title: Bid and calpains cooperate to trigger oxaliplatin-induced apoptosis of cervical carcinoma HeLa cells.
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.
authors.affiliation: Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland.
authors.forename: Sergio
authors.initials: S
authors.lastname: Anguissola
authors.forename: Barbara
authors.initials: B
authors.lastname: Köhler
authors.forename: Robert
authors.initials: R
authors.lastname: O'Byrne
authors.forename: Heiko
authors.initials: H
authors.lastname: Düssmann
authors.forename: Mary D
authors.initials: MD
authors.lastname: Cannon
authors.forename: Frank E
authors.initials: FE
authors.lastname: Murray
authors.forename: Caoimhin G
authors.initials: CG
authors.lastname: Concannon
authors.forename: Markus
authors.initials: M
authors.lastname: Rehm
authors.forename: Donat
authors.initials: D
authors.lastname: Kögel
authors.forename: Jochen H M
authors.initials: JH
authors.lastname: Prehn
mesh_terms: D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D051023:BH3 Interacting Domain Death Agonist Protein / Q000037:antagonists & inhibitors / Q000502:physiology*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000502:physiology*; D005260:Female; D055785:Gene Knockdown Techniques / Q000379:methods; D045325:HCT116 Cells; D006367:HeLa Cells; D006801:Humans; D009944:Organoplatinum Compounds / Q000494:pharmacology* / Q000627:therapeutic use; D000077150:Oxaliplatin; D002583:Uterine Cervical Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology
chemical_list: D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D009944:Organoplatinum Compounds; D000077150:Oxaliplatin; D002154:Calpain