Isothiocyanates Enhance the Anti-melanoma Effect of Entinostat Through Modulation of Apoptosis and HDACs’ Expression Patterns in an In Vitro Model of Human Malignant Melanoma

Background/Aim: This study aimed to characterize the anti-melanoma efficacy of Entinostat (ENT), either as a single agent or in combination with various isothiocyanates (ITCs). Materials and Methods: We utilized an in vitro model of human malignant melanoma consisting of melanoma (A375, Colo-679) and immortalized non-tumorigenic keratinocyte (HaCaT) cells. Cytotoxicity was determined using the Alamar blue assay while assessment of apoptosis was determined using a multiplex activity assay (measuring levels of activated caspase 3) as well as using real time-quantitative polymerase chain reaction (RT-qPCR) analysis (determining the expression profile of several apoptotic genes). Protein expression levels of major histone deacetylases (HDACs) were determined using western blotting. Results: ENT caused dose- and time-dependent cytotoxicity against melanoma cells, a response mediated by apoptosis, while non-tumorigenic cells were shown to be more resistant. Also, combined treatment caused a further decline in viability and a more significant induction of apoptosis associated with the reactivation of genes implicated in both intrinsic and extrinsic apoptosis. Moreover, combination treatments further decreased the protein levels of HDAC6 and Sirt1 when compared to the effect of ENT alone. Conclusion: We show evidence of an optimized experimental protocol capable of potentiating the anti-melanoma efficacy of ENT while maintaining a non-cytotoxic profile in non-tumorigenic cells.