title: Molecular evidence of Zn chelation of the procaspase activating compound B-PAC-1 in B cell lymphoma.
abstract: The resistance of apoptosis in cancer cells is pivotal for their survival and is typically ruled by mutations or dysregulation of core apoptotic cascade. Mantle cell lymphoma (MCL) is a non-Hodgkin's B-cell malignancy expressing higher anti-apoptotic proteins providing survival advantage. B-PAC-1, a procaspase activating compound, induces apoptosis by sequestering Zn bound to procaspase-3, but the amino acids holding Zn in Caspase-3 is not known. Here we show that reintroduction of WT caspase-3 or 7 in Caspase3-7 double knock-out (DKO) mouse embryonic fibroblasts (MEF) promoted B-PAC-1 to induce apoptosis (27-43%), but not in DKO MEFs or MEFs expressing respective Casp3-7 catalytic mutants (12-13%). Using caspase-6 and -9 exosite analysis, we identified and mutated predicted Zn-ligands in caspase-3 (H108A, C148S and E272A) and overexpressed into DKO MEFs. Mutants carrying E272A abrogated Zn-reversal of apoptosis induced by B-PAC-1 via higher XIAP and smac expressions but not in H108A or C148S mutants. Co-immunoprecipitation analysis revealed stronger XIAP-caspase-3 interaction suggesting a novel mechanism of impulsive apoptosis resistance by disrupting predicted Zn-ligands in caspase-3. B-PAC-1 sponsored apoptosis in MCL cell lines (30-73%) via caspase-3 and PARP cleavages accompanied by loss of Mcl-1 and IAPs including XIAP while Zn substantially abrogated B-PAC-1-driven apoptosis (18-36%). In contrary, Zn is dispensable to inhibit staurosporin, bendamustine, ABT199 or MK206-induced apoptosis. Consistent to cell lines, B-PAC-1 stimulated cell death in primary B-lymphoma cells via caspase-3 cleavage with decline in both Mcl-1 and XIAP. This study underscores the first genetic evidence that B-PAC-1 driven apoptosis is mediated via Zn chelation.
authors.affiliation: Department of Experimental Therapeutics, The University of Texas Health Science Center, Houston, Texas, USA.
authors.forename: Aloke
authors.initials: A
authors.lastname: Sarkar
authors.affiliation: Department of Experimental Therapeutics, The University of Texas Health Science Center, Houston, Texas, USA.|Department of Leukemia, UT MD Anderson Cancer Center, Houston, Texas, USA.|Graduate School of Biomedical Sciences, The University of Texas Health Science Center, Houston, Texas, USA.
authors.forename: Kumudha
authors.initials: K
authors.lastname: Balakrishnan
authors.affiliation: Department of Experimental Therapeutics, The University of Texas Health Science Center, Houston, Texas, USA.
authors.forename: Jefferson
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Experimental Therapeutics, The University of Texas Health Science Center, Houston, Texas, USA.
authors.forename: Viralkumar
authors.initials: V
authors.lastname: Patel
authors.affiliation: Department of Lymphoma and Myeloma, The University of Texas Health Science Center, Houston, Texas, USA.
authors.forename: Sattva S
authors.initials: SS
authors.lastname: Neelapu
authors.affiliation: Department of Experimental Therapeutics, The University of Texas Health Science Center, Houston, Texas, USA.|Graduate School of Biomedical Sciences, The University of Texas Health Science Center, Houston, Texas, USA.
authors.forename: John S
authors.initials: JS
authors.lastname: McMurray
authors.affiliation: Department of Experimental Therapeutics, The University of Texas Health Science Center, Houston, Texas, USA.|Department of Leukemia, UT MD Anderson Cancer Center, Houston, Texas, USA.|Graduate School of Biomedical Sciences, The University of Texas Health Science Center, Houston, Texas, USA.
authors.forename: Varsha
authors.initials: V
authors.lastname: Gandhi
mesh_terms: D000328:Adult; D000368:Aged; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D020169:Caspases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002614:Chelating Agents / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D005260:Female; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D006835:Hydrazones / Q000494:pharmacology*; D016393:Lymphoma, B-Cell / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D020522:Lymphoma, Mantle-Cell / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D008297:Male; D051379:Mice; D008875:Middle Aged; D009367:Neoplasm Staging; D010879:Piperazines / Q000494:pharmacology*; D011379:Prognosis; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015032:Zinc / Q000378:metabolism*
chemical_list: C000597017:2-(4-(4-(benzyloxy)benzyl)piperazin-1-yl)-N'-(3,5-di-tert-butyl-2-hydroxybenzylidene)acetohydrazide; D002614:Chelating Agents; D006835:Hydrazones; D010879:Piperazines; D012333:RNA, Messenger; D020169:Caspases; D015032:Zinc
keywords: B-PAC-1; MCL; Zn-ligands; lymphoma; pro-caspase-3