Inhibition Of Proteasome To Disrupt Unfolded Protein Response And Autophagy In Multiple Myeloma Cells

Background and Objectives:Multiple myeloma(MM) , which produces and secretes abundant immunoglobulins (Igs), is a kind of malignant counterpart of the plasma cells malignacy. Because of heavy load of protein translation and abrupt accumulation of unfoaded or misfoaded protein in endoplasmic reticulum, MM cells are subjected to endoplasmic reticulum stress (ERS). To alleviate ER stress, MM cells activate a signaling pathway called the unfolded protein response (UPR), which aims to reestablish homeostasis by transmitting information to the nucleus about the protein-folding status in the ER lumen, triggering adaptive responses. BiP, XBP-1 and CHOP are activated by IRE1αand PERK/eIFαsignal. BiP and XBP-1 are prosurvival components, and CHOP is proapoptotic one. Disequilibrium between cytoprotective outputs and proapoptotic ones results in cell death. Besides UPR, autophagy is also an adaptive response in eukaryocyte. Autophagy, which mediates mainly the bulk degradation of long-lived cytoplasmic proteins, large protein compounds or organelles by lysosomes, is an adaptive catabiolic reaction when eukaryocytes meet insufficient of nutrients and growth factors, hypoxia, endoplasmic reticulum stress, and other harmful stimuli. The formation of autophagosome, an important procession, depends on not only Atg5-Atg12 and Agt8 (LC3), but also the fuction of Beclin1-PI3K compounds. It was reported that UPR can't only trigger ERS, but induce autophagy in yeast cells. Whereas it is unclear that the relationship between UPR and autophagy in MM cells.As a new kind of proteasome inhibitor (PI), Bortezomib, a potent and specific 26s proteasome inhibitor has been shown to selectively induce apoptosis of MM cells, has remarkable effect on refractory MM in clinic. 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, suggesting that NF-κB inhibitor cannot completely explain the nature of the selectivity of Bortezomib for multiple myeloma cells. Recently, ERS-induced MM cells death is considered as an important mechanism of Bortezomib. However, the mechanism by which this compound acts remains unknown. On account of the relationship of ERS, UPR and autophagy, we assume that Bortezomib maybe affect the signals regulated by UPR and autophagy.Methods and Results:To investigate the relationship between UPR and autophagy induced by ERS in MM cells, we exammed the expression of UPR related proteins and autophagy related ones by western blot. Our data showed: (1) After cells were treated by classical autophagy inducer, Rapamycin, for different time, the apoptotic rates of H929 or RPMI8226 and the expression levels of XBP-1s,XBP-1u,BiP and CHOP rose in time-dependent manner; (2) After cells were treated by classical ERS stressors, BFA or Tg, for different time, the apoptotic rates of H929 or RPMI8226 and the expression levels of UPR related proteins, and autophagy-related rose in time-dependent manner; (3) Rapamycin disrupted the expressions of XBP-1s,XBP-1u and BiP , but promote the expression of CHOP induced by BFA or Tg; (4) BFA or Tg disrupted the expressions of autophagy-related. Above data strongly suggest that there should be close relationship beween UPR and autophagy in MM cells.To further study whether Bortezomib results in disequilibriumin of UPR, annexin V-FITC/PI staining followed by flow cytometry analyzed the apoptotic rates of target cells, and western blot showed the expression of UPR related proteins. Our data showed: (1) After H929 and RPMI8226 cells were treated by the classical endoplasmic reticulum stressor BFA or Tg for different time, the apoptotic rates of target cells and the expression levels of UPR related proteins rose in time-dependent manner. The tendency was coincident with Bortezomib induced; (2) Bortezomib inhibited classic ER stressors BFA or Tg-induced up-regulation of prosuevival UPR components XBP-1s,XBP-1u and BiP and disrupted the proapoptotic outputs CHOP. In other words: Bortezomib induced ERS eventually to apoptosis that was closely associated with the regulatory imbalance between proapoptotic and prosurvival UPR.To investigate whether Bortezomib affects on autophagy procession, western blot, electron microscope and co-immunoprecipitation showed: (1) There had few autophagosomes or autolysosomes induced by Bortezomib; (2) After cells were treated by Bortezomib for different time, the apoptotic rates of H929 or RPMI8226 and the expression levels of autophagy-related proteins decreased in time-dependent manner; (3) Bortezomib disrupted the expressions of autophagy-related proteins induced by Rapamycin; (4) Bortezomib inhibited the formation of Beclin-1-PI3KC3 compounds; (5) Induction autophagy by Rapamycin prompted Bortezomib-induced MM cell apoptosis. Our data inferred that Bortezomib did not only disrupt UPR to induc MM cell apoptosis, but disrupted endogenous autophagy to eliminate the protection mechanisms in MM cells.Conclusions:1. There is close relationship between UPR and regulative procession of autophagy in MM cells.2. Bortezomib could induce ERS, but it induces proapoptotic UPR outputs, such as CHOP, whereas inhibits cytoprotective componets for instance BiP and XBP-1.3. Bortezomib could disrupt endogenous autophagy in MM cells, as maybe involve in inhibition the formation of Beclin-1-PI3KC3 compounds.4. Bortezomib could not only disrupt UPR to induce MM cell apoptosis, but disrupt endogenous autophagy to eliminate the protection mechanisms in MM cells. It is the important mechanism that Bortezomib outweigh to treat multiple myeloma compared with other chemotherapeutics.