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Comparative Study
. 2009 Jan 1;77(1):76-85.
doi: 10.1016/j.bcp.2008.09.011. Epub 2008 Sep 17.

VSports在线直播 - The role of GSH efflux in staurosporine-induced apoptosis in colonic epithelial cells

Magdalena L Circu  1 , Sarah Stringer, Carol Ann Rhoads, Mary Pat Moyer, Tak Yee Aw
Affiliations
Comparative Study

The role of GSH efflux in staurosporine-induced apoptosis in colonic epithelial cells

Magdalena L Circu et al. Biochem Pharmacol. .

Abstract

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 VSports手机版. 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. .

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Figures

Figure 1
Figure 1. STP induces HT29 cells apoptosis through cellular GSH efflux
A: HT29 cells were exposed to 2μM STP for various times and apoptosis was determined by DAPI staining as described in Materials and Methods. Results are expressed as mean ± S.E. GSH and GSSG contents in cells and extracellular media were determined at 0-10h after 2μM STP treatment. B: Intracellular GSH or GSSG expressed as nmoles/mg protein are presented as mean ± S.E. for 4 separate experiments. C: GSH or GSSG in extracellular media are expressed as total contents in nmoles and presented as mean ± S.E. for 4 separate experiments. *p< 0.05 versus 0h control.
Figure 2
Figure 2. Inhibitors of cellular GSH efflux attenuates STP-induced apoptosis
HT29 cells were treated for 8h with 2μM STP without or with 30min pre-treatment with 10mM of each of the GSH transport inhibitors, γ-glutamyl glutamate (γGG) or ophthalmic acid (OPA). Total GSH in the extracellular media and cell apoptosis were determined at 8h and 24h, respectively, post STP treatment. A: Percent GSH efflux. B: Percent cell apoptosis. Results are mean ± S.E. for 4 separate experiments * p< 0.05 versus untreated control; # p< 0.05 versus STP alone. C: HT29 cells were treated with 2μM STP and cell apoptosis was determined at 24h. In some experiments, γGG (10mM) were added either at 30min before or at 8h post-STP treatment. Results are mean ± S.E. * p< 0.05 versus untreated control; # p< 0.05 versus STP alone, ° p< 0.05 versus 30min pretreatment with γ-GG before STP exposure.
Figure 3
Figure 3. Inhibitor of cellular GSH synthesis attenuates STP-induced GSH efflux and HT29 cell apoptosis
HT29 cells were treated with 2μM STP without or with 24h pre-treatment with 2mM BSO. Cellular GSH and total GSH in the extracellular media were determined at 8h, and cell apoptosis at 24h post STP treatment. A: Cellular GSH, expressed as nmol/mg protein. B: Total GSH in the extracellular media, expressed as nmoles. C: Percent HT29 cell apoptosis. Data are expressed as mean ± S.E. for 4 separate experiments. * p< 0.05 versus untreated control; # p< 0.05 versus STP alone. 24h pretreatment of cells with BSO alone decreased cellular GSH by 10-fold (panel A).
Figure 4
Figure 4. Effect of acivicin on time course of loss of cellular GSH and GSSG and their accumulation in extracellular media
HT29 cells were pretreated for 30min with 0.25mM acivicin (Av) followed by exposure to 2μM STP. Cellular and extracellular contents of GSH and GSSG were determined at specified times between 0-8h, and are expressed as nmoles/mg protein and nmoles, respectively. Results are mean ± S.E. for 3 separate experiments. * p< 0.05 versus corresponding times with STP alone.
Figure 5
Figure 5. Effect of acivicin and γ-glutamyl glutamate on loss of cellular GSH and GSSG and their accumulation in extracellular media
HT29 cells were pretreated with either acivicin (Av, 0.25 mM), γ-glutamyl glutamate (γGG, 10 mM), or combined Av and γGG before STP addition as described in Materials and Methods. Cellular and extracellular contents of GSH and GSSG were determined at 8h, and are expressed as nmoles/mg protein and nmoles, respectively. Results are mean ± S.E. for 3 separate experiments. * p< 0.05 versus untreated control; # p< 0.05 versus STP alone.
Figure 6
Figure 6. STP induces caspase-3 activation that is independent of caspase-8 or caspase-9
HT29 cells were incubated with 2μM STP for 0-8h, and total protein extracts were prepared. A: Top panel, western blot analysis of procaspase-3 (CPP32) expression; Middle panel, caspase-3 activity as determined by a fluorometric assay (see Materials and Methods). The results at each time point represent all data from two different assays performed in duplicates; Bottom panel, western blot analyses of caspases-8 and -9 expression. The membranes of each immunoblot were stripped and reprobed for β-actin to verify equal protein loading in each lane. One representative of 3 western blots is shown for each procaspase. B: HT29 cells were incubated with 2μM STP without or with 30min pretreatment with 10μM of pan caspase inhibitors of caspases-8 (Cas-8I, IETD-CHO) and -9 (Cas-9I, LEHD-CHO). Apoptosis was determined at 24h by DAPI staining. Results are mean ± S.E. for 4 separate experiments * p< 0.05 versus untreated control.
Figure 7
Figure 7. STP-induced NCM460 cell apoptosis: Association with GSH efflux and caspase-3 activation
NCM460 cells were exposed to 1μM STP without or with pretreatment (30min) with 10mM γ-glutamyl glutamate (γGG). Apoptosis at 24h (A) and extracellular GSH contents at 6h (C) were determined. In other experiments, NCM460 cells were treated with 1μM STP, and at designated times between 0-6h, aliquots of extracellular media were sampled for determination of kinetics of GSH efflux (B). Cellular GSH and total extracellular GSH are expressed as expressed as nmol/mg protein and nmoles, respectively. Results are mean ± S.E for 4 separate experiments. *p< 0.05 versus untreated control; #p< 0.05 versus STP alone. D: NCM460 cells were incubated with 1μM STP for 0-8h, and total protein extracts were prepared for western blot analyses. Left panels, expression of procaspase-3 (CPP-32, Top) and cleaved caspase-3 (19 and 17 kDa bands, Bottom). Right panel, expression of procaspase-8 and procaspase-9. The membranes of each immunoblot were stripped and reprobed for β-actin to verify equal protein loading in each lane. One representative of 3 western blots is shown for each procaspase.
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