Isothiocyanates Enhance the Anti-Melanoma Effect of Zebularine Through Modulation of Apoptosis and Regulation of DNMTs' Expression, Chromatin Configuration and Histone Posttranslational Modifications Associated with Altered Gene Expression Patterns.

In the present study, we aimed to characterize the cytotoxic efficacy of Zebularine either as a single agent or in combination with various isothiocyanates in an in vitro model consisting of human melanoma (A375, Colo-679) as well as non-tumorigenic immortalized keratinocyte (HaCaT) cells. In this model, we have evaluated the anti-melanoma effect of Zebularine (in single and combinatorial protocols) in terms of cell viability, apoptotic induction and alterations in ultrastructural chromatin configuration, protein expression levels of DNA methyltransferases (DNMTs) and associated histone epigenetic marks capable of mediating gene expression. Exposure to Zebularine resulted in dose- and time-dependent cytotoxicity through apoptotic induction in malignant melanoma cells, while neighboring non-tumorigenic keratinocytes remained unaffected. A more profound response was observed in combinational protocols, as evidenced by a further decline in cell viability leading to an even more robust apoptotic induction followed by a differential response (i.e., activation/de-activation) of various apoptotic genes. Furthermore, combined exposure protocols caused a significant decrease of DNMT1, DNMT3A and DNMT3B protein expression levels together with alterations in ultrastructural chromatin configuration and protein expression levels of specific histone modification marks capable of modulating gene expression. Overall, we have developed a novel experimental approach capable of potentiating the cytotoxic efficacy of Zebularine against human malignant melanoma cells while at the same time maintaining a non-cytotoxic profile against neighboring non-tumorigenic keratinocyte (HaCaT) cells.