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.
Highlights
Mantle cell lymphoma (MCL) is a fatal B-cell nonHodgkin’s malignancy with a tendency to disseminate throughout the body, including lymphoid tissues, bone marrow and peripheral blood there by offering a broad spectrum of clinical, pathological, and biological significance
MCL cell lines treated with B-Procaspase-activating compound-1 (PAC-1) for 24 hr in a dose-dependent manner and their respective IC50 values were determined as ~9.0, ~5.0 or ~7.0 μM for Granta-519, Jeko-1 and Mino cells, respectively (Figure 1A)
Co-incubation of B-PAC-1 with Zn rescued cell death in these cells entailing that Zn plays a role in B-PAC-1-induced programmed cell death (PCD) (Figure 1B)
Summary
Mantle cell lymphoma (MCL) is a fatal B-cell nonHodgkin’s malignancy with a tendency to disseminate throughout the body, including lymphoid tissues, bone marrow and peripheral blood there by offering a broad spectrum of clinical, pathological, and biological significance. Cyclin D1 over-expression owing to reciprocal translocation event t(11;14)(q13;q32) [1], accompanied by constitutively activated PI3K/AKT/ mTOR network, aberrant WNT, Hedgehog and NFκB pathways are the major forces that drive MCL cell proliferation and survival [2,3,4,5]. Targeting cancer cells expressing higher level of pro-casp over normal cells would be advantageous This strategy of direct caspase activation and exterminating upstream regulators of PCD that otherwise help MCL cells to acquire a strong survival advantage is becoming a powerful tool in recent years
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