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Chemotherapy using platinum drugs has severe cachectic effects, however, their influence on cellular proteolysis is unknown. Because, the ATP-dependent ubiquitin (ub)-proteasome pathway degrades a great number of abnormal and native proteins, we investigated the overall activity of this pathway in cisplatin, carboplatin and oxaliplatin (30 uM, 3 h)-treated human cancer cell lines. The mechanisms by which the Pt drugs may affect the enzymes of ub-pathway were also examined. An assay involving the degradation of 14C-labeled native cellular proteins in the presence of ATP and ub was set up. Extracts from HT-29 cells that had been treated with Pt drugs showed a consistent and time-dependent inhibition (25-35%) of ub- proteolysis; this inhibition was relieved several hours after drug withdrawal. HT-29 cell extracts exhibited a similar pattern of inhibition and regeneration of ub-proteolysis in other in vitro assays using biotinylated ub as the substrate and the detection of ubiquitinated proteins by streptavidin-linked HRP. Further, the levels of ubiquitination of endogenous proteins were consistent with the results of in vitro enzyme assays. Because of the established interaction of cisplatin with GSH and the possible oxidative stress the cells may encounter with these drugs, we examined the likelihood of S-thiolation of proteins, in particular, of the ub-pathway enzymes, which share an SH-dependent catalysis. Glutathionylation is a reversible posttranslational modification comprising disulfide formation between reactive cysteines in proteins and low MW thiols. It is emerging as a central mechanism by which changes in the intracellular redox state may be transduced into a functional cellular response, and may protect the enzymes from irreversible oxidation of SH-groups during nitrosative and oxidative stress. GSH or GSSG-Sepharose chromatography offers a simple means to examine changes in the redox status of proteins (Niture S et al., ABB, 445, 2005, In Press). Consistent with the above rationale, we observed that specific proteins in cisplatin-treated cells became more susceptible for glutathionylation, as reflected by their increased binding with GSH-Sepharose, and their ability to incorporate biotinylated GSH or GSSG. Immunoprecipitation combined with immunoblotting confirmed that proteasome subunits and the ubiquitin-E1 activating enzyme become highly glutathionylated in cisplatin-treated tumor cells. Cisplatin also induced the S-thiolation of transcription factors and other metabolic enzymes. Our findings, for the first time, demonstrate that platinum drugs induce protein S-thiolation, and such a modification of the enzymes in ubiquitin-proteasome pathway is responsible for the reversible modulation of protein degradation. The clinical significance of our observations to chemotherapy remains to be elucidated.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]