Abstract
Susceptibility to apoptosis is an essential prerequisite for successful eradication of tumor cells by chemotherapy. Consequently, resistance to apoptosis has been established as one of the mechanisms responsible for the failure of therapeutic approaches in many types of cancers. In the present study, we investigated the susceptibility of human lung cancer H460 cells to apoptotic cell death induced by cisplatin and determined its regulatory mechanisms. Treatment of the cells with cisplatin induced rapid generation of multiple oxidative species and a concomitant increase in apoptotic cell death. Apoptosis induced by cisplatin was mediated through the mitochondrial death pathway, which requires caspase-9 activation and is regulated by Bcl-2. Cisplatin induced down-regulation of Bcl-2 through a process that involves dephosphorylation and ubiquitination of the protein, which facilitates its degradation by proteasome. This down-regulation was inhibited by antioxidant enzymes catalase and glutathione peroxidase (H2O2 scavenger), but not by superoxide dismutase ( scavenger) or deferoxamine (OH· inhibitor). Electron spin resonance and flow cytometric analyses showed the formation of H2O2 along with and OH· radicals after cisplatin treatment. H2O2 was generated in part by dismutation of and served as a precursor for OH·. Together, our results indicate an essential role of H2O2 in the regulation of Bcl-2 and apoptotic cell death induced by cisplatin. Because aberrant expression of Bcl-2 has been associated with death resistance of cancer cells to chemotherapy, the results of this study could be used to aid the design of more effective strategies for cancer treatment.
Footnotes
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This work was supported by the National Institutes of Health grants HL071545 and HL076340 (to Y.R.).
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ABBREVIATIONS: cisplatin, cis-diamminedichloroplatinum; ROS, reactive oxygen species; ESR, electron spin resonance; GPx, glutathione peroxidase; CAT, catalase; SOD, superoxide dismutase; DMPO, 5,5-dimethyl-1-pyrroline-N-oxide; DCFH-DA, 2′,7′-dichlorodihydro-fluorescein diacetate; DHE, dihydroethidine; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate; ELISA, enzyme-linked immunosorbent assay; AMC, amino-4-methyl coumarin; z-LEHD-fmk, N-benzyloxycarbonyl-Leu-Glu-His-Asp-fluoromethyl ketone; z-IETD-fmk, N-benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethyl ketone.
- Received August 13, 2007.
- Accepted October 2, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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