ROLE OF ANTIOXIDANTS AND INTRACELLULAR FREE-RADICALS IN RETINAMIDE-INDUCED CELL-DEATH

The cancer chemopreventive synthetic retinoid N-(4-hydroxyphenyl)retin amide (HPR) possesses antiproliferative and apoptotic activity at phar macological doses, In this study we show that addition of antioxidants to HL-60 cells cultured in the presence of 3 mu M HPR, markedly suppr esses the apoptopic effect of the retinoid and significantly prolongs cell survival (48-96 h). We also show, by the use of the oxidation-sen sitive probe 2',7'-dichlorofluorescin diacetate (DCF-DA) and in combin ation with flow cytometric and spectrofluorimetric analysis, that trea tment of cells with 3 mu M HPR results in an immediate and sustained p roduction of intracellular free radicals, most likely hydroperoxides. Interestingly, the formation of these HPR-induced free radicals is eff ectively blocked by the water soluble antioxidants L-ascorbic acid and N-acetyl-L-cysteine. Neither 3-15 mu M N-(4-methoxyphenyl) retinamide (MPR), the structurally similar but biologically inert analog of HPR, nor 3 mu M doses of the retinoids all-trans retinoic acid, 9-cis-reti noic acid, TTNPB and SR11237 induce intracellular free radicals, thus indicating that the specificity of this phenomenon is restricted to HP R, Altogether, we provide the first direct evidence that HPR stimulate s the generation of intracellular free radicals, which appear to have a causative role in the induction of apoptosis in vitro. Our findings raise the possibility that the therapeutic efficacy of HPR may, at lea st in part, depend on these apoptosis-inducing oxidative phenomena.