Bioactivation of luteolin by tyrosinase selectively inhibits glutathione S-transferase.

ABSTRACT

Glutathione S-transferase (GST) plays a significant role in the metabolism and detoxification of drugs used in treatment of melanoma, resulting in a decrease in drug efficacy. Tyrosinase is an abundant enzyme found in melanoma. In this study, we used a tyrosinase targeted approach to selectively inhibit GST. In the presence of tyrosinase, luteolin (10 µM) showed 87% GST inhibition; whereas in the absence of tyrosinase, luteolin led to negligible GST inhibition. With respect to GSH, both luteolin-SG conjugate and luteolin-quinone inhibited ≥90% of GST activity via competitive reversible and irreversible mixed mechanisms with Ki of 0.74 µM and 0.02 µM, respectively. With respect to CDNB, the luteolin-SG conjugate inhibited GST activity via competitive reversible mechanism and competitively with Ki of 0.58 µM, whereas luteolin-quinone showed irreversible mixed inhibition of GST activity with Ki of 0.039 µM. Luteolin (100 µM) inhibited GST in mixed manner with Ki of 53 µM with respect to GSH and non-competitively with respect to CDNB with Ki of 38 µM. Luteolin, at a concentration range of 5-80 µM, exhibited 78-99% GST inhibition in human SK-MEL-28 cell homogenate. Among the 3 species of intact luteolin, luteolin-SG conjugate, and luteoline-quinone, only the latter two have potential as drugs with Ki < 1 µM, which is potentially achievable in-vivo as therapeutic agents. The order of GST inhibition was luteolin-quinone >> luteolin-SG conjugate >>> luteolin. In summary, our results suggest that luteolin was bioactivated by tyrosinase to form a luteolin-quinone and luteolin-glutathione conjugate, which inhibited GST. For the first time, in addition to intracellular GSH depletion, we demonstrate that luteolin acts as a selective inhibitor of GST in the presence of tyrosinase. Such strategy could potentially be used to selectively inhibit GST, a drug detoxifying enzyme, in melanoma cells.