Multiple myeloma (MM) is an incurable B cell malignancy, characterized by accumulation of plasma cells with low proliferative capacity and defective apoptotic mechanisms. A mean life expectancy of 3-5 years, even with existing intensive chemotherapy, highlights the need for development of novel therapies. Such agents should ideally selectively induce malignant cell death via mechanisms distinct from existing chemotherapies. Activation of alternative death pathways is a prerequisite for malignancies displaying resistance to conventional therapies. Development of novel therapeutic approaches also provides tools to dissect the mechanisms of MM pathogenesis.
PBOX compounds are novel agents, which we have developed, that display anti-cancer activity against a variety of malignant cell types. Importantly, PBOX compounds display minimal toxicity against normal blood and bone marrow cells. We have recently identified tubulin as a molecular target of pro-apoptotic PBOX compounds. However, the mechanism(s) by which these agents induce cell death are incompletely defined. Tubulin-interacting agents are widely used in the treatment of cancer. Specifically, the microtubule destabilizing agent vincristine, is used in initial treatment of MM. Here, we investigate the molecular events involved in PBOX-induced apoptosis in MM cells.
Two MM cell lines (H929 and U266) were tested for sensitivity to PBOX-15 using an MTT proliferation and cytotoxicity assay. PBOX-15 arrested both cell lines in the G2/M phase of the cell cycle, however only H929 cells underwent apoptosis as assessed by DNA laddering and AnnexinV/propidium iodide staining. After 24hrs, PBOX-15 induced ~40% apoptosis, comparable to that induced by dexamethasone or vincristine. IL-6, an in vivo MM growth and survival factor, afforded the cells no protection against PBOX-15-induced apoptosis. PBOX-15-induced apoptosis was found to be caspase-dependent, with complete inhibition of apoptosis observed in cells pre-treated with a caspase-8 inhibitor. Time-dependent activation of caspase-8 confirmed its relevance in H929 apoptosis. Depolarization of the mitochondrial membrane was detected after 2hrs of treatment with PBOX-15, indicating a role for the intrinsic apoptotic pathway. However, caspase-8 inhibition did not prevent membrane depolarization. A decrease in expression of pro-apoptotic Bim was detected in PBOX-15-treated cells after 6hrs, and preceded downregulation of anti-apoptotic Mcl-1 and Bcl-2, and pro-apoptotic Bax and Bid.
This study highlights PBOX-15 as a potential novel anti-myeloma agent, and identifies downregulation of Bim as a key early event in PBOX-induced apoptosis. Ongoing investigations in primary cells and animal models are assessing PBOX compounds as novel therapies for MM and other B-cell malignancies.
1. Mulligan JM et al., (2006) Molecular Pharmacology 70(1):60-70
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA