Bortezomib induces Bak expression and cell death in non-Hodgkin's lymphoma cell lines that are both rituximab- and chemotherapy-resistant Free

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Development of resistant phenotypes in cancer is a natural response to therapeutic intervention. The focus of our laboratory is on studying mechanisms of action and resistance to rituximab and chemotherapy in B-cell non-Hodgkin’s lymphoma (NHL). To understand the pathways of and establish biomarkers of rituximab resistance we developed several rituximab resistant cell lines (RRCL) from two rituximab sensitive (RSCL) cell lines (Raji and RL). Disruption of the balance between pro-apoptotic (Bax, Bak) and anti-apoptotic (Bcl-2, Bcl-xL, Mcl-1) Bcl-2 family proteins is necessary for the maintenance of B-cell lymphoma. We previously demonstrated that development of chemotherapy resistance occurred in RRCL due to a post-transcriptional down-regulation of Bax and Bak proteins. Further characterization of RRCL revealed an increase in proteasome activity and expression of E1 ubiquitin-activating and E2 ubiquitin-conjugating enzymes. In vitro exposure of RRCL to the proteasome inhibitor bortezomib (PS-341, Velcade) led to a significant increase in cell death not seen with standard chemotherapeutic agents. We therefore hypothesized that proteasome inhibition could alter the balance between pro-apoptotic and anti-apoptotic Bcl-2 family proteins leading to cell death in RRCL. Western blot analysis of bortezomib-treated cells revealed a significant up-regulation of Bak in RRCL 24 hours following exposure. Interestingly, cell death and caspase activation kinetics were delayed when comparing RRCL to RSCL. The kinetics of bortezomib-induced Bak expression correlated with delayed caspase activation and cell death. Furthermore, transient expression of Bak led to spontaneous caspase-dependent apoptosis in RRCL, whereas expression of a BH3-mutant Bak (D83A) did not. Our data suggest that bortezomib can lead to increased Bak expression and cell death in B-cell lymphoma cell lines resistant to rituximab and chemotherapy. Several studies have suggested that inhibition of NF-κB activity may be responsible for the activity of bortezomib against cancer cells while others have implicated ER-stress induced apoptosis or accumulation of “BH3-only” proteins. Here we propose a novel mechanism-of-action of bortezomib found in RRCL: induction of Bak expression. Our data suggest that proteasome inhibition could be a potential strategy to overcome acquired rituximab and chemotherapy resistance in B-cell lymphomas.

This work was supported by NCI PO1 #: CA103985-1.