**4. Cytotoxic effect of nisin on malignant cells**

Over the last few decades, great strides have been made in cancer treatment and therapies, leading to the steady decline of cancer death rates [53]. Despite these developments, many current cancer therapies are still associated with high cytotoxicity and lack specificity. There is consequently still a need for the development of novel anti-cancer therapies. AMPs, especially bacteriocins, display selectivity towards cancer cells [10]. These AMPs are, therefore, potential alternative candidates to current chemotherapeutic agents. AMPs can also be applied as adjuvants to chemotherapeutic agents to lower the therapeutic doses needed with the intention of quelling the toxicity of these treatments.

Studies have previously investigated the anti-tumour potential of nisin *in vitro* and *in vivo* for head and neck squamous cell carcinoma (HNSCC) [52]. The study by Joo and co-workers indicated that nisin has the ability to selectively induce apoptosis, cell cycle arrest and reduce cell proliferation in HNSCC cells, compared to primary keratinocytes *in vitro* [51]. *In vivo*, nisin treatment reduced the overall tumour burden compared to non-nisin treated groups, in a floor-of-mouth oral cancer xenograft mouse model. Also, to examine the mechanism by which nisin facilitates its anti-proliferative and pro-apoptotic effects on HNSCC cells, the effect of nisin-treatment on the expression of 39,000 genes was examined by using Affymetrix gene arrays. The expression of multiple genes was altered, including those in the apoptotic and cell cycle pathways, membrane physiology, energy and nutrient pathways, ion transport and signal transduction and protein binding pathways. The *CHAC1* gene, a cation transport regulator and apoptosis mediator were dramatically up-regulated. This study was the first to show that the antibacterial food preservative nisin could effectively reduce and prevent tumorigenesis both *in vitro* and *in vivo.*

### **4.1. Cytotoxic effects of nisin Z on melanoma cells**

We also evaluated the potential of nisin Z to induce selective cytotoxicity towards human melanoma cells *in vitro*. Melanoma is the leading cause of skin cancer-related deaths [54, 55]. Contrary to most types of cancer, the frequency of melanoma has been increasing over the last three decades [54]. In addition to a high mortality rate, Melanoma cells also have a sinister tendency to rapidly develop resistance to mainstream chemotherapeutic agents [12, 13]. *In vitro* cytotoxicity of the nisin Z was determined by employing the MTT assay, LDH assay and flow cytometric apoptosis and necrosis analyses. The non-malignant human keratinocyte (HaCat) cell line was used as a control. The MTT and LDH assays were performed, as described previously [56]. The flow cytometric FITV Annexin V apoptosis assay (BD PharmingenTM, BD Biosciences, San Jose, CA, USA) was employed for the detection of apoptotic cytotoxicity. FITC Annexin emits green fluorescence and its presentation indicates early apoptotic events, while propidium iodide (PI) emanates red fluorescence and is associated with late apoptotic or necrotic cells.

Z concentrations. Indicating that nisin Z did not negatively affect the cell viability of HaCat cells. The LDH assay also showed that there was no significant increase in the release of LDH, indicating that nisin Z did not cause any measurable membrane damage. On the other hand, both the MTT and LDH assays showed that relatively low concentrations of melittin led to a

These *in vitro* results eco many of the recent *in vivo* findings, showing that nisin exposure to non-malignant cells has very little to no cytotoxic effects. Even at concentrations of nisin Z that exceeds the MICs for *S. aureus* and *S. epidermidis* (**Figure 1**) no toxicity was observed. Keeping in mind that nisin is an effective antimicrobial agent against several Gram-positive bacterial species, including clinical important and resistant pathogens, this AMP shows promising

Over the last few decades, great strides have been made in cancer treatment and therapies, leading to the steady decline of cancer death rates [53]. Despite these developments, many current cancer therapies are still associated with high cytotoxicity and lack specificity. There is consequently still a need for the development of novel anti-cancer therapies. AMPs, especially bacteriocins, display selectivity towards cancer cells [10]. These AMPs are, therefore, potential alternative candidates to current chemotherapeutic agents. AMPs can also be applied as adjuvants to chemotherapeutic agents to lower the therapeutic doses needed with the intention of

Studies have previously investigated the anti-tumour potential of nisin *in vitro* and *in vivo* for head and neck squamous cell carcinoma (HNSCC) [52]. The study by Joo and co-workers indicated that nisin has the ability to selectively induce apoptosis, cell cycle arrest and reduce cell proliferation in HNSCC cells, compared to primary keratinocytes *in vitro* [51]. *In vivo*, nisin treatment reduced the overall tumour burden compared to non-nisin treated groups, in a floor-of-mouth oral cancer xenograft mouse model. Also, to examine the mechanism by which nisin facilitates its anti-proliferative and pro-apoptotic effects on HNSCC cells, the effect of nisin-treatment on the expression of 39,000 genes was examined by using Affymetrix gene arrays. The expression of multiple genes was altered, including those in the apoptotic and cell cycle pathways, membrane physiology, energy and nutrient pathways, ion transport and signal transduction and protein binding pathways. The *CHAC1* gene, a cation transport regulator and apoptosis mediator were dramatically up-regulated. This study was the first to show that the antibacterial food preservative nisin could effectively reduce and prevent

We also evaluated the potential of nisin Z to induce selective cytotoxicity towards human melanoma cells *in vitro*. Melanoma is the leading cause of skin cancer-related deaths [54, 55].

considerable increase in cytotoxicity in HaCat cells.

**4. Cytotoxic effect of nisin on malignant cells**

potential for clinical application.

28 Cytotoxicity

quelling the toxicity of these treatments.

tumorigenesis both *in vitro* and *in vivo.*

**4.1. Cytotoxic effects of nisin Z on melanoma cells**

The quantitative colourimetric MTT assay was used to investigate the cytotoxic effect of nisin Z on cultured melanoma cells as well as non-malignant keratinocytes. There is a clear concentrationdependent decline in cell viability observed in melanoma cells exposed to nisin Z (**Figure 3A**).

**Figure 3.** Cytotoxic effects of nisin Z on melanoma (A375) cells. (A) Cell viability was determined using the MTT assay. (B) LDH release from cells following treatment with nisin Z. Keratinocytes (HaCat) were used as a non-malignant control. \* p< 0.05 and \*\*\* p< 0.001 compared to the control groups.

A significant increase in cytotoxicity is observed in melanoma cells after exposure to relatively low concentrations of nisin Z. The IC50 value of melanoma cells exposed to nisin Z is approximately 180 μM. Conversely, the non-malignant keratinocytes exposed to nisin Z presented with considerably higher cell viability, with an IC50 value more than double that of its malignant counterpart. To examine whether the observed cytotoxicity of melanoma cells exposed to nisin Z is the result of membrane damage, the LDH assay was performed. This assay measures the release of lactate dehydrogenase, the cytosolic enzyme, as a result of cellular plasma membrane damage. Results suggest that the exposure of melanoma cells to nisin Z concentrations of 150 μM and higher (**Figure 3B**) lead to in a significant increase in LDH release. No significant LDH release was detected in the non-malignant keratinocytes after nisin Z exposure, indicating very little membrane damage. Both, the basic cytotoxicity assays (MTT and LDH assay) suggest that nisin Z is selectively more toxic towards cultured melanoma cells compared to non-malignant cells.

**4.2. The potential of nisin Z to increase the cytotoxicity and selectivity of** 

functional agents to achieve synergistic interactions [14, 15].

Due to the toxicity associated with some conventional chemotherapeutic agents, as well as the constant threat of malignancies evolving chemotherapy resistance [11–13], there is a necessity for the development of novel anti-cancer therapies. To combat chemotherapy resistance, the efficacy of chemotherapeutic agents can be enhanced by the co-administration of multi-

The Cytotoxic, Antimicrobial and Anticancer Properties of the Antimicrobial Peptide Nisin Z…

http://dx.doi.org/10.5772/intechopen.71927

31

As stated earlier, there is an abundance of studies which investigated the use of nisin as an adjuvant to conventional antibiotics [4, 40–42, 57]. It has been shown that nisin displays anticancer properties; however, inadequate focus has been given to applying nisin as an adjuvant for chemotherapeutic agents. The ability of nisin to increase the activity of the chemotherapeutic drug, doxorubicin, was investigated *in vivo* by Preet and co-workers [16]. Doxorubicin (Adriamycin) is traditionally employed to treat breast cancer, bladder cancer, lymphoma, and acute lymphocytic leukaemia, to name a few. When combining nisin with doxorubicin, enhanced anti-cancer activities were observed and apoptosis could be detected upon treatment of mice with induced skin carcinogenesis as well as a slight increase in oxidative stress. However, the exact mechanism by which nisin exerts its anti-cancer activities was not determined [16]. It is suggested that AMPs, which display anticancer activity, should be used in combination with conventional chemotherapeutic agents to enhance the effectiveness of these treatments, prevent recurrence of cancer following treatment and possibly reduce instances of chemotherapy resistance [58, 59]. Other studies have also shown that AMPs have the potential to enhance the effectiveness of conventional chemotherapeutic agents. The cytotoxicity of etoposide and cisplatin could be enhanced through the combination with magainin A and magainin G, respectively [60]. More recently, it was shown that the combination of melittin and 5-Fluorouracil enhanced cytotoxic effects against squamous skin cancer cells, while simultaneously reducing the toxicity to normal keratinocytes [61]. There are currently no AMPs that have entered into clinical trials or that are in preclinical development as cancer therapeutics. However, peptide-derived therapies are being recognised for the selectivity and anticancer effectiveness and have been investigated in clinical trials [59]. For example, the peptide asparagine-glycine arginine tumour homing peptide (NGR-hTNF) has completed phase 1 clinical trials and is waiting to enter phase 2 clinical trials to test its effectiveness when used in combination with cisplatin for the treatment of several refractory solid tumours

Based on the findings that nisin Z is more selectively cytotoxic to melanoma cells, the cytotoxic effect of the combination of nisin Z with conventional chemotherapeutic agents was investigated in cultured melanoma cells. The effect of combinations of nisin Z with conventional chemotherapeutic agents (5-Fluorouracil, etoposide, hydroxyurea) on A375 (melanoma) and HaCat (non-malignant keratinocyte) cells was determined by the MTT assay. Cells were exposed to different concentrations of the respective chemotherapeutic agents independently and in combination with 150 μM of nisin Z for 24 h. Following exposure, the MTT assay was performed as described earlier. Blank and background measurements were subtracted and cell viability is expressed as a percentage relative to the untreated control, which was set as

**conventional chemotherapeutic agents**

including melanomas [62].

Flow cytometry was used to investigate whether the cytotoxicity observed in melanoma cells was of apoptotic or necrotic origin. For the non-malignant keratinocyte cells, the flow cytometric analysis indicated that >98% of the cells exposed to 50 μM nisin Z could be considered viable and is comparable to the untreated control (**Figure 4**).

A small increase in cytotoxicity is observed at higher concentration. Melanoma cells exposed to 50 μM nisin Z showed a much larger early apoptosis (>17%) population than their nonmalignant counterparts. A significant increase in cytotoxicity is observed in melanoma cells exposed to higher concentrations of nisin Z, resulting in approximately half of the cancer cells undergoing apoptosis/necrosis after being exposed to nisin Z concentrations of 100 μM or higher. These results confirm the basic viability data that nisin Z is more selectively cytotoxic to melanoma cells and give an indication that the cell death observed in these cells is probably due to the activation of an apoptotic pathway.

**Figure 4.** Pie graphs representing the cell population sizes of viable, apoptotic and necrotic non-malignant keratinocyte (HaCat) and melanoma (A375) cells after exposure to 50–200 μM of nisin Z for 24 h.
