Dual Proteasome and mTOR Inhibition Promotes Apoptosis in Non-Hodgkin Lymphoma Cell Lines

Abstract

Background: Bortezomib is a selective, reversible inhibitor of the ubiquitinproteasome pathway whose action leads to tumor growth arrest and induction of apoptosis. Bortezomib has demonstrated clinical activity in Non-Hodgkin lymphoma (NHL) and is approved in the treatment of mantle cell lymphoma (MCL). The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that plays a key role in the PI3K/AKT pathway that regulates tumor cell growth and proliferation. Temsirolimus is an mTOR inhibitor that has shown activity in preclinical and clinical studies in various NHL subtypes. The objective of this study was to determine the growth inhibitory and apoptotic effects of the novel combination of bortezomib and temsirolimus in selected NHL cell lines including: Granta-519 (MCL), Toledo (large B-cell lymphoma), and EHEB (chronic B-cell leukemia). Materials and Methods: Cell viability was determined by the colorimetric MTS assay after 24 and 48 hours of treatment with bortezomib (1 – 10 nM) and temsirolimus (0.1 – 5 μM). Drug interactions were examined by the combinationindex (CI) isobologram method of median dose-effect analysis. Apoptosis was assessed by measuring cytochrome c and caspase-3 activity after 24 hours of treatment. In addition, the expression of several key factors involved in pro-apoptotic signaling (Bax and FasL) were evaluated by western blot analysis. Results: The MTS assays revealed a significant time and dose-dependent growth inhibition with either agent alone in all three NHL subtypes. A synergistic interaction was demonstrated with bortezomib and temsirolimus in the EHEB and Granta-519 cell lines, while an additive effect was seen at lower drug doses in the Toledo cell line. The LD50 ranged from 2.7 – 9.2 nM for bortezomib alone and 0.3 – 10.6 μM for temsirolimus alone. These levels were reduced to 0.4 – 4.5 nM and 0.04 – 2.25 μM for bortezomib and temsirolimus, respectively, when these drugs were used in combination. Growth inhibition in these cell lines was due in large part to the induction of the intrinsic apoptotic cascade. A 4 to 10-fold (bortezomib) and 2 to 8-fold (temsirolimus) increase in caspase-3 activity was seen, which was significantly increased 8 to 24-fold (p < 0.001) using the drug combination. In addition, bortezomib was shown to induce mitochondrial cytochrome c release in the 3 cell lines, while temsirolimus initiated release in the EHEB and Granta-519 cells. Increased expression of Bax, a pro-apoptotic member of the BCL-2 family of proteins, was observed predominantly in the MCL subtype (Granta-519) following treatment with either agent. This expression was further enhanced using both drugs. Additionally, an increase in FasL expression in all cell lines with the drug combination suggested an activation of the extrinsic apoptotic pathway. Conclusion: Targeting both the ubiquitin-proteasome and mTOR pathways, which are critical for tumor cell growth and survival, may be a promising new therapeutic strategy in NHL. This in vitro study demonstrates a significant increase in growth inhibition and apoptosis with the novel combination of bortezomib and temsirolimus, when compared to either drug alone, in selected NHL subtypes. These results provide a rationale for the use of this drug combination in NHL clinical trials.