>>Clinical Cancer Research Vol. 11, 4259-4265, June 1, 2005
Cooperative Cytotoxicity of Proteasome Inhibitors and Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand in Chemoresistant Bcl-2-Overexpressing Cells
Alessio Nencioni1,2, Lucia Wille1, Giovanna Dal Bello2, Davide Boy2, Gabriella Cirmena2, Sebastian Wesselborg3, Claus Belka4, Peter Brossart5, Franco Patrone2 and Alberto Ballestrero2
Authors' Affiliations: 1 Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts; 2 Department of Internal Medicine, University of Genova, Genova, Italy; and Departments of 3 Internal Medicine I, 4 Radiation Oncology, and 5 Hematology, Oncology and Immunology, University of Tubingen, Tubingen, Germany
Requests for reprints: Alessio Nencioni, Department of Internal Medicine, University of Genova, V.le Benedetto XV 6, 16132 Genova, Italy. Phone: 39-10-353-8990; Fax: 39-10-353-8650; E-mail: A.Nencioni@gmx.net.
Purpose: Bcl-2 overexpression is frequently detected in lymphoid malignancies, being associated with poor prognosis and reduced response to therapy. Here, we evaluated whether Bcl-2 overexpression affects the cytotoxic activity of proteasome inhibitors taken alone or in association with conventional anticancer drugs or tumor necrosis factor–related apoptosis-inducing ligand (TRAIL).
Experimental Design: Jurkat cells engineered to overexpress Bcl-2 were treated with proteasome inhibitors (MG132, epoxomicin, and bortezomib), anticancer drugs (etoposide and doxorubicin), TRAIL, or combinations of these compounds. Cell death and loss of mitochondrial transmembrane potential were detected by flow cytometry. Cytosolic relocalization of cytochrome c and SMAC/Diablo, caspase cleavage, and Bcl-2 and Mcl-1 levels were determined by immunoblotting. Nuclear factor-B inhibition was done by retroviral transduction with a dominant-negative mutant of IB.
Results: Bcl-2 overexpression results in significant inhibition of apoptosis in response to proteasome inhibitors, antiblastics, and TRAIL. Addition of TRAIL to proteasome inhibitors results in a synergistic cytotoxic effect in Bcl-2-overexpressing cells, whereas this result is not reproduced by the combination of proteasome inhibitors with antiblastic drugs. Importantly, proteasome inhibitors plus TRAIL induce mitochondrial dysfunction irrespective of up-regulated Bcl-2. Bcl-2 cleavage to a fragment with putative proapoptotic activity and elimination of antiapoptotic Mcl-1 may both play a role in proteasome inhibitors-TRAIL cooperation. Conversely, nuclear factor-B inhibition by proteasome inhibitors is per se insufficient to explain the observed synergy.
Conclusions: Combined proteasome inhibitors and TRAIL overcome the apoptotic threshold raised by Bcl-2 and may prove useful in the treatment of chemoresistant malignancies with up-regulated Bcl-2.<<
Cheers, Tuck |
