title: Calpain-mediated Bid cleavage and calpain-independent Bak modulation: two separate pathways in cisplatin-induced apoptosis.
abstract: Calpain is a ubiquitous protease with potential involvement in apoptosis. We report that in human melanoma cells, cisplatin-induced calpain activation occurs early in apoptosis. Calpain activation and subsequent apoptosis were inhibited by calpeptin and PD150606, two calpain inhibitors with different modes of action. Furthermore, cisplatin induced cleavage of the BH3-only protein Bid, yielding a 14-kDa fragment similar to proapoptotic, caspase-cleaved Bid. However, Bid cleavage was inhibited by inhibitors of calpain, but not by inhibitors of caspases or of cathepsin L. Recombinant Bid was cleaved in vitro by both recombinant calpain and by lysates of cisplatin-treated cells. Cleavage was calpeptin sensitive, and the cleavage site was mapped between Gly70 and Arg71. Calpain-cleaved Bid induced cytochrome c release from isolated mitochondria. While calpeptin did not affect cisplatin-induced modulation of Bak to its proapoptotic conformation, a dominant-negative mutant of MEKK1 (dnMEKK) inhibited Bak modulation. dnMEKK did not, however, block Bid cleavage. The combination of dnMEKK and calpeptin had an additive inhibitory effect on apoptosis. In summary, calpain-mediated Bid cleavage is important in drug-induced apoptosis, and cisplatin induces at least two separate apoptotic signaling pathways resulting in Bid cleavage and Bak modulation, respectively.
authors.affiliation: Cancer Center Karolinska, Department of Oncology-Pathology, Karolinska Institute and Hospital, S-171 76 Stockholm, Sweden.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Mandic
authors.forename: Kristina
authors.initials: K
authors.lastname: Viktorsson
authors.forename: Linda
authors.initials: L
authors.lastname: Strandberg
authors.forename: Thomas
authors.initials: T
authors.lastname: Heiden
authors.forename: Johan
authors.initials: J
authors.lastname: Hansson
authors.forename: Stig
authors.initials: S
authors.lastname: Linder
authors.forename: Maria C
authors.initials: MC
authors.lastname: Shoshan
mesh_terms: D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects*; D001120:Arginine / Q000378:metabolism; D051023:BH3 Interacting Domain Death Agonist Protein; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D056668:Cathepsin L; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000378:metabolism; D002945:Cisplatin / Q000494:pharmacology*; D003546:Cysteine Endopeptidases; D003574:Cytochrome c Group / Q000378:metabolism; D004151:Dipeptides / Q000494:pharmacology; D004789:Enzyme Activation; D005998:Glycine / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D015398:Signal Transduction; D013997:Time Factors; D014407:Tumor Cells, Cultured; D050998:bcl-2 Homologous Antagonist-Killer Protein
chemical_list: C494772:BAK1 protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D002352:Carrier Proteins; D003574:Cytochrome c Group; D004151:Dipeptides; D008565:Membrane Proteins; D050998:bcl-2 Homologous Antagonist-Killer Protein; C071482:calpeptin; D001120:Arginine; D002403:Cathepsins; D002154:Calpain; D003546:Cysteine Endopeptidases; C534281:CTSL protein, human; D056668:Cathepsin L; D002945:Cisplatin; D002118:Calcium; D005998:Glycine