Topotecan triggers apoptosis in p53-deficient cells by forcing degradation of XIAP and survivin thereby activating caspase-3-mediated Bid cleavage.
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-x(L) 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-alpha, 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.
Tomicic
Maja T
MT
Department of Toxicology, University Medical Center of the Johannes Gutenberg University of Mainz, Mainz, Germany.
Christmann
Markus
M
Kaina
Bernd
B
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053477:Apoptotic Protease-Activating Factor 1 / Q000235:genetics; D051023:BH3 Interacting Domain Death Agonist Protein / Q000037:antagonists & inhibitors*; D015153:Blotting, Western; D053143:Caspase 2 / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism*; D045744:Cell Line, Tumor; D015536:Down-Regulation; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D006657:Histones / Q000378:metabolism; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008869:Microtubule-Associated Proteins / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000235:genetics; D012097:Repressor Proteins; D000077022:Survivin; D059004:Topoisomerase I Inhibitors; D019772:Topotecan / Q000494:pharmacology*; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000172:deficiency* / Q000235:genetics; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000037:antagonists & inhibitors*
D053477:Apoptotic Protease-Activating Factor 1; D051023:BH3 Interacting Domain Death Agonist Protein; D053143:Caspase 2; D053148:Caspase 3; D006657:Histones; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D000077022:Survivin; D059004:Topoisomerase I Inhibitors; D019772:Topotecan; D016159:Tumor Suppressor Protein p53; D051636:X-Linked Inhibitor of Apoptosis Protein; C506593:Apaf1 protein, mouse; C107489:BIRC5 protein, human; C117749:Birc5 protein, mouse; C475961:gamma-H2AX protein, mouse