Proteasome Inhibition By Bortezomib In The Regulation Of NK Cell Function

Backgroud:Ubiquitin-proteasome pathway is the major proteolytic system in the mammal cells, involved in the regulation of cell proliferation, differentiation and apoptosis. The activation of the proteasome is curial for maintaining cell survival and function. The proteasome inhibitors induce apoptosis of several kinds of tumor cells. Bortezomib (velcade), a specific and potent proteasome inhibitor, has been at present widely used in the clinic of the treatment of multiple myeloma and other solid tumors. However, toxic effects of bortezomib on immune-competent cells such as T cells and dendritic cells (DCs) have also been revealed, recent studies have demonstrated that proteasome inhibition by bortezomib induced apoptosis in activated human T cells and suppressed DC function priming T cell activation or polarization. Natural killer (NK) cells are important innate immune component cells playing curial roles in immune-surveillance against tumors and viral infected cells. To date, little is known about potential toxic property of proteasome inhibition by bortezomib on NK cell function.Objective:To study the regulatory effects of proteasome inhibitor bortezomib on NK cell function.Methods:Human peripheral blood mononuclear cells (PBMCs) from blood donors were separated by Ficoll-Paque gradient centrifugation. Peripheral blood lymphocytes (PBLs) were harvested after incubation of PBMCs in a plastic flask for 1h. To generate LAK cells, PBLs were cultured in the presence of IL-2 for 48h, and then treated by variant doses of Bortezomib for another 6h.To detect the expression of tumor necrosis-related apoptosis-inducing ligand (TRAIL) after NK or LAK cells were exposed to Bortezomib for 6h in culture, the cells were stained with CD56 and CD3 to define NK cells (CD56+CD3) and specific anti-TRAIL antibody, Flow cytometry (FCM) approach was applied for the quantification of TRAIL expression on NK cells; CD 107a was used as a hall marker for NK cell degranulation; intracellular staining with anti-perforin antibody was used to detect perforin expression. Primary resting NK cells were purified from PBMCs of health donors by negative selection method. Purity of these NK cells was determined by FCM, NK cells were cultured in the presence of IL-2 for 48h, and then treated by different doses of Bortezomib or NF-κB inhibitor Bay-11 for another 6h, and FCM was applied to detect surface TRAIL expression. NK cell cytolytic activity against K562 cell, RPMI8226 cell and U266 cell were evaluated using standard 4h chromium (Cr)-51 release assays or optimized 12h 51Cr-released assay.Results:a) Bortezomib reduced cell surface-bound TRAIL expression in human NK cells. Bortezomib treatment with the doses applied for 6h significantly reduced cell surface expression of TRAIL in a dose dependent manner, but did not affect viability of NK cells.b) Bortezomib downregulated TRAIL expression on NK cells via NF-κB pathway. IL-2 activated purified NK cells were treated either by bortezomib at doses of 4 nm,8 nm和16 nm or NF-κB inhibitor Bay-11 for 6h, the TRAIL expression of these NK cells was significantly reduced on both protein and mRNA level.c) Bortezomib did not affect intracellular perforin expression and NK degranulation. Our study has showed that Bortezomib treatment did not significantly affect perforin expression in NK cells and NK cell degranulation activity, as determined by FCM and CD 107a translocation assay, respectively.d) Bortezomib impaired death receptor-mediated cytotoxicity by LAK cells. To evaluate whether bortezomib treatment affects activated NK cell-mediated killing activity, a modified 12 h 51Cr release assay was employed. Using this modified cytotoxicity assay, we observed that bortezomib treatment of LAK cells (16 ng/ml for 6 h) significantly reduced the percentage of lysis of the two MM cell lines RPMI8226 and U266 as compared to control (untreated) LAK cells. To further dissect the potential mechanism underlying the killing defect, and to determine a role of TRAIL-dependent killing in this assay, we employed a specific anti-TRAIL antibody to block the TRAIL-mediated killing pathway. The anti-TRAIL antibody blocked NK cell-mediated lysis of RPMI 8226 cells, but did not affect the lysis of U266 cells, indicating that TRAIL is involved in the NK cell-mediated killing of RPMI8226 cells under the condition in which perforin-dependent killing is blocked.Conclusions:Bortezomib disrupted NK cell TRAIL expression and TRAIL mediated-killing of target cells via the inhibition of NF-κB pathway.