Ethyl 4-hydroxybenzoate toxicity towards human SK-MEL-28 melanoma cell line involves mitochondrial toxicity, intracellular GSH depletion, quinone and reactive oxygen species formation

3968 In search of a novel drug for melanoma therapy, we set to investigate the biochemical basis of ethyl 4-hydroxybenzoate (4-EHB) toxicity against human SK-MEL-28 melanoma cells. 4-EHB was investigated for its metabolism by tyrosinase, an abundant enzyme found in melanoma, and rat liver microsomal P450 enzymes. The extent of GSH depletion was used as a surrogate marker for 4-EHB metabolism by these enzymes. A number of biochemical modulators were used to investigate the mechanism of the 4-EHB toxicity towards human SK-MEL-28 melanoma cells using thiazolyl blue tetrazolium bromide (MTT) assay. The reactive oxygen species (ROS) formation was measured using 2’,7’-dichlorofluorescein diacetate assay. The intracellular GSH was investigated. 4-EHB was metabolized 60% by tyrosinase/O2 at 2 h. The extent of 4-EHB metabolism by CYP2E1 induced rat liver microsomes/NADPH/O2 was 26% at 2h. 4-Hydroxyanisole (4-HA), an analogue of 4-EHB, was metabolized by tyrosinase/O2 and microsomes/NADPH/O2 for 100% and 88%, respectively. The LD50s (day 2) of 4-EHB and 4-HA towards SK-MEL-28 human melanoma cells were 75 and 50 μM, respectively. It was found that dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH reactant and intracellular GSH depletory agent, increased 4-EHB toxicity towards SK-MEL-28 melanoma cells indicating that quinone formation played an important role in 4-EHB’s mechanism of toxicity towards melanoma cells. Cyclosporin A, an inhibitor of mitochondrial permeability transition pore (MPTP) protein, was significantly potent in inhibiting 4-EHB toxicity towards melanoma cells. In addition, trifluoperazine was equally effective at inhibiting MPTP opening in melanoma cells. 4-EHB also led to ROS formation when it was incubated with melanoma cells. Dicoumarol and 1-bromoheptane increased the extent of ROS formation when co-incubated with 4-EHB whereas trifluperazine and cyclosporin A significantly inhibited the ROS formation by 4-EHB. We also showed that 4-EHB (75 μM) significantly depleted the intracellular GSH without a significant change in viability after 2 h incubation, indicating that the GSH depletion precedes 4-EHB cytotoxicity. In summary, the mechanisms of 4-EHB toxicity towards melanoma cells were found to be due to quinone reactive intermediate formation, ROS formation, intracellular GSH depletion and induced mitochondrial toxicity.