Tyrosinase activation of acetylsalicylic acid (Aspirin) leads to acetylsalicylic acid selective toxicity towards melanocytic melanoma cell lines

AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA 1287 Purpose: In the current work, we investigated the biochemical toxicity of acetylsalicylic acid (ASA; Aspirin) in human melanoma cell lines using tyrosinase enzyme as a selective molecular cancer therapeutic target. Because tyrosinase is found abundantly and selectively in melanoma cells we hypothesized that the intracellular tyrosinase enzyme can selectively bioactivate ASA, as a prodrug, to quinone intermediate species, which in turn can lead to selective toxicity against melanoma. Experimental procedures: UV-VIS spectra was used to investigate the enzymatic oxidation of ASA by tyrosinase enzyme. GSH depletion was used to measure the extent of ASA metabolism by tyrosinase and rat liver microsomes. The toxicity of ASA was investigated in four melanocytic melanoma cell lines (which express functional tyrosinase), and in four non-melanoma cell lines and one amelanocytic melanoma cell line (which do not express functional tyrosinase) using MTT assay. The biochemical mechanism of ASA was investigated in human SK-MEL-28 melanocytic melanoma cell line, which expresses functional tyrosinase, using a number of modulators. Results: At 2h, ASA was oxidized 44% by tyrosinase. Ascorbic acid and NADH, quinone reducing agents, were significantly depleted during the enzymatic oxidation of ASA by tyrosinase to quinone. The IC50 (48h) of ASA and salicylic acid towards SK-MEL-28 cells were determined to be 100 μM and 5.2 mM, respectively. ASA at 100 μM was selectively toxic towards human SK-MEL-28, MeWo, and SK-MEL-5 and murine B16-F0 melanocytic melanoma cell lines. However, ASA was not significantly toxic towards human C32 amelanocytic melanoma cell line and human BJ, SW-620, Saos and PC-3 non-melanoma cells (which do not express tyrosinase enzyme). Dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depletory agent, increased ASA toxicity towards SK-MEL-28 cells indicating quinone formation and intracellular GSH depletion played important mechanistic roles in ASA induced melanoma toxicity. Ascorbic acid, a quinone reducing agent, and GSH, an antioxidant and quinone trap substrate, prevented ASA cell toxicity. Trifluoperazine and cyclosporine A, inhibitors of permeability transition pore in mitochondria, prevented ASA toxicity. ASA caused time- and dose-dependent decline in intracellular GSH concentration, that preceded cell toxicity. ASA also led to reactive oxygen species (ROS) formation in melanoma cells which was exacerbated by dicoumarol and 1-bromoheptane. Conclusion: Our investigation suggests that ASA as a prodrug was selectively bioactivated by tyrosinase to reactive quinones, and that quinone species, intracellular GSH depletion, ROS formation and mitochondrial toxicity significantly contributed towards ASA selective toxicity in melanoma cells.