Gelzo, Monica (2014) 7-dehydrocholesterol and its oxidative compounds: stability study and their effects on melanoma cell lines. [Tesi di dottorato]

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Abstract

The 7-dehydrocholesterol (7-DHC), the precursor of cholesterol biosynthesis, is highly reactive and easily modifiable to produce 7-DHC oxidative compounds. Evidences in vitro demonstrated that the instability of 7-DHC in solution and in liposomes is due to its susceptibility to peroxidation. In addition, 7-DHC is also converted in vitamin D3 by photo-induced synthesis which occurs in the skin. 7-DHC is present at relatively high concentrations in skin where it is exposed to exogenous radical sources and oxygen. Recently, it has been reported that 7-DHC oxidative compounds can have deleterious effects on cellular functionality and viability. Ultraviolet radiation is the main cause of skin cancers, and melanoma is the most serious form of tumor. Today, there is no therapy for advanced-stage melanoma and its metastasis due to their high resistance to various anticancer therapies. In this study, we evaluated the effect on melanoma cell lines of 7-DHC as such, and for this aim much care to minimize 7-DHC modifications was used. Therefore, we evaluated the 7-DHC stability in the vehicle suspension used to transfer this compound from the culture medium into the cells. We also measured the intracellular levels of 7-DHC and its oxidative compounds after different treatment times. The stability study showed no significant changes of 7-DHC levels from baseline values in the suspension up to 90 days of storage at 4 °C. We found that from 12 to 72 hours of treatment 82-86% of 7-DHC entered the cells, and the levels of 7-DHC-derived compounds were not significant. Simultaneously, ROS production was significantly increased already after 2 hours. After 24 hours and up to 72 hours, 7-DHC treated melanoma cells showed a reduction of cell growth and viability. The cytotoxic effect of 7-DHC was associated with the increase of Bax levels, the decrease of Bcl-2/Bax ratio, the reduction of mitochondrial membrane potential, the increase of apoptosis inducing factor (AIF) levels, the unchanged caspase-3 activity, and uncleavage of PARP-1. These findings could explain the mechanism through which 7-DHC exerts its cytotoxic effect. The results of this study show that the 7-DHC has a potential pro-apoptotic on melanoma cell lines, shed light on the possible mechanisms through which this molecule exerts its cytotoxic effects and, at same time, may give new insights in the therapeutic perspective of cancer.

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