs are obtained mainly by steam distillation [3]. EOs have several activities, such as antimicrobial, anti-inflammatory, bactericidal, antiviral, fungicidal, antiangiogenic, and antitumor activities [4].

The Lamiaceae family comprises 240 genera and 7200 species distributed around the world. Most members of this family are perennial or annual herbs with square stems and are woody shrubs or subshrubs. This family is characterized by aromatic plants, which are widely used as culinary herbs, such as basil, mint, oregano, and sage. Species of this family are important ornamental and medicinal plants and are considered one of the most important sources of EOs of economic importance. In fact, different studies suggest that several EOs obtained from this family have demonstrated cytotoxic activity against different cell cancer lines and could be used as a preventive and alternative treatment for cancer [5]. Several EOs obtained from plants of this family contain high amounts of monoterpenes, such as thymol, carvacrol, 1,8-cineole, and limonene, among others, and the cytotoxic activity of some of these compounds has been studied.

The aim of this review is to provide a critical overview of the research on the traditional medicine basis, cytotoxic properties, cancer cell lines targeted, and composition of EOs isolated from plants belonging to the Lamiaceae family.

#### **2.** *Satureja*

*Satureja* L., which includes approximately 38 species in the Mediterranean region, is used in folk medicine to treat various ailments, such as cramps, muscle pains, nausea, indigestion, diarrhea, and infectious diseases, as well as for its antioxidant, cytotoxic, antidiabetes, anti-HIV, antihyperlipidemic, reproductionstimulating, expectorant, and vasodilatory effects.

*S. cilicica* P.H. Davis is an endemic species of Turkey. EO from aerial parts of *S. cilicica* collected in Turkey was obtained in a yield of 0.69% v/w. The main identified compounds were *p*-cymene (17.68%), carvacrol (14.02%), γ-terpinene (11.23%), and thymol (8.76%). The cytotoxic activity of the EO was determined in the MCF-7 (breast cancer) cell line, revealing an IC50 value of 268 μg/mL [6].

*S. sahandica* Bornm. aerial parts were collected in Iran. The EO yield was 0.52% (w/w). Thymol (40%), γ-terpinene (28%), and ρ-cymene (22%) were the main

**31**

mL, respectively) [12].

**3.** *Nepeta*

*Cytotoxic Activity of Essential Oils of Some Species from Lamiaceae Family*

values of 15.6, 15.6, 125, and 250 μg/mL, respectively [7].

compounds. The cytotoxicity of the EO in MCF-7, Vero, SW480 (adenocarcinoma cell), and JET 3 (choriocarcinoma cells) cell lines was dose-dependent, with IC50

*S. intermedia* L*.* is used to treat diarrhea, nausea, cramps, muscle pain, indigestion, and infectious diseases. A sample was collected in Iran, and the major components were thymol (34.5%), γ-terpinene (18.2%), and *p*-cymene (10.5%). The cytotoxic activity was tested in the 5637 (urinary bladder carcinoma) and KYSE (human Asian squamous cell carcinoma) cell lines, and an IC50 value of 156 μg/mL

*S. intermedia* C.A. Mey EO was obtained from the aerial parts collected in Fars Province, Iran. The main components of the EO were γ-terpinene (37.1%), thymol (30.2%), p-cymene (16.2%), limonene (3.9%), α-terpinene (3.3%), and myrcene (2.5%). The EO showed an IC50 ≥ 50 μg/mL in the Hep-G2 (hepatocellular carcinoma) and MCF-7 cell lines. The effect was evaluated by the crystal violet staining

*S. khuzistanica* Jamzad is used as analgesic and antiseptic in Iran. The aerial parts were collected in southern Iran. The EO was analyzed by GC/FID and GC/MS. The EO yield was 0.42% (w/w), and the main component was carvacrol (92.87%). MTT cytotoxicity assay was employed. The EO reduced the viability of Vero, SW480 (colon adenocarcinoma), MCF-7, and JET 3 cell lines, with IC50 values of 31.2, 62.5,

*S. montana* subsp. *pisidica* L. is used for its antiseptic, aromatic, carminative, digestive, and expectorant properties and in the treatment of insect bites. The major compounds of the EOs obtained from the aerial parts collected in Korab and Galicica were carvacrol, thymol, carvacrol methyl ether, and β-linalool. The cytotoxic effect of the EOs was tested against MDA-MB-361, MDA-MB-453 (human mammary metastatic carcinoma), HeLa, LS174 (human colorectal adenocarcinoma), and MRC5 (fibroblast of lung cells) cell lines. The EO from Korab had higher activity than the oil from Galicica, particularly against the HeLa and MDA-MB-453 cell lines, with IC50 values of 63.5 and 72.3 μg/mL, respectively [11]. *S. bakhtiarica* Bunge. is traditionally used for its antiseptic, carminative, stimulant, diaphoretic, diuretic, anesthetic, antispasmodic, analgesic, antioxidant, sedative, and antimicrobial properties. *S. bakhtiarica* is an endemic plant in the southern region of Iran. Leaves of *S. bakhtiarica* were collected in the Fars Province of Iran. The chemical composition of the EO was determined by GC/MS, and the main components were phenol (56.35%), thymol (13.82%), p-cymene (8.79%), and carvacrol (2.88%). An MTT cytotoxicity assay was used to test the effect of the EO on HEK (human normal embryonic kidney cells), MDA-MB-231, and SKOV3 (human ovary cancer cells) cell lines. The EO showed antitumor activity against the SKOV3 and MDA-MB-231 cell lines (IC50 values of 74.6 μg/mL and IC50 of 83.7 μg/

*N. schiraziana* Boiss possesses medicinal properties such as antitussive, diaphoretic, antispasmodic, antiasthmatic, diuretic, emmenagogue, and antipyretic effects. The aerial parts of *N. schiraziana* were collected in Iran. The compounds

identified in the EO were 1,8-cineole (33.67%), germacrene D (11.45%), β-caryophyllene (9.88%), caryophyllene oxide (7.34%), *α*-pinene (4.59%), and camphor (3.75%). The EO was tested against Hep-G2 (IC50 of 85.74 μg/mL) and

MCF-7 (IC50 of 32.56 μg/mL) cell lines [13].

*DOI: http://dx.doi.org/10.5772/intechopen.86392*

was obtained in both cases [8].

125, and 125 μg/mL, respectively [10].

method [9].

*Cytotoxic Activity of Essential Oils of Some Species from Lamiaceae Family DOI: http://dx.doi.org/10.5772/intechopen.86392*

compounds. The cytotoxicity of the EO in MCF-7, Vero, SW480 (adenocarcinoma cell), and JET 3 (choriocarcinoma cells) cell lines was dose-dependent, with IC50 values of 15.6, 15.6, 125, and 250 μg/mL, respectively [7].

*S. intermedia* L*.* is used to treat diarrhea, nausea, cramps, muscle pain, indigestion, and infectious diseases. A sample was collected in Iran, and the major components were thymol (34.5%), γ-terpinene (18.2%), and *p*-cymene (10.5%). The cytotoxic activity was tested in the 5637 (urinary bladder carcinoma) and KYSE (human Asian squamous cell carcinoma) cell lines, and an IC50 value of 156 μg/mL was obtained in both cases [8].

*S. intermedia* C.A. Mey EO was obtained from the aerial parts collected in Fars Province, Iran. The main components of the EO were γ-terpinene (37.1%), thymol (30.2%), p-cymene (16.2%), limonene (3.9%), α-terpinene (3.3%), and myrcene (2.5%). The EO showed an IC50 ≥ 50 μg/mL in the Hep-G2 (hepatocellular carcinoma) and MCF-7 cell lines. The effect was evaluated by the crystal violet staining method [9].

*S. khuzistanica* Jamzad is used as analgesic and antiseptic in Iran. The aerial parts were collected in southern Iran. The EO was analyzed by GC/FID and GC/MS. The EO yield was 0.42% (w/w), and the main component was carvacrol (92.87%). MTT cytotoxicity assay was employed. The EO reduced the viability of Vero, SW480 (colon adenocarcinoma), MCF-7, and JET 3 cell lines, with IC50 values of 31.2, 62.5, 125, and 125 μg/mL, respectively [10].

*S. montana* subsp. *pisidica* L. is used for its antiseptic, aromatic, carminative, digestive, and expectorant properties and in the treatment of insect bites. The major compounds of the EOs obtained from the aerial parts collected in Korab and Galicica were carvacrol, thymol, carvacrol methyl ether, and β-linalool. The cytotoxic effect of the EOs was tested against MDA-MB-361, MDA-MB-453 (human mammary metastatic carcinoma), HeLa, LS174 (human colorectal adenocarcinoma), and MRC5 (fibroblast of lung cells) cell lines. The EO from Korab had higher activity than the oil from Galicica, particularly against the HeLa and MDA-MB-453 cell lines, with IC50 values of 63.5 and 72.3 μg/mL, respectively [11].

*S. bakhtiarica* Bunge. is traditionally used for its antiseptic, carminative, stimulant, diaphoretic, diuretic, anesthetic, antispasmodic, analgesic, antioxidant, sedative, and antimicrobial properties. *S. bakhtiarica* is an endemic plant in the southern region of Iran. Leaves of *S. bakhtiarica* were collected in the Fars Province of Iran. The chemical composition of the EO was determined by GC/MS, and the main components were phenol (56.35%), thymol (13.82%), p-cymene (8.79%), and carvacrol (2.88%). An MTT cytotoxicity assay was used to test the effect of the EO on HEK (human normal embryonic kidney cells), MDA-MB-231, and SKOV3 (human ovary cancer cells) cell lines. The EO showed antitumor activity against the SKOV3 and MDA-MB-231 cell lines (IC50 values of 74.6 μg/mL and IC50 of 83.7 μg/ mL, respectively) [12].

#### **3.** *Nepeta*

*Cytotoxicity - Definition, Identification, and Cytotoxic Compounds*

new cancer cases will rise to 23.6 million [2].

lence and population age.

quality of life of the patients.

tine, were obtained from natural sources.

activity of some of these compounds has been studied.

stimulating, expectorant, and vasodilatory effects.

genic, and antitumor activities [4].

where 65% of cancer deaths occurred. In 2030, it is expected that the number of

In 2017, it was estimated that in the USA, national expenditures for cancer care were \$147.3 billion dollars, and the cost will rise with the increase in cancer preva-

Currently, many types of cancer treatment are used. Most patients with cancer undergo a combination of treatments, such as surgery with chemotherapy, radiation, immunotherapy, targeted therapy, or hormone therapy. Chemotherapy is one of the most common cancer treatments, but the drugs used produce severe side effects, such as nausea, vomiting, and alopecia, among others, which diminish the

The use of plants in the treatment of many diseases is an ancient practice that has an increased use in recent years. Medicinal plants are a source of compounds with biological activities as anticancer agents, and over 50% of the drugs used in the clinical treatment of cancer, such as Taxol, camptothecin, vincristine, and vinblas-

Essential oils (EOs) are a highly complex, volatile, and odorous mixture. The main components are monoterpenes, sesquiterpenes, and aromatic compounds. EOs are obtained mainly by steam distillation [3]. EOs have several activities, such as antimicrobial, anti-inflammatory, bactericidal, antiviral, fungicidal, antiangio-

The Lamiaceae family comprises 240 genera and 7200 species distributed around the world. Most members of this family are perennial or annual herbs with square stems and are woody shrubs or subshrubs. This family is characterized by aromatic plants, which are widely used as culinary herbs, such as basil, mint, oregano, and sage. Species of this family are important ornamental and medicinal plants and are considered one of the most important sources of EOs of economic importance. In fact, different studies suggest that several EOs obtained from this family have demonstrated cytotoxic activity against different cell cancer lines and could be used as a preventive and alternative treatment for cancer [5]. Several EOs obtained from plants of this family contain high amounts of monoterpenes, such as thymol, carvacrol, 1,8-cineole, and limonene, among others, and the cytotoxic

The aim of this review is to provide a critical overview of the research on the traditional medicine basis, cytotoxic properties, cancer cell lines targeted, and composition of EOs isolated from plants belonging to the Lamiaceae family.

*Satureja* L., which includes approximately 38 species in the Mediterranean region, is used in folk medicine to treat various ailments, such as cramps, muscle pains, nausea, indigestion, diarrhea, and infectious diseases, as well as for its antioxidant, cytotoxic, antidiabetes, anti-HIV, antihyperlipidemic, reproduction-

*S. cilicica* P.H. Davis is an endemic species of Turkey. EO from aerial parts of *S. cilicica* collected in Turkey was obtained in a yield of 0.69% v/w. The main identified compounds were *p*-cymene (17.68%), carvacrol (14.02%), γ-terpinene (11.23%), and thymol (8.76%). The cytotoxic activity of the EO was determined in the MCF-7 (breast cancer) cell line, revealing an IC50 value of 268 μg/mL [6].

*S. sahandica* Bornm. aerial parts were collected in Iran. The EO yield was 0.52% (w/w). Thymol (40%), γ-terpinene (28%), and ρ-cymene (22%) were the main

**30**

**2.** *Satureja*

*N. schiraziana* Boiss possesses medicinal properties such as antitussive, diaphoretic, antispasmodic, antiasthmatic, diuretic, emmenagogue, and antipyretic effects. The aerial parts of *N. schiraziana* were collected in Iran. The compounds identified in the EO were 1,8-cineole (33.67%), germacrene D (11.45%), β-caryophyllene (9.88%), caryophyllene oxide (7.34%), *α*-pinene (4.59%), and camphor (3.75%). The EO was tested against Hep-G2 (IC50 of 85.74 μg/mL) and MCF-7 (IC50 of 32.56 μg/mL) cell lines [13].

*N. rtanjensis* Diklić & Milojević is found only in a few localities of Mt. Rtanj in northeast Serbia. This plant has antibacterial, antifungal, allelopathic, and phytotoxic activities. *N. rtanjensis* was cultivated at the University of Belgrade, Serbia. Chemical analysis revealed that *N. rtanjensis* EO contains *trans*,*cis*-nepetalactone (71.66%), *cis*,*trans*-nepetalactone (17.21%), α-pinene (3.28%), 2-methoxy-paracresol (1.85%), and α-copaene (0.86%). Cytotoxic assays were performed on five tumor cell lines (HeLa, A549, lung adenocarcinoma cells; LS-174, human colon cancer cells; K562, human myelogenous leukemia cells; and MDA-MB-231), and the IC50 values were 0.050, 0.064, 0.097, 0.052, and 0.097 μL/mL, respectively [14].

*N. sintenisii* Bornm. The EO is used in Iranian folk medicine as a diuretic, antitussive, and antispasmodic treatment. The plant was collected in Neyshabur, Khorasan-Razavi, Iran. The yield of EO was 0.5% v/w, and the composition was determined by using GC/MS. The major compounds were 4aα,7α,7aβ-nepetalactone (51.74%), β-farnesene (12.26), 4aα,7α,7aα-nepetalactone (8.01%), germacrene D (5.01%), and 4α,7α,7aα-nepetelactone (3.71%). The cytotoxicity was evaluated against four cell lines, A2780, HeLa, LS180, and MCF-7. The IC50 values were 51.98, 20.37, 42.64, and 43.75 μg/mL, respectively [15].

*N. menthoides* Boiss & Buhse is an herbaceous aromatic plant endemic to northwest Iran and has been used to treat gastrodynia, insomnia, high blood pressure, bone pain, and rheumatism. The aerial parts were collected in Ardabil, Iran. The EO was analyzed by GC/MS. The major components were 4a-α,7β,7a-α-nepetalactone (18.39%), 4a-α,7α,7a-α-nepetalactone (17.57%), 1,8-cineol (16.66%), and geranyl acetate (7.0%). The cytotoxic activity was evaluated against HT-29 (colon carcinoma), Caco-2 (colorectal adenocarcinoma), T47D (breast ductal carcinoma), and NIH-3 T3 cell lines using the MTT method. The IC50 values were 30.7, 19.37, and 32.24 μg/mL, respectively [16].

#### **4.** *Thymus*

The genus *Thymus* consists of approximately 215 species distributed throughout Europe, Asia, and North Africa. Most of these plants are important in food, pharmaceutical, and cosmetic fields. Many species have been investigated for their preservative effects on foods, protecting the food from lipid peroxidation. In traditional medicine, the leaves and flowering aerial parts of *Thymus* species have been used extensively for their tonic, antiseptic, antitussive, and carminative properties in the treatment of colds, coughs, sore throats, cystitis, insomnia, bronchitis, and indigestion.

*T. munbyanus* Boiss. & Reut. is an endemic species of North Africa and is used as an antimicrobial, antioxidant, and antiproliferative agent*.* Fresh aerial parts of *T. munbyanus* were collected in Hennaya, Algeria. The EO was analyzed by GC and GC/MS. The main components of the oil were carvacrol (71%), *p*-cymene (8.3%), and ϒ-terpinene (5.9%). The *T. munbyanus* EO showed antiproliferative activity against the human acute monocytic leukemia cell line (THP-1, 100 μg/mL) using MTT assay [17].

*T. munbyanus* subsp. *coloratus* Boiss. and Reut. has been reported to be effective against cough, colds, influenza, sore throat, abdominal bloating, and endocrine gland diseases and as a depurative agent. Inflorescences and vegetative parts (stems + leaves) of *T. munbyanus* subsp. *coloratus* collected in Algeria yielded 0.2% and 0.1% w/w EO, respectively. The principal components of the EO from flowers were borneol (44.8%), camphor (5.7%), 1,8-cineole (6.0%), and germacrene D (5.0%). The major constituents of the EO from aerial parts were borneol (31.2%), camphor (13.6%), and camphene (7.5%). The cytotoxic activity was tested, and the EO from flowers showed higher activity against the A375 (IC50 of 46.95 μg/mL), T98G (human glioblastoma multiforme; IC50 of 51.54 μg/mL), and MDA-MB-231 (IC50 of 97.27 μg/mL) cell lines than that from leaves and stems. The EO from leaves and

**33**

**6.** *Ocimum*

*Cytotoxic Activity of Essential Oils of Some Species from Lamiaceae Family*

84.77 μg/mL), and A375 (IC50 > 100 μg/mL) cell lines [18].

stems was cytotoxic against T98G (IC50 of 91.83 μg/mL), MDA-MB-231 (IC50 of

*T. carmanicus* Jalas is used in Iranian folk medicine in the treatment of rheumatism and skin disorders and as an antibacterial agent. Aerial parts were collected from Iran, and the EO was obtained in 2.67% w/w yield. The main components were carvacrol (51.0%), thymol (20.84%), borneol (6.80%), cymene (6.25%), γ-terpinene (5.50%), and β-myrcene (1.63%). The IC50 value was 0.44 μL/mL in KB

*T. vulgaris* L. is reported to have antiseptic, antispasmodic, antimicrobial, antioxidant, anti-inflammatory, and anticancer effects. This plant was cultivated in Helwan University Cairo/Egypt. EO was obtained from the fresh and aerial parts with a yield of 0.21% v/w. The major constituents were p-cymene (31.62% v/w), γ-terpenine (17.72% v/w), thymyl methyl ether (9.83% v/w), and thymol (7.38% v/w). The cytotoxic effect of EO was tested in four cell lines: A-549, IC50 of 7.22 μg/mL; HCT-116, IC50 of 3.61 μg/mL; CaCo-2, IC50 of 1.93 μg/mL; and MCF-7, IC50 of 9.52 μg/mL [20].

The genus *Mentha* includes 20 species found all over the world. Most *Mentha* species are perennial, contain essential oils, and are widely cultivated as industrial crops for essential oil production. Many EO chemotypes have a distinct aromatic flavor conferred by different terpene. The whole herb of these species has been used to extract many compounds that have been evaluated as antifungal, antiviral, antimicrobial, insecticidal, antioxidant, antiamoebic, antihemolytic, antiallergenic,

*M. spicata* L. is a medicinal plant, and its EO inhibits free radical reactions, retards the oxidative rancidity of lipids, and shows antimicrobial and antitumor activities. The major compounds in the EO from *M. spicata* collected in China were carvone (65.33%), limonene (18.19%), dihydrocarvone (2.97%), and camphene (2.34%). The cytotoxicity was evaluated in a HeLa cell line, and an IC50 value of

*M. piperita* L. is commonly known as peppermint. It is widely grown in temperate areas of the world, particularly Europe, North America, and North Africa. The EO extracted from its aerial parts was analyzed by GC/MS, and the main component was menthol (47.5%). The cytotoxic activity of the EO was tested against HeLa, A549, and MRC-5 (human fibroblast lung cells) using MTT assay. The IC50 values were 165.24, 183.00, and 197.08 μg/mL, respectively. EO was obtained from *M. piperita* collected in Guatemala in a yield of 0.50% w/w. The IC50 values of the EO against the AGS, A375,

*M. pulegium* L. is commonly known as pennyroyal. This plant is traditionally used in the treatment of infectious diseases. Analysis of the EO by GC/MS revealed pulegone (68.7%) as the main component. The cytotoxic activity of the EO was tested against HeLa, A549, and MRC-5 cell lines using MTT assay. The IC50 values of

The genus *Ocimum* includes approximately 150 species, comprising annual and perennial herbs and shrubs native to the tropical and subtropical regions of Asia, Africa, and Central and South America. *Ocimum* species are commercially cultivated aromatic crops in India and other countries for the EO and high-value

and A431 cell lines were 0.35, 0.40, and 0.23 μL/mL, respectively [22].

the EO were 168.58, 253.64, and 189.48 μg/mL, respectively [23].

*DOI: http://dx.doi.org/10.5772/intechopen.86392*

cell line (oral carcinoma) [19].

**5.** *Mentha*

and antitumoral agents.

approximately 2.08 μg/mL was obtained [21].

*Cytotoxic Activity of Essential Oils of Some Species from Lamiaceae Family DOI: http://dx.doi.org/10.5772/intechopen.86392*

stems was cytotoxic against T98G (IC50 of 91.83 μg/mL), MDA-MB-231 (IC50 of 84.77 μg/mL), and A375 (IC50 > 100 μg/mL) cell lines [18].

*T. carmanicus* Jalas is used in Iranian folk medicine in the treatment of rheumatism and skin disorders and as an antibacterial agent. Aerial parts were collected from Iran, and the EO was obtained in 2.67% w/w yield. The main components were carvacrol (51.0%), thymol (20.84%), borneol (6.80%), cymene (6.25%), γ-terpinene (5.50%), and β-myrcene (1.63%). The IC50 value was 0.44 μL/mL in KB cell line (oral carcinoma) [19].

*T. vulgaris* L. is reported to have antiseptic, antispasmodic, antimicrobial, antioxidant, anti-inflammatory, and anticancer effects. This plant was cultivated in Helwan University Cairo/Egypt. EO was obtained from the fresh and aerial parts with a yield of 0.21% v/w. The major constituents were p-cymene (31.62% v/w), γ-terpenine (17.72% v/w), thymyl methyl ether (9.83% v/w), and thymol (7.38% v/w). The cytotoxic effect of EO was tested in four cell lines: A-549, IC50 of 7.22 μg/mL; HCT-116, IC50 of 3.61 μg/mL; CaCo-2, IC50 of 1.93 μg/mL; and MCF-7, IC50 of 9.52 μg/mL [20].

#### **5.** *Mentha*

*Cytotoxicity - Definition, Identification, and Cytotoxic Compounds*

20.37, 42.64, and 43.75 μg/mL, respectively [15].

*N. rtanjensis* Diklić & Milojević is found only in a few localities of Mt. Rtanj in northeast Serbia. This plant has antibacterial, antifungal, allelopathic, and phytotoxic activities. *N. rtanjensis* was cultivated at the University of Belgrade, Serbia. Chemical analysis revealed that *N. rtanjensis* EO contains *trans*,*cis*-nepetalactone (71.66%), *cis*,*trans*-nepetalactone (17.21%), α-pinene (3.28%), 2-methoxy-paracresol (1.85%), and α-copaene (0.86%). Cytotoxic assays were performed on five tumor cell lines (HeLa, A549, lung adenocarcinoma cells; LS-174, human colon cancer cells; K562, human myelogenous leukemia cells; and MDA-MB-231), and the IC50 values were 0.050, 0.064, 0.097, 0.052, and 0.097 μL/mL, respectively [14]. *N. sintenisii* Bornm. The EO is used in Iranian folk medicine as a diuretic, antitussive, and antispasmodic treatment. The plant was collected in Neyshabur, Khorasan-Razavi, Iran. The yield of EO was 0.5% v/w, and the composition was determined by using GC/MS. The major compounds were 4aα,7α,7aβ-nepetalactone (51.74%), β-farnesene (12.26), 4aα,7α,7aα-nepetalactone (8.01%), germacrene D (5.01%), and 4α,7α,7aα-nepetelactone (3.71%). The cytotoxicity was evaluated against four cell lines, A2780, HeLa, LS180, and MCF-7. The IC50 values were 51.98,

*N. menthoides* Boiss & Buhse is an herbaceous aromatic plant endemic to northwest Iran and has been used to treat gastrodynia, insomnia, high blood pressure, bone pain, and rheumatism. The aerial parts were collected in Ardabil, Iran. The EO was analyzed by GC/MS. The major components were 4a-α,7β,7a-α-nepetalactone (18.39%), 4a-α,7α,7a-α-nepetalactone (17.57%), 1,8-cineol (16.66%), and geranyl acetate (7.0%). The cytotoxic activity was evaluated against HT-29 (colon carcinoma), Caco-2 (colorectal adenocarcinoma), T47D (breast ductal carcinoma), and NIH-3 T3 cell lines using the MTT method. The IC50 values were 30.7, 19.37, and 32.24 μg/mL, respectively [16].

The genus *Thymus* consists of approximately 215 species distributed throughout Europe, Asia, and North Africa. Most of these plants are important in food, pharmaceutical, and cosmetic fields. Many species have been investigated for their preservative effects on foods, protecting the food from lipid peroxidation. In traditional medicine, the leaves and flowering aerial parts of *Thymus* species have been used extensively for their tonic, antiseptic, antitussive, and carminative properties in the treatment of colds, coughs, sore throats, cystitis, insomnia, bronchitis, and indigestion. *T. munbyanus* Boiss. & Reut. is an endemic species of North Africa and is used as an antimicrobial, antioxidant, and antiproliferative agent*.* Fresh aerial parts of *T. munbyanus* were collected in Hennaya, Algeria. The EO was analyzed by GC and GC/MS. The main components of the oil were carvacrol (71%), *p*-cymene (8.3%), and ϒ-terpinene (5.9%). The *T. munbyanus* EO showed antiproliferative activity against the human acute monocytic leukemia cell line (THP-1, 100 μg/mL) using MTT assay [17].

*T. munbyanus* subsp. *coloratus* Boiss. and Reut. has been reported to be effective against cough, colds, influenza, sore throat, abdominal bloating, and endocrine gland diseases and as a depurative agent. Inflorescences and vegetative parts (stems + leaves) of *T. munbyanus* subsp. *coloratus* collected in Algeria yielded 0.2% and 0.1% w/w EO, respectively. The principal components of the EO from flowers were borneol (44.8%), camphor (5.7%), 1,8-cineole (6.0%), and germacrene D (5.0%). The major constituents of the EO from aerial parts were borneol (31.2%), camphor (13.6%), and camphene (7.5%). The cytotoxic activity was tested, and the EO from flowers showed higher activity against the A375 (IC50 of 46.95 μg/mL), T98G (human glioblastoma multiforme; IC50 of 51.54 μg/mL), and MDA-MB-231 (IC50 of 97.27 μg/mL) cell lines than that from leaves and stems. The EO from leaves and

**32**

**4.** *Thymus*

The genus *Mentha* includes 20 species found all over the world. Most *Mentha* species are perennial, contain essential oils, and are widely cultivated as industrial crops for essential oil production. Many EO chemotypes have a distinct aromatic flavor conferred by different terpene. The whole herb of these species has been used to extract many compounds that have been evaluated as antifungal, antiviral, antimicrobial, insecticidal, antioxidant, antiamoebic, antihemolytic, antiallergenic, and antitumoral agents.

*M. spicata* L. is a medicinal plant, and its EO inhibits free radical reactions, retards the oxidative rancidity of lipids, and shows antimicrobial and antitumor activities. The major compounds in the EO from *M. spicata* collected in China were carvone (65.33%), limonene (18.19%), dihydrocarvone (2.97%), and camphene (2.34%). The cytotoxicity was evaluated in a HeLa cell line, and an IC50 value of approximately 2.08 μg/mL was obtained [21].

*M. piperita* L. is commonly known as peppermint. It is widely grown in temperate areas of the world, particularly Europe, North America, and North Africa. The EO extracted from its aerial parts was analyzed by GC/MS, and the main component was menthol (47.5%). The cytotoxic activity of the EO was tested against HeLa, A549, and MRC-5 (human fibroblast lung cells) using MTT assay. The IC50 values were 165.24, 183.00, and 197.08 μg/mL, respectively. EO was obtained from *M. piperita* collected in Guatemala in a yield of 0.50% w/w. The IC50 values of the EO against the AGS, A375, and A431 cell lines were 0.35, 0.40, and 0.23 μL/mL, respectively [22].

*M. pulegium* L. is commonly known as pennyroyal. This plant is traditionally used in the treatment of infectious diseases. Analysis of the EO by GC/MS revealed pulegone (68.7%) as the main component. The cytotoxic activity of the EO was tested against HeLa, A549, and MRC-5 cell lines using MTT assay. The IC50 values of the EO were 168.58, 253.64, and 189.48 μg/mL, respectively [23].

#### **6.** *Ocimum*

The genus *Ocimum* includes approximately 150 species, comprising annual and perennial herbs and shrubs native to the tropical and subtropical regions of Asia, Africa, and Central and South America. *Ocimum* species are commercially cultivated aromatic crops in India and other countries for the EO and high-value aromatic chemicals used extensively in food, perfumery, cosmetic and pharmaceutical preparations, and as spices. *Ocimum* species are known for their diverse use in folk medicine for the treatment of various gastric and urinary diseases, insomnia, inflammation, and constipation due to their diverse biological actions, such as carminative, stimulant, antiseptic, antimicrobial, antioxidant, antipyretic, insecticidal, and antispasmodic activities.

*Ocimum basilicum* L. grows in several regions all over the world and is traditionally used to treat anxiousness, grippe, infectious diseases, headaches, coughs, acne, diarrhea, constipation, warts, worms, and kidney malfunction. EO from its leaves has insecticidal, pesticidal, antibacterial, antioxidant, antiviral, antifungal, antiulcer, cytotoxic, and larvicidal activities.

Plants were collected in Guatemala, and the yield of EO was 0.33% w/w. The main components of the EO were methyl cinnamate (70.1%), linalool (17.5%), β-elemene (2.6%), and camphor (1.52%). The IC50 values of the EO against AGS (epithelial gastric adenocarcinoma), A375 (epithelial malignant melanoma), and A431 (epithelial squamous carcinoma) cell lines were 0.39, 0.36, and 0.34 μL/mL, respectively [22].

This oil was also tested against HeLa (cervical adenocarcinoma cells; IC50 of 90.5 μg/mL) and HEp-2 (human epithelioma; IC50 of 96.3 μg/mL) cell lines [24]. In *O basilicum* L. collected in Egypt (yield of 0.85% v/w), the major components were estragole (75.45%), 1,8-cineole (7.56%), linalool (5.01%), trans-anethole (3.72%), and methyleugenol (3.48%). The anticancer activity was assessed in HL-60 (promyelocytic leukemia) and NB4 (acute promyelocytic leukemia) cell lines. The EO was tested at doses of 200 μg/mL in both cell lines and killed 82.33% of HL-60 cells and 73.38% of NB4 cells [25].

*O. canum* Sims. is used in traditional Indian medicinal for treating diabetes, cold, fever, inflammation, and headaches. The EO was obtained from its leaves. The main components were camphor (39.77%), naphtalene (7.37%), valencene (5.80%), α-pinene (5.59%), camphene (5.20%), and caryophyllene (5.62%). The cytotoxic activity of the EO was tested against MCF-7 cells (IC50 of 60 μg/mL) [26].

*O. kilimandscharicum* Guerke, popularly known as "Basil African blue," is a semi-evergreen shrub native to East Africa and used in traditional medicine for the treatment of constipation, abdominal pain, cough, and diarrhea. The chemical composition of the EO was determined by GC/MS, and the main components were camphor (51.81%), 1,8-cineole (20.13%), and limonene (11.23%). The EO was evaluated against OVCAR-03 cell line (human epithelial ovarian adenocarcinoma) using a sulforhodamine B (SRB) colorimetric assay, with an IC50 of 31.90 μg/mL [27].

#### **7.** *Salvia*

*The Salvia* genus comprises more than 960 species, which are known as Sage in folk medicine and have been used in the treatment of different ailments, such as stomach pain, diarrhea, fever, inflammation, headaches, bruises, and sprains.

Aerial parts of these plants usually contain flavonoids, triterpenoids, and essential oils. Diterpenoids are the main compounds in the roots. These compounds show a variety of activities, and different pharmacological models have been used to explain their mechanisms of activity.

*S. officinalis* L. is used in traditional medicine to treat microbial infections, cancer, malaria, and inflammation and to disinfect homes after sickness. This plant was collected in south-central Italy in 2008–2009. The leaves were used to obtain an EO, the composition of which was determined by GC/MS, with a yield of 0.55–2.2% on a dry mass basis. The main components were α-thujone, camphor, borneol,

**35**

**9.** *Origanum*

*Cytotoxic Activity of Essential Oils of Some Species from Lamiaceae Family*

γ-muurolene, and sclareol. The anticancer activity was tested in the cell lines M14, A375, and A2058. The IC50 values were 8.2, 12.1, and 11.7 μg/mL, respectively [28]. *S. macrosiphon* Boiss is used for treating infection; rheumatoid arthritis; chronic

*S. lavandulifolia* Vahl is a medicinal plant native to the Iberian Peninsula. It is used to treat gastric problems and inflammatory disorders. The composition of its EO was determined by GC/MS, and camphor (29.1%) was the main component. The cytotoxic activity of the EO was tested against HeLa, A549, and MRC-5 cell lines using MTT assay. The IC50 values of the EO were 133.56, 140.10, and 131.50

The genus *Lavandula* includes more than 20 species, and the EOs from the species of this genus have been applied in food, pharmaceutical, and agricultural industries as biological products. Four medicinal plants of this genus (*L. vera* DC, *L. angustifolia* Miller, *L. latifolia* Medikus, and *L. hybrida* Rev) were collected in Italy. The EO constituents were analyzed, and the major compounds were as follows: linalool (36.15%), linalyl acetate (17.08%), and terpinen-4-ol (16.13%) in *L. vera* DC; linalool (56.57%) and camphor (10.01%) in *L. angustifolia* Miller; linalool (34.43%), linalyl acetate (24.36%), and camphor (8.84%) in *L. latifolia* Medikus; and linalool (39.24%), linalyl acetate (22.88%), and 1,8-cineole (6.74%) in *L. hybrida* Rev. The EOs were tested in Caco-2 cell line (epithelial colorectal adenocarcinoma), and the

*L. angustifolia* Mill*.* was collected in the southeastern region of Brazil. The yield obtained for its EO was 0.28% (w/w), and the major components were borneol (22.4%), epi-α-muurolol (13.4%), α-bisabolol (13.1%), precocene I (13%), and eucalyptol (7.9%). The cytotoxic activity was tested in the cell line GM07492-A and

*L. angustifolia* Mill was collected in Bulgaria. Analysis of the EO extracted from its aerial parts revealed linalool (40.3%) as the main component. The cytotoxic activity of the EO was tested against HeLa (IC50 of 80.62 μg/mL), A549 (88.90 of

*Origanum* species are herbaceous perennial shrubs native to Europe and North Africa. These plants have aromatic leaves. This genus includes important culinary

*O. onites* Elmalı is used in Turkey as a condiment or aromatic tea, and a sample of this species was collected in Antalya, Turkey. EO was obtained from the herbal parts of the plant. The composition was determined by GC/MS; the main compo-

was observed only at a high concentration (IC50 of 243.7 μg/mL) [32].

μg/mL), and MRC-5 (75.19 of μg/mL) cell lines using MTT assay [30].

nents were carvacrol (24.52%), thymol (15.66%), and linalool (50.53%).

pain; inflammatory, cardiovascular, and cerebrovascular diseases; and as an antioxidant, acetylcholinesterase-inhibiting, antinociceptive, anti-inflammatory, antidepressant, anxiolytic, antitumor, and cytotoxic agent. *S. macrosiphon* was collected in Iran. The aerial parts yielded 0.2% v/w of EO. Analysis of the EO showed that linalool (19%), β-cedrene (14.64%), and β-elemene (13.33%) were the major components. The effect of the EO on the proliferation of cell lines MCF-7, MDA-MB-231, and T47D was assessed, and the IC50 values were 0.155, 0.145, and

*DOI: http://dx.doi.org/10.5772/intechopen.86392*

0.093 μg/mL, respectively [29].

μg/mL, respectively [30].

cytotoxic effect of EOs was very low [31].

plants, such as marjoram and oregano.

**8.** *Lavandula*

#### *Cytotoxic Activity of Essential Oils of Some Species from Lamiaceae Family DOI: http://dx.doi.org/10.5772/intechopen.86392*

γ-muurolene, and sclareol. The anticancer activity was tested in the cell lines M14, A375, and A2058. The IC50 values were 8.2, 12.1, and 11.7 μg/mL, respectively [28].

*S. macrosiphon* Boiss is used for treating infection; rheumatoid arthritis; chronic pain; inflammatory, cardiovascular, and cerebrovascular diseases; and as an antioxidant, acetylcholinesterase-inhibiting, antinociceptive, anti-inflammatory, antidepressant, anxiolytic, antitumor, and cytotoxic agent. *S. macrosiphon* was collected in Iran. The aerial parts yielded 0.2% v/w of EO. Analysis of the EO showed that linalool (19%), β-cedrene (14.64%), and β-elemene (13.33%) were the major components. The effect of the EO on the proliferation of cell lines MCF-7, MDA-MB-231, and T47D was assessed, and the IC50 values were 0.155, 0.145, and 0.093 μg/mL, respectively [29].

*S. lavandulifolia* Vahl is a medicinal plant native to the Iberian Peninsula. It is used to treat gastric problems and inflammatory disorders. The composition of its EO was determined by GC/MS, and camphor (29.1%) was the main component. The cytotoxic activity of the EO was tested against HeLa, A549, and MRC-5 cell lines using MTT assay. The IC50 values of the EO were 133.56, 140.10, and 131.50 μg/mL, respectively [30].

#### **8.** *Lavandula*

*Cytotoxicity - Definition, Identification, and Cytotoxic Compounds*

cidal, and antispasmodic activities.

cer, cytotoxic, and larvicidal activities.

and 73.38% of NB4 cells [25].

to explain their mechanisms of activity.

aromatic chemicals used extensively in food, perfumery, cosmetic and pharmaceutical preparations, and as spices. *Ocimum* species are known for their diverse use in folk medicine for the treatment of various gastric and urinary diseases, insomnia, inflammation, and constipation due to their diverse biological actions, such as carminative, stimulant, antiseptic, antimicrobial, antioxidant, antipyretic, insecti-

*Ocimum basilicum* L. grows in several regions all over the world and is traditionally used to treat anxiousness, grippe, infectious diseases, headaches, coughs, acne, diarrhea, constipation, warts, worms, and kidney malfunction. EO from its leaves has insecticidal, pesticidal, antibacterial, antioxidant, antiviral, antifungal, antiul-

Plants were collected in Guatemala, and the yield of EO was 0.33% w/w. The main components of the EO were methyl cinnamate (70.1%), linalool (17.5%), β-elemene (2.6%), and camphor (1.52%). The IC50 values of the EO against AGS (epithelial gastric adenocarcinoma), A375 (epithelial malignant melanoma), and A431 (epithelial squamous carcinoma) cell lines were 0.39, 0.36, and 0.34 μL/mL, respectively [22]. This oil was also tested against HeLa (cervical adenocarcinoma cells; IC50 of 90.5 μg/mL) and HEp-2 (human epithelioma; IC50 of 96.3 μg/mL) cell lines [24]. In *O basilicum* L. collected in Egypt (yield of 0.85% v/w), the major components were estragole (75.45%), 1,8-cineole (7.56%), linalool (5.01%), trans-anethole (3.72%), and methyleugenol (3.48%). The anticancer activity was assessed in HL-60 (promyelocytic leukemia) and NB4 (acute promyelocytic leukemia) cell lines. The EO was tested at doses of 200 μg/mL in both cell lines and killed 82.33% of HL-60 cells

*O. canum* Sims. is used in traditional Indian medicinal for treating diabetes, cold, fever, inflammation, and headaches. The EO was obtained from its leaves. The main components were camphor (39.77%), naphtalene (7.37%), valencene (5.80%), α-pinene (5.59%), camphene (5.20%), and caryophyllene (5.62%). The cytotoxic

activity of the EO was tested against MCF-7 cells (IC50 of 60 μg/mL) [26]. *O. kilimandscharicum* Guerke, popularly known as "Basil African blue," is a semi-evergreen shrub native to East Africa and used in traditional medicine for the treatment of constipation, abdominal pain, cough, and diarrhea. The chemical composition of the EO was determined by GC/MS, and the main components were camphor (51.81%), 1,8-cineole (20.13%), and limonene (11.23%). The EO was evaluated against OVCAR-03 cell line (human epithelial ovarian adenocarcinoma) using a

sulforhodamine B (SRB) colorimetric assay, with an IC50 of 31.90 μg/mL [27].

*The Salvia* genus comprises more than 960 species, which are known as Sage in folk medicine and have been used in the treatment of different ailments, such as stomach pain, diarrhea, fever, inflammation, headaches, bruises, and sprains. Aerial parts of these plants usually contain flavonoids, triterpenoids, and essential oils. Diterpenoids are the main compounds in the roots. These compounds show a variety of activities, and different pharmacological models have been used

*S. officinalis* L. is used in traditional medicine to treat microbial infections, cancer, malaria, and inflammation and to disinfect homes after sickness. This plant was collected in south-central Italy in 2008–2009. The leaves were used to obtain an EO, the composition of which was determined by GC/MS, with a yield of 0.55–2.2% on a dry mass basis. The main components were α-thujone, camphor, borneol,

**34**

**7.** *Salvia*

The genus *Lavandula* includes more than 20 species, and the EOs from the species of this genus have been applied in food, pharmaceutical, and agricultural industries as biological products. Four medicinal plants of this genus (*L. vera* DC, *L. angustifolia* Miller, *L. latifolia* Medikus, and *L. hybrida* Rev) were collected in Italy. The EO constituents were analyzed, and the major compounds were as follows: linalool (36.15%), linalyl acetate (17.08%), and terpinen-4-ol (16.13%) in *L. vera* DC; linalool (56.57%) and camphor (10.01%) in *L. angustifolia* Miller; linalool (34.43%), linalyl acetate (24.36%), and camphor (8.84%) in *L. latifolia* Medikus; and linalool (39.24%), linalyl acetate (22.88%), and 1,8-cineole (6.74%) in *L. hybrida* Rev. The EOs were tested in Caco-2 cell line (epithelial colorectal adenocarcinoma), and the cytotoxic effect of EOs was very low [31].

*L. angustifolia* Mill*.* was collected in the southeastern region of Brazil. The yield obtained for its EO was 0.28% (w/w), and the major components were borneol (22.4%), epi-α-muurolol (13.4%), α-bisabolol (13.1%), precocene I (13%), and eucalyptol (7.9%). The cytotoxic activity was tested in the cell line GM07492-A and was observed only at a high concentration (IC50 of 243.7 μg/mL) [32].

*L. angustifolia* Mill was collected in Bulgaria. Analysis of the EO extracted from its aerial parts revealed linalool (40.3%) as the main component. The cytotoxic activity of the EO was tested against HeLa (IC50 of 80.62 μg/mL), A549 (88.90 of μg/mL), and MRC-5 (75.19 of μg/mL) cell lines using MTT assay [30].

#### **9.** *Origanum*

*Origanum* species are herbaceous perennial shrubs native to Europe and North Africa. These plants have aromatic leaves. This genus includes important culinary plants, such as marjoram and oregano.

*O. onites* Elmalı is used in Turkey as a condiment or aromatic tea, and a sample of this species was collected in Antalya, Turkey. EO was obtained from the herbal parts of the plant. The composition was determined by GC/MS; the main components were carvacrol (24.52%), thymol (15.66%), and linalool (50.53%).

The cytotoxicity of the EO, thymol, and carvacrol was determined against hepatoma G2 cells (Hep G2), and the IC50 values were 149.12, 53.09, and 60.1 μg/mL, respectively [33].

*O. vulgare* L. is used in traditional medicine for treating colds, indigestion, and upset stomach. The plant was collected in Guatemala, and the yield of EO was 0.66% w/w. The IC50 values of the EO against epithelial gastric adenocarcinoma, epithelial malignant melanoma, and epithelial squamous carcinoma were 0.18, 0.09, and 0.08 μL/mL, respectively [22].

*O. vulgare* L. is commonly known as oregano and *O. majorana* L. as sweet marjoram. The EOs from both species have antioxidant and antimicrobial properties; these plants were collected in Faisalabad, Pakistan, in July–August 2008. The EOs were analyzed by GC and GC/MS. The main components of the EO from *O. vulgare* were terpinen-4-ol (20.9%), linalool (15.7%), linalyl acetate (13.9%), limonene (13.4%), and α-terpineol (8.57%). The major compounds in the EO from *O. majorana* were thymol (21.6%), carvacrol (18.8%), and α-terpineol (8.57%).

The cytotoxic activity of the EO from *O. vulgare* was tested against MCF-7, prostate cancer (LNCaP), and NIH3T3 cell lines. The IC50 values were 70, 85.3, and 300.5 μg/mL, respectively. The IC50 values for the EO from *O. majorana* were 100, 90.1, and 320.3 μg/mL, respectively [34].

The EO obtained from *O. vulgare* collected in Córdoba, Argentina, was analyzed by GC/MS. Analysis of the chemical composition showed carvacrol and thymol as the predominant compounds. The cytotoxicity activity was evaluated in cultured A549 cells, and this oil reduced the viability of the cells (IC50 of 2.25 μg/mL) [35].

*Tetradenia riparia* Hochst Codd is used in traditional medicinal to treat cough, dropsy, diarrhea, fever, headaches, malaria, and toothaches. The main components of its EO were *E*,*E*-farnesol (15%), aromadendrene oxide (14.7%), and dronabinol (11%). The cytotoxic activity was tested against HT29 (IC50 of 6.93 μg/mL), MCF-7 (IC50 of 129.57 μg/mL), HeLa (IC50 of 155 μg/mL), HepG-2 (IC50 of 149.97 μg/mL), glioblastoma (M059; IC50 of 217.97 μg/mL), U343 (IC50 of 221.30 μg/mL), and U251 (IC50 of 109.90 μg/mL) cells using an XTT-based toxicology assay kit [36].

*T. riparia* leaves were collected in Umuarama, Brazil. 9β,13β-Epoxy-7-abietene (1) was isolated from the EO. The cytotoxic activities of the EO and (1) were determined by MTT assay in the MDA-MB-435, HCT-8, SF-295, and HL-60 cell lines. The EO and compound (1), at concentrations of 50 μg/mL and 25 μg/mL, respectively, showed high cytotoxic potential against the cell lines SF-295 (78.06% and 94.80%), HCT-8 (85.00% and 86.54%), and MDA-MB-435 (59.48% and 45.43%) [37].

*Ajuga chamaepitys* L. Schreb grows in the Mediterranean region and is used as a diuretic and emmenagogue. This plant was collected in Rocca Mattei, Italy, and the composition of the EO isolated from the aerial parts was determined by GC and GC/MS. Ethyl linoleate (13.7%), germacrene D (13.4%), kaurene (8.4%), β-pinene (6.8%), and phytol (5.3%) were the major components. The EO had moderate cytotoxic activity against the MDA-MB-231 cell line (IC50 of 36.88 μg/mL) and an IC50 value of 60.48 μg/mL against the human colon carcinoma cell line (HCT116) [38].

*Ziziphora tenuior* L. is used in Jordan for the treatment of stomachache, dysentery, and fever. The EO was obtained in 0.72% yield, and the composition was determined using GC/MS; pulegone (46.8%) and *p*-menth-3-en-8-ol (12.5%) were the major compounds. The EO was tested against HepG2 cell line, and cytotoxicity was determined using MTT assay. The IC50 value obtained for the EO was 1.25 μL/mL [39].

*Sideritis montana L. subsp*. *montana* is used as a diuretic and digestive aid. *S. montana* was collected in Camerino, Italy. The EO (yield of 0.07%) was analyzed using GC/MS, and the major compounds were germacrene D (20.8%), bicyclogermacrene (13.3%), and 8,13-abietadien-18-ol (10.2%). The cytotoxicity was tested

**37**

*Cytotoxic Activity of Essential Oils of Some Species from Lamiaceae Family*

against MDA-MB-231 and HCT116 cell lines with IC50 values of 32.32 and 31.84

*Stachys annua* L. is a perennial herb and small shrub. In the folk medicine of central Italy, its aerial parts have been used as anticatarrhal, antipyretic, tonic, and vulnerary (wound healing) agents. The EO isolated from its aerial parts was analyzed by GC/MS, and the major components were phytol (9.8%), germacrene D (9.2%), spathulenol (8.5%), and bicyclogermacrene (5.8%). The cytotoxic activity of the EO was determined by MTT assay. Analysis of the cytotoxicity against the HCT116, A375, and MDA-MB-231 cell lines showed IC50 values of 23.5, 37.2, and

*Plectranthus amboinicus* Lour Spreng is cultivated in home gardens and is used in India for the treatment of cough, chronic asthma, and hiccough. The leaves of *P. amboinicus* were collected from a medicinal plant garden in Tamil, India. The cytotoxic activity of the EO was tested using MTT assay against the MCF-7 and HT-29

*Zhumeria majdae* Rech. F. & Wendelbo is a medicinal plant endemic to Iran that is under the threat of extinction. This plant has been used as a curative agent for stomachache, flatulence, diarrhea, indigestion, cold, and headache, for wound healing and treatment of painful menstruation, and as an antiseptic. The aerial parts of *Z. majdae* were collected at five locations in Iran (S1, S2, S3, S4, and S5). The major components were trans-linalool oxide (18.7%), linalool (29.6%), and camphor (27.4%) at S1; trans-linalool oxide (28.6%), linalool (24.4%), and camphor (27.2%) at S2; trans-linalool oxide (16.2%), linalool (34.2%), and camphor (27.7%) at S3; trans-linalool oxide (14.6%), linalool (33.9%), and camphor (26.1%) at S4; and trans-linalool oxide (7.6%), linalool (34.6%), and camphor (34.7%) at S5. The cytotoxicity of the EOs was measured using MTT assay against A375 and MCF7 cell lines, with IC50 values of 746 (S1), 666 (S2), 624 (S3), 779 (S4), and 718 (S5) μg/mL and 674 (S1), 717 (S2), 732 (S3), 646 (S4), and 642 (S5), μg/mL, respectively [43]. *Cedronella canariensis* L. Webb & Berthel. (syn. *Dracocephalum canariense* L.) is present in the Canary Islands. It is a perennial herb, sometimes shrubby. The plant is used in traditional medicine as an anticatarrhal, tonic, antimicrobial, analgesic, carminative, diuretic, hypoglycemiant, hypotensive, and anti-inflammatory agent and decongestant of the respiratory tract. The EO was obtained from the aerial parts of *C. canariensis* collected in El Monte de las Mercedes, Canary Islands, Spain, in 2013 (yield of 2.5%). The EO was analyzed by GC/FID and GC/MS; pinocarvone (58.0%) and α-pinene (10.8%) were the main constituents. The cytotoxicity of the EO was evaluated against A345 (IC50 of 4.3 μg/mL), MDA-MB-231 (IC50 of 7.3 μg/

cell lines, and the IC50 values were 53 and 87 μg/mL, respectively [42].

mL), and HCT 116 (IC50 of 11.4 μg/mL) cell lines by MTT assay [44].

*Rosmarinus officinalis* L. EO is used as an antibacterial, cytotoxic, antimutagenic, antioxidant, antiphlogistic, and chemopreventive agent. The EO (yield of 0.23% w/w) was extracted from the plant collected in Guatemala. The IC50 values of the EO against AGS, A375, and A431 cell lines were 0.21, 0.24, and 0.41 μL/mL, respectively [22]. *Teucrium yemense* Delfi. possesses antifungal, antibacterial, larvicidal, antispasmodic, antioxidant, anti-inflammatory, antiulcer, hypoglycemic, antiacetylcholinesterase, and hepatoprotective activities. Its leaves were collected in two different provinces of Yemen: Dhamar (TY-d) and Taiz (TY-t). The EOs were analyzed, and the most abundant constituents of TY-d were (*E*)-caryophyllene (11.2%), α-humulene (4.0%), γ-selinene (5.5%), 7-epi-α-selinene (20.1%), and caryophyllene oxide (20.1%). The major compounds in TY-t were α-pinene (6.6%), (*E*)-caryophyllene (19.1%), α-humulene (6.4%), δ-cadinene (6.5%), caryophyllene oxide (4.3%), α-cadinol (9.5%), and shyobunol (4.6%). TY-d was active against the HT-29 cell line with an IC50 value of 43.7 μg/mL. TY-t was active against the MCF-7 and MDA-MB-231 cell lines (IC50 of 24.4 and 59.9 μg/mL, respectively) [45].

*DOI: http://dx.doi.org/10.5772/intechopen.86392*

μg/mL, respectively [40].

41.5 μg/mL, respectively [41].

*Cytotoxic Activity of Essential Oils of Some Species from Lamiaceae Family DOI: http://dx.doi.org/10.5772/intechopen.86392*

*Cytotoxicity - Definition, Identification, and Cytotoxic Compounds*

μg/mL, respectively [33].

0.09, and 0.08 μL/mL, respectively [22].

90.1, and 320.3 μg/mL, respectively [34].

The cytotoxicity of the EO, thymol, and carvacrol was determined against hepatoma G2 cells (Hep G2), and the IC50 values were 149.12, 53.09, and 60.1

*O. vulgare* L. is used in traditional medicine for treating colds, indigestion, and upset stomach. The plant was collected in Guatemala, and the yield of EO was 0.66% w/w. The IC50 values of the EO against epithelial gastric adenocarcinoma, epithelial malignant melanoma, and epithelial squamous carcinoma were 0.18,

*O. vulgare* L. is commonly known as oregano and *O. majorana* L. as sweet marjoram. The EOs from both species have antioxidant and antimicrobial properties; these plants were collected in Faisalabad, Pakistan, in July–August 2008. The EOs were analyzed by GC and GC/MS. The main components of the EO from *O. vulgare* were terpinen-4-ol (20.9%), linalool (15.7%), linalyl acetate (13.9%), limonene (13.4%), and α-terpineol (8.57%). The major compounds in the EO from *O. majo-*

*rana* were thymol (21.6%), carvacrol (18.8%), and α-terpineol (8.57%).

(IC50 of 109.90 μg/mL) cells using an XTT-based toxicology assay kit [36].

HCT-8 (85.00% and 86.54%), and MDA-MB-435 (59.48% and 45.43%) [37].

*T. riparia* leaves were collected in Umuarama, Brazil. 9β,13β-Epoxy-7-abietene (1) was isolated from the EO. The cytotoxic activities of the EO and (1) were determined by MTT assay in the MDA-MB-435, HCT-8, SF-295, and HL-60 cell lines. The EO and compound (1), at concentrations of 50 μg/mL and 25 μg/mL, respectively, showed high cytotoxic potential against the cell lines SF-295 (78.06% and 94.80%),

*Ajuga chamaepitys* L. Schreb grows in the Mediterranean region and is used as a diuretic and emmenagogue. This plant was collected in Rocca Mattei, Italy, and the composition of the EO isolated from the aerial parts was determined by GC and GC/MS. Ethyl linoleate (13.7%), germacrene D (13.4%), kaurene (8.4%), β-pinene (6.8%), and phytol (5.3%) were the major components. The EO had moderate cytotoxic activity against the MDA-MB-231 cell line (IC50 of 36.88 μg/mL) and an IC50 value of 60.48 μg/mL against the human colon carcinoma cell line (HCT116) [38]. *Ziziphora tenuior* L. is used in Jordan for the treatment of stomachache, dysentery, and fever. The EO was obtained in 0.72% yield, and the composition was determined using GC/MS; pulegone (46.8%) and *p*-menth-3-en-8-ol (12.5%) were the major compounds. The EO was tested against HepG2 cell line, and cytotoxicity was determined using MTT assay. The IC50 value obtained for the EO

*Sideritis montana L. subsp*. *montana* is used as a diuretic and digestive aid. *S. montana* was collected in Camerino, Italy. The EO (yield of 0.07%) was analyzed using GC/MS, and the major compounds were germacrene D (20.8%), bicyclogermacrene (13.3%), and 8,13-abietadien-18-ol (10.2%). The cytotoxicity was tested

The cytotoxic activity of the EO from *O. vulgare* was tested against MCF-7, prostate cancer (LNCaP), and NIH3T3 cell lines. The IC50 values were 70, 85.3, and 300.5 μg/mL, respectively. The IC50 values for the EO from *O. majorana* were 100,

The EO obtained from *O. vulgare* collected in Córdoba, Argentina, was analyzed by GC/MS. Analysis of the chemical composition showed carvacrol and thymol as the predominant compounds. The cytotoxicity activity was evaluated in cultured A549 cells, and this oil reduced the viability of the cells (IC50 of 2.25 μg/mL) [35]. *Tetradenia riparia* Hochst Codd is used in traditional medicinal to treat cough, dropsy, diarrhea, fever, headaches, malaria, and toothaches. The main components of its EO were *E*,*E*-farnesol (15%), aromadendrene oxide (14.7%), and dronabinol (11%). The cytotoxic activity was tested against HT29 (IC50 of 6.93 μg/mL), MCF-7 (IC50 of 129.57 μg/mL), HeLa (IC50 of 155 μg/mL), HepG-2 (IC50 of 149.97 μg/mL), glioblastoma (M059; IC50 of 217.97 μg/mL), U343 (IC50 of 221.30 μg/mL), and U251

**36**

was 1.25 μL/mL [39].

against MDA-MB-231 and HCT116 cell lines with IC50 values of 32.32 and 31.84 μg/mL, respectively [40].

*Stachys annua* L. is a perennial herb and small shrub. In the folk medicine of central Italy, its aerial parts have been used as anticatarrhal, antipyretic, tonic, and vulnerary (wound healing) agents. The EO isolated from its aerial parts was analyzed by GC/MS, and the major components were phytol (9.8%), germacrene D (9.2%), spathulenol (8.5%), and bicyclogermacrene (5.8%). The cytotoxic activity of the EO was determined by MTT assay. Analysis of the cytotoxicity against the HCT116, A375, and MDA-MB-231 cell lines showed IC50 values of 23.5, 37.2, and 41.5 μg/mL, respectively [41].

*Plectranthus amboinicus* Lour Spreng is cultivated in home gardens and is used in India for the treatment of cough, chronic asthma, and hiccough. The leaves of *P. amboinicus* were collected from a medicinal plant garden in Tamil, India. The cytotoxic activity of the EO was tested using MTT assay against the MCF-7 and HT-29 cell lines, and the IC50 values were 53 and 87 μg/mL, respectively [42].

*Zhumeria majdae* Rech. F. & Wendelbo is a medicinal plant endemic to Iran that is under the threat of extinction. This plant has been used as a curative agent for stomachache, flatulence, diarrhea, indigestion, cold, and headache, for wound healing and treatment of painful menstruation, and as an antiseptic. The aerial parts of *Z. majdae* were collected at five locations in Iran (S1, S2, S3, S4, and S5). The major components were trans-linalool oxide (18.7%), linalool (29.6%), and camphor (27.4%) at S1; trans-linalool oxide (28.6%), linalool (24.4%), and camphor (27.2%) at S2; trans-linalool oxide (16.2%), linalool (34.2%), and camphor (27.7%) at S3; trans-linalool oxide (14.6%), linalool (33.9%), and camphor (26.1%) at S4; and trans-linalool oxide (7.6%), linalool (34.6%), and camphor (34.7%) at S5. The cytotoxicity of the EOs was measured using MTT assay against A375 and MCF7 cell lines, with IC50 values of 746 (S1), 666 (S2), 624 (S3), 779 (S4), and 718 (S5) μg/mL and 674 (S1), 717 (S2), 732 (S3), 646 (S4), and 642 (S5), μg/mL, respectively [43].

*Cedronella canariensis* L. Webb & Berthel. (syn. *Dracocephalum canariense* L.) is present in the Canary Islands. It is a perennial herb, sometimes shrubby. The plant is used in traditional medicine as an anticatarrhal, tonic, antimicrobial, analgesic, carminative, diuretic, hypoglycemiant, hypotensive, and anti-inflammatory agent and decongestant of the respiratory tract. The EO was obtained from the aerial parts of *C. canariensis* collected in El Monte de las Mercedes, Canary Islands, Spain, in 2013 (yield of 2.5%). The EO was analyzed by GC/FID and GC/MS; pinocarvone (58.0%) and α-pinene (10.8%) were the main constituents. The cytotoxicity of the EO was evaluated against A345 (IC50 of 4.3 μg/mL), MDA-MB-231 (IC50 of 7.3 μg/ mL), and HCT 116 (IC50 of 11.4 μg/mL) cell lines by MTT assay [44].

*Rosmarinus officinalis* L. EO is used as an antibacterial, cytotoxic, antimutagenic, antioxidant, antiphlogistic, and chemopreventive agent. The EO (yield of 0.23% w/w) was extracted from the plant collected in Guatemala. The IC50 values of the EO against AGS, A375, and A431 cell lines were 0.21, 0.24, and 0.41 μL/mL, respectively [22].

*Teucrium yemense* Delfi. possesses antifungal, antibacterial, larvicidal, antispasmodic, antioxidant, anti-inflammatory, antiulcer, hypoglycemic, antiacetylcholinesterase, and hepatoprotective activities. Its leaves were collected in two different provinces of Yemen: Dhamar (TY-d) and Taiz (TY-t). The EOs were analyzed, and the most abundant constituents of TY-d were (*E*)-caryophyllene (11.2%), α-humulene (4.0%), γ-selinene (5.5%), 7-epi-α-selinene (20.1%), and caryophyllene oxide (20.1%). The major compounds in TY-t were α-pinene (6.6%), (*E*)-caryophyllene (19.1%), α-humulene (6.4%), δ-cadinene (6.5%), caryophyllene oxide (4.3%), α-cadinol (9.5%), and shyobunol (4.6%). TY-d was active against the HT-29 cell line with an IC50 value of 43.7 μg/mL. TY-t was active against the MCF-7 and MDA-MB-231 cell lines (IC50 of 24.4 and 59.9 μg/mL, respectively) [45].

**Figure 1.**

*Plants whose EOs present remarkable cytotoxic activity. Images taken from [48]. Image of N. rtanjensis taken from [49].*

*Premna microphylla* Turcz. is broadly distributed in the eastern, middle, and southern regions of China. Leaves of *P. microphylla* are used to treat dysentery, appendicitis, and infections for their antioxidant and cytotoxic activities. The plant was collected in China. The EO was obtained with a yield of 0.31% w/w. The major components were blumenol C (49.7%), β-cedrene (6.1%), limonene (3.8%), α-guaiene (3.3%), cryptone (3.1%), and α-cyperone (2.7%). The EO was tested for its cytotoxic activity against HepG2 and MCF-7 cells, with IC50 values of 0.072 and 0.188 mg/mL, respectively [46].

*Dracocephalum kotschyi* Boiss is native to Iran. In traditional medicine, it is used to treat headaches, congestion, and liver disorders and for its antihyperlipidemic and anti-epimastigotic effects. The herb *D. kotschyi* is locally known as "Sama" in Lorestan Province. Aerial parts of wild *D. kotschyi* Boiss were collected in western Iran. A yellowish fragrant oil was obtained (yield of 0.16% w/w). The principal components of the EO were geranial (12.08%), α-pinene (10.34%), geraniol acetate (10.27%), geraniol (9.55%), neral (8.9%), limonene (6.95%), β-myrcene (3.42%), and β-pinene (2.18%). The IC50 against HeLa cell line was 26.4 μg/mL [47].

The National Cancer Institute of the USA (NCI) has screened approximately 100,000 compounds and 50,000 natural product extracts for potential anticancer agents [47]. The NCI considers a compound or an extract to have potential anticancer activity if it has an IC50 value of 4 or 30 μg/mL, respectively. Therefore, according to the NCI, the EOs described in this review with remarkable cytotoxic activity are those obtained from *O. basilicum, S. sahandica, O. vulgare, N. rtanjensis, M. spicata, S. macrosiphon, Z. tenuior, C. canariensis, R. officinalis*, and *T. carmanicus* (**Figure 1**).

**39**

**Author details**

provided the original work is properly cited.

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Cuauhtémoc Pérez-González, Julia Pérez-Ramos, Carlos Alberto Méndez-Cuesta, Roberto Serrano-Vega, Miguel Martell-Mendoza and Salud Pérez-Gutiérrez\* Universidad Autónoma Metropolitana-Xochimilco, Ciudad de México, Mexico

\*Address all correspondence to: msperez@correo.xoc.uam.mx

*Cytotoxic Activity of Essential Oils of Some Species from Lamiaceae Family*

*DOI: http://dx.doi.org/10.5772/intechopen.86392*

*Cytotoxic Activity of Essential Oils of Some Species from Lamiaceae Family DOI: http://dx.doi.org/10.5772/intechopen.86392*

### **Author details**

*Cytotoxicity - Definition, Identification, and Cytotoxic Compounds*

0.188 mg/mL, respectively [46].

**Figure 1.**

*from [49].*

*Premna microphylla* Turcz. is broadly distributed in the eastern, middle, and southern regions of China. Leaves of *P. microphylla* are used to treat dysentery, appendicitis, and infections for their antioxidant and cytotoxic activities. The plant was collected in China. The EO was obtained with a yield of 0.31% w/w. The major components were blumenol C (49.7%), β-cedrene (6.1%), limonene (3.8%), α-guaiene (3.3%), cryptone (3.1%), and α-cyperone (2.7%). The EO was tested for its cytotoxic activity against HepG2 and MCF-7 cells, with IC50 values of 0.072 and

*Plants whose EOs present remarkable cytotoxic activity. Images taken from [48]. Image of N. rtanjensis taken* 

*Dracocephalum kotschyi* Boiss is native to Iran. In traditional medicine, it is used to treat headaches, congestion, and liver disorders and for its antihyperlipidemic and anti-epimastigotic effects. The herb *D. kotschyi* is locally known as "Sama" in Lorestan Province. Aerial parts of wild *D. kotschyi* Boiss were collected in western Iran. A yellowish fragrant oil was obtained (yield of 0.16% w/w). The principal components of the EO were geranial (12.08%), α-pinene (10.34%), geraniol acetate (10.27%), geraniol (9.55%), neral (8.9%), limonene (6.95%), β-myrcene (3.42%),

and β-pinene (2.18%). The IC50 against HeLa cell line was 26.4 μg/mL [47].

The National Cancer Institute of the USA (NCI) has screened approximately 100,000 compounds and 50,000 natural product extracts for potential anticancer agents [47]. The NCI considers a compound or an extract to have potential anticancer activity if it has an IC50 value of 4 or 30 μg/mL, respectively. Therefore, according to the NCI, the EOs described in this review with remarkable cytotoxic activity are those obtained from *O. basilicum, S. sahandica, O. vulgare, N. rtanjensis, M. spicata, S. macrosiphon, Z. tenuior, C. canariensis, R. officinalis*, and *T. carmanicus* (**Figure 1**).

**38**

Cuauhtémoc Pérez-González, Julia Pérez-Ramos, Carlos Alberto Méndez-Cuesta, Roberto Serrano-Vega, Miguel Martell-Mendoza and Salud Pérez-Gutiérrez\* Universidad Autónoma Metropolitana-Xochimilco, Ciudad de México, Mexico

\*Address all correspondence to: msperez@correo.xoc.uam.mx

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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S0102-695X2011005000165

10.1007/s11130-014-0441-x

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695X2 013000600004

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**42**

html#a1

[39] Venditti A, Bianco A, Frezza C, Serafini M, Giacomello G, Giuliani C, et al. Secondary metabolites, glandular trichomes and biological activity of *Sideritis montana* L. subsp. Montana from Central Italy. Chemistry & Biodiversity. 2016;**13**:1380-1390. DOI: 10.1002/cbdv.201600082

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Research. 2015;**29**(17):1641-1649. DOI: 10.1080/14786419.2014.994213

[44] Awadh Ali NA, Chhetri BK, Dosoky NS, Shari K, Al-Fahad AJA, Wessjohann L, et al. Antimicrobial, antioxidant, and cytotoxic activities of *Ocimum forskolei* and *Teucrium yemense* (Lamiaceae) essential oils. Medicine. 2017;**4**(17):1-14. DOI: 101155/2019/8928306

[45] Han-Yu Z, Yang G, Peng-Xiang L. Chemical composition, antioxidant, antimicrobial and cytotoxic activities of essential oil from *Premna microphylla* Turczaninow. Molecules. 2017;**22**(381):1-11. DOI: 10.3390/ molecules22030381

[46] Ashrafi B, Ramak P, Ezatpour B, Talei GR. Investigation on chemical composition, antimicrobial, antioxidant, and cytotoxic properties of essential oil from *Dracocephalum kotschyi* Boiss. African Journal of Traditional, Complementary and Alternative Medicines. 2017;**14**(3): 209-217. DOI: 10.21010/ajtcam.v14i3.23

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[49] JSTOR. Global Plants. Available from: https://plants.jstor.org/plants/ browse Consulted 11-04-2019

**45**

**Chapter 4**

**Abstract**

Cytotoxic Effect and Mechanisms

from Some Plant-Derived

*and María del Consuelo Gómez-García*

Compounds in Breast Cancer

develop new and more effective drugs against different types of BrC.

in vivo model, flavonoids, terpenoid, alkaloids, nanoparticles

**1. Breast cancer (BrC)**

**Keywords:** breast cancer, cytotoxic agents, natural products, in vitro model,

Cancer is the uncontrolled and unregulated growth of cells that generally invade

and destroy normal cells [1]. Breast cancer [BrC] is the deadliest malignancy in women worldwide and also an important public health problem being the second leading cause of cancer in women accounting for more than 626,679 deaths only in 2018 [2]. In many cases this disease has become fatal because of its multifactorial origin and also because it has a great number of exogenous and endogenous factors that can stimulate different pathways [3]. In early BrC, gene expression profiles are determined for hormone receptor (HR)-positive disease and human epidermal growth factor type 2 receptor (HER2) status which define if a patient is likely to receive systemic therapy and are therefore used to guide their treatment [4]. Approximately

*Elvia Pérez-Soto, Cynthia Carolina Estanislao-Gómez,* 

*David Guillermo Pérez-Ishiwara, Crisalde Ramirez-Celis* 

Breast cancer (BrC) is a major health problem in women all around the world. A growing knowledge about these alterations and their associated molecular signaling pathways offers opportunities for therapeutic strategies; chemotherapy is one of the most utilized treatments; however, because of the adverse side effects and multidrug resistance that patients may present, there has been great advancement in search of new alternatives as the use of plant-derived natural compounds. This review describes information on the progress and development of cytotoxic compounds against BrC belonging to the families of flavonoids, terpenes, and alkaloids that through in vitro and in vivo studies have demonstrated to induce cellular death mainly through apoptosis, activating the intrinsic pathway. The in vitro IC50 and the in vivo EC50 dose-response relationship can vary depending on various factors, including the choice of cell line and/or the model used. Also, the association of some of these compounds with nanoparticles or paclitaxel with antibodies has clearly shown a potential improvement in its effect. The clinical studies that are being conducted with some of them show promising results; however, it is necessary to continue with the effort to

#### **Chapter 4**

## Cytotoxic Effect and Mechanisms from Some Plant-Derived Compounds in Breast Cancer

*Elvia Pérez-Soto, Cynthia Carolina Estanislao-Gómez, David Guillermo Pérez-Ishiwara, Crisalde Ramirez-Celis and María del Consuelo Gómez-García*

### **Abstract**

Breast cancer (BrC) is a major health problem in women all around the world. A growing knowledge about these alterations and their associated molecular signaling pathways offers opportunities for therapeutic strategies; chemotherapy is one of the most utilized treatments; however, because of the adverse side effects and multidrug resistance that patients may present, there has been great advancement in search of new alternatives as the use of plant-derived natural compounds. This review describes information on the progress and development of cytotoxic compounds against BrC belonging to the families of flavonoids, terpenes, and alkaloids that through in vitro and in vivo studies have demonstrated to induce cellular death mainly through apoptosis, activating the intrinsic pathway. The in vitro IC50 and the in vivo EC50 dose-response relationship can vary depending on various factors, including the choice of cell line and/or the model used. Also, the association of some of these compounds with nanoparticles or paclitaxel with antibodies has clearly shown a potential improvement in its effect. The clinical studies that are being conducted with some of them show promising results; however, it is necessary to continue with the effort to develop new and more effective drugs against different types of BrC.

**Keywords:** breast cancer, cytotoxic agents, natural products, in vitro model, in vivo model, flavonoids, terpenoid, alkaloids, nanoparticles

#### **1. Breast cancer (BrC)**

Cancer is the uncontrolled and unregulated growth of cells that generally invade and destroy normal cells [1]. Breast cancer [BrC] is the deadliest malignancy in women worldwide and also an important public health problem being the second leading cause of cancer in women accounting for more than 626,679 deaths only in 2018 [2]. In many cases this disease has become fatal because of its multifactorial origin and also because it has a great number of exogenous and endogenous factors that can stimulate different pathways [3]. In early BrC, gene expression profiles are determined for hormone receptor (HR)-positive disease and human epidermal growth factor type 2 receptor (HER2) status which define if a patient is likely to receive systemic therapy and are therefore used to guide their treatment [4]. Approximately

60–70% of primary BrC cases express positive estrogen receptors (ER+) or positive progesterone receptors (PR+) or both and are hormone responsive. However, about 15–20% of BrC cases are in the category of triple-negative phenotype owing to their lack of ER, PR, and amplified HER2. Commonly, ER+ hormone-dependent BrCs have a better prognosis and are often responsive to antihormone therapy [4].

Because of these reasons, the survival rates for BrC have decreased significantly; however, there have been great advancements in new alternative therapies which not only are safer but are also more effective and inexpensive and have minimal side effects [5]. Therapeutic drugs derived from natural compounds have become of great interest since more than 75% of anticancer drugs were designed and developed from plant-derived natural ingredients which have been proven to have anticancer properties with novel mechanisms [6]. In the last 50 years, nearly 200 new chemical compounds have been approved to fight cancer, of which around 50% are molecules of unmodified natural products and their semisynthetic or synthetic derivatives that are safe and profitable [7, 8]. Small organic molecules such as terpenes, flavonoids, alkaloids, lignans, saponins, vitamins, minerals, glycosides, oils, and other secondary metabolites play a significant role in either the inhibition of proliferation, induction of apoptosis, or other mechanisms that may be altered [9, 10]. The structural diversity of natural products and their wide application in therapeutics have always been recognized by pharmaceutical industries [1].

This chapter summarizes the small novel organic molecules obtained from plants and their derivatives, some of which are on the market and others are found in preclinical studies with encouraging results. We also describe some interesting biotechnological associations between some compounds and nanoparticles or other molecules as antibodies that show a novel potential in the treatment of BrC.

#### **2. In vitro models for studying plant-derived compounds in breast cancer**

The evaluation of the therapeutic potential of novel plant-derived compounds or secondary metabolites, either pure compounds or the mixture of active constituents, can serve as chemotherapeutic agents in BrC. The biological models used for the development of new drugs include in vitro models using BrC cell lines and are divided into estrogen receptor positive (ER+, T47, MCF-7) and ER negative (ER-, MDA-MB-231, MDA-MB-453, SKBR3). MDA-MB-231 or triple-negative breast cancer cell (TNBC) line, estrogen negative (ER-) and progesterone negative (PR-), and HER- are known to be models for metastasis, which are more aggressive, containing a high potential to metastasize, and are unresponsive to antiestrogens [5, 8]. TNBC line is used to investigate the mechanism underlying migration and invasion. It is important to find cancer therapeutic compounds which possess multi-targeted and multifunctional potential with anti-metastasis activity [8, 11]. MCF-7 is the most used cell line with a great number of publications due to the presence of ER+ [9].

In vitro experiments have also been used in different studies, in order to characterize and identify compounds derived from extracts, essential oils, and other extractions. Trypan blue dye, MTT, sulforhodamine B, and lactic dehydrogenase assays constitute some of the most utilized assays used to evaluate the cytotoxic effect of essential oils and/or pure compounds in different cell lines of BrC. In order to characterize morphological changes, biochemical and molecular levels of cell death, proteins that are modified (expression and activation), gene regulation, migration, invasion, and cell division, among other changes, experiments such as staining with hematoxylin and eosin, Western blot, TUNEL, annexin V, qRT-PCR, scratch assay, and cell cycle assays are performed [12].

**47**

*4.1.1 D-limonene*

*Cytotoxic Effect and Mechanisms from Some Plant-Derived Compounds in Breast Cancer*

derived compounds in different stages, such as cancer initiation, promotion progression, invasion, and metastasis. These models are also used to comprehend therapeutic response, which represents an essential step between in vitro systems and clinical studies [8, 13]. The in vivo models are also used to investigate the capability of plant formulation to induce an anti-BrC effect where it is sought to optimize dose, bioavailability, administration routes, and selective delivery and reduce toxic effects, among others [8, 14]. The two animal species that will be mentioned in this review are those involving mice and rats [14]; however, BrC mouse models

**3. In vivo models for studying plant-derived compounds in breast cancer**

Wide varieties of animal model systems are now available to investigate plant-

There are different types of in vivo models of BrC, such as cell line-derived xenografts (MDA-MB-231 line) that are implanted into immunocompromised animals (cell-derived xenografts, CDX). CDX models represent a relatively homogenous mass of transformed breast epithelial cells, and depending on where the cells are inoculated, they are classified as ectopic CDX (models advanced disease only, subcutaneous injection of human tumor cells), orthotopic CDX (in mammary gland/fat pad), metastatic CDX (following tail vein or intra-cardiac injection in specific sites, i.e., bone or lung), syngeneic (mouse tissue implanted to strainmatched host) or metastasis with syngeneic model (usually fast-growing tumors and microenvironment derive from the same species, i.e., 4T1 cells), and genetically engineered mouse models (GEMMs) to address early events of tumorigenesis [13]. Nonetheless, researchers need to consider the limitations of each model and the mechanism of action of the compound previously investigated in in vitro models.

Plants produce bioactive secondary metabolites such as flavonoids [15], terpenoids [1], alkaloids [16], tannins, and others, which have profusely been studied for BrC (1, 8). Here, we describe some terpenoid compounds such as *D*-limonene, camptothecin (CPT), paclitaxel, and ursolic acid (UA) and some flavonoids such as cynaroside, isoflavones as Biochanin A (BA) or ginsenoside R2, naringenin, and other novel cytotoxic compounds from different natural sources as shown in **Figure 1** and **Table 1**. The anti-BrC activities of plant-derived compounds discussed in this chapter are taken from published articles that demonstrated an anti-BrC activity against specific cancer cell lines (see **Table 2**) and in vivo models (see **Table 3**) or clinical

Terpenoids are organic compounds derived from five-carbon units (isoprene) assembled and modified in different ways. The classification of terpenoids is based on the isoprene units which are commonly classified as monoterpenes (C10) and diterpenes (C20), i.e., paclitaxel, and triterpenes as ursolic acid [1]. Interestingly, essential oils are a rich and complex composition of monoterpenes with anti-BrC

*D*-limonene, (1-methyl-4-(1-methylethenyl-cyclohexene) a monocyclic monoterpene, with a molecular mass of 136.23 g/mol [18], is found in the peels of citrus

*DOI: http://dx.doi.org/10.5772/intechopen.87177*

are used in a variety of preclinical studies [13].

**4. Cytotoxicity of plant-derived compounds**

studies with their mechanism of action.

**4.1 Terpenoids as cytotoxic compounds**

activity such as *Decatropis bicolor* essential oil (*DBEO*) [17].

*Cytotoxic Effect and Mechanisms from Some Plant-Derived Compounds in Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.87177*

#### **3. In vivo models for studying plant-derived compounds in breast cancer**

Wide varieties of animal model systems are now available to investigate plantderived compounds in different stages, such as cancer initiation, promotion progression, invasion, and metastasis. These models are also used to comprehend therapeutic response, which represents an essential step between in vitro systems and clinical studies [8, 13]. The in vivo models are also used to investigate the capability of plant formulation to induce an anti-BrC effect where it is sought to optimize dose, bioavailability, administration routes, and selective delivery and reduce toxic effects, among others [8, 14]. The two animal species that will be mentioned in this review are those involving mice and rats [14]; however, BrC mouse models are used in a variety of preclinical studies [13].

There are different types of in vivo models of BrC, such as cell line-derived xenografts (MDA-MB-231 line) that are implanted into immunocompromised animals (cell-derived xenografts, CDX). CDX models represent a relatively homogenous mass of transformed breast epithelial cells, and depending on where the cells are inoculated, they are classified as ectopic CDX (models advanced disease only, subcutaneous injection of human tumor cells), orthotopic CDX (in mammary gland/fat pad), metastatic CDX (following tail vein or intra-cardiac injection in specific sites, i.e., bone or lung), syngeneic (mouse tissue implanted to strainmatched host) or metastasis with syngeneic model (usually fast-growing tumors and microenvironment derive from the same species, i.e., 4T1 cells), and genetically engineered mouse models (GEMMs) to address early events of tumorigenesis [13]. Nonetheless, researchers need to consider the limitations of each model and the mechanism of action of the compound previously investigated in in vitro models.

#### **4. Cytotoxicity of plant-derived compounds**

Plants produce bioactive secondary metabolites such as flavonoids [15], terpenoids [1], alkaloids [16], tannins, and others, which have profusely been studied for BrC (1, 8). Here, we describe some terpenoid compounds such as *D*-limonene, camptothecin (CPT), paclitaxel, and ursolic acid (UA) and some flavonoids such as cynaroside, isoflavones as Biochanin A (BA) or ginsenoside R2, naringenin, and other novel cytotoxic compounds from different natural sources as shown in **Figure 1** and **Table 1**.

The anti-BrC activities of plant-derived compounds discussed in this chapter are taken from published articles that demonstrated an anti-BrC activity against specific cancer cell lines (see **Table 2**) and in vivo models (see **Table 3**) or clinical studies with their mechanism of action.

#### **4.1 Terpenoids as cytotoxic compounds**

Terpenoids are organic compounds derived from five-carbon units (isoprene) assembled and modified in different ways. The classification of terpenoids is based on the isoprene units which are commonly classified as monoterpenes (C10) and diterpenes (C20), i.e., paclitaxel, and triterpenes as ursolic acid [1]. Interestingly, essential oils are a rich and complex composition of monoterpenes with anti-BrC activity such as *Decatropis bicolor* essential oil (*DBEO*) [17].

#### *4.1.1 D-limonene*

*D*-limonene, (1-methyl-4-(1-methylethenyl-cyclohexene) a monocyclic monoterpene, with a molecular mass of 136.23 g/mol [18], is found in the peels of citrus

*Cytotoxicity - Definition, Identification, and Cytotoxic Compounds*

60–70% of primary BrC cases express positive estrogen receptors (ER+) or positive progesterone receptors (PR+) or both and are hormone responsive. However, about 15–20% of BrC cases are in the category of triple-negative phenotype owing to their lack of ER, PR, and amplified HER2. Commonly, ER+ hormone-dependent BrCs have a better prognosis and are often responsive to antihormone therapy [4].

Because of these reasons, the survival rates for BrC have decreased significantly; however, there have been great advancements in new alternative therapies which not only are safer but are also more effective and inexpensive and have minimal side effects [5]. Therapeutic drugs derived from natural compounds have become of great interest since more than 75% of anticancer drugs were designed and developed from plant-derived natural ingredients which have been proven to have anticancer properties with novel mechanisms [6]. In the last 50 years, nearly 200 new chemical compounds have been approved to fight cancer, of which around 50% are molecules of unmodified natural products and their semisynthetic or synthetic derivatives that are safe and profitable [7, 8]. Small organic molecules such as terpenes, flavonoids, alkaloids, lignans, saponins, vitamins, minerals, glycosides, oils, and other secondary metabolites play a significant role in either the inhibition of proliferation, induction of apoptosis, or other mechanisms that may be altered [9, 10]. The structural diversity of natural products and their wide application in therapeutics have always been recognized by pharmaceutical industries [1]. This chapter summarizes the small novel organic molecules obtained from plants and their derivatives, some of which are on the market and others are found in preclinical studies with encouraging results. We also describe some interesting biotechnological associations between some compounds and nanoparticles or other

molecules as antibodies that show a novel potential in the treatment of BrC.

The evaluation of the therapeutic potential of novel plant-derived compounds or secondary metabolites, either pure compounds or the mixture of active constituents, can serve as chemotherapeutic agents in BrC. The biological models used for the development of new drugs include in vitro models using BrC cell lines and are divided into estrogen receptor positive (ER+, T47, MCF-7) and ER negative (ER-, MDA-MB-231, MDA-MB-453, SKBR3). MDA-MB-231 or triple-negative breast cancer cell (TNBC) line, estrogen negative (ER-) and progesterone negative (PR-), and HER- are known to be models for metastasis, which are more aggressive, containing a high potential to metastasize, and are unresponsive to antiestrogens [5, 8]. TNBC line is used to investigate the mechanism underlying migration and invasion. It is important to find cancer therapeutic compounds which possess multi-targeted and multifunctional potential with anti-metastasis activity [8, 11]. MCF-7 is the most used cell line with a great number of publications due to the presence of ER+ [9]. In vitro experiments have also been used in different studies, in order to characterize and identify compounds derived from extracts, essential oils, and other extractions. Trypan blue dye, MTT, sulforhodamine B, and lactic dehydrogenase assays constitute some of the most utilized assays used to evaluate the cytotoxic effect of essential oils and/or pure compounds in different cell lines of BrC. In order to characterize morphological changes, biochemical and molecular levels of cell death, proteins that are modified (expression and activation), gene regulation, migration, invasion, and cell division, among other changes, experiments such as staining with hematoxylin and eosin, Western blot, TUNEL, annexin V, qRT-PCR,

**2. In vitro models for studying plant-derived compounds** 

scratch assay, and cell cycle assays are performed [12].

**in breast cancer**

**46**

**Figure 1.** *Molecular targets of plant-derived compounds in BrC.*

fruits [18–20]. The main mechanism described by *D*-limonene is the inhibition of the posttranslational isoprenylation of cell growth-regulatory proteins such as Ras, inducing cell death. It has also demonstrated to decrease the viability of cancer cells in a dose-dependent manner by inducing apoptotic cell death. It induced the activation of caspase-3 and caspase-9, PARP cleavage, and Bax protein and cytosolic release of cytochrome c from the mitochondria and through the attenuation of the expression of Bcl-2 protein, suggesting that *D*-limonene induces apoptosis via the mitochondrial pathway and through the suppression of the PI3K/AKT pathway [21, 22]. The interest in this compound as a potential cancer chemotherapeutic agent was stimulated by pronounced chemopreventive and chemotherapeutic efficacy in spontaneous and carcinogen-induced animal tumor models with little toxicity [19, 22]. In the in vivo models, it has been reported to exhibit various effects on several hallmarks of cancer (i.e., proliferation, apoptosis, inflammation) [23, 24]. 7,12-Dimethylbenz(a)anthracene (DMBA) and *N*-methyl-*N*-nitrosourea (MNU) induced mammary carcinogenesis in rats and induced regression of carcinomas [24–26]. Dietary feeding of *D*-limonene also inhibited the development of Ras oncogene in mammary carcinomas in rats [25].

**49**

*4.1.2 Paclitaxel*

**Table 1.**

*Cytotoxic Effect and Mechanisms from Some Plant-Derived Compounds in Breast Cancer*

Ursolic acid (UA) MCF-7,

Biochanin (BA) MCF-7,

Ginsenoside (Rh2) MCF-7,

Soy products Genistein (GEN) MCF-7,

Lycopene (LYC-WPI-NPs)

Perillyl alcohol a hydroxylated product of

*D*-limonene

nanospheres

*Camptotheca accuminata* Camptothecin MDA-MB-231

*Curcuma longa* Curcumin MCF-7

Vinca alkaloids Vincristine MCF-7,

*Cytotoxic effect of plant-derived compounds in in vitro models of BrC.*

*Taxus brevifolia* Paclitaxel MCF-7

*Taxus brevifolia* Paclitaxel-loaded

Ginsenoside + biochanin MDA-MB-231

Lycopene (LYC) MDA-MB-231

**Source Compound Study type Concentration Ref**

MDA-MB-231

MDA-MB-231

MDA-MB-231

MDA-MB-231 /ERβ1

KPL-1, MCF-7, MKL-F, MDA-MB-231

MDA-MB-435, ZR.75.1

Resveratrol MCF-7 10–150 μm [51]

100 nm IC50:0.65 nm

IC50: 29 μg/ml IC50: 35 μm IC50: 30 μm

T47-D, MBA-MB-231, MDA-MB-435

MCF-7

MCF-7 MDA-MB-231

HeLa

MCF-7

MCF-7

Cynaroside MCF-7 IC50:3.98 μg/ml [42]

Naringenin MDA-MB-231 40 μg/ml [43]

221.39 μg/ml 239.47 μg/

63.76 μm and 59.76 μm,

57.53 μm and 52.53 μm,

27.68, 25.41 μm; 25.2 μm,

100 μm [48]

500 μm [26]

100–500 nm [28]

0.06–0.6 ng/ml [50]

IC50: 170 and 50 nmol/L [56]

respectively

respectively

and 22.75 μm

≥1.25 μm to 5 μl in TNBC. 50 μm

MCF-7 25–50 μm/ml [49]

[33]

[44]

[44]

[44]

[11, 12]

[52, 53]

[54, 55]

ml

Paclitaxel is a complex diterpene, with a molecular structure of C47H51NO14 and a molecular mass of 853.91 g/mol [18]. This compound induces mitotic arrest and also apoptosis by activating extrinsic or intrinsic pathway. In MCF-7 cells it decreased levels of Bcl-2 protein and increased proapoptotic proteins such as Bax, cytochrome c, caspase-9, and caspase-3 [27, 28]. Likewise, it has also been reported

*DOI: http://dx.doi.org/10.5772/intechopen.87177*

Apples, herbs and spices including rosemary

Apiaceae family, *Cuminum cyminum*

legumes

legumes

legumes

fruits

Soybeans, soy foods,

Soybeans, soy foods,

Soybeans, soy foods,

Tomatoes (*Solanum lycopersicum*), citrus fruits and grapes

Tomatoes (*Solanum lycopersicum*), other red

LYC incorporated biopolymeric nanoparticles with whey protein isolate

A derivative from *D*-limonene

Grapes, wine, nuts,

berries

*Cytotoxic Effect and Mechanisms from Some Plant-Derived Compounds in Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.87177*


#### **Table 1.**

*Cytotoxicity - Definition, Identification, and Cytotoxic Compounds*

fruits [18–20]. The main mechanism described by *D*-limonene is the inhibition of the posttranslational isoprenylation of cell growth-regulatory proteins such as Ras, inducing cell death. It has also demonstrated to decrease the viability of cancer cells in a dose-dependent manner by inducing apoptotic cell death. It induced the activation of caspase-3 and caspase-9, PARP cleavage, and Bax protein and cytosolic release of cytochrome c from the mitochondria and through the attenuation of the expression of Bcl-2 protein, suggesting that *D*-limonene induces apoptosis via the mitochondrial pathway and through the suppression of the PI3K/AKT pathway [21, 22]. The interest in this compound as a potential cancer chemotherapeutic agent was stimulated by pronounced chemopreventive and chemotherapeutic efficacy in spontaneous and carcinogen-induced animal tumor models with little toxicity [19, 22]. In the in vivo models, it has been reported to exhibit various effects on several hallmarks of cancer (i.e., proliferation, apoptosis, inflammation) [23, 24]. 7,12-Dimethylbenz(a)anthracene (DMBA) and *N*-methyl-*N*-nitrosourea (MNU) induced mammary carcinogenesis in rats and induced regression of carcinomas [24–26]. Dietary feeding of *D*-limonene also inhibited the development of Ras oncogene in mammary carcinomas in rats [25].

**48**

**Figure 1.**

*Molecular targets of plant-derived compounds in BrC.*

*Cytotoxic effect of plant-derived compounds in in vitro models of BrC.*

#### *4.1.2 Paclitaxel*

Paclitaxel is a complex diterpene, with a molecular structure of C47H51NO14 and a molecular mass of 853.91 g/mol [18]. This compound induces mitotic arrest and also apoptosis by activating extrinsic or intrinsic pathway. In MCF-7 cells it decreased levels of Bcl-2 protein and increased proapoptotic proteins such as Bax, cytochrome c, caspase-9, and caspase-3 [27, 28]. Likewise, it has also been reported


#### **Table 2.**

*Compounds evaluated in in vivo model of BrC.*

that the induction of apoptosis was independent of caspases [29]. The combination of paclitaxel with a compound that inhibits the mitotic slippage such as phenylethyl isothiocyanate (PEITC) induced apoptosis in MDA-MB-231 cells which are drug resistance [30]. Also, additional activities of taxol have been described including the effect on cell signaling and gene expression and activation of mitogen-activated protein kinases (MAPKs), Raf-1, and protein tyrosine kinases [29]. Paclitaxel has been approved by the FDA to be used alone, or in combination with other anticancer treatments, to treat BrC and other cancers [31, 32].

#### *4.1.3 Ursolic acid*

Ursolic acid (3-β-hydroxy-urs-12-en-28-oic acid) is a pentacyclic triterpenoid natural product and a member of the cyclosqualenoid family, commonly named as UA with a molecular structure of C30H48C3 and a molecular mass of 456.7 g/mol [18]. UA is derived from diverse plants and fruits, such as rosemary (*Rosmarinus officinalis*), apple (*Malus domestica*), makino, cranberries (*Vaccinium macrocarpon*), pears (*Pyrus pyrifolia*), prunes (*Prunus domestica*), bearberries (*Arctostaphylos alpina*), loquat (*Eriobotrya japonica*), scotch heather (*Calluna vulgaris*), basil (*Ocimum sanctum*), and jamun (*Eugenia jambolana*) [9, 11]. UA posed different activity against BrC via several molecular mechanisms [7, 11, 33–35], and UA

**51**

**Table 3.**

*Cytotoxic Effect and Mechanisms from Some Plant-Derived Compounds in Breast Cancer*

Camptothecin PubChem CID:24360

Lycopene PubChem CID: 446925

Biochanin PubChem CID: 5280373

Paclitaxel PubChem CID:36314

Cynaroside PubChem CID: 5280637

Ginsenoside Rh2 PubChem CID: 119307

Curcumin PubChem CID:969516

is relatively nontoxic to normal cells [11]. Administration of UA demonstrates inhibitory efficacy against cell proliferation rate and induces apoptosis via both the mitochondrial death pathway (cleavage of caspase-9, caspase-3, and PARP, Bax upregulation and Bcl-2 downregulation, release of cytochrome c to the cytosol, decreased mitochondrial membrane potential) and extrinsic death receptordependent pathway (Fas receptor) in MDA-MB-231 cells [9, 11]. The treatment

*Structures of plant-derived compounds with potential effect in the BrC treatment.*

Resveratrol PubChem CID: 445154

*DOI: http://dx.doi.org/10.5772/intechopen.87177*

*D*-Limonene PubChem CID:22311

Ursolic acid PubChem CID: 64945

Naringenin PubChem CID: 932

Genistein PubChem CID: 5280961 *Cytotoxic Effect and Mechanisms from Some Plant-Derived Compounds in Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.87177*

#### **Table 3.**

*Cytotoxicity - Definition, Identification, and Cytotoxic Compounds*

induced mammary carcinogenesis in rats

Mammary rat tumor induced with KPL-1

DMBA mammary

Tumor xenograft model in mice

mammary cancer

estrogen-induced breast carcinoma in

mouse breast cancer

cells

tumor

in rats

rats

model

tumor model

*D*-Limonene DMBA and NMU-

Resveratrol DMBA induced

Curcumin MCF-7 xenografts

Camptothecin Mouse 4T1 breast

*Compounds evaluated in in vivo model of BrC.*

Perillyl alcohol a hydroxylated product of *D*-limonene

Paclitaxelencapsulated liposomes

Paclitaxel-loaded nanospheres

**Compound Study type Dose and activity Ref**

secondary tumors

mouse system

paclitaxel doses

tumor.

pathways

10% of limonene diet. Induced complete regression of primary rat mammary tumors and prevented the development of

75 mg/kg. Suppressed orthotopically transplanted KPL-1 tumor cell growth and regional lymph node metastasis in a nude

20 mg/kg. In combination with 500 mg/ kg of *Eruca sativa* extract reduced NF-κB, COX-2 and Bcl-2 gene expression

100 μg/rat. Suppressed COX-2 and matrix metalloprotease-9 expression in the breast

50 mg. Inhibits breast carcinogenesis via induction of NRF2-mediated protective

100 mg/kg in combination with mitomycin 1–2 mg/kg. The combined treatment inhibited tumor growth, induced G1 arrest, and decreased cyclin D1, cyclin E,

2 mg/kg in combination with 1.05 mg/kg of doxorubicin. Induced 70% of tumor volume reduction and caspase-3 induction

cyclin A, CDK2, and CDK4

5–50 mg/kg. Showed an equivalent antitumor efficacy to the clinical formulation and provided a superior safety and improved tolerability to higher [24]

[26]

[97]

[50]

[67, 98]

[99]

[100]

that the induction of apoptosis was independent of caspases [29]. The combination of paclitaxel with a compound that inhibits the mitotic slippage such as phenylethyl isothiocyanate (PEITC) induced apoptosis in MDA-MB-231 cells which are drug resistance [30]. Also, additional activities of taxol have been described including the effect on cell signaling and gene expression and activation of mitogen-activated protein kinases (MAPKs), Raf-1, and protein tyrosine kinases [29]. Paclitaxel has been approved by the FDA to be used alone, or in combination with other antican-

Ursolic acid (3-β-hydroxy-urs-12-en-28-oic acid) is a pentacyclic triterpenoid natural product and a member of the cyclosqualenoid family, commonly named as UA with a molecular structure of C30H48C3 and a molecular mass of 456.7 g/mol [18]. UA is derived from diverse plants and fruits, such as rosemary (*Rosmarinus officinalis*), apple (*Malus domestica*), makino, cranberries (*Vaccinium macrocarpon*), pears (*Pyrus pyrifolia*), prunes (*Prunus domestica*), bearberries (*Arctostaphylos alpina*), loquat (*Eriobotrya japonica*), scotch heather (*Calluna vulgaris*), basil (*Ocimum sanctum*), and jamun (*Eugenia jambolana*) [9, 11]. UA posed different activity against BrC via several molecular mechanisms [7, 11, 33–35], and UA

cer treatments, to treat BrC and other cancers [31, 32].

**50**

*4.1.3 Ursolic acid*

**Table 2.**

*Structures of plant-derived compounds with potential effect in the BrC treatment.*

is relatively nontoxic to normal cells [11]. Administration of UA demonstrates inhibitory efficacy against cell proliferation rate and induces apoptosis via both the mitochondrial death pathway (cleavage of caspase-9, caspase-3, and PARP, Bax upregulation and Bcl-2 downregulation, release of cytochrome c to the cytosol, decreased mitochondrial membrane potential) and extrinsic death receptordependent pathway (Fas receptor) in MDA-MB-231 cells [9, 11]. The treatment

of BrC cells with UA induced changes in glycolytic pathway leading to cytotoxic autophagy, also at low doses (5–20 μm) caused a G0/G1 cell cycle arrest, increased p21 levels, oxidative stress, and DNA damage [36]. UA has been demonstrated to exhibit strong anti-BrC potential by inducing cell cycle arrest and inhibition of proliferation, angiogenesis, and metastasis in both in vitro and in vivo models [7, 11, 33–35]. Finally, UA inhibited BrC growth by inducing cell death via the inhibition of inflammatory responses through the NF-κB, PI3K/AKT signaling pathways [9, 33]. Therefore, UA could be used as a potential anti-BrC strategy in clinical studies.

#### *4.1.4 Lycopene alone*

Lycopene (LYC) (trans-lycopene) is a terpene assembled from eight isoprene units and is a rich antioxidant compound, a major carotenoid present in tomatoes (*Solanum lycopersicum*), apricots, red oranges, pink grapefruit, watermelon, rose hips, guava, vegetables, and photosynthetic algae [37]. LYC has a molecular structure of C40H56 and a molecular mass of 536.888 g/mol [18]. LYC is a compound that has displayed antiproliferative, anti-migration, anti-invasive, anti-metastatic, and antioxidant characteristics in numerous in vitro and in vivo studies in BrC [9, 37–39]. Also, LYC induces apoptosis and activates caspase-9 enzyme in human BrC cells [9]. Finally, LYC has inhibited the multiplication of cancer cells by arresting cell cycle at different phases (G1, S, and M phases) and by sustained activation of the ERK½ with suppression of cyclin D1 and upregulation of p21 [39]. Other mechanism of action of LYC is through the inhibition of IκBα phosphorylation and decrease in the expression of NF-κB [40].

#### *4.1.5 Decatropis bicolor essential oil: a mixture of monoterpenes*

Essential oils are a complex mixture of secondary metabolites such as monoterpenes that are responsible for their biological activity that includes anti-BrC effect. However, some studies suggested a synergistic activity of the compounds [17]. For example, DBEO was studied on MDA-MB-231 cells.

It had a cytotoxic effect with an IC50 of 53.81 μg/ml. It induced DNA fragmentation and apoptosis via intrinsic pathways due to the activation of Bax, caspase-9, and caspases-3, suggesting a synergistic activity of compounds present in the essential oil, such as 1,5-cyclooctadiene, 3-(methyl-2)propenyl, β-terpineol, 1-(3-methylcyclopent-2-enyl)-cyclohexene, *D*-limonene, pinene, and linalool [41].

#### **4.2 Flavonoids as cytotoxic compounds**

Flavonoids which are polyphenolic substances found in different plant-derived food are divided into flavones (cynaroside), flavonols, flavanones (naringenin), flavanols, isoflavones (genistein (GEN), biochanin A), anthocyanidins, and nonflavonoids [10]. Flavonoids have been reported to have an effect on BrC through numerous mechanisms such as antioxidant, anti-inflammatory, antiproliferative, cytotoxic, anti-angiogenic, and anti-metastatic effects in numerous in vitro and in vivo experiments in estrogen-dependent or estrogen-independent BrC [15, 37] (**Tables 1**–**3**).

#### *4.2.1 Cynaroside*

Cynaroside (luteolin-7-*O*-glucoside) is a glycosyloxyflavone or a glycoside form. It derives from luteolin. Cynaroside has a molecular structure of C21H20O11 and a molecular mass of 448.38 g/mol [18]. Cynaroside is a constituent of the leaves of

**53**

*4.2.4 Genistein*

*Cytotoxic Effect and Mechanisms from Some Plant-Derived Compounds in Breast Cancer*

*Capsicum annuum* (red pepper) and seeds and fruits of *Cuminum cyminum*, an old famous medicinal and culinary plant from the Apiaceae family. Currently, Goodarzi and collaborators demonstrated that luteolin-7-*O*-glucoside plays a significant role in cytotoxic effect of *C. cyminum* against MCF-7 cell line (IC50 of 3.98 μg/ml) and can be introduced as a candidate for chemopreventive and chemotherapeutic drugs [42]. More in vitro or in vivo studies are necessary to elucidate its mechanism of action.

Naringenin (4',5,7-trihydroxyflavanone) is a flavanone and member of 4'hydroxyflavanones; it has a molecular structure of C15H12O5 and a molecular mass of 272.256 g/mol [18]. This bioflavonoid is a constituent of tomatoes, citrus fruits, and grapes. Naringenin is a phytoestrogen which is also an important anti-BrC, reported to be involved in decreasing the number of ER-α-positive cells by modulating p38 MAPK signaling pathway [9]. Recently, investigators reported that naringenin has antiproliferative effects by arresting the cell cycle at the G2 phase and caused an inhibitory effect on MDA-MB-231 cells via induction of apoptosis

Biochanin or 4'-methylgenistein (B5,7-dihydroxy-4'-methoxyisoflavone) is an *O*-methylated isoflavone that is isolated from red clover *Trifolium pratense* and the root of *Astragalus membranaceus*, a traditional Chinese herbal medicine. BA has a molecular structure of C16H12O5 and a molecular mass of 284.267 g/mol [18, 44]. The phytoestrogen BA caused antiproliferative activity by stabilizing and activating p53 through the upregulated expression of phospho-p53, phospho-p38, and p-ASK1 and downregulated expression of TRAF2 in MDA-MB-231 and MCF-7 cells [44] and apoptosis through upregulated expression of mRNA levels of ER-α, Bcl-2, and

Also, BA stopped cell growth by blocking the activity of aromatase enzyme which is encoded by the gene CYP19 [5]. On the other hand, ginsenoside Rh2 (protopanaxadiol-type) is the major type of saponin ginsenoside that is separated from *Panax ginseng* and other species. Rh2 has a molecular structure of C36H62O8 and a molecular mass of 622.884 g/mol [18, 44]. Rh2 exhibits antitumor activity in ER+(MCF-7) and ER-(MDA-MB-231). However, Ren and collaborators determined that BA plus Rh2 synergistically enhanced the antiproliferative effect in both BrC cells, with decreased EC50 values of both the compounds and a mechanism through the stabilization and activation of p53, p38, and ASK1 proteins (**Table 1**) [44].

Genistein (4',5,7-trihydroxyisoflavone) is an isoflavonoid derived from soy products [46]. It has a molecular structure of C15H10O5 and a molecular mass of 270.24 g/mol [18]. This agent has an antineoplastic effect in BrC [47]. GEN inhibits the growth of MDA-MB-231 cells by altering the phosphorylation of proteins included in cell cycle regulation and DNA damage response predominantly, and GEN induced apoptosis via the upregulation of Bax and p21WAF1 proteins in MDA-MB-231 cells and downregulating the expression of caspase-3 [5, 47]. In a recent study, GEN increases cell cycle arrest in G2/M phase in MDA-MB-231/ERβ1 cells, even though there is a high dose of GEN-arrested cells in G0/G1, just like in the MCF-7 cells. Thus, the combinatorial effect of GEN and overexpressed ERβ1 resulted in an active blockade of cell cycle progression and a dramatic inhibition

and inhibition of caspase-3 and caspase-9 activities [37, 43].

*4.2.3 Biochanin and ginsenoside Rh2 as pure compounds or mixture*

miR-375 in ER+ BrC cells, that is to say T47D and MCF-7 [37, 44, 45].

*DOI: http://dx.doi.org/10.5772/intechopen.87177*

*4.2.2 Naringenin*

*Capsicum annuum* (red pepper) and seeds and fruits of *Cuminum cyminum*, an old famous medicinal and culinary plant from the Apiaceae family. Currently, Goodarzi and collaborators demonstrated that luteolin-7-*O*-glucoside plays a significant role in cytotoxic effect of *C. cyminum* against MCF-7 cell line (IC50 of 3.98 μg/ml) and can be introduced as a candidate for chemopreventive and chemotherapeutic drugs [42]. More in vitro or in vivo studies are necessary to elucidate its mechanism of action.

#### *4.2.2 Naringenin*

*Cytotoxicity - Definition, Identification, and Cytotoxic Compounds*

*4.1.4 Lycopene alone*

decrease in the expression of NF-κB [40].

**4.2 Flavonoids as cytotoxic compounds**

*4.1.5 Decatropis bicolor essential oil: a mixture of monoterpenes*

example, DBEO was studied on MDA-MB-231 cells.

of BrC cells with UA induced changes in glycolytic pathway leading to cytotoxic autophagy, also at low doses (5–20 μm) caused a G0/G1 cell cycle arrest, increased p21 levels, oxidative stress, and DNA damage [36]. UA has been demonstrated to exhibit strong anti-BrC potential by inducing cell cycle arrest and inhibition of proliferation, angiogenesis, and metastasis in both in vitro and in vivo models [7, 11, 33–35]. Finally, UA inhibited BrC growth by inducing cell death via the inhibition of inflammatory responses through the NF-κB, PI3K/AKT signaling pathways [9, 33]. Therefore, UA could be used as a potential anti-BrC strategy in clinical studies.

Lycopene (LYC) (trans-lycopene) is a terpene assembled from eight isoprene units and is a rich antioxidant compound, a major carotenoid present in tomatoes (*Solanum lycopersicum*), apricots, red oranges, pink grapefruit, watermelon, rose hips, guava, vegetables, and photosynthetic algae [37]. LYC has a molecular structure of C40H56 and a molecular mass of 536.888 g/mol [18]. LYC is a compound that has displayed antiproliferative, anti-migration, anti-invasive, anti-metastatic, and antioxidant characteristics in numerous in vitro and in vivo studies in BrC [9, 37–39]. Also, LYC induces apoptosis and activates caspase-9 enzyme in human BrC cells [9]. Finally, LYC has inhibited the multiplication of cancer cells by arresting cell cycle at different phases (G1, S, and M phases) and by sustained activation of the ERK½ with suppression of cyclin D1 and upregulation of p21 [39]. Other mechanism of action of LYC is through the inhibition of IκBα phosphorylation and

Essential oils are a complex mixture of secondary metabolites such as monoterpenes that are responsible for their biological activity that includes anti-BrC effect. However, some studies suggested a synergistic activity of the compounds [17]. For

It had a cytotoxic effect with an IC50 of 53.81 μg/ml. It induced DNA fragmentation and apoptosis via intrinsic pathways due to the activation of Bax, caspase-9, and caspases-3, suggesting a synergistic activity of compounds present in the essential oil, such as 1,5-cyclooctadiene, 3-(methyl-2)propenyl, β-terpineol, 1-(3-methyl-

Flavonoids which are polyphenolic substances found in different plant-derived food are divided into flavones (cynaroside), flavonols, flavanones (naringenin), flavanols, isoflavones (genistein (GEN), biochanin A), anthocyanidins, and

nonflavonoids [10]. Flavonoids have been reported to have an effect on BrC through numerous mechanisms such as antioxidant, anti-inflammatory, antiproliferative, cytotoxic, anti-angiogenic, and anti-metastatic effects in numerous in vitro and in vivo experiments in estrogen-dependent or estrogen-independent BrC [15, 37]

Cynaroside (luteolin-7-*O*-glucoside) is a glycosyloxyflavone or a glycoside form. It derives from luteolin. Cynaroside has a molecular structure of C21H20O11 and a molecular mass of 448.38 g/mol [18]. Cynaroside is a constituent of the leaves of

cyclopent-2-enyl)-cyclohexene, *D*-limonene, pinene, and linalool [41].

**52**

(**Tables 1**–**3**).

*4.2.1 Cynaroside*

Naringenin (4',5,7-trihydroxyflavanone) is a flavanone and member of 4'hydroxyflavanones; it has a molecular structure of C15H12O5 and a molecular mass of 272.256 g/mol [18]. This bioflavonoid is a constituent of tomatoes, citrus fruits, and grapes. Naringenin is a phytoestrogen which is also an important anti-BrC, reported to be involved in decreasing the number of ER-α-positive cells by modulating p38 MAPK signaling pathway [9]. Recently, investigators reported that naringenin has antiproliferative effects by arresting the cell cycle at the G2 phase and caused an inhibitory effect on MDA-MB-231 cells via induction of apoptosis and inhibition of caspase-3 and caspase-9 activities [37, 43].

#### *4.2.3 Biochanin and ginsenoside Rh2 as pure compounds or mixture*

Biochanin or 4'-methylgenistein (B5,7-dihydroxy-4'-methoxyisoflavone) is an *O*-methylated isoflavone that is isolated from red clover *Trifolium pratense* and the root of *Astragalus membranaceus*, a traditional Chinese herbal medicine. BA has a molecular structure of C16H12O5 and a molecular mass of 284.267 g/mol [18, 44]. The phytoestrogen BA caused antiproliferative activity by stabilizing and activating p53 through the upregulated expression of phospho-p53, phospho-p38, and p-ASK1 and downregulated expression of TRAF2 in MDA-MB-231 and MCF-7 cells [44] and apoptosis through upregulated expression of mRNA levels of ER-α, Bcl-2, and miR-375 in ER+ BrC cells, that is to say T47D and MCF-7 [37, 44, 45].

Also, BA stopped cell growth by blocking the activity of aromatase enzyme which is encoded by the gene CYP19 [5]. On the other hand, ginsenoside Rh2 (protopanaxadiol-type) is the major type of saponin ginsenoside that is separated from *Panax ginseng* and other species. Rh2 has a molecular structure of C36H62O8 and a molecular mass of 622.884 g/mol [18, 44]. Rh2 exhibits antitumor activity in ER+(MCF-7) and ER-(MDA-MB-231). However, Ren and collaborators determined that BA plus Rh2 synergistically enhanced the antiproliferative effect in both BrC cells, with decreased EC50 values of both the compounds and a mechanism through the stabilization and activation of p53, p38, and ASK1 proteins (**Table 1**) [44].

#### *4.2.4 Genistein*

Genistein (4',5,7-trihydroxyisoflavone) is an isoflavonoid derived from soy products [46]. It has a molecular structure of C15H10O5 and a molecular mass of 270.24 g/mol [18]. This agent has an antineoplastic effect in BrC [47]. GEN inhibits the growth of MDA-MB-231 cells by altering the phosphorylation of proteins included in cell cycle regulation and DNA damage response predominantly, and GEN induced apoptosis via the upregulation of Bax and p21WAF1 proteins in MDA-MB-231 cells and downregulating the expression of caspase-3 [5, 47]. In a recent study, GEN increases cell cycle arrest in G2/M phase in MDA-MB-231/ERβ1 cells, even though there is a high dose of GEN-arrested cells in G0/G1, just like in the MCF-7 cells. Thus, the combinatorial effect of GEN and overexpressed ERβ1 resulted in an active blockade of cell cycle progression and a dramatic inhibition

of proliferation in vitro in MCF7 and MDA-MB-231 cells [48]. GEN could be a potential therapeutic agent for ERβ1-positive cancer, which merits further clinical research in the future.

#### *4.2.5 Resveratrol*

Resveratrol (3,5,4′-trihydroxy-*trans*-stilbene) is a natural nonflavonoid polyphenol with a molecular formula of C14H12O3 [18]. These compounds are isolated from more than 72 species of plants including peanuts, grapes, mulberries, bilberries, and blueberries [9].

Numerous in vitro studies have shown that resveratrol has multiple anticancer effects, which protect the cells against both tumor initiation and cancer progression pathway [9, 57]. The activity of this compound was described in hormonedependent or non-hormone-dependent BrC cells, in which it was found to induce apoptosis by intrinsic pathway through the upregulation of Bax, Bak, caspase-3, p53, and Akt pathway in different breast cancer cell lines and downregulated Bcl-2 and NF-kB and VEGF [51, 58–62]. Also, it can induce the extrinsic pathway through the expression of CD95 receptor [63]. A cell surface resveratrol receptor on the extracellular domain of heterodimeric αVβ3-integrin in MCF-7 human BrC cells induces extracellular-regulated kinases 1 and 2 (ERK1/2) and serine-15-p53 dependent phosphorylation leading to a p53-dependent apoptosis [57]. In several in vivo models, resveratrol supplementation was shown to decrease the incidence of mammary tumor formation, tumor volume, metastasis, and induced apoptosis [64–66]. The effect of resveratrol demonstrated lower tumor growth, decreased angiogenesis, and increased apoptotic index in ERα− and ERβ+ [62]. Also, the following can suppress mammary carcinogenesis in rats induced by DMBA: dietary administration of resveratrol (10 ppm), downregulation of NF-kB, cyclooxygenase-2 and matrix metalloprotease-9 expression in the breast tumor, and decreased tumor incidence [67, 68].

The effect of resveratrol in cancer patients has been investigated in a few clinical trials. The first clinical trial dealing with resveratrol and cancer was performed by Nguyen and collaborators in 2009, through the administration of 0.07 mg/day of resveratrol which resulted in the reduction of Wnt target gene expression, indicating that it may play a beneficial role in the prevention of cancer. These clinical trials have demonstrated resveratrol to be a promising therapeutic and chemopreventive agent [64, 69, 70].

#### *4.2.6 Curcumin*

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is an orange-yellow component of turmeric or curry powder; it is a polyphenol natural product isolated from the rhizome of *Curcuma longa* [9, 71]. Curcumin has antiproliferative and proapoptotic effects against a variety of cancer cells in vitro. The anticancer effects observed by activating intrinsic apoptotic pathway by interacting with reactive oxygen species can release cytochrome C; upregulate caspase-9, caspase-3, Bax, and Bad; downregulate Bcl-2 antiapoptotic proteins; and induce DNA fragmentation in different BrC cell lines [72–75]. Also, it was found that curcumin inhibits the expression of Ki-67, proliferating cell nuclear antigen (PCNA), p53, and VEGF in BrC cells [54, 76]. Curcumin prevents carcinogen-induced cancers in rodents [77]. Banerjee et al. [79] reported that curcumin-induced G2/M arrest and apoptosis inhibited cell proliferation in MCF-7 cells, leading to an accumulation in the G1 phase, and suppressed the expression of zeste homolog 2 (EZH2) gene via MAPK pathway [78, 79].

**55**

*Cytotoxic Effect and Mechanisms from Some Plant-Derived Compounds in Breast Cancer*

Alkaloids are a highly diverse group of compounds containing an organic nitrogen atom and a ring structure. Additionally, in most alkaloids the nitrogen atom is located inside the heterocyclic ring structure, which gives them a great biological diversity [16]. The structural diversity of this family is due to the wide number of amino acids used as building blocks [80]. Indeed, the peptide ring that they contain has one or more of its hydrogen atoms replaced with various alkyl radicals, most of which contain oxygen [81, 82]. Consequently, alkaloids can interact with a wide spectrum of molecules. They have a wide distribution in the plant kingdom and are a chemically heterogeneous group of ~17,000 molecules which have displayed pronounced biological and pharmacological activities. Furthermore, several alkaloids exhibit significant biological activities, with their unlimited supply of variable structures as well as their relatively low toxicity and well-documented stability; therefore, alkaloids are being used for their anticancer activity against various cancers [83].

Camptothecin is a monoterpene indole alkaloid that consists of five rings [18], commonly named as CPT with a molecular structure of C20H16N2C4 and a molecular mass of 348.35 g/mol [18]. The antitumor activity of this compound is mainly due to its interaction with topoisomerase I (Top1), an enzyme involved in the regulation of DNA topology during replication, recombination, and transcription. It induces cell death by stabilizing a covalent complex between DNA topoisomerase I and the

CPT antitumoral activity has been reported in different cancer cell lines. Low doses of this compound lead to cell cycle arrest in the G2/M phase and inhibit DNA synthesis but at higher doses cause cell cycle arrest in S phase [52]. Also, the expression of some genes as c-Myc, Bax, BFL1, Bak, pRb2, c-Jun, and Jun-B was upregulated, and Cdk4, cyclin B1, Wee1, CRAF1, and DP1 were downregulated. Among these derivatives, camptothecin-20(s)-*O*-(2-pyrazolyl-1)acetic ester exhibited antitumor activity which demonstrated cytotoxicity, DNA fragmentation, and apoptosis toward MCF-7 cell line [53]. Also, in different studies in vivo, where it was delivered using an intralipid formulation through intramuscular (IM) route, CPT showed nearly 100% growth inhibition and regression in the colon, lung, breast, stomach, and ovary and malignant melanoma xenografts [88, 89].

There are some vinca alkaloids in clinical use such as vinblastine, vinorelbine, and vincristine. Many alkaloids have poisonous characteristics but also have physiological effects that make them useful as medications. The oldest group of the plant alkaloids used to treat cancer is the vinca alkaloids. They have a dimeric chemical structure composed of two basic multi-ringed units, an indole nucleus (catharanthine) and a dihydroindole nucleus (vindoline), joined together with other complex systems. Structurally, vincristine and vinblastine are identical except for a single substitution on the vindoline nucleus, where vincristine and vinblastine possess formyl and methyl groups, respectively [90, 91] (**Table 2**). The main mechanisms of vinca alkaloid cytotoxicity is due to their interactions with tubulin and disruption of microtubule function, particularly of microtubules comprising the mitotic spindle apparatus, directly causing metaphase arrest. The disturbing effects occur at drug concentrations below those that decrease microtubule mass [91, 92]. Also, disorganization of the microtubule structure provokes the induction of tumor suppressor gene

*DOI: http://dx.doi.org/10.5772/intechopen.87177*

**4.3 Alkaloids as cytotoxic compounds**

nicked DNA, leading to a DNA lesion [84–87].

*4.3.2 Vinca alkaloids: vinblastine, vincristine, and vinorelbine*

*4.3.1 Camptothecin*

#### **4.3 Alkaloids as cytotoxic compounds**

Alkaloids are a highly diverse group of compounds containing an organic nitrogen atom and a ring structure. Additionally, in most alkaloids the nitrogen atom is located inside the heterocyclic ring structure, which gives them a great biological diversity [16]. The structural diversity of this family is due to the wide number of amino acids used as building blocks [80]. Indeed, the peptide ring that they contain has one or more of its hydrogen atoms replaced with various alkyl radicals, most of which contain oxygen [81, 82]. Consequently, alkaloids can interact with a wide spectrum of molecules. They have a wide distribution in the plant kingdom and are a chemically heterogeneous group of ~17,000 molecules which have displayed pronounced biological and pharmacological activities. Furthermore, several alkaloids exhibit significant biological activities, with their unlimited supply of variable structures as well as their relatively low toxicity and well-documented stability; therefore, alkaloids are being used for their anticancer activity against various cancers [83].

#### *4.3.1 Camptothecin*

*Cytotoxicity - Definition, Identification, and Cytotoxic Compounds*

research in the future.

*4.2.5 Resveratrol*

and blueberries [9].

tumor incidence [67, 68].

agent [64, 69, 70].

*4.2.6 Curcumin*

of proliferation in vitro in MCF7 and MDA-MB-231 cells [48]. GEN could be a potential therapeutic agent for ERβ1-positive cancer, which merits further clinical

Resveratrol (3,5,4′-trihydroxy-*trans*-stilbene) is a natural nonflavonoid polyphenol with a molecular formula of C14H12O3 [18]. These compounds are isolated from more than 72 species of plants including peanuts, grapes, mulberries, bilberries,

Numerous in vitro studies have shown that resveratrol has multiple anticancer effects, which protect the cells against both tumor initiation and cancer progression pathway [9, 57]. The activity of this compound was described in hormonedependent or non-hormone-dependent BrC cells, in which it was found to induce apoptosis by intrinsic pathway through the upregulation of Bax, Bak, caspase-3, p53, and Akt pathway in different breast cancer cell lines and downregulated Bcl-2 and NF-kB and VEGF [51, 58–62]. Also, it can induce the extrinsic pathway through the expression of CD95 receptor [63]. A cell surface resveratrol receptor on the extracellular domain of heterodimeric αVβ3-integrin in MCF-7 human BrC cells induces extracellular-regulated kinases 1 and 2 (ERK1/2) and serine-15-p53 dependent phosphorylation leading to a p53-dependent apoptosis [57]. In several in vivo models, resveratrol supplementation was shown to decrease the incidence of mammary tumor formation, tumor volume, metastasis, and induced apoptosis [64–66]. The effect of resveratrol demonstrated lower tumor growth, decreased angiogenesis, and increased apoptotic index in ERα− and ERβ+ [62]. Also, the following can suppress mammary carcinogenesis in rats induced by DMBA: dietary administration of resveratrol (10 ppm), downregulation of NF-kB, cyclooxygenase-2 and matrix metalloprotease-9 expression in the breast tumor, and decreased

The effect of resveratrol in cancer patients has been investigated in a few clinical trials. The first clinical trial dealing with resveratrol and cancer was performed by Nguyen and collaborators in 2009, through the administration of 0.07 mg/day of resveratrol which resulted in the reduction of Wnt target gene expression, indicating that it may play a beneficial role in the prevention of cancer. These clinical trials have demonstrated resveratrol to be a promising therapeutic and chemopreventive

Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is an orange-yellow component of turmeric or curry powder; it is a polyphenol natural product isolated from the rhizome of *Curcuma longa* [9, 71]. Curcumin has antiproliferative and proapoptotic effects against a variety of cancer cells in vitro. The anticancer effects observed by activating intrinsic apoptotic pathway by interacting with reactive oxygen species can release cytochrome C; upregulate caspase-9, caspase-3, Bax, and Bad; downregulate Bcl-2 antiapoptotic proteins; and induce DNA fragmentation in different BrC cell lines [72–75]. Also, it was found that curcumin inhibits the expression of Ki-67, proliferating cell nuclear antigen (PCNA), p53, and VEGF in BrC cells [54, 76]. Curcumin prevents carcinogen-induced cancers in rodents [77]. Banerjee et al. [79] reported that curcumin-induced G2/M arrest and apoptosis inhibited cell proliferation in MCF-7 cells, leading to an accumulation in the G1 phase, and suppressed the expression of zeste homolog 2 (EZH2) gene via

**54**

MAPK pathway [78, 79].

Camptothecin is a monoterpene indole alkaloid that consists of five rings [18], commonly named as CPT with a molecular structure of C20H16N2C4 and a molecular mass of 348.35 g/mol [18]. The antitumor activity of this compound is mainly due to its interaction with topoisomerase I (Top1), an enzyme involved in the regulation of DNA topology during replication, recombination, and transcription. It induces cell death by stabilizing a covalent complex between DNA topoisomerase I and the nicked DNA, leading to a DNA lesion [84–87].

CPT antitumoral activity has been reported in different cancer cell lines. Low doses of this compound lead to cell cycle arrest in the G2/M phase and inhibit DNA synthesis but at higher doses cause cell cycle arrest in S phase [52]. Also, the expression of some genes as c-Myc, Bax, BFL1, Bak, pRb2, c-Jun, and Jun-B was upregulated, and Cdk4, cyclin B1, Wee1, CRAF1, and DP1 were downregulated. Among these derivatives, camptothecin-20(s)-*O*-(2-pyrazolyl-1)acetic ester exhibited antitumor activity which demonstrated cytotoxicity, DNA fragmentation, and apoptosis toward MCF-7 cell line [53]. Also, in different studies in vivo, where it was delivered using an intralipid formulation through intramuscular (IM) route, CPT showed nearly 100% growth inhibition and regression in the colon, lung, breast, stomach, and ovary and malignant melanoma xenografts [88, 89].

#### *4.3.2 Vinca alkaloids: vinblastine, vincristine, and vinorelbine*

There are some vinca alkaloids in clinical use such as vinblastine, vinorelbine, and vincristine. Many alkaloids have poisonous characteristics but also have physiological effects that make them useful as medications. The oldest group of the plant alkaloids used to treat cancer is the vinca alkaloids. They have a dimeric chemical structure composed of two basic multi-ringed units, an indole nucleus (catharanthine) and a dihydroindole nucleus (vindoline), joined together with other complex systems. Structurally, vincristine and vinblastine are identical except for a single substitution on the vindoline nucleus, where vincristine and vinblastine possess formyl and methyl groups, respectively [90, 91] (**Table 2**). The main mechanisms of vinca alkaloid cytotoxicity is due to their interactions with tubulin and disruption of microtubule function, particularly of microtubules comprising the mitotic spindle apparatus, directly causing metaphase arrest. The disturbing effects occur at drug concentrations below those that decrease microtubule mass [91, 92]. Also, disorganization of the microtubule structure provokes the induction of tumor suppressor gene p53 and activation/inactivation of several protein kinases involved in key signaling pathways, including p21, WAF1/CIP1, Ras/Raf, and PKC/PKA, the apoptosis inhibitor Bcl2 and induction of Bax triggering the process of apoptosis in the cell [93]. These alkaloids demonstrated significant antitumor activity in patients with BrC. Also, xenograft mice models were used to evaluate low doses of vinblastine, which resulted in significant but transient xenograft regression, diminishing tumor vascularity, and direct inhibition of angiogenesis. Also, a combination therapy resulted in full and sustained regressions of large established tumors, without an ensuing increase in host toxicity or any signs of acquired drug resistance during treatment [94].

The risk of side effects and multidrug resistance limited the development of vinca alkaloids for clinical applications. To solve these problems, researchers have developed numerous strategies, such as using liposome-entrapped drugs, chemically modified drugs, and polymeric packaging drugs, to reduce the toxicity and enhance the therapeutic efficiency of vinca alkaloids. Many liposome products are still being tested in clinical trials. Another strategy for reducing chemotherapeutic toxicity involves using chemically modified drugs [95, 96].

#### **5. Biotechnological and clinical advances of plant-derived compounds in breast cancer**

Nanotechnology has been found to potentially improve current methods for disease, diagnosis, disease-state imaging, and treatment in BrC.

Targeted nanoparticle drug delivery is intended to reduce the side effects of anticancer drugs with both decreasing consumption and treatment expenses, which are the major hurdles in conventional cancer treatment. These small entities can be used in combination with a variety of plant-derived compounds in BrC with a variety of formulations being developed, making them a desirable choice of drug formulation [8].

#### **5.1 Use of phytochemical compounds coupled to nanoparticles**

#### *5.1.1 Lycopene with nanoparticles*

Recently, Jain and collaborators designed and synthesized LYC incorporated with biopolymeric nanoparticles with whey protein isolate nanoparticles (WPI NPs) with encouraging results in the compatibility of LYC-WPI-NPs over plain LYC as an optimum delivery system in vitro because encapsulation process did not affect its anticancer activity even in in vivo tumor model of DMBA where ~57% of LYC group of animals developed tumor compared with ~29% of LYC-WPI-NPs. The new formulation (LYC-WPI-NPs) posed higher cytotoxicity and cellular uptake efficacy as compared to the plain LYC in MCF-7 cells. Antitumoral effect of LYC-WPI-NPs and survival data indicate that proposed formulation strategy is a novel approach for the synchronized delivery of bioactive compound, leading to increased bioavailability, therapeutic efficacy, and safety profiling because of improvement in animal survival (100%), in contrast to animals free of LYC (66.67%) and negative control group (16.67%). This will certainly open new avenues to explore cancer treatment [49].

#### *5.1.2 Paclitaxel with nanoparticles*

As a strongly hydrophobic drug, it requires suitable delivery vehicles to effectively distribute into tumor tissues. For efficient distribution of this hydrophobic anticancer drug, paclitaxel is currently formulated and administered to patients via polyethoxylated castor oil (Cremophor EL, CrEL), but it is reported as causing

**57**

*or Abraxane®*

*Cytotoxic Effect and Mechanisms from Some Plant-Derived Compounds in Breast Cancer*

hypersensitivity reactions and neurotoxicity [101]. To date, paclitaxel albuminbound nanoparticles (Abraxane®) have been approved by the FDA for the treatment of metastatic BrC and non-small cell lung cancer [8]. Nanoparticle-based delivery systems can take advantage of the enhanced permeability and retention (EPR) effect for passive tumor targeting; therefore, they can improve the therapeutic index and decrease the side effects of paclitaxel. In addition, there are a number of novel paclitaxel nanoparticle formulations in clinical trials [50, 101–104]. Nanoparticle-assisted chemotherapeutic drug delivery has been used because it enhances therapeutic effectiveness. Studies on metastatic BrC demonstrate the inhibition of metastasis by co-delivering chemotherapeutic agent paclitaxel and

Research have concentrated on the development of potential delivery system to increase the aqueous solubility, stability, and bioavailability as well as controlled delivery of camptothecin at or around cancer tissues. For that purpose nanoencapsulation of drugs in a biodegradable polymer has been reported to protect the drug in the core of the polymeric shell [106–108]. Camptothecin encapsulated in nanoparticles demonstrated antitumor activity in in vitro and in vivo models; in MCF-7 cells

As other compounds, in order to increase photostability and enhance its anticancer activity against BrC cells, scientists have formulated the transferrin-mediated solid lipid nanoparticle, which enhances the anticancer effect of curcumin in BrC cells in vitro [110]. A polymer-drug conjugate called polycurcumins also has advantages of high drug-loading efficiency, fixed drug-loading contents, stabilized curcumin in their backbones, and tailored water solubility. The polycurcumins are cytotoxic to cancer cells, but a polyacetal-based polycurcumin is highly cytotoxic to MCF-7 cells. The effect of these polymers induced cell cycle arrest and apoptosis partially through the caspase-3-dependent pathway. In vivo, this polymer showed antitumor activity in SKOV-3 intraperitoneal xenograft tumor model [111].

**5.2 Novel therapies: antibody-phytopharmaceutical conjugates in breast cancer**

in order to reduce off-target effects and increase therapeutic outcomes [8, 112].

*5.2.1 Plant natural products as components of development ADCs: nab-paclitaxel* 

The plant-derived compounds are secondary metabolites that have a different mechanism of action; although all of them are cytotoxic for BrC cells, new tools are being sought to increase their effectiveness, with less toxic effects. Also, incorporation of paclitaxel in liposomes can facilitate its delivery to cancer cells and eliminate the adverse reactions associated with the Cremophor EL vehicle. The lipid components of

In recent years, natural products and their derivatives have been among the major sources of drugs for the treatment of cancer as well as nanoparticles or antibodies. Furthermore, new treatments for different cases of BrC are necessary; this involves linking each cytotoxic drug concerned with a mAb by a linker group to produce a tripartite drug called an "antibody-drug conjugate" (ADC). A means of selective delivery of highly cytotoxic natural products as "prodrugs" to tumor cells has proven necessary

the IC50 was lower (0.23 μm) than the pure compound (0.57 μm) [106, 109].

*DOI: http://dx.doi.org/10.5772/intechopen.87177*

twist shRNA via complex nanoparticles [105].

*5.1.3 Camptothecin with nanoparticles*

*5.1.4 Curcumin with nanoparticles*

#### *Cytotoxic Effect and Mechanisms from Some Plant-Derived Compounds in Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.87177*

hypersensitivity reactions and neurotoxicity [101]. To date, paclitaxel albuminbound nanoparticles (Abraxane®) have been approved by the FDA for the treatment of metastatic BrC and non-small cell lung cancer [8]. Nanoparticle-based delivery systems can take advantage of the enhanced permeability and retention (EPR) effect for passive tumor targeting; therefore, they can improve the therapeutic index and decrease the side effects of paclitaxel. In addition, there are a number of novel paclitaxel nanoparticle formulations in clinical trials [50, 101–104]. Nanoparticle-assisted chemotherapeutic drug delivery has been used because it enhances therapeutic effectiveness. Studies on metastatic BrC demonstrate the inhibition of metastasis by co-delivering chemotherapeutic agent paclitaxel and twist shRNA via complex nanoparticles [105].

#### *5.1.3 Camptothecin with nanoparticles*

*Cytotoxicity - Definition, Identification, and Cytotoxic Compounds*

p53 and activation/inactivation of several protein kinases involved in key signaling pathways, including p21, WAF1/CIP1, Ras/Raf, and PKC/PKA, the apoptosis inhibitor Bcl2 and induction of Bax triggering the process of apoptosis in the cell [93]. These alkaloids demonstrated significant antitumor activity in patients with BrC. Also, xenograft mice models were used to evaluate low doses of vinblastine, which resulted in significant but transient xenograft regression, diminishing tumor vascularity, and direct inhibition of angiogenesis. Also, a combination therapy resulted in full and sustained regressions of large established tumors, without an ensuing increase in host

The risk of side effects and multidrug resistance limited the development of vinca alkaloids for clinical applications. To solve these problems, researchers have developed numerous strategies, such as using liposome-entrapped drugs, chemically modified drugs, and polymeric packaging drugs, to reduce the toxicity and enhance the therapeutic efficiency of vinca alkaloids. Many liposome products are still being tested in clinical trials. Another strategy for reducing chemotherapeutic

Nanotechnology has been found to potentially improve current methods for

Targeted nanoparticle drug delivery is intended to reduce the side effects of anticancer drugs with both decreasing consumption and treatment expenses, which are the major hurdles in conventional cancer treatment. These small entities can be used in combination with a variety of plant-derived compounds in BrC with a variety of formulations being developed, making them a desirable choice of drug formulation [8].

Recently, Jain and collaborators designed and synthesized LYC incorporated with

As a strongly hydrophobic drug, it requires suitable delivery vehicles to effectively distribute into tumor tissues. For efficient distribution of this hydrophobic anticancer drug, paclitaxel is currently formulated and administered to patients via polyethoxylated castor oil (Cremophor EL, CrEL), but it is reported as causing

biopolymeric nanoparticles with whey protein isolate nanoparticles (WPI NPs) with encouraging results in the compatibility of LYC-WPI-NPs over plain LYC as an optimum delivery system in vitro because encapsulation process did not affect its anticancer activity even in in vivo tumor model of DMBA where ~57% of LYC group of animals developed tumor compared with ~29% of LYC-WPI-NPs. The new formulation (LYC-WPI-NPs) posed higher cytotoxicity and cellular uptake efficacy as compared to the plain LYC in MCF-7 cells. Antitumoral effect of LYC-WPI-NPs and survival data indicate that proposed formulation strategy is a novel approach for the synchronized delivery of bioactive compound, leading to increased bioavailability, therapeutic efficacy, and safety profiling because of improvement in animal survival (100%), in contrast to animals free of LYC (66.67%) and negative control group (16.67%). This will certainly open new avenues to explore cancer treatment [49].

toxicity or any signs of acquired drug resistance during treatment [94].

toxicity involves using chemically modified drugs [95, 96].

**compounds in breast cancer**

*5.1.1 Lycopene with nanoparticles*

*5.1.2 Paclitaxel with nanoparticles*

**5. Biotechnological and clinical advances of plant-derived** 

disease, diagnosis, disease-state imaging, and treatment in BrC.

**5.1 Use of phytochemical compounds coupled to nanoparticles**

**56**

Research have concentrated on the development of potential delivery system to increase the aqueous solubility, stability, and bioavailability as well as controlled delivery of camptothecin at or around cancer tissues. For that purpose nanoencapsulation of drugs in a biodegradable polymer has been reported to protect the drug in the core of the polymeric shell [106–108]. Camptothecin encapsulated in nanoparticles demonstrated antitumor activity in in vitro and in vivo models; in MCF-7 cells the IC50 was lower (0.23 μm) than the pure compound (0.57 μm) [106, 109].

#### *5.1.4 Curcumin with nanoparticles*

As other compounds, in order to increase photostability and enhance its anticancer activity against BrC cells, scientists have formulated the transferrin-mediated solid lipid nanoparticle, which enhances the anticancer effect of curcumin in BrC cells in vitro [110]. A polymer-drug conjugate called polycurcumins also has advantages of high drug-loading efficiency, fixed drug-loading contents, stabilized curcumin in their backbones, and tailored water solubility. The polycurcumins are cytotoxic to cancer cells, but a polyacetal-based polycurcumin is highly cytotoxic to MCF-7 cells. The effect of these polymers induced cell cycle arrest and apoptosis partially through the caspase-3-dependent pathway. In vivo, this polymer showed antitumor activity in SKOV-3 intraperitoneal xenograft tumor model [111].

#### **5.2 Novel therapies: antibody-phytopharmaceutical conjugates in breast cancer**

In recent years, natural products and their derivatives have been among the major sources of drugs for the treatment of cancer as well as nanoparticles or antibodies. Furthermore, new treatments for different cases of BrC are necessary; this involves linking each cytotoxic drug concerned with a mAb by a linker group to produce a tripartite drug called an "antibody-drug conjugate" (ADC). A means of selective delivery of highly cytotoxic natural products as "prodrugs" to tumor cells has proven necessary in order to reduce off-target effects and increase therapeutic outcomes [8, 112].

#### *5.2.1 Plant natural products as components of development ADCs: nab-paclitaxel or Abraxane®*

The plant-derived compounds are secondary metabolites that have a different mechanism of action; although all of them are cytotoxic for BrC cells, new tools are being sought to increase their effectiveness, with less toxic effects. Also, incorporation of paclitaxel in liposomes can facilitate its delivery to cancer cells and eliminate the adverse reactions associated with the Cremophor EL vehicle. The lipid components of

the liposomal formulation were nontoxic, but the intracellular paclitaxel levels were higher when MCF-7 cells were treated with the liposomal paclitaxel formulation; also, liposomal paclitaxel was as effective as conventional paclitaxel in inducing G2/M arrest after 1 day of treatment with 10 mmol/L, increasing the percentage of cells in this population from about 20% in cycling cells to over 60% after 7 days [104].

Abraxane® or nanoparticle albumin-bound paclitaxel (nab-paclitaxel) suspension demonstrated greater efficacy with less toxicity than docetaxel in metastatic BrC. This treatment has been approved to reduce toxicity and increased overall survival rates, compared to the parent compound [8]. Nab-paclitaxel is a neoadjuvant chemotherapy in HER2-negative BrC stages I, II, and III.

The recommended dose of Abraxane® is 260 mg/m2 administered intravenously for 30 minutes, every 3 weeks. The results suggest that Abraxane® is effective in patients with highly proliferative cancers (81).

#### **6. Conclusion**

In nature, plants contain secondary metabolites, which have been used by humans to treat different diseases, since they have a complex diversity of chemical structures that have been specifically related to have anti-BrC activity in several preclinical studies with more than one mechanism of action; as a result, they can provide greater degree of efficacy. Several natural compounds are highlighted in this chapter, and their mechanism of action, synergistic action, nano-formulations, and future potentials are widely discussed, and due to the promising potential they represent, in fact, some of them are already used as treatment for BrC. However, it is necessary to have a greater diversity of drugs to be able to treat each one of the different tumors of BrC, since each BrC is different and many of these drugs may still induce several side effects and the development of mechanisms of resistance to drugs must be avoided. Subsequent from this review, we can conclude that although there are many compounds that have been characterized mainly in in vitro models and only around 10–20% of these were also evaluated in in vivo models and less than 10% are being evaluated already in clinical phases, it is essential to conduct more research on these compounds to learn their mechanism of action. Also, in their cellular, biochemical, and molecular levels, clinical effects as well as their genetic toxicities should be investigated sufficiently. Compounds that meet the eligibility criteria in these tests should be taken into clinical trial phase, and they may be administered in combination with other compounds or materials that make their pharmacological effect more efficient, make their arrival to the target site more selective, and guarantee their stability, bioavailability, pharmacokinetics, etc. In conclusion, addressing the study of these compounds in clinical phase is a pressing need.

#### **Acknowledgements**

This work was supported by the Secretaría de Investigación y Posgrado del Instituto Politécnico Nacional Grants SIP20170567 and SIP20196913. CRC is supported by CONACyT and BEIFI, IPN Fellowships.

**59**

**Author details**

Elvia Pérez-Soto, Cynthia Carolina Estanislao-Gómez, David Guillermo Pérez-Ishiwara, Crisalde Ramirez-Celis and

Laboratorio de Biomedicina Molecular I, Programa Institucional de Biomedicina Molecular, Escuela Nacional de Medicina y Homeopatía-Instituto Politécnico

\*Address all correspondence to: cgomezg@ipn.mx; consuelogg22@yahoo.com.mx

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

María del Consuelo Gómez-García\*

Nacional, Ciudad de México, México

provided the original work is properly cited.

*Cytotoxic Effect and Mechanisms from Some Plant-Derived Compounds in Breast Cancer*

*DOI: http://dx.doi.org/10.5772/intechopen.87177*

#### **Conflict of interest**

The authors declare that they have no competing interests.

*Cytotoxic Effect and Mechanisms from Some Plant-Derived Compounds in Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.87177*

### **Author details**

*Cytotoxicity - Definition, Identification, and Cytotoxic Compounds*

vant chemotherapy in HER2-negative BrC stages I, II, and III. The recommended dose of Abraxane® is 260 mg/m2

patients with highly proliferative cancers (81).

**6. Conclusion**

clinical phase is a pressing need.

ported by CONACyT and BEIFI, IPN Fellowships.

The authors declare that they have no competing interests.

**Acknowledgements**

**Conflict of interest**

the liposomal formulation were nontoxic, but the intracellular paclitaxel levels were higher when MCF-7 cells were treated with the liposomal paclitaxel formulation; also, liposomal paclitaxel was as effective as conventional paclitaxel in inducing G2/M arrest after 1 day of treatment with 10 mmol/L, increasing the percentage of cells in this population from about 20% in cycling cells to over 60% after 7 days [104].

Abraxane® or nanoparticle albumin-bound paclitaxel (nab-paclitaxel) suspension demonstrated greater efficacy with less toxicity than docetaxel in metastatic BrC. This treatment has been approved to reduce toxicity and increased overall survival rates, compared to the parent compound [8]. Nab-paclitaxel is a neoadju-

for 30 minutes, every 3 weeks. The results suggest that Abraxane® is effective in

In nature, plants contain secondary metabolites, which have been used by humans to treat different diseases, since they have a complex diversity of chemical structures that have been specifically related to have anti-BrC activity in several preclinical studies with more than one mechanism of action; as a result, they can provide greater degree of efficacy. Several natural compounds are highlighted in this chapter, and their mechanism of action, synergistic action, nano-formulations, and future potentials are widely discussed, and due to the promising potential they represent, in fact, some of them are already used as treatment for BrC. However, it is necessary to have a greater diversity of drugs to be able to treat each one of the different tumors of BrC, since each BrC is different and many of these drugs may still induce several side effects and the development of mechanisms of resistance to drugs must be avoided. Subsequent from this review, we can conclude that although there are many compounds that have been characterized mainly in in vitro models and only around 10–20% of these were also evaluated in in vivo models and less than 10% are being evaluated already in clinical phases, it is essential to conduct more research on these compounds to learn their mechanism of action. Also, in their cellular, biochemical, and molecular levels, clinical effects as well as their genetic toxicities should be investigated sufficiently. Compounds that meet the eligibility criteria in these tests should be taken into clinical trial phase, and they may be administered in combination with other compounds or materials that make their pharmacological effect more efficient, make their arrival to the target site more selective, and guarantee their stability, bioavailability, pharmacokinetics, etc. In conclusion, addressing the study of these compounds in

This work was supported by the Secretaría de Investigación y Posgrado del Instituto Politécnico Nacional Grants SIP20170567 and SIP20196913. CRC is sup-

administered intravenously

**58**

Elvia Pérez-Soto, Cynthia Carolina Estanislao-Gómez, David Guillermo Pérez-Ishiwara, Crisalde Ramirez-Celis and María del Consuelo Gómez-García\* Laboratorio de Biomedicina Molecular I, Programa Institucional de Biomedicina Molecular, Escuela Nacional de Medicina y Homeopatía-Instituto Politécnico Nacional, Ciudad de México, México

\*Address all correspondence to: cgomezg@ipn.mx; consuelogg22@yahoo.com.mx

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[21] Jia S, Xi G, Zhang M, Chen Y, Lei BO, Dong X, et al. Induction of apoptosis by *D*-limonene is mediated by inactivation of Akt in LS174T human colon cancer cells. Oncology Reports. 2013;**3**(23):349-354

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**60**

*Cytotoxicity - Definition, Identification, and Cytotoxic Compounds*

Biomedicine & Pharmacotherapy. 2018;**108**:752-756. DOI: 10.1016/j.

[8] Agarwal G, Carcache PB, Addo EM, Kinghorn AD. Current status and contemporary approaches to the discovery of antitumor agents from higher plants. Biotechnology Advances. 2019. DOI: 10.1016/j.

biopha.2018.09.096

biotechadv.2019.01.004

[9] Iqbal J, Ahsan B, Mahmood T, Ali B, Talha A, Kanwal S, et al. Potential

phytocompounds for developing breast cancer therapeutics: Nature's healing touch. European Journal of Pharmacology. 2018;**827**:125-148. DOI:

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[11] Kim KH, Seo HS, Choi HS, Choi I, Shin YC, Ko S. Induction of apoptotic cell death by ursolic acid through mitochondrial death pathway and extrinsic death receptor pathway in MDA-MB-231 cells. Archives of Pharmacal Research. 2011;**34**(8):1363- 1372. DOI: 10.1007/s12272-011-0817-5

[12] Tariq A, Sadia S, Pan K, Ullah I, Mussarat S, Sun F, et al. A systematic review on ethnomedicines of anticancer plants. Physical Therapy Research. 2017;**31**(2):202-264. DOI:

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[14] Cekanova M. Animal models and therapeutic molecular targets of

10.1016/j.ejphar.2018.03.007

s10020-018-0032-7

10.1002/ptr.5751

10.1242/dmm.028274

[1] Prakash V. Terpenoids as

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caac.21492

cmet.2015.12.006

pone.0210891

10.1155/2018/8324696

cytotoxic compounds: A perspective.

[2] Bray F, Ferlay J, Soerjomataram I, Siegel R, Torre L, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians. 2018;**68**(6):394-424. DOI: 10.3322/

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[77] Kunnumakkara AB, Anand P, Aggarwal BB. Curcumin inhibits proliferation, invasion, angiogenesis and metastasis of different cancers through interaction with multiple cell signaling proteins. Cancer Letters. 2008;**269**(2):199-225. DOI: 10.1016/j. canlet.2008.03.009

[78] Hua W, Fu Y, Liao Y, Xia W, Chen Y, Zeng Y, et al. Curcumin induces downregulation of EZH2 expression through the MAPK pathway in MDA-MB-435 human breast cancer cells. European Journal of Pharmacology. 2010;**637**(1-3):16-21. DOI: 10.1016/j. ejphar.2010.03.05179

[79] Banerjee M, Singh P, Panda D. Curcumin suppresses the dynamic instability of microtubules, activates the mitotic checkpoint and induces apoptosis in MCF-7 cells. The FEBS Journal. 2010;**277**(16):3437-3448. DOI: 10.1111/j.1742-4658.2010.07750

[80] Habli Z, Toumieh G, Fatfat M, Rahal ON, Gali-Muhtasib H. Emerging cytotoxic alkaloids in the battle against cancer: Overview of molecular mechanisms. Molecules. 2017;**22**(2): 1-22. DOI: 10.3390/molecules22020250

[81] Wink M. Modes of action of herbal medicines and plant secondary metabolites. Medicine. 2015;**2**(3):

251-286. DOI: 10.3390/ medicines2030251

[82] Aniszewski T. Alkaloids. Secrets of Life. Alkaloid Chemistry, Biological Significance, Applications and Ecological Role.Amsterdam: Elsevier; 2007. p. 316

[83] Mohan K, Jeyachandran R. Alkaloids as anticancer agents. Annals of Phytomedicine. 2012;**1**(1):46-53

[84] Legarza K, Yang L. New molecular mechanisms of action of camptothecintype drugs. Anticancer Research. 2006;**26**(5A):3301-3305

[85] Sirikantaramas S, Yamazaki M, Saito K. Mutations in topoisomerase I as a self-resistance mechanism coevolved with the production of the anticancer alkaloid camptothecin in plants. Proceedings of the National Academy of Sciences of the United States of America. 2008;**105**(18):6782-6786. DOI: 10.1073/pnas.0801038105

[86] Liu LF, Desai SD, Li TK, Mao Y, Sun M, Sim S. Mechanism of action of camptothecin. Annals of the New York Academy of Sciences. 2000;**922**:1-10

[87] Burke TG, Xiang TX, Anderson BD, Latus LJ. Recent advances in camptothecin drug design and delivery strategies. camptothecins in cancer therapy. Human Press. 2005:171-190. DOI: 10.1385/1-59259-866-8:171

[88] Giovanella BC, Hinz HR, Kozielski AJ, Stehlin JS, Silber R, Potmesil M. Complete growth inhibition of human cancer inhibition of human cancer xenografts in nude mice by treatment with 20-(5-camptothecin). Cancer Research. 1991;**5**:3052-3056

[89] Venditto VJ, Simanek EE. Cancer therapies utilizing the camptothecins: A review of *in vivo* literature. Molecular Pharmaceutics. 2010;**7**(2):307-349. DOI: 10.1021/mp900243b

[90] Lee C, Huang Y, Yang C, Huang K. Drug delivery systems and combination therapy by using vinca alkaloids. Current Topics in Medicinal Chemistry. 2015;**15**(15):1491-1500. DOI: 10.2174/1568026615666150414120547

[91] Moudi M, Go R, Yien CY, Nazre M. Vinca alkaloids. International Journal of Preventive Medicine. 2013;**4**(11):1231-1235

[92] Downing KH. Structural basis for the interaction of tubulin with proteins and drugs that affect microtubule dynamics. Annual Review of Cell and Developmental Biology. 2000;**16**:89-111

[93] Gregory RK, Smith IE. Vinorelbina: A clinical review. British Journal of Cancer. 2000;**82**:1907-1913

[94] Klement G, Baruchel S, Rak J, Man S, Clark K, Hicklin DJ, et al. Erratum: Continuous low-dose therapy with vinblastine and VEGF receptor-2 antibody induces sustained tumor regression without overt toxicity. The Journal of Clinical Investigation. 2000;**105**:R15-R24. DOI: 10.1172/ JCI08829C1

[95] Sen K, Mandal M. Second generation liposomal cancer therapeutics: Transition from laboratory to clinic. International Journal of Pharmaceutics. 2013;**448**(1):28-43. DOI: 10.1016/j.ijpharm.2013.03.006

[96] Allen TM, Cullis PR. Liposomal drug delivery systems: From concept to clinical applications. Advanced Drug Delivery Reviews. 2013;**65**(1):36-48. DOI: 10.1016/j.addr.2012.09.037

[97] Shaban N, Abdel-Rahman S, Haggag A, Awad D, Bassiouny A, Talaat I. Combination between Taxolencapsulated liposomes and Eruca sativa seed extract suppresses mammary tumors in female rats induced by 7,12-dimethylbenz(α) anthracene. Asian Pacific Journal of

**67**

*Cytotoxic Effect and Mechanisms from Some Plant-Derived Compounds in Breast Cancer*

Preclinical development of drug delivery systems for paclitaxel-based cancer chemotherapy. Journal of Controlled Release. 2017;**267**:100-118. DOI: 10.1016/j.jconrel.2017.09.026

[105] Shen J, Sun H, Xu P, Yin Q, Zhang Z, Wang S, et al. Biomaterials simultaneous inhibition of metastasis and growth of breast cancer by co-delivery of twist shRNA and

biomaterials.2012.10.057

apb.2015.008

paclitaxel using pluronic P85-PEI/TPGS complex nanoparticles. Biomaterials. 2012;**34**(5):1581-1590. DOI: 10.1016/j.

[106] Mahalingam M, Krishnamoorthy K. Selection of a suitable method for the preparation of polymeric nanoparticles:

[107] Acevedo-Morantes CY, Acevedo-Morantes MT, Suleiman-Rosado D, Ramírez-Vick JE. Evaluation of the cytotoxic effect of camptothecin solid lipid nanoparticles on MCF7 cells. Drug Delivery. 2013;**20**(8):338-348. DOI: 10.3109/10717544.2013.834412

[108] Mohanty C, Sahoo SK. The *in vitro* stability and *in vivo* pharmacokinetics of curcumin prepared as an aqueous nanoparticulate formulation.

Biomaterials. 2010;**31**(25):6597-6611. DOI: 10.1016/j.biomaterials.2010.04.062

Pharmacokinetic and pharmacodynamic

encapsulated poly (methacylic acid-comethyl methacrylate) nanoparticles. Journal of Applied Pharmaceutical Science. 2017;**7**(3):9-16. DOI: 10.7324/

[109] Manikandan M, Kannan K.

evaluation of camptothecin

[110] Mulik RS, Mönkkönen J, Juvonen RO, Mahadik KR, Paradkar AR. Transferrin mediated solid lipid nanoparticles containing curcumin: Enhanced *in vitro* anticancer

JAPS.2017.70303

Multi-criteria decision making approach. Advanced Pharmaceutical Bulletin. 2015;**5**(1):57-67. DOI: 10.5681/

*DOI: http://dx.doi.org/10.5772/intechopen.87177*

Cancer Prevention. 2016;**17**(1):

Chatterjee A, Ronghe A, Bhat NK, Dim DC, et al. Resveratrol inhibits estrogen-induced breast carcinogenesis through induction of NRF2-mediated protective pathways. Carcinogenesis. 2014;**35**(8):1872-1880. DOI: 10.1093/

[99] Zhou QM, Wang XF, Liu XJ, Zhang H, Lu YY, Su SB. Curcumin enhanced antiproliferative effect of mitomycin C in human breast cancer MCF-7 cells *in vitro* and *in vivo*. Acta Pharmacologica Sinica. 2011;**32**(11):1402-1410. DOI: 10.1038/

[100] Camacho KM, Kumar S, Menegatti S, Vogus DG, Anselmo AC, Mitragotri S. Synergistic Antitumor Activity of Camptothecin-Doxorubicin

Combinations and their Conjugates with Hyaluronic Acid. Journal of Controlled Release. 2015:1-25. DOI:10.1016/j.

[101] Ping M, Russell M. Paclitaxel

comprehensive review. Journal of Nanomedicine & Nanotechnology.

[102] Sofias AM, Dunne M, Storm G, Allen G. The battle of "nano" paclitaxel. Advanced Drug Delivery Reviews. 2017;**122**:20-30. DOI: 10.1016/j.

[103] Wu C, Gao Y, Liu Y, Xu X. Pure paclitaxel nanoparticles: Preparation, characterization, and antitumor effect for human liver cancer SMMC-7721 cells. International Journal of Nanomedicine. 2018;**13**:6189-6198. DOI:

Konstantopoulos A, Zhang P, Cui H.

Nano-delivery systems: A

2013;**4**(2):1000164. DOI: 10.4172/2157-7439.1000164

addr.2017.02.003

10.2147/IJN.S169209

[104] Wang F, Porter M,

[98] Singh B, Shoulson R,

117-123

carcin/bgu120

aps.2011.97

jconrel.2015.04.031

*Cytotoxic Effect and Mechanisms from Some Plant-Derived Compounds in Breast Cancer DOI: http://dx.doi.org/10.5772/intechopen.87177*

Cancer Prevention. 2016;**17**(1): 117-123

*Cytotoxicity - Definition, Identification, and Cytotoxic Compounds*

[90] Lee C, Huang Y, Yang C,

[91] Moudi M, Go R, Yien CY,

2013;**4**(11):1231-1235

Journal of Preventive Medicine.

Huang K. Drug delivery systems and combination therapy by using vinca alkaloids. Current Topics in Medicinal Chemistry. 2015;**15**(15):1491-1500. DOI: 10.2174/1568026615666150414120547

Nazre M. Vinca alkaloids. International

[92] Downing KH. Structural basis for the interaction of tubulin with proteins and drugs that affect microtubule dynamics. Annual Review of Cell and Developmental Biology. 2000;**16**:89-111

[93] Gregory RK, Smith IE. Vinorelbina: A clinical review. British Journal of

Cancer. 2000;**82**:1907-1913

JCI08829C1

[94] Klement G, Baruchel S, Rak J, Man S, Clark K, Hicklin DJ, et al. Erratum: Continuous low-dose therapy with vinblastine and VEGF receptor-2 antibody induces sustained tumor regression without overt toxicity. The Journal of Clinical Investigation. 2000;**105**:R15-R24. DOI: 10.1172/

[95] Sen K, Mandal M. Second generation liposomal cancer

to clinic. International Journal of Pharmaceutics. 2013;**448**(1):28-43. DOI: 10.1016/j.ijpharm.2013.03.006

[96] Allen TM, Cullis PR. Liposomal drug delivery systems: From concept to clinical applications. Advanced Drug Delivery Reviews. 2013;**65**(1):36-48. DOI: 10.1016/j.addr.2012.09.037

[97] Shaban N, Abdel-Rahman S, Haggag A, Awad D, Bassiouny A, Talaat I. Combination between Taxol-

Eruca sativa seed extract suppresses mammary tumors in female rats induced by 7,12-dimethylbenz(α) anthracene. Asian Pacific Journal of

encapsulated liposomes and

therapeutics: Transition from laboratory

251-286. DOI: 10.3390/ medicines2030251

2007. p. 316

[82] Aniszewski T. Alkaloids. Secrets of Life. Alkaloid Chemistry, Biological

[83] Mohan K, Jeyachandran R. Alkaloids

[84] Legarza K, Yang L. New molecular mechanisms of action of camptothecintype drugs. Anticancer Research.

[85] Sirikantaramas S, Yamazaki M, Saito K. Mutations in topoisomerase I as a self-resistance mechanism coevolved with the production of the anticancer alkaloid camptothecin in plants. Proceedings of the National Academy of Sciences of the United States of America. 2008;**105**(18):6782-6786. DOI:

[86] Liu LF, Desai SD, Li TK, Mao Y, Sun M, Sim S. Mechanism of action of camptothecin. Annals of the New York Academy of Sciences. 2000;**922**:1-10

[87] Burke TG, Xiang TX, Anderson BD,

camptothecin drug design and delivery strategies. camptothecins in cancer therapy. Human Press. 2005:171-190. DOI: 10.1385/1-59259-866-8:171

Potmesil M. Complete growth inhibition of human cancer inhibition of human cancer xenografts in nude mice by treatment with 20-(5-camptothecin). Cancer Research. 1991;**5**:3052-3056

[89] Venditto VJ, Simanek EE. Cancer therapies utilizing the camptothecins: A review of *in vivo* literature. Molecular Pharmaceutics. 2010;**7**(2):307-349. DOI:

Significance, Applications and Ecological Role.Amsterdam: Elsevier;

as anticancer agents. Annals of Phytomedicine. 2012;**1**(1):46-53

2006;**26**(5A):3301-3305

10.1073/pnas.0801038105

Latus LJ. Recent advances in

[88] Giovanella BC, Hinz HR, Kozielski AJ, Stehlin JS, Silber R,

**66**

10.1021/mp900243b

[98] Singh B, Shoulson R, Chatterjee A, Ronghe A, Bhat NK, Dim DC, et al. Resveratrol inhibits estrogen-induced breast carcinogenesis through induction of NRF2-mediated protective pathways. Carcinogenesis. 2014;**35**(8):1872-1880. DOI: 10.1093/ carcin/bgu120

[99] Zhou QM, Wang XF, Liu XJ, Zhang H, Lu YY, Su SB. Curcumin enhanced antiproliferative effect of mitomycin C in human breast cancer MCF-7 cells *in vitro* and *in vivo*. Acta Pharmacologica Sinica. 2011;**32**(11):1402-1410. DOI: 10.1038/ aps.2011.97

[100] Camacho KM, Kumar S, Menegatti S, Vogus DG, Anselmo AC, Mitragotri S. Synergistic Antitumor Activity of Camptothecin-Doxorubicin Combinations and their Conjugates with Hyaluronic Acid. Journal of Controlled Release. 2015:1-25. DOI:10.1016/j. jconrel.2015.04.031

[101] Ping M, Russell M. Paclitaxel Nano-delivery systems: A comprehensive review. Journal of Nanomedicine & Nanotechnology. 2013;**4**(2):1000164. DOI: 10.4172/2157-7439.1000164

[102] Sofias AM, Dunne M, Storm G, Allen G. The battle of "nano" paclitaxel. Advanced Drug Delivery Reviews. 2017;**122**:20-30. DOI: 10.1016/j. addr.2017.02.003

[103] Wu C, Gao Y, Liu Y, Xu X. Pure paclitaxel nanoparticles: Preparation, characterization, and antitumor effect for human liver cancer SMMC-7721 cells. International Journal of Nanomedicine. 2018;**13**:6189-6198. DOI: 10.2147/IJN.S169209

[104] Wang F, Porter M, Konstantopoulos A, Zhang P, Cui H. Preclinical development of drug delivery systems for paclitaxel-based cancer chemotherapy. Journal of Controlled Release. 2017;**267**:100-118. DOI: 10.1016/j.jconrel.2017.09.026

[105] Shen J, Sun H, Xu P, Yin Q, Zhang Z, Wang S, et al. Biomaterials simultaneous inhibition of metastasis and growth of breast cancer by co-delivery of twist shRNA and paclitaxel using pluronic P85-PEI/TPGS complex nanoparticles. Biomaterials. 2012;**34**(5):1581-1590. DOI: 10.1016/j. biomaterials.2012.10.057

[106] Mahalingam M, Krishnamoorthy K. Selection of a suitable method for the preparation of polymeric nanoparticles: Multi-criteria decision making approach. Advanced Pharmaceutical Bulletin. 2015;**5**(1):57-67. DOI: 10.5681/ apb.2015.008

[107] Acevedo-Morantes CY, Acevedo-Morantes MT, Suleiman-Rosado D, Ramírez-Vick JE. Evaluation of the cytotoxic effect of camptothecin solid lipid nanoparticles on MCF7 cells. Drug Delivery. 2013;**20**(8):338-348. DOI: 10.3109/10717544.2013.834412

[108] Mohanty C, Sahoo SK. The *in vitro* stability and *in vivo* pharmacokinetics of curcumin prepared as an aqueous nanoparticulate formulation. Biomaterials. 2010;**31**(25):6597-6611. DOI: 10.1016/j.biomaterials.2010.04.062

[109] Manikandan M, Kannan K. Pharmacokinetic and pharmacodynamic evaluation of camptothecin encapsulated poly (methacylic acid-comethyl methacrylate) nanoparticles. Journal of Applied Pharmaceutical Science. 2017;**7**(3):9-16. DOI: 10.7324/ JAPS.2017.70303

[110] Mulik RS, Mönkkönen J, Juvonen RO, Mahadik KR, Paradkar AR. Transferrin mediated solid lipid nanoparticles containing curcumin: Enhanced *in vitro* anticancer

Chapter 5

Abstract

synthetic, cell survival

to the type of stimuli [1].

69

1. Introduction

Survival

Apoptotic Inhibitors as

El-Shimaa Mohamed Naguib Abdelhafez,

Sara Mohamed Naguib Abdelhafez Ali,

Mohamed Ramadan Eisa Hassan and

Adel Mohammed Abdel-Hakem

Therapeutic Targets for Cell

Apoptosis has revealed an essential function in the development or prevention of oncogenic transformation in the body; however, programmed cell death (PCD) must be tightly controlled since deregulated cell death is involved in the development of a large number of different pathologies. Apoptosis can be decreased in pathological

neurodegeneration, retinal cell death, myocardial and liver ischemia, inflammatory diseases such as sepsis, osteoarthritis (OA), rheumatoid arthritis (RA), and asthma. Different types of apoptotic inhibitors will be discussed in this chapter displaying their mechanism of action, which have been reported to be therapeutic targets for cell survival or at least limiting cell death. These inhibitors are classified according to their nature into natural antiapoptotic proteins that present mainly in the cell and synthetic small molecule inhibitors that are widely used to protect against overexpression of

states such as in cancer and autoimmunity or elevated such as in stroke,

apoptosis mediators and, in turn, to prevent corresponding diseases.

Keywords: antiapoptosis, mechanism, apoptotic inhibitors, endogenous,

Apoptosis is a crucial normal biological process that occurs in the cell as a component of animal development, tissue hemostasis, and immune response. In pathological state, it can be abrogated as in cancer and autoimmune diseases, or over-expressed as in case of stroke, ischemia, psoriasis, and inflammatory diseases. The apoptotic mechanism occurs through either extrinsic pathway or intrinsic pathway, which leads to cell death through different apoptotic cascades, according

These cell survival strategies involve a myriad of coordinated and systematic

There are various inhibitors of these pathways, which have been reported to be helpful in inhibition of the cell death or limiting it. These inhibitors are classified

physiological and genetic changes that serve to ward off death [2].

activity by induction of apoptosis. International Journal of Pharmaceutics. 2010;**398**(1-2):190-203. DOI: 10.1016/j. ijpharm.2010.07.021

[111] Tang H, Murphy CJ, Zhang B, Shen Y, Van Kirk EA, Murdoch WJ, et al. Biomaterials Curcumin polymers as anticancer conjugates. Biomaterials. 2010;**31**(27):7139-7149. DOI: 10.1016/j. biomaterials.2010.06.007

[112] Ducry L, Stump B. Antibody— Drug conjugates: Linking cytotoxic payloads to monoclonal antibodies. Bioconjugate Chemistry. 2010;**21**:5-13. DOI: 10.1021/bc9002019

#### Chapter 5

*Cytotoxicity - Definition, Identification, and Cytotoxic Compounds*

activity by induction of apoptosis. International Journal of Pharmaceutics. 2010;**398**(1-2):190-203. DOI: 10.1016/j.

[111] Tang H, Murphy CJ, Zhang B, Shen Y, Van Kirk EA, Murdoch WJ, et al. Biomaterials Curcumin polymers as anticancer conjugates. Biomaterials. 2010;**31**(27):7139-7149. DOI: 10.1016/j.

[112] Ducry L, Stump B. Antibody— Drug conjugates: Linking cytotoxic payloads to monoclonal antibodies. Bioconjugate Chemistry. 2010;**21**:5-13.

ijpharm.2010.07.021

biomaterials.2010.06.007

DOI: 10.1021/bc9002019

**68**

## Apoptotic Inhibitors as Therapeutic Targets for Cell Survival

El-Shimaa Mohamed Naguib Abdelhafez, Sara Mohamed Naguib Abdelhafez Ali, Mohamed Ramadan Eisa Hassan and Adel Mohammed Abdel-Hakem

#### Abstract

Apoptosis has revealed an essential function in the development or prevention of oncogenic transformation in the body; however, programmed cell death (PCD) must be tightly controlled since deregulated cell death is involved in the development of a large number of different pathologies. Apoptosis can be decreased in pathological states such as in cancer and autoimmunity or elevated such as in stroke, neurodegeneration, retinal cell death, myocardial and liver ischemia, inflammatory diseases such as sepsis, osteoarthritis (OA), rheumatoid arthritis (RA), and asthma. Different types of apoptotic inhibitors will be discussed in this chapter displaying their mechanism of action, which have been reported to be therapeutic targets for cell survival or at least limiting cell death. These inhibitors are classified according to their nature into natural antiapoptotic proteins that present mainly in the cell and synthetic small molecule inhibitors that are widely used to protect against overexpression of apoptosis mediators and, in turn, to prevent corresponding diseases.

Keywords: antiapoptosis, mechanism, apoptotic inhibitors, endogenous, synthetic, cell survival

#### 1. Introduction

Apoptosis is a crucial normal biological process that occurs in the cell as a component of animal development, tissue hemostasis, and immune response. In pathological state, it can be abrogated as in cancer and autoimmune diseases, or over-expressed as in case of stroke, ischemia, psoriasis, and inflammatory diseases.

The apoptotic mechanism occurs through either extrinsic pathway or intrinsic pathway, which leads to cell death through different apoptotic cascades, according to the type of stimuli [1].

These cell survival strategies involve a myriad of coordinated and systematic physiological and genetic changes that serve to ward off death [2].

There are various inhibitors of these pathways, which have been reported to be helpful in inhibition of the cell death or limiting it. These inhibitors are classified

according to their nature into endogenous antiapoptotic proteins that present mainly in the cell to regulate the apoptosis process and synthetic inhibitors that are synthesized to be used in case of overexpression of apoptosis mediators as in some diseases.

1.2 Synthetic apoptotic inhibitors

1.2.1 Tumor necrotic factor (TNF) inhibitors

DOI: http://dx.doi.org/10.5772/intechopen.85465

Apoptotic Inhibitors as Therapeutic Targets for Cell Survival

pared to the control group 5.61 ng/ml [19].

inflammatory cytokine TNF-α by (39.19%) [20].

71

Infliximab (IFX) [12], etanercept (ETN) [13], Adalimumab (ADA), golimumab (GOLI) [14], and certolizumab pegol (CZP) [15] are clinical biologic drugs that act as necrotic factor (TNF)-α inhibitors that were approved by the U.S. Food and Drug Administration (FDA). Other synthetic TNF inhibitors were designed such as:

Compound (1) inhibited the release of TNF in cells and in animals. It was active in a chronic rheumatoid arthritis model (MCIA) when administrated orally and it was advanced for further preclinical evaluation [16]. A novel thalidomide analog was synthesized and characterized for anti-TNF-α activity with up to a 38% inhibi-

Compound (3) is an oleanolic acid analog, characterized by structural modifications at position C-3 and C-28 of oleanane skeleton and tested for anti-inflammatory potential, when C-3 became Indole, and C-28 = cyclohexamine, gave mild inhibition by 51.9% [18]. Compound (4) suppressed serum TNF-α levels by 2.45 ng/ml com-

Compound (5) possessed TNF inhibition with half-maximal inhibitory concentration (IC50) = 0.5 μm [19]. Besides, Compound (6) decreased the level of the pro-

tion for compound (2) with no obvious concentration dependence [17].

#### 1.1 Endogenous antiapoptotic inhibitors

#### 1.1.1 Reduction in the number of apoptotic cells

It was reported that Ginkgo biloba extract (EGb 761) exhibited antiapoptotic effect on different cell types [3], and it particularly inhibits death in human lymphocytes when exposed to gossypol, a toxin that causes cell death via apoptosis. Similar results have been observed in thymus cells pretreated with EGb 761 and then exposed to ferrous sulfate in hydrogen peroxide (H2O2) [4]. Lymphocytes that are isolated from spleen of aged mice and treated with EGb 761 were less susceptible to reactive oxygen species (ROS)-induced apoptosis [5]. Scientists revealed that the posttreatment with EGb 761 in the peripheral nervous system decreased efficiently the number of apoptotic cells in injured rat spinal cord [6]. Moreover, it helps in treatment of the central nervous system reduced neuronal death in the substantia nigra pars compacta from an experimental model of Parkinson's disease [7].

#### 1.1.2 Maintaining the mitochondrial integrity

Rhodiola crenulata extract (RCE) is an edible alcohol extract, conserving greatly the mitochondrial integrity and in turn prohibiting the release of cytochrome C, which leads to cell death. The effective concentration of the most important component, salidroside, was 4% (w/w).

Other herbals mediate its antiapoptotic effect through the same mechanism as they possess a potent reactive oxygen species scavenging function; however, they restore the mitochondrial membrane potential [8, 9].

Some drugs were tested in sympathoadrenal cells that showed obviously another antiapoptotic pathway through inducing the antiapoptotic protein B-cell lymphoma 2 (Bcl-2) transcription rate and B-cell lymphoma-extra-large (Bcl-xL) proteins. The role of these proteins appears crucial, because inhibition of their production by antisense oligonucleotides (directed toward the translation initiation site of the Bcl-2 transcript) resulted in aboliting protective effect. The prosurvival pathway also included activation of the transcription nuclear factors NF-κB (a protein complex that controls transcription of DNA, cytokine production, and cell survival) and CREB (cellular transcription factor). It binds to certain DNA sequences and the antiapoptotic kinase PKCα/β such as dehydroepiandrosterone (DHEAS) and Allo [10].

#### 1.1.3 Decrease in caspase transcription rate and DNA fragmentation

Some natural component reverse such as diosmin induces Bad and Bax, proapoptotic members of the Bcl-2 family, to react with the mitochondrial membrane and prevent the release of apoptotic-inducing factor (AIF) and cytochrome-C. Cytochrome-C in turn inhibits initiator caspase-9, which prevents sequential cascade of activation of caspase-3, and conserves DNA fragment along with no apoptotic cell death [11].

#### 1.2 Synthetic apoptotic inhibitors

according to their nature into endogenous antiapoptotic proteins that present mainly in the cell to regulate the apoptosis process and synthetic inhibitors that are synthesized to be used in case of overexpression of apoptosis mediators as in some

Cytotoxicity - Definition, Identification, and Cytotoxic Compounds

It was reported that Ginkgo biloba extract (EGb 761) exhibited antiapoptotic effect on different cell types [3], and it particularly inhibits death in human

lymphocytes when exposed to gossypol, a toxin that causes cell death via apoptosis. Similar results have been observed in thymus cells pretreated with EGb 761 and then exposed to ferrous sulfate in hydrogen peroxide (H2O2) [4]. Lymphocytes that are isolated from spleen of aged mice and treated with EGb 761 were less susceptible to reactive oxygen species (ROS)-induced apoptosis [5]. Scientists revealed that the posttreatment with EGb 761 in the peripheral nervous system decreased efficiently the number of apoptotic cells in injured rat spinal cord [6]. Moreover, it helps in treatment of the central nervous system reduced neuronal death in the substantia nigra pars compacta from an experimental model of

Rhodiola crenulata extract (RCE) is an edible alcohol extract, conserving greatly the mitochondrial integrity and in turn prohibiting the release of cytochrome C, which leads to cell death. The effective concentration of the most important com-

Other herbals mediate its antiapoptotic effect through the same mechanism as they possess a potent reactive oxygen species scavenging function; however, they

Some drugs were tested in sympathoadrenal cells that showed obviously another antiapoptotic pathway through inducing the antiapoptotic protein B-cell lymphoma 2 (Bcl-2) transcription rate and B-cell lymphoma-extra-large (Bcl-xL) proteins. The role of these proteins appears crucial, because inhibition of their production by antisense oligonucleotides (directed toward the translation initiation site of the Bcl-2 transcript) resulted in aboliting protective effect. The prosurvival pathway also included activation of the transcription nuclear factors NF-κB (a protein complex that controls transcription of DNA, cytokine production, and cell survival) and CREB (cellular transcription factor). It binds to certain DNA sequences and the antiapoptotic kinase PKCα/β such as dehydroepiandrosterone (DHEAS) and

Some natural component reverse such as diosmin induces Bad and Bax, proapoptotic members of the Bcl-2 family, to react with the mitochondrial membrane and prevent the release of apoptotic-inducing factor (AIF) and cytochrome-C. Cytochrome-C in turn inhibits initiator caspase-9, which prevents sequential cascade of activation of caspase-3, and conserves DNA fragment along with no

diseases.

Parkinson's disease [7].

Allo [10].

70

apoptotic cell death [11].

1.1 Endogenous antiapoptotic inhibitors

1.1.1 Reduction in the number of apoptotic cells

1.1.2 Maintaining the mitochondrial integrity

ponent, salidroside, was 4% (w/w).

restore the mitochondrial membrane potential [8, 9].

1.1.3 Decrease in caspase transcription rate and DNA fragmentation

#### 1.2.1 Tumor necrotic factor (TNF) inhibitors

Infliximab (IFX) [12], etanercept (ETN) [13], Adalimumab (ADA), golimumab (GOLI) [14], and certolizumab pegol (CZP) [15] are clinical biologic drugs that act as necrotic factor (TNF)-α inhibitors that were approved by the U.S. Food and Drug Administration (FDA). Other synthetic TNF inhibitors were designed such as:

Compound (1) inhibited the release of TNF in cells and in animals. It was active in a chronic rheumatoid arthritis model (MCIA) when administrated orally and it was advanced for further preclinical evaluation [16]. A novel thalidomide analog was synthesized and characterized for anti-TNF-α activity with up to a 38% inhibition for compound (2) with no obvious concentration dependence [17].

Compound (3) is an oleanolic acid analog, characterized by structural modifications at position C-3 and C-28 of oleanane skeleton and tested for anti-inflammatory potential, when C-3 became Indole, and C-28 = cyclohexamine, gave mild inhibition by 51.9% [18]. Compound (4) suppressed serum TNF-α levels by 2.45 ng/ml compared to the control group 5.61 ng/ml [19].

Compound (5) possessed TNF inhibition with half-maximal inhibitory concentration (IC50) = 0.5 μm [19]. Besides, Compound (6) decreased the level of the proinflammatory cytokine TNF-α by (39.19%) [20].

Compound (7) had 56% inhibition of TNF-α at 10 μm [21]. Betulinic acid (8) had a significant decrease in IL1β, IL6, and TNFα in the neuronal tissues [22].

1.2.2 Fatty acid synthase (Fas) inhibitors

KR-33493 (9) was used as a potent inhibitor for ischemia Fas-mediated cell death 68 [23]. Compound (10a, b) RKTS-33,34 with 10 μm ED50 value (concentration causing 50% of maximum effect) selectively inhibited apoptosis induced by FasL as well as ECH (epoxycyclohexenone derivative) (11), which is produced by fungus [24].

Compound (16) is the active form of vitamin D that inhibited Fas ligandinduced apoptosis in human osteoblasts by regulating components of both the mitochondrial and Fas-related pathways [30]. Estrogen (17) also inhibited Fas-

Cilazapril (18) acts as an angiotensin-converting enzyme inhibitor along with protection against apoptosis through downregulating Fas protein during the induction of apoptosis in cardiomyocytes in rat hearts when subjected to reperfusion after ischemia [32]. M50054 (19) (cell-permeable inhibitor of the activation of caspase-3) inhibited apoptosis induced by a variety of apoptotic stimuli such as the Fas/Fas ligand system and etoposide. Thus, it might be effective for hepatitis when administered orally and chemotherapy-induced alopecia when administered topically [33].

mediated apoptosis in experimental stroke [31].

Apoptotic Inhibitors as Therapeutic Targets for Cell Survival

DOI: http://dx.doi.org/10.5772/intechopen.85465

73

Glycine (12) was tested as a cytoprotector against ischemic damage by downregulation of FasL/Fas and caspase3 and upregulation of Bcl2 and Bcl2-bax (apoptosis regulator BAX) [25]. Compound (13) was designed as a novel class of ischemic cell death inhibitors targeting Fas-mediated cell death pathway with EC50 = 0.557 μm (the concentration of a drug that gives half-maximal response), and cell survival = 92.98% at 10 μm [26]. It was found that (dichlorovinyl dimethyl phosphate) DDVP (14) significantly decreased the expression of Fas antigen on YAC-1S target cells and the expression of FasL (Fas ligand) on LAK cells (lymphokineactivated killer cell). These findings provided direct evidences that DDVP impaired the FasL/Fas pathway via downregulating the expression of both Fas and FasL [27]. Geldanamycin (15), which was originally discovered in Streptomyces hygroscopicus [28], inhibited Fas signaling pathway and protected neurons against ischemia [29].

Compound (7) had 56% inhibition of TNF-α at 10 μm [21]. Betulinic acid (8) had a significant decrease in IL1β, IL6, and TNFα in the neuronal tissues [22].

Cytotoxicity - Definition, Identification, and Cytotoxic Compounds

KR-33493 (9) was used as a potent inhibitor for ischemia Fas-mediated cell death 68 [23]. Compound (10a, b) RKTS-33,34 with 10 μm ED50 value (concentration causing 50% of maximum effect) selectively inhibited apoptosis induced by FasL as well as ECH (epoxycyclohexenone derivative) (11), which is produced by fungus [24].

Glycine (12) was tested as a cytoprotector against ischemic damage by downregulation of FasL/Fas and caspase3 and upregulation of Bcl2 and Bcl2-bax (apoptosis regulator BAX) [25]. Compound (13) was designed as a novel class of ischemic cell death inhibitors targeting Fas-mediated cell death pathway with

EC50 = 0.557 μm (the concentration of a drug that gives half-maximal response), and cell survival = 92.98% at 10 μm [26]. It was found that (dichlorovinyl dimethyl phosphate) DDVP (14) significantly decreased the expression of Fas antigen on YAC-1S target cells and the expression of FasL (Fas ligand) on LAK cells (lymphokineactivated killer cell). These findings provided direct evidences that DDVP impaired the FasL/Fas pathway via downregulating the expression of both Fas and FasL [27]. Geldanamycin (15), which was originally discovered in Streptomyces hygroscopicus [28], inhibited Fas signaling pathway and protected neurons against ischemia [29].

1.2.2 Fatty acid synthase (Fas) inhibitors

72

Compound (16) is the active form of vitamin D that inhibited Fas ligandinduced apoptosis in human osteoblasts by regulating components of both the mitochondrial and Fas-related pathways [30]. Estrogen (17) also inhibited Fasmediated apoptosis in experimental stroke [31].

Cilazapril (18) acts as an angiotensin-converting enzyme inhibitor along with protection against apoptosis through downregulating Fas protein during the induction of apoptosis in cardiomyocytes in rat hearts when subjected to reperfusion after ischemia [32]. M50054 (19) (cell-permeable inhibitor of the activation of caspase-3) inhibited apoptosis induced by a variety of apoptotic stimuli such as the Fas/Fas ligand system and etoposide. Thus, it might be effective for hepatitis when administered orally and chemotherapy-induced alopecia when administered topically [33].

73

SC79 (20) (AKT activator) prevented acute hepatic failure induced by Fasmediated apoptosis of hepatocytes [34]. Vit K2 (21) significantly suppressed both Fas expression and Fas-mediated apoptosis of the cells in a dose-dependent fashion. The maximum effect was observed when 6–10 mol/L of vitamin K2 was added to the culture, a concentration comparable to that attained during therapy with vitamin K2 [35].

2,4-dione (27) protected neural cells against glutamate- and tBid-induced toxicity

1.2.4 Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) inhibitors

TWEAK also known as (Apo3L or TNFSF12) was first described as an inducer of apoptosis in transformed cell lines [47]. It is a member of the tumor necrosis factor (TNF) receptor family that is induced in a variety of cell types in situations of tissue injury. It is a crucial player in muscle atrophy, cerebral ischemia, kidney injury, atherosclerosis, and infarction, as well as in various autoimmune scenarios including experimental autoimmune encephalitis, rheumatoid arthritis, and inflammatory bowel disease [48]. Aurintricarboxylic acid (ATA) (28) was a potent inhibitor of the TWEAK-Fn14 signaling axis and could potentially be utilized to enhance the therapeutic response in glioblastoma (GBM) [49]. L524-0366 (29) was a specific dose-dependent inhibitor of TWEAK-Fn14 interaction [50] and it was found to be a complete suppressor for TWEAK-induced T98G cell migration at dose equal to

with an EC50 = 6.78 μm [46].

Apoptotic Inhibitors as Therapeutic Targets for Cell Survival

DOI: http://dx.doi.org/10.5772/intechopen.85465

10 μm [51].

75

Vanillic acid (22) inhibited Fas-receptor and caspase-mediated apoptosis signaling pathway and so acted as cardioprotective [36]. NCX-1000 (NOreleasing derivative of ursodeoxycholic acid) (23) is a nitric oxide (NO) derivative of ursodeoxycholic acid (UDCA). When an NO-releasing moiety is added to UDCA, the effectiveness in preventing Fas-mediated liver injury increased [37].

1.2.3 BH3 interacting-domain death agonist (BID) inhibitors

BID plays a central role in the apoptotic machinery mediating cytochrome C and SMAC/DIABLO (mitochondrial protein that potentiates some forms of apoptosis) released from mitochondria, a crucial event for caspase activation and cell death [38]. Pharmacological inhibition of BID could therefore provide a protective benefit against pathological cell death, occurring in cerebral ischemia [39], neurodegenerative diseases [40, 41], liver inflammation [42], or other illnesses where BID has been implicated [43].

BI-6C9 (24) was effective in inhibiting the carboxyl-terminal fragment (tBid) (truncated protein) association with isolated mitochondria at 20 μm [39]. TC9-305 (2-sulfonyl-pyrimidinyl derivatives) (25) had an EC50 = 0.23 nm [44]. BI-11A7 (26) was much more effective in this assay when compared with BI-6C9 (24) but showed some toxicity at higher concentrations (20 μm) [45]. 3-o-tolylthiazolidine-

SC79 (20) (AKT activator) prevented acute hepatic failure induced by Fasmediated apoptosis of hepatocytes [34]. Vit K2 (21) significantly suppressed both Fas expression and Fas-mediated apoptosis of the cells in a dose-dependent fashion. The maximum effect was observed when 6–10 mol/L of vitamin K2 was added to the culture, a concentration comparable to that attained during therapy with vita-

Cytotoxicity - Definition, Identification, and Cytotoxic Compounds

Vanillic acid (22) inhibited Fas-receptor and caspase-mediated apoptosis signaling pathway and so acted as cardioprotective [36]. NCX-1000 (NO-

the effectiveness in preventing Fas-mediated liver injury increased [37].

1.2.3 BH3 interacting-domain death agonist (BID) inhibitors

been implicated [43].

74

releasing derivative of ursodeoxycholic acid) (23) is a nitric oxide (NO) derivative of ursodeoxycholic acid (UDCA). When an NO-releasing moiety is added to UDCA,

BID plays a central role in the apoptotic machinery mediating cytochrome C and SMAC/DIABLO (mitochondrial protein that potentiates some forms of apoptosis) released from mitochondria, a crucial event for caspase activation and cell death [38]. Pharmacological inhibition of BID could therefore provide a protective benefit against pathological cell death, occurring in cerebral ischemia [39], neurodegenerative diseases [40, 41], liver inflammation [42], or other illnesses where BID has

BI-6C9 (24) was effective in inhibiting the carboxyl-terminal fragment (tBid) (truncated protein) association with isolated mitochondria at 20 μm [39]. TC9-305 (2-sulfonyl-pyrimidinyl derivatives) (25) had an EC50 = 0.23 nm [44]. BI-11A7 (26) was much more effective in this assay when compared with BI-6C9 (24) but showed some toxicity at higher concentrations (20 μm) [45]. 3-o-tolylthiazolidine-

min K2 [35].

2,4-dione (27) protected neural cells against glutamate- and tBid-induced toxicity with an EC50 = 6.78 μm [46].

1.2.4 Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) inhibitors

TWEAK also known as (Apo3L or TNFSF12) was first described as an inducer of apoptosis in transformed cell lines [47]. It is a member of the tumor necrosis factor (TNF) receptor family that is induced in a variety of cell types in situations of tissue injury. It is a crucial player in muscle atrophy, cerebral ischemia, kidney injury, atherosclerosis, and infarction, as well as in various autoimmune scenarios including experimental autoimmune encephalitis, rheumatoid arthritis, and inflammatory bowel disease [48]. Aurintricarboxylic acid (ATA) (28) was a potent inhibitor of the TWEAK-Fn14 signaling axis and could potentially be utilized to enhance the therapeutic response in glioblastoma (GBM) [49]. L524-0366 (29) was a specific dose-dependent inhibitor of TWEAK-Fn14 interaction [50] and it was found to be a complete suppressor for TWEAK-induced T98G cell migration at dose equal to 10 μm [51].

doses of irradiation [58, 59]. TPP-6-ISA (35) was an effective inhibitor of the peroxidase function of cyt c/CL complexes with a significant antiapoptotic activity that realized in mouse embryonic cells exposed to ionizing irradiation [60, 61].

Apoptotic Inhibitors as Therapeutic Targets for Cell Survival

DOI: http://dx.doi.org/10.5772/intechopen.85465

1.2.6 P53 upregulated modulator of apoptosis (PUMA) inhibitors

not published due to intellectual property protection [62].

1 μm [63].

77

It is a Bcl-2 homology 3 (BH3)-only Bcl-2 family member and a key mediator of apoptosis induced by a wide variety of stimuli 106. PUMA inhibitors may provide radiation protection and mitigation, there were three compounds that had a strong PUMA inhibition and that were designed by Gabriela Mustata et al., and data were

CLZ-8 (36) was capable of targeting a PUMA protein and has very good physicochemical properties, very good apoptosis resistance, and radiation protection effects. It was found to protect cells from DNA damage under the concentration of

#### 1.2.5 Cytochrome C inhibitors

Cytochrome C is the specific and efficient electron transfer mediator between the two last redox complexes of the mitochondrial respiratory chain [52]. The release of cytochrome C from the mitochondria into the cytoplasm results in caspase-9 activation leading to cell death [43]. Minocycline (30) directly inhibited the release of cytochrome C from mitochondria. Therefore, it was beneficial in experimental models of stroke, traumatic brain and spinal cord injury, Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Parkinson's disease, and multiple sclerosis [53, 54]. Methazolamide (31) was FDA approved for the treatment of glaucoma, while melatonin (32) inhibited oxygen/glucose deprivation—induced cell death, loss of mitochondrial membrane potential, release of mitochondrial factors, pro-IL-1β processing, and activation of caspase-1 and -3 in primary cerebrocortical neurons. Furthermore, compounds (32, 33) decreased infarct size and improved neurological scores after middle cerebral artery occlusion in mice [54–56]. Gamma-tocotrienol (GTT) (33) had the antiapoptotic effects by preventing the activation of caspase-3 and caspase-9, reducing the release of cytochrome C from the mitochondria and preventing H2O2-induced apoptosis in human diploid fibroblasts (HDFs), and delayed cellular aging [57].

3-hydroxypropyl-triphenylphosphonium (TPP)-conjugated imidazolesubstituted oleic acid (TPP-IOA(34a)) and stearic acid (TPP-ISA(34b)) exerted strong specific liganding of heme-iron in cytochrome C/cardiolipin (CL) complex and effectively suppressed its peroxidase activity and CL peroxidation, thus preventing cytochrome C release and cell death, and protecting mice against lethal doses of irradiation [58, 59]. TPP-6-ISA (35) was an effective inhibitor of the peroxidase function of cyt c/CL complexes with a significant antiapoptotic activity that realized in mouse embryonic cells exposed to ionizing irradiation [60, 61].

1.2.6 P53 upregulated modulator of apoptosis (PUMA) inhibitors

It is a Bcl-2 homology 3 (BH3)-only Bcl-2 family member and a key mediator of apoptosis induced by a wide variety of stimuli 106. PUMA inhibitors may provide radiation protection and mitigation, there were three compounds that had a strong PUMA inhibition and that were designed by Gabriela Mustata et al., and data were not published due to intellectual property protection [62].

CLZ-8 (36) was capable of targeting a PUMA protein and has very good physicochemical properties, very good apoptosis resistance, and radiation protection effects. It was found to protect cells from DNA damage under the concentration of 1 μm [63].

1.2.5 Cytochrome C inhibitors

76

Cytochrome C is the specific and efficient electron transfer mediator between the two last redox complexes of the mitochondrial respiratory chain [52]. The release of cytochrome C from the mitochondria into the cytoplasm results in caspase-9 activation leading to cell death [43]. Minocycline (30) directly inhibited the release of cytochrome C from mitochondria. Therefore, it was beneficial in experimental models of stroke, traumatic brain and spinal cord injury, Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Parkinson's disease, and multiple sclerosis [53, 54]. Methazolamide (31) was FDA approved for the treatment of glaucoma, while melatonin (32) inhibited oxygen/glucose deprivation—induced cell death, loss of mitochondrial membrane potential, release of mitochondrial factors, pro-IL-1β processing, and activation of caspase-1 and -3 in primary cerebrocortical neurons. Furthermore, compounds (32, 33) decreased infarct size and improved neurological scores after middle cerebral artery occlusion in mice [54–56]. Gamma-tocotrienol (GTT) (33) had the antiapoptotic effects by

preventing the activation of caspase-3 and caspase-9, reducing the release of cytochrome C from the mitochondria and preventing H2O2-induced apoptosis in human

3-hydroxypropyl-triphenylphosphonium (TPP)-conjugated imidazolesubstituted oleic acid (TPP-IOA(34a)) and stearic acid (TPP-ISA(34b)) exerted strong specific liganding of heme-iron in cytochrome C/cardiolipin (CL) complex and effectively suppressed its peroxidase activity and CL peroxidation, thus preventing cytochrome C release and cell death, and protecting mice against lethal

diploid fibroblasts (HDFs), and delayed cellular aging [57].

Cytotoxicity - Definition, Identification, and Cytotoxic Compounds

#### 1.2.7 Bax inhibitors

Xanthan gum (XG) (37) is an extracellular polysaccharide secreted by microorganisms and was first discovered during fermentation process using Xanthomonas campestris. It could protect subchondral trabecular in bone subchondral, decrease the apoptosis of chondrocytes, downregulate the expressions of active caspase-9, active caspase-3 and bax, and upregulate the expression of bcl-2. Lower range of molecular weight of xanthan gum (LRWXG) could upregulate the expression of cytochrome C in mitochondria while downregulating the expression of cytochrome C in cytoplasm. These findings showed that LRWXG could inhibit cartilage degradation via an intrinsic bax-mitochondria cytochrome C-caspase pathway [64].

2. Conclusion

Conflict of interest

Author details

Minia, Egypt

79

El-Shimaa Mohamed Naguib Abdelhafez<sup>1</sup>

provided the original work is properly cited.

Mohamed Ramadan Eisa Hassan<sup>3</sup> and Adel Mohammed Abdel-Hakem<sup>1</sup>

\*Address all correspondence to: shimaanaguib\_80@mu.edu.eg

1 Department of Medicinal Chemistry, Faculty of Pharmacy, Minia University,

2 Department of Histology, Faculty of Medicine, Minia University, Minia, Egypt

3 Department of Organic Chemistry, Faculty of Pharmacy, Azhar University, Egypt

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Apoptotic inhibitors regulate cell proliferation by promoting cell survival. One

No financial or commercial conflicts of interest were declared by all authors.

\*, Sara Mohamed Naguib Abdelhafez Ali<sup>2</sup>

,

promising area of research that has been covered extensively in this review is displaying the recent developed apoptotic inhibitors and their significance to functional therapies for a number of diseases and pathophysiologies. These inhibitors are working through numerous built-in avenues' mechanisms, including inhibition of pro-apoptotic and apoptotic factors. Our perspectives are to develop new therapeutic strategies aiming to participate in treatment of serious diseases such as stroke, neurodegeneration, retinal cell death, myocardial and liver ischemia, sepsis, osteoarthritis (OA), rheumatoid arthritis (RA), and asthma or to reduce the adverse effects accompanied with long-term therapy of cancer and autoimmunity. Hopefully, scientists will soon be able to provide every patient suffering from imbalanced

apoptotic disease with a more specified and suitable therapy.

Apoptotic Inhibitors as Therapeutic Targets for Cell Survival

DOI: http://dx.doi.org/10.5772/intechopen.85465

PD98059 (38) showed inhibition of staurosporine-, UV-, anticancer druginduced apoptosis in vitro and protected brain against cell death through inhibition of BAX and other factors [65, 66]. Vitamin E (39) significantly reduced the effects of gentamicin on BAX and BCL-2 expression levels [67].

Tanshinone (40) could inhibit the expression of Bax and stimulated the expression of Bcl-2 in cardiomyocytes in the ischemia-reperfusion rat model [68].

### 2. Conclusion

1.2.7 Bax inhibitors

78

Xanthan gum (XG) (37) is an extracellular polysaccharide secreted by microorganisms and was first discovered during fermentation process using Xanthomonas campestris. It could protect subchondral trabecular in bone subchondral, decrease the apoptosis of chondrocytes, downregulate the expressions of active caspase-9, active caspase-3 and bax, and upregulate the expression of bcl-2. Lower range of molecular weight of xanthan gum (LRWXG) could upregulate the expression of cytochrome C in mitochondria while downregulating the expression of cytochrome C in cytoplasm. These findings showed that LRWXG could inhibit cartilage degradation via an intrinsic bax-mitochondria cytochrome C-caspase pathway [64].

Cytotoxicity - Definition, Identification, and Cytotoxic Compounds

PD98059 (38) showed inhibition of staurosporine-, UV-, anticancer druginduced apoptosis in vitro and protected brain against cell death through inhibition of BAX and other factors [65, 66]. Vitamin E (39) significantly reduced the effects

sion of Bcl-2 in cardiomyocytes in the ischemia-reperfusion rat model [68].

Tanshinone (40) could inhibit the expression of Bax and stimulated the expres-

of gentamicin on BAX and BCL-2 expression levels [67].

Apoptotic inhibitors regulate cell proliferation by promoting cell survival. One promising area of research that has been covered extensively in this review is displaying the recent developed apoptotic inhibitors and their significance to functional therapies for a number of diseases and pathophysiologies. These inhibitors are working through numerous built-in avenues' mechanisms, including inhibition of pro-apoptotic and apoptotic factors. Our perspectives are to develop new therapeutic strategies aiming to participate in treatment of serious diseases such as stroke, neurodegeneration, retinal cell death, myocardial and liver ischemia, sepsis, osteoarthritis (OA), rheumatoid arthritis (RA), and asthma or to reduce the adverse effects accompanied with long-term therapy of cancer and autoimmunity. Hopefully, scientists will soon be able to provide every patient suffering from imbalanced apoptotic disease with a more specified and suitable therapy.

### Conflict of interest

No financial or commercial conflicts of interest were declared by all authors.

### Author details

El-Shimaa Mohamed Naguib Abdelhafez<sup>1</sup> \*, Sara Mohamed Naguib Abdelhafez Ali<sup>2</sup> , Mohamed Ramadan Eisa Hassan<sup>3</sup> and Adel Mohammed Abdel-Hakem<sup>1</sup>

1 Department of Medicinal Chemistry, Faculty of Pharmacy, Minia University, Minia, Egypt

2 Department of Histology, Faculty of Medicine, Minia University, Minia, Egypt

3 Department of Organic Chemistry, Faculty of Pharmacy, Azhar University, Egypt

\*Address all correspondence to: shimaanaguib\_80@mu.edu.eg

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### References

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Cytotoxicity - Definition, Identification, and Cytotoxic Compounds

[8] Singh BK, Tripathi M, Chaudhari BP, Pandey PK, Kakkar P. Natural terpenes prevent mitochondrial dysfunction, oxidative stress and release of apoptotic

hepatotoxicity in rats. PLoS ONE. 2012;

[9] Wang J-m, Qu Z-q, Wu J-l, Chung P, Zeng Y-S. Mitochondrial protective and anti-apoptotic effects of Rhodiola crenulata extract on hippocampal neurons in a rat model of Alzheimer's disease. Neural Regeneration Research.

[10] Charalampopoulos I, Tsatsanis C, Dermitzaki E, Alexaki V-I, Castanas E,

sympathoadrenal medulla cells against apoptosis via antiapoptotic Bcl-2 proteins. Proceedings of the National Academy of Sciences. 2004;101:

[11] Dholakiya SL, Benzeroual KE. Protective effect of diosmin on LPSinduced apoptosis in PC12 cells and inhibition of TNF-α expression.

Toxicology in Vitro. 2011;25:1039-1044

[12] Danese S. Mechanisms of action of infliximab in inflammatory bowel disease: An anti-inflammatory multitasker. Digestive and Liver Disease. 2008;40:S225-S228

[13] Couderc M, Mathieu S, Tournadre A, Dubost J-J, Soubrier M. Acute ocular myositis occurring under etanercept for rheumatoid arthritis. Joint, Bone, Spine.

[14] Renna S, Mocciaro F, Ventimiglia M, Orlando R, Macaluso FS, Cappello M, et al. A real life comparison of the effectiveness of adalimumab and golimumab in moderate-to-severe

2014;81(5):445-446

proteins during nimesulide-

7:e34200

2017;12:2025

8209-8214

Margioris AN, et al.

Dehydroepiandrosterone and allopregnanolone protect

[2] Portt L, Norman G, Clapp C, Greenwood M, Greenwood MT. Antiapoptosis and cell survival: A review. Biochimica et Biophysica Acta (BBA)- Molecular Cell Research. 2011;1813:

[3] Ergun U, Yurtcu E, Ergun MA. Protective effect of Ginkgo biloba against gossypol-induced apoptosis in human lymphocytes. Cell Biology International. 2005;29:717-720

[4] Tian Y-M, Tian H-J, Zhang G-Y, Dai Y-R. Effects of Ginkgo biloba extract (EGb 761) on hydroxyl radical-induced thymocyte apoptosis and on age-related thymic atrophy and peripheral immune dysfunctions in mice. Mechanisms of Ageing and Development. 2003;124:

[6] Ao Q, Sun X, Wang A, Fu P, Gong K, Zuo H, et al. Protective effects of extract of Ginkgo biloba (EGb 761) on nerve cells after spinal cord injury in rats.

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[34] Liu W, Jing Z-T, Wu S-X, He Y, Lin Y-T, Chen W-N, et al. Novel AKT

activator, SC79, prevents acute hepatic failure induced by Fas-mediated apoptosis of hepatocytes. The American Journal of Pathology. 2018;188(5): 1171-1182

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Apoptotic Inhibitors as Therapeutic Targets for Cell Survival

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[50] Tran N, Meurice N, Dhruv H. FN14 antagonists and therapeutic uses

[51] Dhruv H, Loftus JC, Narang P, Petit JL, Fameree M, Burton J, et al. Structural basis and targeting of the interaction between fibroblast growth factorinducible 14 and tumor necrosis factorlike weak inducer of apoptosis. The Journal of Biological Chemistry. 2013;

[52] Maneg O, Malatesta F, Ludwig B, Drosou V. Interaction of cytochrome C with cytochrome oxidase: Two different

[53] Zhu S, Stavrovskaya IG, Drozda M,

[54] Wang X, Zhu S, Pei Z, Drozda M, Stavrovskaya IG, Del Signore SJ, et al. Inhibitors of cytochrome C release with therapeutic potential for Huntington's disease. The Journal of Neuroscience.

[55] Wang X, Figueroa BE, Stavrovskaya IG, Zhang Y, Sirianni AC, Zhu S, et al. Methazolamide and melatonin inhibit mitochondrial cytochrome C release and are neuroprotective in experimental models of ischemic injury. Stroke. [Epub ahead of print]. 2009;14:1877-1885

docking scenarios. Biochimica et Biophysica Acta (BBA)-Bioenergetics.

Kim BYS, Ona V, Li M, et al. Minocycline inhibits cytochrome C release and delays progression of amyotrophic lateral sclerosis in mice. Nature. 2002;417(6884):74-78

2008;28(38):9473-9485

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288(45):32261-32276

2004;1655:274-281

748-764

hippocampal slice cultures and modulates tissue inflammation in a transient focal cerebral ischemia model without changing lesion volume. Frontiers in Cellular Neuroscience; Feb 3 2016;10:14. DOI: 10.3389/fncel.

[42] Brenner C, Galluzzi L, Kepp O, Kroemer G. Decoding cell death signals in liver inflammation. Journal of Hepatology. 2013;59(3):583-594

[43] Wang C, Youle RJ. The role of mitochondria in apoptosis. Annual Review of Genetics. 2009;43:95-118

[44] Li L, Jiang X, Huang S, Ying Z, Zhang Z, Pan C, et al. Discovery of highly potent 2-sulfonyl-pyrimidinyl derivatives for apoptosis inhibition and ischemia treatment. ACS

Medicinal Chemistry Letters. 2017;8(4):

[45] Becattini B, Culmsee C, Leone M, Zhai D, Zhang X, Crowell KJ, et al. Structure–activity relationships by interligand NOE-based design and synthesis of antiapoptotic compounds targeting bid. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(33):

[46] Oppermann S, Schrader FC,

[47] Maecker H, Varfolomeev E,

W, et al. TWEAK attenuates the transition from innate to adaptive immunity. Cell. 2005;123(5):931-944

Kischkel F, Lawrence D, LeBlanc H, Lee

Elsässer K, Dolga AM, Kraus AL, Doti N, et al. Novel N-phenyl–substituted thiazolidinediones protect neural cells against glutamate- and tBid-induced toxicity. The Journal of Pharmacology and Experimental Therapeutics. 2014;

2016.00014

407-412

12602-12606

350(2):273-289

83

[35] Urayama S, Kawakami A, Nakashima T, Tsuboi M, Yamasaki S, Hida A, et al. Effect of vitamin K2 on osteoblast apoptosis: Vitamin K2 inhibits apoptotic cell death of human osteoblasts induced by Fas, proteasome inhibitor, etoposide, and staurosporine. Journal of Laboratory and Clinical Medicine. 2000;136(3):181-193

[36] Stanely Mainzen Prince P, Dhanasekar K, Rajakumar S. Vanillic acid prevents altered ion pumps, ions, inhibits Fas-receptor and caspase mediated apoptosis-signaling pathway and cardiomyocyte death in myocardial infarcted rats. Chemico-Biological Interactions. 2015;23:268-276

[37] Fiorucci S, Mencarelli A, Palazzetti B, Del Soldato P, Morelli A, Ignarro LJ. An NO derivative of ursodeoxycholic acid protects against Fas-mediated liver injury by inhibiting caspase activity. Proceedings of the National Academy of Sciences of the United States of America. 2001;98(5):2652-2657

[38] Adrain C, Creagh EM, Martin SJ. Apoptosis-associated release of Smac/ DIABLO from mitochondria requires active caspases and is blocked by Bcl-2. The EMBO Journal. 2001;20(23): 6627-6636

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Apoptotic Inhibitors as Therapeutic Targets for Cell Survival DOI: http://dx.doi.org/10.5772/intechopen.85465

[41] Martin NA, Bonner H, Elkjær ML, D'Orsi B, Chen G, König HG, et al. BID mediates oxygen-glucose deprivationinduced neuronal injury in organotypic hippocampal slice cultures and modulates tissue inflammation in a transient focal cerebral ischemia model without changing lesion volume. Frontiers in Cellular Neuroscience; Feb 3 2016;10:14. DOI: 10.3389/fncel. 2016.00014

T lymphocytes and lymphokineactivated killer cells via the Fas-ligand/ Fas pathway in perforin-knockout (PKO) mice. Toxicology. 2004;204(1):

[28] He W, Wu L, Gao Q, Du Y, Wang Y. Identification of AHBA biosynthetic genes related to geldanamycin biosynthesis in Streptomyces hygroscopicus 17997. Current Microbiology. 2006;52(3):197-203

Cytotoxicity - Definition, Identification, and Cytotoxic Compounds

activator, SC79, prevents acute hepatic failure induced by Fas-mediated

apoptosis of hepatocytes. The American Journal of Pathology. 2018;188(5):

Nakashima T, Tsuboi M, Yamasaki S, Hida A, et al. Effect of vitamin K2 on osteoblast apoptosis: Vitamin K2 inhibits

osteoblasts induced by Fas, proteasome inhibitor, etoposide, and staurosporine. Journal of Laboratory and Clinical Medicine. 2000;136(3):181-193

[37] Fiorucci S, Mencarelli A, Palazzetti B, Del Soldato P, Morelli A, Ignarro LJ. An NO derivative of ursodeoxycholic acid protects against Fas-mediated liver injury by inhibiting caspase activity. Proceedings of the National Academy of

Sciences of the United States of America. 2001;98(5):2652-2657

6627-6636

469-477

[38] Adrain C, Creagh EM, Martin SJ. Apoptosis-associated release of Smac/ DIABLO from mitochondria requires active caspases and is blocked by Bcl-2. The EMBO Journal. 2001;20(23):

[39] Becattini B, Sareth S, Zhai D, Crowell KJ, Leone M, Reed JC, et al. Targeting apoptosis via chemical design: Inhibition of bid-induced cell death by small organic molecules. Chemistry &

Biology. 2004;11(8):1107-1117

[40] Yuan J. Neuroprotective strategies targeting apoptotic and necrotic cell death for stroke. Apoptosis. 2009;14(4):

[35] Urayama S, Kawakami A,

apoptotic cell death of human

[36] Stanely Mainzen Prince P, Dhanasekar K, Rajakumar S. Vanillic acid prevents altered ion pumps, ions, inhibits Fas-receptor and caspase mediated apoptosis-signaling pathway and cardiomyocyte death in myocardial infarcted rats. Chemico-Biological Interactions. 2015;23:268-276

1171-1182

[29] Yin X-H, Han Y-L, Zhuang Y, Yan J-Z, Li C. Geldanamycin inhibits Fas signaling pathway and protects neurons

[30] Duque G, El Abdaimi K, Henderson JE, Lomri A, Kremer R. Vitamin D inhibits Fas ligand-induced apoptosis in human osteoblasts by regulating components of both the mitochondrial and Fas-related pathways. Bone. 2004;

[31] Jia J, Guan D, Zhu W, Alkayed NJ, Wang MM, Hua Z, et al. Estrogen inhibits Fas-mediated apoptosis in

Experimental Neurology. 2009;215(1):

[32] Xie Z, Koyama T, Abe K. Effects of an angiotensin-converting enzyme inhibitor on the expression of Fas protein and on apoptosis in rat

ventricles subjected to reperfusion after

[33] Tsuda T, Ohmori Y, Muramatsu H, Hosaka Y, Takiguchi K, Saitoh F, et al. Inhibitory effect of M50054, a novel inhibitor of apoptosis, on anti-Fas-antibody-induced hepatitis and chemotherapy-induced alopecia. European Journal of Pharmacology.

[34] Liu W, Jing Z-T, Wu S-X, He Y, Lin Y-T, Chen W-N, et al. Novel AKT

ischemia. Current Therapeutic Research. 2000;61(6):358-366

2001;433(1):37-45

82

against ischemia. Neuroscience Research. 2017;12:433-439

41-50

35(1):57-64

48-52

experimental stroke.

[42] Brenner C, Galluzzi L, Kepp O, Kroemer G. Decoding cell death signals in liver inflammation. Journal of Hepatology. 2013;59(3):583-594

[43] Wang C, Youle RJ. The role of mitochondria in apoptosis. Annual Review of Genetics. 2009;43:95-118

[44] Li L, Jiang X, Huang S, Ying Z, Zhang Z, Pan C, et al. Discovery of highly potent 2-sulfonyl-pyrimidinyl derivatives for apoptosis inhibition and ischemia treatment. ACS Medicinal Chemistry Letters. 2017;8(4): 407-412

[45] Becattini B, Culmsee C, Leone M, Zhai D, Zhang X, Crowell KJ, et al. Structure–activity relationships by interligand NOE-based design and synthesis of antiapoptotic compounds targeting bid. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(33): 12602-12606

[46] Oppermann S, Schrader FC, Elsässer K, Dolga AM, Kraus AL, Doti N, et al. Novel N-phenyl–substituted thiazolidinediones protect neural cells against glutamate- and tBid-induced toxicity. The Journal of Pharmacology and Experimental Therapeutics. 2014; 350(2):273-289

[47] Maecker H, Varfolomeev E, Kischkel F, Lawrence D, LeBlanc H, Lee W, et al. TWEAK attenuates the transition from innate to adaptive immunity. Cell. 2005;123(5):931-944

[48] Wajant H. The TWEAK-Fn14 system as a potential drug target. British Journal of Pharmacology. 2013;170(4): 748-764

[49] Roos A, Dhruv HD, Mathews IT, Inge LJ, Tuncali S, Hartman LK, et al. Identification of aurintricarboxylic acid as a selective inhibitor of the TWEAK-Fn14 signaling pathway in glioblastoma cells. Oncotarget. 2017;8(7):12234-12246

[50] Tran N, Meurice N, Dhruv H. FN14 antagonists and therapeutic uses thereof. 2016. US9238034B2

[51] Dhruv H, Loftus JC, Narang P, Petit JL, Fameree M, Burton J, et al. Structural basis and targeting of the interaction between fibroblast growth factorinducible 14 and tumor necrosis factorlike weak inducer of apoptosis. The Journal of Biological Chemistry. 2013; 288(45):32261-32276

[52] Maneg O, Malatesta F, Ludwig B, Drosou V. Interaction of cytochrome C with cytochrome oxidase: Two different docking scenarios. Biochimica et Biophysica Acta (BBA)-Bioenergetics. 2004;1655:274-281

[53] Zhu S, Stavrovskaya IG, Drozda M, Kim BYS, Ona V, Li M, et al. Minocycline inhibits cytochrome C release and delays progression of amyotrophic lateral sclerosis in mice. Nature. 2002;417(6884):74-78

[54] Wang X, Zhu S, Pei Z, Drozda M, Stavrovskaya IG, Del Signore SJ, et al. Inhibitors of cytochrome C release with therapeutic potential for Huntington's disease. The Journal of Neuroscience. 2008;28(38):9473-9485

[55] Wang X, Figueroa BE, Stavrovskaya IG, Zhang Y, Sirianni AC, Zhu S, et al. Methazolamide and melatonin inhibit mitochondrial cytochrome C release and are neuroprotective in experimental models of ischemic injury. Stroke. [Epub ahead of print]. 2009;14:1877-1885

[56] Li M, Wang W, Mai H, Zhang X, Wang J, Gao Y, et al. Methazolamide improves neurological behavior by inhibition of neuron apoptosis in subarachnoid hemorrhage mice. Scientific Reports. 2016;6:35055

[57] Makpol S, Abdul Rahim N, Kien Hui C, Ngah W, Zurinah W. Inhibition of mitochondrial cytochrome C release and suppression of caspases by gammatocotrienol prevent apoptosis and delay aging in stress-induced premature senescence of skin fibroblasts. Oxidative Medicine and Cellular Longevity. 2012; 2012:1-13. Available from: https://www. hindawi.com/journals/omcl/2012/ 785743/ [Accessed: September 24, 2018]

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[61] Ghosh AP, Walls KC, Klocke BJ, Toms R, Strasser A, Roth KA. The proapoptotic BH3-only, Bcl-2 family member Puma is critical for acute ethanol-induced neuronal apoptosis. Journal of Neuropathology and Experimental Neurology. 2009;68(7): 747-756

[62] Mustata G, Li M, Zevola N, Bakan A, Zhang L, Epperly M, et al. Development of small-molecule PUMA inhibitors for mitigating radiationinduced cell death. Current Topics in Medicinal Chemistry. 2011;11(3): 281-290

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[64] Shao X, Chen Q, Dou X, Chen L, Wu J, Zhang W, et al. Lower range of molecular weight of xanthan gum inhibits cartilage matrix destruction via intrinsic bax-mitochondria cytochrome C-caspase pathway. Carbohydrate Polymers. 2018;198:354-363

[65] Sawatzky DA, Willoughby DA, Colville-Nash PR, Rossi AG. The involvement of the apoptosismodulating proteins ERK 1/2, Bcl-xL and Bax in the resolution of acute inflammation in vivo. The American Journal of Pathology. 2006;168(1):33-41

[66] Nguyen Thi PA, Chen M-H, Li N, Zhuo X-J, Xie L. PD98059 protects brain against cells death resulting from ROS/ ERK activation in a cardiac arrest rat model. Oxidative Medicine and Cellular Longevity. 2016;2016:3723762. Available from: https://www.hindawi.c om/journals/omcl/2016/3723762/ [Accessed: December 10, 2018]

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**85**

Section 2

Methods in Cytotoxicity

Assessment

Section 2
