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  1. 1. Melanoma is currently the 5th most common cancer in the UK, having become more prevalent in recent years due to increased UV light exposure. Therefore it is essential we focus research on more targeted treatments as opposed to the regularly used approaches such as chemotherapy and radiotherapy. Naturally occurring compounds are being investigated thanks to reports publishing data that suggests diets high in cruciferous vegetables can lower your risk of several cancers. This is likely due to their high content of a group of organic molecules named isothiocyanates (ITCs). A specific ITC – phenethyl ITC has previously been shown to induce apoptosis in human osteogenic sarcoma cells via a reactive oxygen species mediated mechanism. It has also reportedly induced apoptosis in several other malignancies including non-small lung cancer, colon cancer, breast cancer and prostate cancer cells. Since cancer cells already maintain a relatively high level of reactive oxygen species (ROS), it is thought their antioxidative system may already be slightly suppressed thus rendering them more susceptible to attack by further oxidative stress. Healthy human cells should have a comparable low level of ROS therefore assisting in targeting the cancerous cells with a ROS inducer, whilst illiciting the least damage possible to normal cells. This suggests we can manipulate ROS levels in cancer cells to elevate them past the apoptotic threshold using ITCs. This research is therefore focused on the mechanisms involved in PEITC inhibition of human malignant melanoma A375.S2 cells and H-Ras transformed epithelial cells and whether it has a targeted effect on malignant cells. Results suggest PEITC may act not as an antioxidant but can instead increase oxidative stress in cells resulting in apoptosis. Introduc)on     The human malignant melanoma A375.S2 cell line was cultured on 12-well plates and allowed to grow for 24 hours. Half the cells were then treated with 10µM PEITC and all cells left for a range of time periods (0.5 – 48hrs). A number of cells were pre-treated with a ROS scavenger to ensure the fluorescence measured was due to ROS molecules. Cells were collected, washed with PBS twice, resuspended in the dye 2’,7’-dichlorofluorescin diacetate (DCF-DA) and incubated for 30 minutes. ROS levels were determined using flow cytometry. The cells to be transformed were transfected with H-Ras and some cultured in media containing catalase as a pre-treatment, some without. Both transformed and non-transformed cells were analysed to provide their basal ROS levels. They were then treated with 10µM PEITC for a range of time periods (1-5 hours), incubated with DCF-DA for 60 minutes and ROS levels measured using flow cytometry. Pre-treated cells were tested using a similar method but 5µM PEITC was used. Methods   Effects of PEITC on ROS Production in A375.S2 Cells After being treated with 10µM PEITC, cells had a significantly larger concentration of ROS molecules from as little as 30 minutes after application and seemed to follow a time- dependant trend (Figure 2A). Cells pre-treated with N-acetyl-L-cysteine – a ROS scavenger, had a significantly lower level of ROS when compared to cells treated only with PEITC (Figure 2B). Effects of PEITC on ROS Production in T72 and T72-Ras Cells Treatment of the T72Ras cells with 10µM PEITC induced a considerable increase in DCF- DA fluorescence, corresponding to the concentration of ROS molecules in the cells. As the values increased as time continued, PEITC had a time-dependant effect on the cells reaching a maximum 16-fold increase of the control at the last time point of 5 hours (Figure 3A). The untransformed cells however, produced a lower difference in ROS compared to the control and did not increase at the same rate as the transformed cells at each further time point (Figure 3B). Cells pre-treated with catalase had a lower increase in ROS compared to those treated with PEITC alone (Figure 4A). Quantitative analysis showed the cells with catalase had significantly lower levels of ROS at every time point excluding 0 hours (Figure 4B). Results   Discussion  and  Conclusions   Apoptosis is known to be a key event in preventing carcinogenesis progression and can be initiated by high oxidative stress in cells. Therefore these studies have focused on assessing levels of oxidative stress in the hope that it could be possible to manipulate ROS concentrations in cells to induce apoptosis in malignant cells using PEITC and other similar compounds. This particular compound was capable of inducing ROS production to significantly larger levels than untreated cells. Cells were transfected with H-Ras in order to increase their basal ROS levels to allow comparison of cells under greater oxidative stress than those with lower levels and how they each cope with further stress. As the transformed cells were much more susceptible to the actions of PEITC, this suggests previously stressed cells are less equipped to cope with additional oxidative stress and will respond greatly to a second applications of stressors. We can use this information in the treatment of cancer cells by administrating a double dose of ITC compounds after the initial dose has been applied and left to ensure the cells antioxidative system becomes exacerbated and hopefully, suppressed. The cells with a lower basal ROS concentration exhibited a reduced increase in ROS compared to the control; as these cells may be comparable to healthy cells, these results bode well for the use of PEITC as a targeted anticancer agent. To conclude, PEITC has proven successful in elevating ROS levels in two different cell lines and could provide a selective mechanism to inhibit growth and induce apoptosis in cancerous cells. References   Huang SH, Hsu MH, Hsu SC, Yang JS, Huang WW, Huang aC, Chung JG. 2014. Phenethyl isothiocyanate triggers apoptosis in human malignant melanoma A375.S2 cells through reactive oxygen species and the mitochondria- dependent pathways. Human & Experimental Toxicology 33(3):270–283. Trachootham D, Zhou Y, Zhang H, Demizu Y, Chen Z, Pelicano H, Huang P. 2006. Selective killing of oncogenically transformed cells through a ROS-mediated mechanism by beta-phenylethyl isothiocyanate. Cancer Cell 10(3): 241–252. School  of  Life  Sciences,  Heriot  Wa3  University,  Edinburgh,  UK   Robyn  Adams   The  Role  of  ROS  Molecules  within  PEITC  as  an  An)cancer  Therapeu)c   Figure 1. Chemical Structure of phenethyl isothiocyanate A B Figure 2. Effects of ROS production by 10µM PEITC on A375.S2 cells (A) Effects of pre-treatment with NAC on PEITC treated cells (B). Values are percentage of the control +/- SD, * = p<0.05. Measured using flow cytometry. Figure 3. Effects of ROS production by 10µM PEITC on T72Ras (A) and T72 (B) cells as measured by flow cytometry. Figure 4. Effects of pre-treatment with catalase compared with no pre-treatment on 5µM PEITC treated T72Ras cells. Measured using flow cytometry (A) . Effects of pre-treatment with catalase on 5µM PEITC treated T72Ras cells (quantified) (B). Values are in mean +/- SD, * = p<0.05. A B