The Combination of Casticin and Vemurafenib Enhances Apoptotic Cell Death Through the Mitochondria- and Caspases-Mediated Pathways in Human Melanoma A375.S2 Cells In Vitro

Vemurafenib (PLX4032) targets the most common BRAF mutation, V600E, in human skin melanoma. Casticin, a polymethoxylated flavonoid compound, is extracted from Viticis fructus (a traditional Chinese medicine) and presents many pharmacological activities, in particular, anticancer activities. However, no report shows that both combinations induce cytotoxic effects in human cancer cells. This study is the first report to illustrate the effects of PLX4032, casticin, or casticin combined with PLX4032 on inducing apoptotic cell death in human skin melanoma A375.S2 cells. Results showed that the treatment of PLX4032 combined with casticin caused cytotoxic activities via suppressing cell viability and inducing DNA condensation and then resulted in cell death in A375.S2 cells. The two-drug combination enhanced the amounts of reactive oxygen species (ROS) and Ca²⁺ release; however, it diminished the level of mitochondrial membrane potential (ΔΨm). Moreover, the levels of caspase-3, caspase-8, caspase-9, and mitochondria-associated proteins (cytochrome c, AIF, and Endo G) were higher in casticin combined with PLX4032 treatment than that in PLX4032 or casticin treatment only by western blotting. The levels of Fas, FasL, FADD, GADD153, and caspase-4 were elevated in the combined treatment. Confocal laser microscopy also confirms that cytochrome c, AIF, and Endo G had higher expression in the combined drug treatments than in single-drug treatment. The casticin combined with PLX4032 treatment decreased cell viability by triggering apoptotic cell death via both mitochondria- and caspase-dependent pathways in human skin melanoma A375.S2 cells.