The proteasome inhibitor, bortezomib (PS-341/ Velcade) is a dipeptidyl boronic acid that has recently been approved for use in patients with refractory multiple myeloma. Bortezomib (BZ) inhibits the activity of the 26S proteasome and induces cell death in a variety of tumor cells, however the mechanism of cytotoxicity is not well understood. Gene expression profiling suggested an endoplasmic reticulum (ER)-stress related mechanism in BZ-induced cell death. The ER is the major organelle involved in intracellular Ca2+ homeostasis and signaling, including Ca2+ signals that mediate apoptosis. In this study we utilized fluorescent imaging and Ca2+ modulating agents to examine the effects of BZ exposure on Ca2+ homeostasis in myeloma cells. Additionally, we studied the effects of pharmacological manipulation of Ca2+ fluxes on BZ cytotoxicity. Acute exposure to BZ elicited a rapid and transient increase in cytosolic Ca2+ that quickly recovered to baseline. To identify the Ca2+ homeostatic mechanisms affected by BZ, a standard protocol was used in which Ca2+ was removed from the media and plasma membrane efflux pathways were selectively blocked. Following depletion of intracellular Ca2+ stores, Ca2+ was added back and the capacitative calcium influx (CCI) analyzed. Both the magnitude and the rate of CCI were significantly increased in BZ treated cells compared to controls, suggesting that BZ alters either the number of store operated Ca2+ channels, or the length of time such channels remain open following store depletion. Co-treatment of myeloma cells with the mitochondrial uniporter inhibitor Ru360 substantially decreased the rate and magnitude of CCI in both control and treated cells. However, the peak [Ca2+]i attained in cells treated with both BZ and Ru360 were significantly less than that seen with BZ alone, suggesting that a [Ca2+]i threshold may be the critical determinant of BZ induced cell death. To further investigate the relationship between BZ mediated apoptosis and CCI, we treated 8226 myeloma cells with BZ and specific channel blockers. Treatment of myeloma cells with the uniporter inhibitors Ru360 or ruthenium red prevented caspase activation, and almost entirely inhibited BZ induced cell death. Inhibition of non-selective cation channels (including store operated channels) using SKF96365 reduced BZ-mediated cell death, although to a lesser degree than inhibition with ruthenium containing agents. The voltage-gated channel inhibitor, Nifedipine, had no significant effect, nor did additional Ca2+ modulating agents including 2-APB, dantrolene, BAPTA-AM, or Cyclosporine A. These data suggest that disregulation of Ca2+ homeostasis by altered CCI channel activity may play a key role in BZ cytotoxicity. Further definition of the mediators in this unique apoptotic pathway will promote the design of strategies to enhance drug activity, reduce toxicity and overcome drug resistance.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]