Oncotarget

Research Papers:

Molecular evidence of Zn chelation of the procaspase activating compound B-PAC-1 in B cell lymphoma

Aloke Sarkar, Kumudha Balakrishnan, Jefferson Chen, Viralkumar Patel, Sattva S. Neelapu, John S. McMurray and Varsha Gandhi _

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Oncotarget. 2016; 7:3461-3476. https://doi.org/10.18632/oncotarget.6505

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Abstract

Aloke Sarkar1, Kumudha Balakrishnan1,3,4, Jefferson Chen1, Viralkumar Patel1, Sattva S. Neelapu2, John S. McMurray1,4, Varsha Gandhi1,3,4

1Department of Experimental Therapeutics, The University of Texas Health Science Center, Houston, Texas, USA

2Department of Lymphoma and Myeloma, The University of Texas Health Science Center, Houston, Texas, USA

3Department of Leukemia, UT MD Anderson Cancer Center, Houston, Texas, USA

4Graduate School of Biomedical Sciences, The University of Texas Health Science Center, Houston, Texas, USA

Correspondence to:

Varsha Gandhi, e-mail: vgandhi@mdanderson.org

Keywords: lymphoma, MCL, pro-caspase-3, Zn-ligands, B-PAC-1

Received: November 13, 2015     Accepted: November 17, 2015     Published: December 07, 2015

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.


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