Proteasome Inhibition By Bortezomib In The Regulation Of NK Cell Survival And Function

Backgroud Ubiquitin-proteasome pathway is the major proteolytic system in the mammal cells, involved in cell proliferation , differentiation and apoptosis. 26S proteasome is a ATP-dependent proteolytic complex that ubiquitously exists in the cytoplasm and nucleus of almost all eukaryotic cells. 26S proteasome consisting of a 20S core catalytic cylindrical complex capped at both ends by 19S regulatory subunits is responsible for the degradation of the proteins regulating cell proliferation, differentiation and apoptosis. Bortezomib, a specific and potent 26S proteasome inhibitor, has recently received much attention by hematologists/oncologists due to its potent apoptosis induction proterty in tumor cells. It was first approved by FDA of America to treat multiple myeloma (MM), but now it has been applied to treat other hematological malignancies such as lymphoma, and certain types of solid rumors. More interestingly, Bortezomib also is shown to sensitize tumor cells to NK cell mediated-lysis through TRAIL and/or Fas/Fas ligand pathways. Apart from its treatment effects on tumors, toxic effects of bortezomib on immune-competent cells such as T cells and dendritic cells have also been revealed. Recent studies have demonstrated that proteasome inhibition by bortezomib induced apoptosis in activated human T cells and suppressed the expression of activation-associated cell surface receptors and cytokines in T cells and DCs, implicating that proteasome inhibition by bortezomib may affect function of T cells and DCs. However, to date little is known about its regulatory effects of proteasome inhibition by bortezomib on natural keller (NK) cells.Objective The aim of the study was to investigate the effects of bortezomib on natural killer cell survival and function, to explore underling mechanisms of bortezomib mediated apoptosis of resting NK cells; To investigate its inhibitory effect on NK cell-mediated antitumor activity in a mouse model of MM. Combination therapy with bortezomib and IL-12 was designed to test whether IL-12 had a synergistic antitumor effect with bortezomib in vivo and protected NK cells from bortezomib treatment-associated damage.Methods1. To investigate the effects of bortezomib on NK cells survival and function in vitro.1.1 The effects of bortezomib on PBLs or LAK cells survival 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 72h. PBLs or LAK cells were cultured in a 24-well plate in the presence or absence of bortezomib at different concentrations defined for 12h, 24h, and/or 48h, and the apoptotic cells were quantified by dual labeling of recombinant Annexin V(AV) and propidium iodide (PI), and flow cytometry(FCM).1.2 The effects of bortezomib on NK cells survival and function. Primary resting natural killers were purified from peripheral blood mononuclear cells of health donors by negative selection. Purity of these NK cells was determined by FCM. Cells were cultured in a 24-well plate in the presence, absence of bortezomib or combined with GSH or zVAD at different concentrations defined for 12h or 24h,respectively. The percentages of apoptotic cells were quantified by dual labeling of AV and PI , and flow cytometry. Caspase-3-like activity was measured by fluoroscan II plate reader; Functional mitochondria were labeled by Mitotracker Red, mitochondria membrane potential were detected by FCM; Intracelluar staining for perforin and cell surface staining for NK cells activation receptors were assayed by FCM. NK cell cytolytic activity against K562 cell and P815 cell were evaluated using a standard chromium-51 release assay.2. The effects of bortezomib on NK cells survival and function in vivo2.1 The establishment of SCID mouse model bearing MM tumor ( RPMI 8226; a human multiple myeloma cell line): The five-to-six week old female SCE) mice were injected subcutaneously with 1×10⁷ logarithmically growing RPMI 8226 cells to right forelegs back. Treatment began about 14 days post-inoculation when tumor reached 0.5cm in diameter.2.2 To investigate inhibitory effects of antitumor activity, quantify NK cell numbers in blood and to evaluate killing activity of spleen cells in the mouse model bearing tumor treated with NS, bortezomib and/or IL-12. The mice were randomly divided into NS, IL-12, bortezomib and bortezomib + IL-12 groups when tumor formed. NS, bortezomib (0.75mg/kg) and/or IL-12(0.4ug/each) were administered intravenously or intraperitonealy, respectively. The tumor growth (tumor mass) was monitored after the beginning of treatment. The absolute numbers of NK cells in peripheral blood were calculated by blood routine and FCM before and after treatment. The cytotoxicity of NK cells in a spleen was evaluated through LDH release assay.Result1. Study the effects of bortezomib on NK cells survival and function in vitro.1.1 Bortezomib induced PBLs and LAK cell apoptosis: The results show that bortezomib induced apoptosis in primary PBLs in a dose and/or incubation time-dependent manner. When exposure to the lowest dose of bortezomib for 12h, the apoptotic cells were found in PBLs. When prolonged the incubation time to 48h, bortezomib induced over 80% of apoptosis. Moreover, LAK cells were also markedly compromised to apoptosis induced by bortezomib, as compared with untreated group (P<0.05).1.2 Bortezomib induced NK cell apoptosis and suppressed NK cell killing activity. Our results demonstrated that Bortezomib markedly induced apoptosis in NK cells in a time-and dose-dependent manner. The percentages of total apoptotic cells were significantly increased in the NK cells treated by bortezomib with the dose of 4.7ng/ml and 18.8ng/ml for 12h and 24h. Bortezomib treatment for 12 and 24h at the dose of 18.8ng/ml induced more pronounced apoptosis in NK cells than 4.7ng/ml, respectively ( P<0.01 for 12h and P<0.001 for 24h, respectively). When the same dose of bortezomib (4.7ng/ml or 18.8ng/ml) was applied, 24h treatment triggered more apoptosis in NK cells than 12h treatment (P<0.0001).Our results showed that bortezomib at dose of 18.8ng/ml induced about 4 fold increasement of caspase-3 activity. GSH almost completely abolished caspase activity of NK cells induced by bortezomib, moreover the percentages of early and total apoptotic cells in NK cells induced by bortezomib for 24h in the presence of GSH were significantly decreased as compared with those of bortezomib(4.7ng/ml) alone group (P<0.05 ) . Blocking caspase activation by zVAD did not effectively block total apoptosis in NK cells induced by bortezomib. These data indicated that caspase activation is ROS-dependent, and ROS generation contributes to apoptosis in NK cells induced by bortezomib.Mitochondria play pivotal roles in the process of apoptosis. To determine a role of mitochondria in the bortezomib-induced apoptosis in NK cells, mitochondrial membrane potential (MMP) was measured by Mitotracker Red. The results showed that bortezomib markedly induced the loss of Mitotracker Red fluorescence intensity in NK cells in a dose-dependent manner. Statistical analysis showed that the differences in mean fluorescence intensity of Mitotracker Red between bortezomib (4.7ng/ml or 18.8ng/ml)-treated and untreated NK cell were significant. Furthermore, our studies showed that GSH partially and significantly prevented the loss of MMP induced by bortezomib (4.7ng/ml). In contrast, zVAD did not show any protective effects on MMP, suggesting that the dissipation of MMP is upstream of caspase activation in bortezomib-treated NK cells.To explore whether bortezomib affects NK cells function, intracellular staining of perforin and cell surface staining of NK cell activation receptors were quantified by flow cytometry. The results showed that expression of intracellular perforin on bortezomib-treated NK cells was not significantly different from untreated NK cells. Moreover expression of DNAM-1, NKG2D and NKp30 was compared between bortezomib treated and untreated NK cells, and no difference was found between these two groups. By contrast, NKp46 expression in NK cells was significantly reduced upon treatment with bortezomib in a dose-dependent manner. We further found blocking NF-kB activity by a NF-kB blocker Bay-11 induced significant loss of NKp46 expression on NK cells, indicating that NF-kB may be involved in the regulation of NKp46 expression and bortezomib-induced decrease in NKp46 expression may be in part attributable to its blocking effects on NF-kB activity. It is known that NKp46 is one of the important activating receptors for NK cell function. To investigate functional consequence of decreased NKp46 expression on NK cells induced by bortezomib, redirected NK cell cytotoxicity was assayed by ⁵¹Cr release test. The results showed that the lysis of P815 cells in the presence of anti-NKp46 antibody by the NK cells exposed to a low dose of bortezomib (4.7ng/ml) for 12h was significantly decreased when compared with controls at E.T ratios of 20:1 and 10:1. Moreover, blocking NF-kB activation by Bay-11 for 12h substantially inhibited NK cell-mediated lysis of P815 cells in the presence of anti-NKp46 antibody. By contrast, NK cells treated by bortezomib for 12h were able to kill K562 target cells as efficiently as the untreated NK cells. These data implied that the bortezomib-induced impairment of NK cell killing mediated through the NKp46 activation pathway.2. The effect of bortezomib on NK cells survival and function in vivo (aninal study) .2.1 Tumor formed in all the mice 14 days after implantation. After given different treatments, growth inhibition on the xenografted tumors was monitored and compared. Our results demonstrated that tumor volumes in the mice that treated with NS and IL-12 increased in a time-dependent manner, A slight suppression on tumor growth from IL-12-treated mice was observed when compared with NS-treated mice, however difference in tumor mass between these two groups did not reach significance. The tumor volumes of SCID mice were markedly reduced after treatment with bortezomib alone and bortezomib plus IL-12 as compared with control group (P<0.01), respectively. Bortezomib treatment alone induced markedly reduction of RPMI 8226 tumors in all of the treated mice. However, tumor re-growth was observed after termination of bortezomib treatment. As expected, treatment with bortezomib plus IL-12 resulted in a significance delay of tumor re-growth as compared with either Bortezomib or IL-12 alone treated mice. These data indicated a synergistic anti-MM effect of IL-12 and bortezomib.To determine whether bortezomib affected the numbers and function of NK cells in these mice in vivo, the numbers of NK cells in peripheral blood of the mice before and after the treatment were quantified by blood routine test and FCM. The results showed that NK cell number was markedly decreased after bortezomib treatment and combined treament of Bortezomib with IL-12 did not result in an increasement of NK cell number. In addition, NK cell numbers after NS and IL-12-treated mice were not significantly different from the mice before treatment. Cytotoxictity of spleen NK cells from mice treated by Bortezomib alone was significantly decreased when compared with the control and IL-12 alone -treated mice (P<0.01) , as determined by LDH release assay. In contrast, combined treatment of bortezomib with IL-12 resulted in a significance increasement of NK cell killing activity, suggesting that IL-12 may restore NK cell cytolytic activity deficiency induced by Bortezomib.Conclusion1. Bortezomib induces apoptosis in PBLs, LAK cells, primary resting natural killer cells in a dose- and time dependent manner, ROS-dependent and independent pathways are implicated in the apoptosis machinery of resting NK cells induced by bortezomib.2. Bortezomib decreased NKp46 receptor expression on primary resting NK cells, resulting in decreased NK cell-mediated lysis of target cells through the NKp46 activation pathway.3. Bortezomib reduces absolute numbers of NK cells in peripheral blood, and affects NK cell cytotoxicity in SCID mice. IL-12 showed a synergistic antitumor activity with bortezomib and restored NK cell killing defects induced by bortezomib.