Abstract
The topoisomerase I inhibitor topotecan (TPT) is used in the therapy of different tumors including high-grade gliomas. We previously showed that TPT-induced apoptosis depends on p53 with p53 wild-type (wt) cells being more resistant because of p53-controlled degradation of topoisomerase I. Here, we show that p53-deficient (p53−/−) fibroblasts undergo excessive mitochondrial apoptosis featuring H2AX phosphorylation, Bcl-xL decline, cytochrome c release, caspase-9/-3/-2 activation, and cleavage of Bid. In wt and apaf-1−/− cells, caspase-2 did not become activated and Bid was not cleaved. In addition, p53−/− cells cotreated with TPT and caspase-3 inhibitor showed neither caspase-2 activation nor Bid cleavage, implying that caspase-2 is processed downstream of the apoptosome by caspase-3. Although processing of caspase-9/-3 was similar in wt and p53−/− cells, only p53−/− cells displayed active caspase-3. This was due to the proteasomal degradation of X-chromosome-linked inhibitor of apoptosis (XIAP) and survivin that inhibits caspase-3 activity. Accordingly, TPT-induced apoptosis in wt cells was increased after XIAP/survivin knockdown. Silencing of Bid led to reduction of TPT-triggered apoptosis. Data obtained with mouse fibroblasts could be extended to human glioma cells. In U87MG (p53wt) cells cotreated with TPT and pifithrin-α, or transfected with p53-siRNA, caspase-2 and Bid were significantly cleaved and XIAP/survivin was degraded. Furthermore, the knockdown of XIAP and survivin led to increased TPT-triggered apoptosis. Overall, the data show that p53-deficient/depleted cells are hypersensitive to TPT because they down-regulate XIAP and survivin, and thus amplify the intrinsic apoptotic pathway via caspase-3-mediated Bid cleavage. Therefore, in gliomas harboring wild-type p53, TPT-based therapy might be improved by targeted down-regulation of XIAP and survivin.
Footnotes
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This work was supported by the German Research Foundation [Grants DFG KA724, DFG CH665/2-1].
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.109.159962
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The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- TPT
- topotecan
- IAP
- inhibitor of apoptosis protein
- DMEM
- Dulbecco's minimal essential medium
- DSB
- double-strand break
- FBS
- fetal bovine serum
- mAb
- monoclonal antibody
- MEF
- mouse embryonic fibroblast
- pAb
- polyclonal antibody
- PIDD
- p53-induced protein with a death domain
- PI
- propidium iodide
- Pth
- pifithrin-α
- siRNA
- small interfering RNA
- topoI
- topoisomerase I
- XIAP
- X-chromosome linked inhibitor of apoptosis
- zVAD-fmk
- benzyloxy-carbonyl-Val-Ala-Asp-fluoromethylketone
- zDEVD-fmk
- benzyloxy-carbonyl-Asp-Glu-Val-Asp-fluoromethylketone
- zLEHD-fmk
- benzyloxy-carbonyl-Leu-Glu-His-Asp-fluoromethylketone.
- Received August 5, 2009.
- Accepted October 6, 2009.
- © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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