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

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

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

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

**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.

melanoma cells compared to non-malignant cells.

30 Cytotoxicity

due to the activation of an apoptotic pathway.

viable and is comparable to the untreated control (**Figure 4**).

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

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 including melanomas [62].

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 100% viable. Possible synergistic interactions were evaluated by comparing the cytotoxicity of combination treatment with mono-treatment on melanoma cells.

The chemotherapeutic agent 5-Fluorouracil can inhibit RNA and DNA synthesis leading to cell death. The combination of nisin Z with 5-Fluorouracil increased the cytotoxicity to melanoma cells over the entire concentration range tested compared to the mono-treatment of 5-Fluorouracil (p < 0.05) (**Figure 5A**), with no significant increase in toxicity to non-malignant keratinocytes (**Figure 5B**).

The 5-Fluorouracil treatment is initially cytotoxic at 50 μM (p < 0.01 compared to the control), whereas the combination of 5-Fluorouracil and nisin Z only begins to induce toxicity at 200 μM (p < 0.001 compared to the control) in the non-malignant keratinocytes. Results indicate that the 5-Fluorouracil-nisin Z combination is more cytotoxic to melanoma cells compared to the mono-treatment. The anti-cancer activity of 5-Fluorouracil may, therefore, be enhanced by combination treatment with nisin Z. Etoposide is a chemotherapeutic agent that is able to induce DNA strand breaks in cancer cells by interfering with type II topoisomerase, ultimately inducing apoptosis. When combining etoposide with nisin Z it was found that the activity towards melanoma cells was enhanced compared to mono-treatment across the entire concentration range (p < 0.001) (**Figure 5C**), with no increase in cytotoxicity to non-malignant keratinocytes (**Figure 5D**). In melanoma cells, the combination of nisin Z with etoposide had a higher level of activity at the lowest concentration tested compared to the highest concentration for mono-treatment (p < 0.001). The anti-cancer activity of etoposide can, therefore, be significantly enhanced through the combination of nisin Z. Hydroxyurea is able to induce DNA damage and inhibit DNA synthesis. The combination of nisin Z with hydroxyurea was able to increase the cytotoxicity to melanoma cells at concentrations of between 25 and 400 μM compared to the mono-treatment of hydroxyurea (p < 0.01) (**Figure 5E**), with no significant increase in toxicity to non-malignant keratinocytes (**Figure 5F**).

To evaluate if possible synergistic interactions occurred between the chemotherapeutic agents and nisin Z, the cytotoxicity of melanoma cells following the mono-treatment of the respective chemotherapeutic agents (50 μM) was compared to that of the mono-treatment of nisin Z (150 μM), followed by that of the combination (50 μM chemotherapeutic agent +150 μM nisin Z). Synergism occurs when the combined effects of the different components are greater than their individual effects. The cell viability of melanoma cells was significantly lower for all combinations compared to mono-treatment with the chemotherapeutic agent alone (p < 0.05) (**Figure 6**). However, the only combination that displayed synergism was the combination of nisin Z with etoposide.

The AMP nisin, which is considered safe for human consumption, not only displays antibacterial properties, but also anti-cancer activities. Although the use of nisin as an adjuvant for conventional antibiotics has been investigated extensively, there are few studies investigating nisin as an adjuvant for conventional chemotherapeutic agents. Nisin also exhibits immune-modulatory properties. We have shown that nisin Z induces selective cytotoxicity towards melanoma cells through an apoptotic pathway. These properties make nisin Z an attractive anti-cancer agent to be used alone or in combination with current chemotherapeutic agents to enhance anti-cancer properties of these agents, while also potentially combatting chemotherapy resistance. Here,

it was shown that combinations of nisin Z with 5-Fluorouracil, hydroxyurea and etoposide was able to enhance the cytotoxicity to melanoma cells, while no significant increase in toxicity toward non-malignant keratinocytes were observed. Especially of interest is the consequence of nisin Z on the effectiveness of etoposide, seeing as etoposide resistance is known in melanoma [63, 64]. The combination of nisin Z with etoposide was able to significantly and selectively

n = 4. \*p < 0.05, \*\*p < 0.01 and \*\*\* p < 0.001 for combination compared to chemotherapeutic agent alone.

**Figure 5.** Cytotoxicity of chemotherapeutic agents in combination with nisin Z on melanoma (A375) cells and nonmalignant keratinocytes (HaCat) as determined by the MTT assay. (A) Melanoma cells exposed to 5-Fluorouracil (FU) combinations. (B) HaCat exposed to 5-FU combinations. (C) Melanoma cells exposed to etoposide combinations. (D) HaCat exposed to etoposide combinations. (E) Melanoma cells exposed to hydroxyurea combinations. (F) HaCat exposed to hydroxyurea combinations. Vehicle control groups were included and are represented by 0 μM. Results are expressed relative to the untreated controls which were set as being 100% viable. Bars represent the standard deviation,

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The Cytotoxic, Antimicrobial and Anticancer Properties of the Antimicrobial Peptide Nisin Z… http://dx.doi.org/10.5772/intechopen.71927 33

100% viable. Possible synergistic interactions were evaluated by comparing the cytotoxicity

The chemotherapeutic agent 5-Fluorouracil can inhibit RNA and DNA synthesis leading to cell death. The combination of nisin Z with 5-Fluorouracil increased the cytotoxicity to melanoma cells over the entire concentration range tested compared to the mono-treatment of 5-Fluorouracil (p < 0.05) (**Figure 5A**), with no significant increase in toxicity to non-malignant

The 5-Fluorouracil treatment is initially cytotoxic at 50 μM (p < 0.01 compared to the control), whereas the combination of 5-Fluorouracil and nisin Z only begins to induce toxicity at 200 μM (p < 0.001 compared to the control) in the non-malignant keratinocytes. Results indicate that the 5-Fluorouracil-nisin Z combination is more cytotoxic to melanoma cells compared to the mono-treatment. The anti-cancer activity of 5-Fluorouracil may, therefore, be enhanced by combination treatment with nisin Z. Etoposide is a chemotherapeutic agent that is able to induce DNA strand breaks in cancer cells by interfering with type II topoisomerase, ultimately inducing apoptosis. When combining etoposide with nisin Z it was found that the activity towards melanoma cells was enhanced compared to mono-treatment across the entire concentration range (p < 0.001) (**Figure 5C**), with no increase in cytotoxicity to non-malignant keratinocytes (**Figure 5D**). In melanoma cells, the combination of nisin Z with etoposide had a higher level of activity at the lowest concentration tested compared to the highest concentration for mono-treatment (p < 0.001). The anti-cancer activity of etoposide can, therefore, be significantly enhanced through the combination of nisin Z. Hydroxyurea is able to induce DNA damage and inhibit DNA synthesis. The combination of nisin Z with hydroxyurea was able to increase the cytotoxicity to melanoma cells at concentrations of between 25 and 400 μM compared to the mono-treatment of hydroxyurea (p < 0.01) (**Figure 5E**), with no significant increase in toxic-

To evaluate if possible synergistic interactions occurred between the chemotherapeutic agents and nisin Z, the cytotoxicity of melanoma cells following the mono-treatment of the respective chemotherapeutic agents (50 μM) was compared to that of the mono-treatment of nisin Z (150 μM), followed by that of the combination (50 μM chemotherapeutic agent +150 μM nisin Z). Synergism occurs when the combined effects of the different components are greater than their individual effects. The cell viability of melanoma cells was significantly lower for all combinations compared to mono-treatment with the chemotherapeutic agent alone (p < 0.05) (**Figure 6**). However, the only combination that displayed synergism was the combination of

The AMP nisin, which is considered safe for human consumption, not only displays antibacterial properties, but also anti-cancer activities. Although the use of nisin as an adjuvant for conventional antibiotics has been investigated extensively, there are few studies investigating nisin as an adjuvant for conventional chemotherapeutic agents. Nisin also exhibits immune-modulatory properties. We have shown that nisin Z induces selective cytotoxicity towards melanoma cells through an apoptotic pathway. These properties make nisin Z an attractive anti-cancer agent to be used alone or in combination with current chemotherapeutic agents to enhance anti-cancer properties of these agents, while also potentially combatting chemotherapy resistance. Here,

of combination treatment with mono-treatment on melanoma cells.

keratinocytes (**Figure 5B**).

32 Cytotoxicity

ity to non-malignant keratinocytes (**Figure 5F**).

nisin Z with etoposide.

**Figure 5.** Cytotoxicity of chemotherapeutic agents in combination with nisin Z on melanoma (A375) cells and nonmalignant keratinocytes (HaCat) as determined by the MTT assay. (A) Melanoma cells exposed to 5-Fluorouracil (FU) combinations. (B) HaCat exposed to 5-FU combinations. (C) Melanoma cells exposed to etoposide combinations. (D) HaCat exposed to etoposide combinations. (E) Melanoma cells exposed to hydroxyurea combinations. (F) HaCat exposed to hydroxyurea combinations. Vehicle control groups were included and are represented by 0 μM. Results are expressed relative to the untreated controls which were set as being 100% viable. Bars represent the standard deviation, n = 4. \*p < 0.05, \*\*p < 0.01 and \*\*\* p < 0.001 for combination compared to chemotherapeutic agent alone.

it was shown that combinations of nisin Z with 5-Fluorouracil, hydroxyurea and etoposide was able to enhance the cytotoxicity to melanoma cells, while no significant increase in toxicity toward non-malignant keratinocytes were observed. Especially of interest is the consequence of nisin Z on the effectiveness of etoposide, seeing as etoposide resistance is known in melanoma [63, 64]. The combination of nisin Z with etoposide was able to significantly and selectively

cytotoxic to melanoma cells compared to non-malignant keratinocytes. It was shown that nisin Z disrupts the cell membrane integrity of melanoma cells, while also inducing apoptosis in the majority of exposed malignant cells (**Figure 7B**). Taking into account these anticancer properties of nisin Z, the cytotoxicity of nisin Z-chemotherapeutic agent combinations to melanoma cells was compared to the mono-treatment with selected conventional chemotherapeutic agents. This study indicated that when used in combination with the conventional chemotherapeutic agents (5-Fluorouracil, hydroxyurea and etoposide), nisin Z has the potential to enhance the cytotoxicity of these conventional chemotherapeutic agents against cultured melanoma cells. Synergism was observed between the nisin Z and etoposide combination. However, this study was only limited to the *in vitro* effect in melanoma cells with regards to cytotoxicity as measured by the MTT assay. For future *in vitro* studies, it is suggested that more cancer cell lines be included. The mechanistic interaction between nisin Z and the chemotherapeutic agents should also be investigated. It is also suggested that *in vivo* studies be conducted similarly to that by Preet and co-workers to assess whether the combination of nisin Z with these conventional chemotherapeutic agents are able to reduce melanoma tumorigenesis *in vivo* [16]. The effective dosages also need to be determined with *in vivo* assays. Nisin Z has great potential for clinical application considering its low cytotoxicity to non-malignant cells and the effectiveness of this AMP against Gram-positive bacterial strains and certain cancers. However, detailed antimicrobial and anticancer mechanistic interaction studies analysis are lacking and many *in vitro* results must still be confirmed within *in vivo*

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**Figure 7.** Summary of the antimicrobial and anticancer properties of nisin Z alone and in combination with conventional therapies. (A) The antimicrobial effects and mechanisms of action of nisin Z and selected antibiotics alone and in combination on gram-positive bacteria. (B) The cytotoxic effect of nisin Z on cultured melanoma cells and combinations

of this AMP with conventional chemotherapeutic agents.

systems.

**Figure 6.** Cytotoxicity results for mono-treatment and combinations of chemotherapeutic agents (50 μM) and nisin Z (150 μM) as determined by the MTT assay. Nisin Z was combined with (A) etoposide, (B) 5-Fluorouracil and (C) hydroxyurea. Bars represent the average and error bars the standard deviation, n = 4. \*\*p < 0.01 and \*\*\*p < 0.001 for combination compared to chemotherapeutic agent alone. #p < 0.05 and ##p < 0.01 for combination compared to nisin Z alone.

enhance the cytotoxic effect etoposide to melanoma cells. Synergism was also observed when combining nisin Z and etoposide with regards to the cytotoxic effect in melanoma cells. Based on all the properties of nisin Z and its GRAS status it could, therefore, be considered as an adjuvant for conventional chemotherapeutic agents.

### **5. Conclusion**

The majority of AMPs exhibit direct microbial killing activity and occur in all living species as an important part of their innate immune system. Due to the co-evolution of AMPs and bacteria, bacterial species are less likely to develop resistance to these peptides compared to conventional antibiotics. The lantibiotic, nisin, has a promising potential for clinical application as it exhibits very low cytotoxicity to mammalian cells, while displaying potent antimicrobial activity against several common foods borne and clinically important Gram-positive bacteria. The use of nisin against Gram-negative bacteria still remains limited. Nisin can be considered as a promising adjuvant for antibiotics in the treatment of Gram-positive bacteria. Antibioticnisin combinations can potentially be used to lower the therapeutic dose of antibiotic treatments, while also enhancing the antimicrobial activity by employing multiple modes of action. With multiple antimicrobial mechanisms concurrently in play, these combinations can hinder the development of antibiotic resistance. We have demonstrated that nisin Z displays synergism when combined with novobiocin against *S. aureus*. This bacterial species is associated with mastitis. Both nisin-based products and novobiocin are used for the treatment of mastitis. The synergistic interactions between nisin and novobiocin make this combination, especially of interest for developing novel formulations for the treatment of mastitis (**Figure 7A**).

Apart from the antimicrobial properties of nisin, this AMP also displays promising anticancer potential towards several types of malignancies. This chapter discussed the anti-cancer potential of nisin Z towards cultured melanoma cells. Results showed that this AMP is more cytotoxic to melanoma cells compared to non-malignant keratinocytes. It was shown that nisin Z disrupts the cell membrane integrity of melanoma cells, while also inducing apoptosis in the majority of exposed malignant cells (**Figure 7B**). Taking into account these anticancer properties of nisin Z, the cytotoxicity of nisin Z-chemotherapeutic agent combinations to melanoma cells was compared to the mono-treatment with selected conventional chemotherapeutic agents. This study indicated that when used in combination with the conventional chemotherapeutic agents (5-Fluorouracil, hydroxyurea and etoposide), nisin Z has the potential to enhance the cytotoxicity of these conventional chemotherapeutic agents against cultured melanoma cells. Synergism was observed between the nisin Z and etoposide combination. However, this study was only limited to the *in vitro* effect in melanoma cells with regards to cytotoxicity as measured by the MTT assay. For future *in vitro* studies, it is suggested that more cancer cell lines be included. The mechanistic interaction between nisin Z and the chemotherapeutic agents should also be investigated. It is also suggested that *in vivo* studies be conducted similarly to that by Preet and co-workers to assess whether the combination of nisin Z with these conventional chemotherapeutic agents are able to reduce melanoma tumorigenesis *in vivo* [16]. The effective dosages also need to be determined with *in vivo* assays. Nisin Z has great potential for clinical application considering its low cytotoxicity to non-malignant cells and the effectiveness of this AMP against Gram-positive bacterial strains and certain cancers. However, detailed antimicrobial and anticancer mechanistic interaction studies analysis are lacking and many *in vitro* results must still be confirmed within *in vivo* systems.

enhance the cytotoxic effect etoposide to melanoma cells. Synergism was also observed when combining nisin Z and etoposide with regards to the cytotoxic effect in melanoma cells. Based on all the properties of nisin Z and its GRAS status it could, therefore, be considered as an adju-

**Figure 6.** Cytotoxicity results for mono-treatment and combinations of chemotherapeutic agents (50 μM) and nisin Z (150 μM) as determined by the MTT assay. Nisin Z was combined with (A) etoposide, (B) 5-Fluorouracil and (C) hydroxyurea. Bars represent the average and error bars the standard deviation, n = 4. \*\*p < 0.01 and \*\*\*p < 0.001 for combination compared to chemotherapeutic agent alone. #p < 0.05 and ##p < 0.01 for combination compared to nisin Z

The majority of AMPs exhibit direct microbial killing activity and occur in all living species as an important part of their innate immune system. Due to the co-evolution of AMPs and bacteria, bacterial species are less likely to develop resistance to these peptides compared to conventional antibiotics. The lantibiotic, nisin, has a promising potential for clinical application as it exhibits very low cytotoxicity to mammalian cells, while displaying potent antimicrobial activity against several common foods borne and clinically important Gram-positive bacteria. The use of nisin against Gram-negative bacteria still remains limited. Nisin can be considered as a promising adjuvant for antibiotics in the treatment of Gram-positive bacteria. Antibioticnisin combinations can potentially be used to lower the therapeutic dose of antibiotic treatments, while also enhancing the antimicrobial activity by employing multiple modes of action. With multiple antimicrobial mechanisms concurrently in play, these combinations can hinder the development of antibiotic resistance. We have demonstrated that nisin Z displays synergism when combined with novobiocin against *S. aureus*. This bacterial species is associated with mastitis. Both nisin-based products and novobiocin are used for the treatment of mastitis. The synergistic interactions between nisin and novobiocin make this combination, especially

of interest for developing novel formulations for the treatment of mastitis (**Figure 7A**).

Apart from the antimicrobial properties of nisin, this AMP also displays promising anticancer potential towards several types of malignancies. This chapter discussed the anti-cancer potential of nisin Z towards cultured melanoma cells. Results showed that this AMP is more

vant for conventional chemotherapeutic agents.

**5. Conclusion**

alone.

34 Cytotoxicity

**Figure 7.** Summary of the antimicrobial and anticancer properties of nisin Z alone and in combination with conventional therapies. (A) The antimicrobial effects and mechanisms of action of nisin Z and selected antibiotics alone and in combination on gram-positive bacteria. (B) The cytotoxic effect of nisin Z on cultured melanoma cells and combinations of this AMP with conventional chemotherapeutic agents.
