Abstract

The dietary isothiocyanate and cancer chemopreventive agent, phenethyl isothiocyanate, induced apoptosis of human leukaemia HL60 and human myeloblastic leukaemia ML-1 cells in vitro. Cytotoxicity was associated with an initial decrease in GSH and GSSG, with a concomitant formation of the GSH adduct S-( N-phenethylthiocarbamoyl)glutathione inside cells, which was then exported from cells. After 12 hr, the cellular concentration of GSH recovered and then declined after 24 hr. Buthionine sulphoximine prevented the recovery of cellular GSH concentration and potentiated the cytotoxicity of phenethyl isothiocyanate. S-( N-phenethylthiocarbamoyl)glutathione spontaneously fragmented to GSH and phenethyl isothiocyanate, GSH oxidized to GSSG and glutathionyl–protein disulphides, and phenethyl isothiocyanate hydrolyzed to phenylethylamine. GSH and GSSG depletion was more marked in ML-1 cells than in HL60 cells. Studies with [ 14C]-labelled phenethyl isothiocyanate gave evidence of phenethylthiocarbamoylation of cells that maximized after 2–3 hr. This occurred later than the maximum concentration of S-( N-phenethylthiocarbamoyl)glutathione, but coincided with the commitment to apoptosis and cytotoxicity which developed later. The cytotoxicity of phenethyl isothiocyanate was prevented by a high concentration of GSH (15 mM) and delayed by the antioxidant and c-Jun N-terminal kinase signalling pathway inhibitor curcumin. GSH prevented and curcumin partly prevented the decrease in cellular GSH. These studies show that the cysteinyl thiol group of GSH is an important site of thiocarbamoylation by phenethyl isothiocyanate during induction of apoptosis and that this may lead to depletion of cellular GSH by efflux of the GSH conjugate. Thiocarbamoylation also occurred at other sites. The recent demonstration of a critical role for activation of caspase-8 in phenethyl isothiocyanate-induced apoptosis suggests that this thiocarbamoylation directly or indirectly leads to functional activation of a cell death receptor/adaptor protein complex.

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