Protesome Inhibitors Induce Unfolded Protein Response And Apoptosis In Multiple Myeloma Cells

Background and Objectives: As a new kind of antitumor drug, proteasome inhibitor (PI) has remarkable effects on refractory MM in clinic. However, the mechanism by which these compounds act remains unknown. Therefore, investigating the signal transduction pathway by which PI triggers MM cell apoptosis will be beneficial to understanding the action mechanism of PI and the developing of more effective targeting agents and treatment programmes. It had been constantly thought that PI killed tumor cells by inhibiting nuclear factor (NF)-κB activation. But recently, it was discovered that the apoptotic MM cells induced by the inhibitor of NF-κB were obviously less than those by PI. This phenomenon fiercely suggests that there would be a new pathway involved in the PI-induced apoptosis of MM cells. Based on the background that the endoplasmic reticulum stress induced cell response (or unfolded protein response, UPR) plays important role in apoptosis of many secreting tumor cells, in this study, we studied the effects of Bortezomib on NCI-H929 and RPMI8226 cell lines by detecting the the expression levels of XBP-1s,XBP-1u,BiP,CHOP mRNA and protein to investigate the effects of PI on the apoptosis and ERS in human multiple myeloma cells.Methods and Results: After treatment of MM cells with different approaches, apoptosis was assayed by Annexin V-FITC/PI staining followed by flow cytometry analysis, and the expression levels of XBP-1s,XBP-1u,BiP,CHOP mRNA and protein were detected by RT-PCR and Western blotting analysis. The Corresponding changes were observed after the XBP-1 gene was silenced by ViraPowerTM Lentiviral Expression System mediated RNAi. The results showed that: (1) After cells were treated by different concentrations of Bortezomib for 24h, the apoptotic rates of target cells and the expression levels of XBP-1s,XBP-1u,BiP and CHOP rose in dose-dependent manner; (2) After cells were treated by Bortezomib for different time, the apoptotic rates of target cells and the expression levels of XBP-1s,XBP-1u,BiP and CHOP rose in time-dependent manner; (3) After cells were treated by the classical endoplasmic reticulum stressor Brefeldin A for different time, the apoptotic rates of target cells and the expression levels of XBP-1s,XBP-1u,BiP and CHOP rose in time-dependent manner. The tendency was coincident with Bortezomib induced; (4) Bortezomib could inhibit the expressions of XBP-1s,XBP-1u and BiP and promote the expression of CHOP induced by Brefeldin A. In other words: Bortezomib could inhibit the expressions of molecules protecting cells and promote the expressions of molecules promoting apoptosis. (5) When XBP-1 was silenced, the target cells were more susceptive to Bortezomib which demonstrated that XBP-1 could protect cells from apoptosis.Conclusions: 1. PI can induce apoptosis of MM cells in a dose- and time-dependent manner. 2. PI-induced apoptosis of MM cells is associated with UPR. 3. Bortezomib can induce ERS by inhibiting ERAD, but it can also interrupt the expressions of molecules protecting cells from apoptosis in UPR. 4. XBP-1, the key molecule of UPR, plays important role in protecting MM cells in ERS.