Triptolide Induce Endoplasmic Reticulum Stress And Enhance The Pro-apoptotic Activity Of Bortezomib In Multiple Myeloma Cells.

Background and Aim: Triptolide is the principal active ingredient in extracts from the Chinese herb Tripterygium wilfordii Hook.F(TwHF),and has various functions such as immunosuppression ,anti-inflammatory and antitumor properties.In diverse hematological tumors triptolide exerts antitumor activity. Triptolide can induce apoptosis in multiple myeloma cells,but the potential antitumor mechanisms are still unknown. Multiple myeloma(MM)cells ,malignant counterpart of the plasma cells produce and secrete abundant immunoglobulins (Igs) .Because of heavy load of protein translation and abrupt accumulation of unfoaded or misfoaded protein in endoplasmic reticulum, MM cells are susceptible to endoplasmic reticulum stress (ERS).To alleviate ER stress, MM cells activate a sigalling pathway called the unfolded protein response(UPR), which aim to restablish homeostasis by transmitting information to the nucleus about the protein-folding status in the ER lumen,triggering adaptive response. BiP, XBP-1 and CHOP are activated by IRE1αand PERK/eIFαsignal. BiP and XBP-1 are prosurvival components, while CHOP is proapoptotic one. Disequilibrium between cytoprotective outputs and proapoptotic ones results in cell death. Bortezomib,a potent inhibitor of the 26S proteasome ,has been shown to selectively induce apoptosis of multiple myeloma(MM) cells and used for treatment of relapsed refractory MM. Bortezomib induced apoptosis in myeloma cells by that evokes ER stress but disrupts UPR . Based on the background that the endoplasmic reticulum stress induced cell response (or unfolded protein response, UPR) plays an important role in apoptosis of many secreting tumor cells. in this study, we studied the effects of Triptolide on model of myeloma cells (NCI-H929 and RPMI8226 and primary cells), we try to explore the effects on MM cells apoptosis induced by Triptolide and the correlateing mechanisms about the Triptolide enhance the pro-apoptotic activity of Bortezomib in multiple myeloma cells.Methods and Results: 1. In our work, MTT assays showed that Triptolide could inhibit proliferation of RPMI8226 and H929 cells in a time-and dose-dependent manner.2. MTT method confirmed that the non-inhibitory concentrations of triptolide (5-10ng/mL) add proteasome inhibitor bortezomib can sensitizes the two cell lines and primary cells. 3. Transmission electron microscopy confirmed: triptolide induced apoptosis in H929 cells. 4. Western blot showed:①After cells were treated by Triptolide for different time, the apoptotic rates of target cells and the protein expression levels of XBP-1s,XBP-1u,BiP and CHOP in time-dependent manner;②After cells were treated with different concentrations of Triptolide for 24h, the apoptotic rates of target cells and the expression levels of XBP-1s,XBP-1u,BiP and CHOP rose in a dose-dependent manner;③After cells were treated with the classical endoplasmic reticulum stressor Brefeldin A and Tg for different time, the apoptotic rates of target cells and the expression levels of XBP-1s,XBP-1u,BiP and CHOP in a time-dependent manner. The tendency was coincident with Triptolide induced; 5. XBP-1 protein levels of MM cells were inhibited effectively by the PLL3.7 lentiviral vector mediated expression XBP-1 shRNA , and increased the apoptosis rates which induced by Triptolide. . Conclusions: 1. Triptolide can induce apoptosis of MM cells in a time-and dose-dependent manner. 2. Non-inhibitory concentration of triptolide combined bortezomib can significantly enhance the pro-apoptotic activity of Bortezomib in multiple myeloma cells.4. XBP-1 silencing can induce the apoptosis in MM cells and cansignificantly enhance the pro-apoptotic activity of Bortezomib in multiple myeloma cells. XBP-1, the key molecule of UPR, plays important role in protecting MM cells .