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Bid and Calpains Cooperate to Trigger Oxaliplatin-Induced Apoptosis of Cervical Carcinoma HeLa Cells

Sergio Anguissola, Barbara Köhler, Robert O'Byrne, Heiko Düssmann, Mary D. Cannon, Frank E. Murray, Caoimhin G. Concannon, Markus Rehm, Donat Kögel and Jochen H. M. Prehn
Molecular Pharmacology November 2009, 76 (5) 998-1010; DOI: https://doi.org/10.1124/mol.109.058156
Sergio Anguissola
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Barbara Köhler
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Robert O'Byrne
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Heiko Düssmann
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Mary D. Cannon
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Frank E. Murray
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Caoimhin G. Concannon
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Markus Rehm
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Donat Kögel
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Jochen H. M. Prehn
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Abstract

The Bcl-2 homology 3-only protein Bid is an important mediator of death receptor-induced apoptosis. Recent reports and this study suggest that Bid may also mediate genotoxic drug-induced apoptosis of various human cancer cells. Here, we characterized the role of Bid and the mechanism of Bid activation during oxaliplatin-induced apoptosis of HeLa cervical cancer cells. Small hairpin RNA-mediated silencing of Bid protected HeLa cells against both death receptor- and oxaliplatin-induced apoptosis. Expression of a Bid mutant in which caspase-8 cleavage site was mutated (D59A) reactivated oxaliplatin-induced apoptosis in Bid-deficient cells but failed to reactivate death receptor-induced apoptosis, suggesting that caspase-8-mediated Bid cleavage did not contribute to oxaliplatin-induced apoptosis. Overexpression of bcl-2 or treatment with the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone abolished caspase-2, -8, -9, and -3 activation as well as Bid cleavage in response to oxaliplatin, suggesting that Bid cleavage occurred downstream of mitochondrial permeabilization and was predominantly mediated by caspases. We also detected an early activation of calpains in response to oxaliplatin. Calpain inhibition reduced Bid cleavage, mitochondrial depolarization, and activation of caspase-9, -3, -2, and -8 in response to oxaliplatin. Further experiments, however, suggested that Bid cleavage by calpains was not a prerequisite for oxaliplatin-induced apoptosis: single-cell imaging experiments using a yellow fluorescent protein-Bid-cyan fluorescent protein probe demonstrated translocation of full-length Bid to mitochondria that was insensitive to calpain or caspase inhibition. Moreover, calpain inhibition showed a potent protective effect in Bid-silenced cells. In conclusion, our data suggest that calpains and Bid act in a cooperative, but mutually independent, manner to mediate oxaliplatin-induced apoptosis of HeLa cells.

  • BH, Bcl-2 homology
  • TRAIL, tumor necrosis factor-related apoptosis-inducing ligand
  • TNF, tumor necrosis factor
  • Ac-DEVD-AMC, N-acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin
  • zVAD-fmk, N-benzyloxycarbonyl-Val-Ala-Asp(O-methyl)-fluoromethylketone
  • tet, tetracycline
  • FRET, fluorescence resonance energy transfer
  • FITC, fluorescein isothiocyanate
  • TMRM, tetra-methyl-rhodamine-methyl-ester
  • YFP, yellow fluorescent protein
  • CYP, cyan fluorescent protein
  • GFP, green fluorescent protein
  • PAGE, polyacrylamide gel electrophoresis
  • PARP, poly(ADP-ribose) polymerase
  • CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate
  • STS, staurosporine
  • wt, wild type
  • MOMP, mitochondrial outer membrane permeabilization
  • BI-6C9, N-[4-[(4-aminophenyl)thio]phenyl]-4-[[(4-methoxyphenyl)sulfonyl]amino]-butanamide.

Footnotes

  • ↵Embedded Image The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

  • This study was supported by the Science Foundation Ireland [Grants 03/RP1/B344, 08/IN1/1949, 06/UR/B920]; the European Commission Seventh Framework Prorogramme (APO-SYS); the Higher Education Authority [PRTLI Cycle 4, National Biophotonics and Imaging Platform Ireland] (to J.H.M.P.); and the Deutsche Forschungsgemeinschaft [Grants PR 338/9-3, 9-4] (to D.K. and J.H.M.P.). M.D.C. was supported by a Clinician Scientist Fellowship from the Higher Education Authority.

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

    doi:10.1124/mol.109.058156

  • ABBREVIATIONS:

  • S.A. and B.K. contributed equally to this work.

    • Received May 29, 2009.
    • Accepted August 27, 2009.
  • © 2009 The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 76 (5)
Molecular Pharmacology
Vol. 76, Issue 5
November 2009
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Bid and Calpains Cooperate to Trigger Oxaliplatin-Induced Apoptosis of Cervical Carcinoma HeLa Cells

Sergio Anguissola, Barbara Köhler, Robert O'Byrne, Heiko Düssmann, Mary D. Cannon, Frank E. Murray, Caoimhin G. Concannon, Markus Rehm, Donat Kögel and Jochen H. M. Prehn
Molecular Pharmacology November 1, 2009, 76 (5) 998-1010; DOI: https://doi.org/10.1124/mol.109.058156

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Bid and Calpains Cooperate to Trigger Oxaliplatin-Induced Apoptosis of Cervical Carcinoma HeLa Cells

Sergio Anguissola, Barbara Köhler, Robert O'Byrne, Heiko Düssmann, Mary D. Cannon, Frank E. Murray, Caoimhin G. Concannon, Markus Rehm, Donat Kögel and Jochen H. M. Prehn
Molecular Pharmacology November 1, 2009, 76 (5) 998-1010; DOI: https://doi.org/10.1124/mol.109.058156
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