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¹ Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina and ² Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia

Requests for reprints: Andrew S. Kraft, Director Hollings Cancer Center, Medical University of South Carolina, 86 Jonathan Lucas Street, Charleston, SC 29425. E-mail: kraft{at}musc.edu

Previously, we showed that the proteasome inhibitor bortezomib/Velcade (formerly PS-341) synergizes with the protein tumor necrosis factor –related apoptosis-inducing ligand (TRAIL), a ligand for certain death receptors, to induce apoptosis in cell lines derived from prostate and colon cancers. Because apoptosis is often triggered by BH3-only proteins of the Bcl-2 family, we have explored the hypothesis that bortezomib contributes to the apoptosis by up-regulating their levels. Indeed, bortezomib induced increases of Bik and/or Bim in multiple cell lines but not notably of two other BH3-only proteins (Puma and Bid) nor other family members (Bax, Bak, Bcl-2, and Bcl-x_L). The increase in Bik levels seems to reflect inhibition by bortezomib of its proteasome-mediated degradation. Importantly, both Bik and Bim seem central to the proapoptotic function of bortezomib, because mouse embryo fibroblasts in which the genes for both Bik and Bim had been disrupted were refractory to its cytotoxic action. Similarly, the synergy between bortezomib and TRAIL in killing human prostate cancer cells was impaired in cells in which both Bik and Bim were down-regulated by RNA interference. Further evidence that bortezomib acts through the mitochondrial pathway regulated by the Bcl-2 family is that deficiency for APAF-1, which acts downstream of Bcl-2, also blocked its apoptotic effect. These results implicate BH3-only proteins, in particular both Bik and Bim, as important mediators of the antitumor action of bortezomib and establish their role in its enhancement of TRAIL-induced apoptosis.

Key Words: Bortezomib • Bim • Bik • TRAIL • RNAi

Grant support: Department of Defense grant DAMD 17-01-0045 (A.S. Kraft), NIH grant CA10471 (A.S. Kraft), Leukemia and Lymphoma Society Specialized Center of Research grant (J.M. Adams), and National Health and Medical Research Council program grant (J.M. Adams).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: M. Nikrad, T. Johnson, and A.S. Kraft contributed equally to this work. M. Nikrad is currently at the Department of Molecular and Cellular Development, University of Colorado, Campus Box 347, Boulder, CO, 80309-0347. T. Johnson is currently at the Department of Surgery, University of Colorado Health Sciences Center, Denver, CO 80262.

³ P. Bouillet et al., unpublished results.

⁴ L. Coultas et al., unpublished results.

Received 9/24/04; revised 11/30/04; accepted 1/ 5/05.