Abstract 3347: L14R8, a Pac derivative, induces casapase-3 mediated apoptosis in mantle cell lymphoma.

Abstract

Abstract The resistance of cancer cells to natural apoptotic signals is pivotal for their survival and is typically ruled by either up- or down-regulation or mutations of key proteins in the apoptotic cascade. Mantle cell lymphoma (MCL) is an incurable B-cell non-Hodgkin's malignancy and expresses high levels of anti-apoptotic proteins resulting in survival advantage. Given the potential role(s) of pro-apoptotic compounds for treating cancer, unremitting efforts are in use to develop therapeutics that target specific proteins in the apoptotic cascade. Activation of zymogen procaspase-3 to caspase-3 is the hallmark feature of both extrinsic and intrinsic cell death pathways. MCL cell lines, Granta-519, JeKo and Mino possess high levels of procaspase-3 thereby providing a rational approach for using agents that can activate procaspase-3. L14R8 is a novel and potent Procaspase Activating Compound (Pac) analogue that forms a stable complex with Zn2+ ion. It directly activates caspase-3 via chelating Zn2+ thereby removes Zn2+-mediated inhibition of caspase-3 activation. In this study, three MCL cells lines, Granta-519, JeKo and Mino were treated for 24 hr with different concentrations of L14R8 and their respective IC50 values were determined (8, 9 and 11 μM) by Annexin V-PI assay. To determine mechanism of cell death, these cell lines were treated with L14R8 (10 μM), Pac1a (10 μM; an inactive analogue) or staurosporin (100 nM; used as a positive control) for 24 hr either alone or in presence of Zn2+ (100 μM; ZnSO4) and cell death was evaluated by FACS analysis. L14R8 treatment resulted in significant cell death (p<0.05) in all three MCL cell lines tested compared with either DMSO or only ZnSO4 treated cells. Addition of Zn2+ completely inhibited L14R8-mediated cell death but did not change staurosporin-induced apoptosis. Western blot analysis from JeKo and Mino cells revealed a strong procaspase-3 to caspase-3 (p17 and p12 fragments) cleavage accompanied by loss of both Mcl-1 and XIAP proteins. This was consistent with PARP cleavage data. Confocal analysis using cleaved caspase-3 antibody reaffirmed L14R8 role on caspase-3 cleavage. To extend cell line data to primary human tumor cells, malignant B-lymphoma cells were obtained from five patients. A 24 hr treatment with 10 μM L14R8 reaffirmed an evocative role on cell death (26% vehicle control versus 61% L14R8 treated; p<0.02). Consistent with the cell line data, a few patient samples also showed a decrease in Mcl-1 and XIAP proteins suggesting a clinical significance of exploiting L14R8 in MCL. Based on these results, we conclude that L14R8 induces cell death via activating procaspase-3 cleavage accompanied by loss of anti-apoptotic proteins such as Mcl-1 and XIAP. Further evidence will be provided on MEF cells deficient in Caspse-3, -7 or both to explicate the mechanism of L14R8 mediated cell death. Citation Format: Aloke Sarker, Jefferson Chen, Sattva S. Neelapu, Kumudha Balakrishnan, Varsha Gandhi. L14R8, a Pac derivative, induces casapase-3 mediated apoptosis in mantle cell lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3347. doi:10.1158/1538-7445.AM2013-3347