**2.1. Cell culture**

The cell culture procedures were carried out in an aseptic condition in a Class II biohazard cabinet according to good cell culture practice (GCCP) guidelines. The MCF-7 breast cancer cell line (ATCC, USA) were grown in 25 cm<sup>2</sup> tissue culture flasks (TPP, Switzerland) and incubated in an incubator (Binder, Germany) at 37°C under a humidified atmosphere of 5% CO<sup>2</sup> . The cells were maintained in RPMI supplemented with 10% FBS and 1% penicillin–streptomycin. Upon reaching about 80% confluency, the cells were washed twice with PBS and 1 mL of trypsin–EDTA solution was added to detach the monolayer cells. The trypsin was inactivated by the addition of the complete cell growth medium and the cells were collected by centrifugation at 1000 rpm (Hettich Universal 32 R centrifuge, DJB Labcare Ltd., UK) for 5 min before discarding the medium. The cell count was determined using a hemocytometer counting chamber (Marienfeld, Germany) and about 0.5 to 1 × 10<sup>6</sup> cells were subcultured into a fresh 25 cm2 tissue culture flask containing 6 ml of fresh medium at the subcultivation ratio of 1:4. The cultures were incubated at 37°C under 5% CO<sup>2</sup> and 95% air.

### **2.2. Cryogenic preservation and recovery**

The cells were preserved in liquid nitrogen to avoid loss of their original characteristics. Briefly, the cells growing in the exponential phase were subcultured. Following detachment and centrifugation, the cells were resuspended in freezing medium containing 90% FBS and 10% DMSO, as a cryoprotective agent, to yield a final cell density of 2 to 5 × 10<sup>6</sup> cells/mL. Subsequently, 1 mL of cell suspension was transferred into each labeled cryotube and allowed to stand at −20°C for 2 hours and at −80°C overnight before storage in liquid nitrogen (−196°C) (CBS Cryosystem). When needed, the cells were recovered by thawing with gentle agitation in water bath at 37°C for approximately 2 min. The cell suspension was transferred into a sterile 15 mL centrifuge tube (TPP, Switzerland) containing prewarmed complete growth medium in a biosafety cabinet and centrifuged at 1000 rpm (Hettich Universal 32 R centrifuge, DJB Labcare Ltd., UK) for 5 min at room temperature to remove the cryoprotective agent (DMSO). Finally, the cell pellets were resuspended in culture media and transferred to a 25cm2 culture flask (TPP, Switzerland), incubated at 37°C in a CO<sup>2</sup> incubator. After 2 days, the medium was replaced with fresh complete growth medium.

#### **2.3. Plating**

the mitochondrial mediated pathway (intrinsic pathway). Caspases are the key regulatory proteins for both pathways [2]. The extrinsic pathway requires the binding of ligands such as tumor necrosis factor α (TNFα), Fas ligand (Fas-L) and TNF-related apoptosis-inducing ligand (TRAIL) to the death receptor on cell surfaces and corresponding transduction of signals leading to apoptosis [3]. The intrinsic pathway, on the other hand, is mediated by the release of cytochrome c by the mitochondrial following DNA damage. The released cytochrome c then induces the formation of apoptosomes composed of apoptotic protease activating factor 1 (Apaf-1), procaspase 9, and either ATP or dATP [4]. Caspase 9 is a downstream factor that activates the executioner caspase-3, which cleaves substrates such as Poly (ADP-ribose) polymerase (PARP) [5]. The activated caspase 3 then initiates the caspase cascade that culminates

In this study, the colorimetric microculture tetrazolium assay [7] was employed to access the cells viability while assays including morphology studies, Annexin V-FITC Assay and DNA

The chemical and reagents used in the study were; MTT (3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (PhytoTechnology Laboratories, USA), DMSO (dimethylsulfoxide) (Fisher Scientific, UK), Dulbecco's Modified Eagle's Medium (DMEM) and Roswell Park Memorial Institute medium (RPMI 1640) powders with L-glutamine (GIBCO, New Zealand), fetal bovine serum (FBS), horse serum, trypsin–EDTA, phosphate-buffered saline (PBS) tablets and penicillin (10,000 U/mL)–streptomycin (10 mg/mL) (Sigma-Aldrich, USA), acridine orange (AO) and propidium iodide (PI), annexin V-FITC (BD Pharmingen), and gel

The cell culture procedures were carried out in an aseptic condition in a Class II biohazard cabinet according to good cell culture practice (GCCP) guidelines. The MCF-7 breast

and incubated in an incubator (Binder, Germany) at 37°C under a humidified atmosphere

cillin–streptomycin. Upon reaching about 80% confluency, the cells were washed twice with PBS and 1 mL of trypsin–EDTA solution was added to detach the monolayer cells. The trypsin was inactivated by the addition of the complete cell growth medium and the cells were collected by centrifugation at 1000 rpm (Hettich Universal 32 R centrifuge, DJB Labcare Ltd., UK) for 5 min before discarding the medium. The cell count was determined using a hemocytometer counting chamber (Marienfeld, Germany) and about 0.5 to 1 × 10<sup>6</sup>

medium at the subcultivation ratio of 1:4. The cultures were incubated at 37°C under 5%

. The cells were maintained in RPMI supplemented with 10% FBS and 1% peni-

tissue culture flasks (TPP, Switzerland)

tissue culture flask containing 6 ml of fresh

fragmentation were thereafter utilized to ascertain the mode of the cell death.

red nucleic acid stain and apoptotic DNA ladder detection kit (Abcam, USA).

cancer cell line (ATCC, USA) were grown in 25 cm<sup>2</sup>

cells were subcultured into a fresh 25 cm2

in the demolition of the cell [6].

22 Current Understanding of Apoptosis - Programmed Cell Death

**2. Materials and methods**

**2.1. Cell culture**

of 5% CO<sup>2</sup>

CO<sup>2</sup>

and 95% air.

At about 80% confluency, the cells were collected and the concentration was determined using a hemocytometer. Briefly, 0.5 × 10<sup>5</sup> cells/mL were dispensed into each well of a 96-well flat bottom tissue culture plate. To obtain the desired plating concentration of 0.5 × 10<sup>5</sup> cells/ mL, the initial cell concentration was adjusted with culture medium using the following formula:

$$\mathbf{M}\_1 \mathbf{V}\_1 = \mathbf{M}\_2 \mathbf{V}\_2 \tag{1}$$

where M1 is the initial cell concentration, V<sup>1</sup> is the initial cell suspension volume, M2 is the final cell concentration, and V<sup>2</sup> is the final cell suspension volume.

Using a multichannel pipette, 100 μL of the cell suspension was dispensed into each well, except the blank wells, which received 200 μL of culture medium. The cell-seeded plate was incubated overnight at 37°C to facilitate attachment.

#### **2.4. Preparation of the test agents**

Artonin E was kindly donated by Dr. Najihah Hashim of the Department of Pharmacy, Faculty of Medicine, University of Malaya. The compound was isolated from *Artocarpus elasticus*, characterized and identified as reported by [8]. The standard agents, Tamoxifen and Paclitaxel were purchase from Sigma Aldrich, St. Louis, MO, USA. These agents were dissolved in DMSO and normal saline respectively and then diluted with respective medium with highest final DMSO concentration of 0.1% for *in vitro* cell culture studies. They were diluted serially using culture medium to obtain concentrations in the range of 1.56–100 μM. About 100 μL of each concentration of Artonin E, Tamoxifen and Paclitaxel solutions was added into appropriate wells in four replicates.

#### **2.5. Microculture tetrazolium assay**

The colorimetric microculture tetrazolium assay ([7] was used to assess breast cancer cell viability. The tetrazolium dye is converted into an insoluble formazan by the action of nicotinamide adenine dinucleotide hydrogenase present in metabolically active cells. This cellular conversion into a purple-colored formazan is directly proportional to the number of viable cells. Briefly, exponentially growing cells were seeded into a 96-well flat bottom tissue culture plate at a density of approximately 0.5 × 10<sup>4</sup> cells/well and allowed to adhere to the plate by incubating at 37°C under 5% CO<sup>2</sup> and 95% air overnight. Following cell attachment, the cells were incubated with the tested compounds at concentrations ranging from 1.56 to 100 μM. Control cells were treated with 0.01% of DMSO, which was equivalent to the amount of DMSO used as vehicle. After each of the 24, 48 and 72 h treatment time period, 20 μL of 5 mg/mL of MTT solution was added to each well and the plate was reincubated for 4 h to facilitate catalysis by mitochondrial dehydrogenases. Next, 100 μL DMSO was added to each well to solubilize the formazan crystals. The absorbances of the resultant solutions were determined colorimetrically at 570 nm. The experiment was performed in triplicate.

We conducted a nonlinear regression analysis and utilized the GraphPad Prism software to fit a dose-response curve. The concentration of the compound that triggered a 50% growth inhibition was indicated at different time interval in **Figure 1**. The percentage viability used in fitting the dose-response curve was calculated using the following formula:

$$\% \text{ of cell viability} = \frac{A\_\text{}}{A\_\text{c}} \times 100\tag{2}$$

Where the absorbance reading of treated samples is indicated as AT while the absorbance of control samples treated with 0.01% of DMSO equivalent to the concentration used in dissolv-

Apoptosis-Inducing Effect of Artonin E in Breast Cancer http://dx.doi.org/10.5772/intechopen.79205 25

Acridine Orange (AO) and propidium iodide (PI) double staining assay was employed to determine the effect of Artonin E in the induction of breast cancer cell death. A total of 3 × 10<sup>5</sup> MCF-7 breast cancer cells were seeded in a six-well plate and exposed to various treatment concentrations of Artonin E (1.56–100 μM) at varying time points (24–72 h). The adherent MCF-7 cells were trypsinized, and centrifuged at 2000 rpm (Hettich Universal 32 R centrifuge, DJB Labcare Ltd., UK) for 5 min. The pellet was washed with ice-cold PBS, re-centrifuged before suspending in 20 μL of PBS. The cells were thereafter stained on ice with 20 μL dye containing 10 μg/mL of AO with 10 μg/mL of PI. Carl Zeiss Axioskop plus-2 fluorescence microscope was used to visualize aliquots of 20 μL of the cell suspension. Three fields each containing at least 200 cells were immediately assessed for viability, early and late apoptosis

Apoptosis was also investigated by detection of externalized phosphatidylserine using the Annexin V Kit (BD Pharmingen, USA). The cell preparation was carried out in accordance with the manufacturer's protocol. The prepared cells were finally subjected to flow cytometric analysis using laser emitting excitation light at 488 nm and a BD flow cytometer equipped with an Argon laser (Cyan ADP, DAKO, Glostrup, Denmark). The data were analyzed using

Qualitative DNA fragmentation (Roche) kit was used in this analysis. Briefly, after trypsin-

by centrifugation at 3000 rpm for 5 min, the supernatant removed and replaced with 200 μL of fresh PBS. The sample was thereafter lysed with 200 μL binding/lysis buffer consisting of 6 M guanidine-HCl, 10 mM urea, 10 mM Tris–HCl, 20% Triton X-100 (v/v), pH 4.4 and incubated for 10 min at 15–25°C. After the incubation, 100 μL of isopropanol was added and the mixture was filtered through a high pure spin filter tube combined with a clean collection tube and centrifuged at 8000 rpm for 1 min. The flow-through was discarded and the filter tube was combined again with the collection tube before washing with 500 μL of washing buffer consisting of 20 mM NaCl, 2 mM Tris–HCl, pH 7.5 in 20 mL with the addition of 80 mL ethanol. This washing step was done twice before spinning the tube dry at 13,000 rpm (Eppendorf 5424 microcentrifuge, USA) for 10 s to remove any residual washing

The extracted DNA was eluted into a fresh collection tube with 200 μL of prewarmed (70°C) elution buffer (10 mM Tris) and recentrifuged at 8000 rpm for 1 min. The quality of the

cells were collected and washed twice with PBS. The cells were then pelleted

as well as necrosis [9]. The experiment was performed in triplicate.

ing the tested agent is depicted as AC.

**2.6. Cell morphological study**

**2.7. Annexin V-FITC assay**

the Summit V4.3 software.

ization, 1 × 10<sup>6</sup>

buffer.

**2.8. DNA fragmentation analysis**

**Figure 1.** Viability of MCF-7 cell line treated with Artonin E. The half maximum growth inhibitory concentrations (IC50) were: 6.90, 5.10 and 3.77 μM Artonin E at 24, 48 and 72 h, respectively.

Where the absorbance reading of treated samples is indicated as AT while the absorbance of control samples treated with 0.01% of DMSO equivalent to the concentration used in dissolving the tested agent is depicted as AC.
