The role of GSH efflux in staurosporine-induced apoptosis in colonic epithelial cells.
Staurosporine (STP) was shown to induce cell apoptosis through formation of reactive oxygen species, but a role for cellular redox has not been defined. In this study, we report that STP (2 microM) caused apoptosis (24+/-3% at 24 h) of human colon adenocarcinoma epithelial cell line HT29 that was preceded by significant glutathione (GSH) and glutathione disulfide (GSSG) efflux (6 h), but independent of changes in cellular glutathione/glutathione disulfide (GSH/GSSG) redox status. The blockade of GSH efflux by gamma-glutamyl glutamate (gamma-GG) or ophthalmic acid was associated with apoptosis attenuation; however, gamma-GG administration after peak GSH efflux (8 h) did not confer cytoprotection. Moreover, lowering cellular GSH through inhibition of its synthesis prevented extracellular GSH accumulation and cell apoptosis, thus validating a link between cellular GSH export and the trigger of cell apoptosis. Inhibition of gamma-glutamyl transferase (GGT1, EC 2.3.2.2)-catalyzed extracellular GSH degradation with acivicin significantly blocked GSH efflux, suggesting that GSH breakdown is a driving force for GSH export. Interestingly, acivicin treatment enhanced extracellular GSSG accumulation, consistent with GSH oxidation. STP-induced HT29 cell apoptosis was associated with caspase-3 activation independent of caspase-8 or caspase-9 activity; accordingly, inhibitors of the latter caspases were without effect on STP-induced apoptosis. STP similarly induced GSH efflux and apoptosis in a non-malignant human NCM460 colonic cell line in association with caspase-3 activation. Collectively, our results demonstrate that STP induction of apoptosis in malignant and non-malignant colonic cells is temporally linked to the export of cellular GSH and the activation of caspase-3 without caspase-8 or -9 involvement.
Department of Molecular & Cellular Physiology, Louisiana State University Health Science Center, 1501 Kings Highway, Shreveport, LA 71130-3932, United States.
Magdalena L
ML
Circu
Sarah
S
Stringer
Carol Ann
CA
Rhoads
Mary Pat
MP
Moyer
Tak Yee
TY
Aw
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D003106:Colon / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D005978:Glutathione / Q000378:metabolism* / Q000502:physiology; D019073:HT29 Cells; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010084:Oxidation-Reduction; D019311:Staurosporine / Q000494:pharmacology*
D005978:Glutathione; D019311:Staurosporine