Glutathione depletion-induced apoptosis of Ha-ras-transformed NIH3T3 cells can be prevented by melatonin

It is well known that intracellular antioxidant glutathione (GSH) plays major roles in the maintenance of redox status and defense of oxidative stress. Ras, a small GTP-binding protein, may send growth-stimulating message to the nucleus through downstream Rac oncoprotein and superoxide (O-2(radical anion)). These findings led us to investigate the effects of GSH and melatonin, a free-radical scavenger, on Ras-Rac-O-2(radical anion)-related growth signal transduction. Our results demonstrate that overexpression of the inducible Ha-ras oncogene by isopropyl-beta-D-thiogalactoside (IPTG) increases the levels of reactive oxygen species (ROS, including O-2(radical anion) and hydrogen peroxide (H2O2)) and GSH in an Ha-ras-transformed NIH/3T3 fibroblast cell line. On the contrary, melatonin significantly suppresses ras-triggered cell growth by inhibiting the increase of ROS and GSH. Moreover, severe apoptosis of this transformed cell line occurred when the cell redox balance between ROS and GSH was dramatically changed in the presence of IPTG and L-buthionine-[S,R]-sulfoximine (BSO, a specific inhibitor of GSH synthetase). That BSO-induced cell apoptosis needs Ras to increase the ROS level was demonstrated by the free-radical scavenger melatonin. It effectively blocked cell apoptosis, but cell growth was also slowed without affecting Ras expression. Based on our studies, two approaches can be applied to treating ras-related cancers. One is utilizing melatonin to suppress cancer cell proliferation, and the other is utilizing BSO to induce cancer-cell apoptosis. Cotreatment of ras-related cancer cells with melatonin and BSO stops cell growth as well as apoptosis. Whether these cancer cells will undergo further regression or become recurrent merits investigation.