| BackgroundHematologic neoplasms refer to malignant tumors that originate from blood,bone marrow or lymph nodes,including leukemia,lymphoma and myeloma.Multiple myeloma(MM)is a kind of malignant plasmacytoma,which usually has an aggressive and incurable course,and its incidence is increasing year by year.MM accounts for about 10%of hematological malignancies and has become the second most common hematological tumor.MM usually causes end-organ damage,including anemia,renal impairment,osteolytic lesions,and hypercalcemia.Since 1990,the global incidence of MM has increased by 126%and typically occurs in the elderly,with approximately 85%and 60%of diagnoses occurring in people aged over 55 and 65 years,respectively.In recent years,high-dose chemotherapy combined with autologous hematopoietic stem cell transplantation(HSCT)has extended the survival of MM patients to a certain extent,and new drugs and new generation drugs have been widely used in MM.The prognosis has greatly improved,with median survival now exceeding 6 years,although this varies according to disease risk factors.Although great progress has been made in the treatment of MM,with significant improvement in survival,MM remains largely incurable due to drug resistance,immune escape of tumors and other factors,and there is a risk of recurrence,which poses a challenge to new treatment options.Natural killer(NK)cells are lymphocytes belonging to the innate immune system that can target infection or tumor cells without prior sensitization,and exert effective cytotoxic activity against malignant cells such as MM cells.The effector functions of NK cells are regulated by a balance of inhibitory and activating receptor expression on the cell surface.Unlike cytotoxic T cells,NK cells can be activated by recognizing tumor cells that have reduced or lack expression of major histocompatibility complex(MHC)and can kill their target cells by releasing perforin or granzyme,than results in apoptosis mediated by death receptors or induced by cytokine release.In addition,NK cells regulate other immune cells by secreting soluble factors,such as tumor necrosis factor-α(TNF-α),interferon-γ(IFN-γ),Interleukin and so on.In addition to their antitumor properties,NK cells do not induce graft versus host disease(GVHD)after transplantation.Thus,they provide a promising approach for the treatment of MM patients.Recent advances in adoptive transfer immune cell therapy have dramatically changed the paradigm of cancer immunotherapy.Therefore,ex vivo expanded/activated NK cells from own patients or allogeneic donors are potential options for NK cells therapy in MM.Although current immunotherapy can cure many patients with MM,relapsed/refractory MM remains challenging in some cases.Glycogen synthase kinase-3β(GSK-3β)regulates many cellular processes and participates in a wide range of signaling pathways.Studies have found that TWS119 is an inhibitor of GSK-3β and promotes the activation of NK cells.In this study,we explored the effect and related mechanism of TWS119 on NK cells killing activity of MM cells and degranulation.Method(1)The mRNA and protein expression of GSK-3β in NK cell lines NK-92,YTS and primary NK cells were detected by polymerase chain reaction(PCR)and western blot(WB),respectively.After NK cells treated with TWS119,the effect of TWS119 on β-catenin mRNA and protein expression in NK cells was detected.(2)Flow cytometry was used to detected the effects of TWS 119 treatment for 48h on the activity of NK92(OμM,10μM,20μM,40μM,80μM)and primary NK cells(0μM,2.5μM,5μM,10μM,20μM,40μM,80μM)were examined.Purity of primary NK cells on day 0 and day 21 were detected.(3)Flow cytometry was used to detect the effect of TWS 119 on the killing of RPMI8266 cells by NK-92 cells,the killing of RPMI8266 cells by primary NK cells and the killing of MM cells by NK-92 cells and degranification.(4)Enzyme linked immunosorbent assay(ELISA)was used to detect the effects of TWS 119 on NK-92 cells and primary NK cells when stimulated with TWS 119 alone or co-cultured with RPMI8226 cells.The changes in the secretion of cytokines IFN-y and TNF-α.(5)Flow cytometry was used to detect the effect of TWS119 on the surface receptors of NK-92 cells.The expression of activating receptors(NKG2D,NKp46,NKp44,DNAM-1)and inhibitory receptors(PD-1,Tim-3)on NK-92 cells were detected after co-culture with RPMI8226 cells.(6)PCR was used to detected the expressions of RAB27A,SNAP23,Syntaxin 11(STX-11),STXBP2 and UNC13D in NK cells.(7)ELISA and PCR were used to detected the effects of p-catenin inhibitor MSAB and NF-κB inhibitor parthenolide(PTL)combined with TWS119 on cytokines IFN-y and TNF-αin NK-92 cells,respectively.The protein expression levels of β-catenin,p-NF-κB,c-Rel and RAB27A were detected by WB.To study the regulation of GSK-3β/β-catenin/NF-κB pathway on cytokines and RAB27A.(8)Co-Immunoprecipitation(Co-IP)and Laser Scanning Confocal microscopy(LSCM)were used to investigate the interaction between β-catenin and NF-κB in NK-92 cells.(9)NOG mice were subcutaneously inoculated with RPMI8226-luciferase to establish a tumor-bearing mouse model.The effect of adoptive transfer of NK cells treated in vitro on tumor size and survival time of mice was monitored.Results(1)PCR and WB showed that the mRNA and protein expression levels of GSK-3β in NK-92 cells were higher than those in other cells.After treatment of NK cells in TWS119 group,β-catenin mRNA and protein levels in NK-92 cells and primary NK cells were increased.(2)When TWS119 concentration was 20μM,there was no effect on NK-92 cells activity.When TWS119 concentration was 2.5μM and 5μM,there was no effect on primary NK cell activity.Therefore,20μM was selected for NK-92 cells in subsequent experiments,and 2.5μM and 5μM were selected for primary NK cells.The purity of primary NK cells was 11.9%and 78.8%on day 0 and day 21,respectively.(3)When NK-92 cells and target cells(RPMI8226)were co-cultured according to E:T=0.25:1,0.5:1 and 1:1,the apoptosis rate of RPMI8226 increased gradually with the increase of E:T ratio.The apoptosis rate of RPMI8226 in TWS119 group was significantly higher than that in DMSO group.The proportion of CD107a+NK-92 cells degranulation was increased.When primary NK cells and RPMI8226 were co-cultured according to E:T=1:1,RPMI8226 apoptosis was significantly increased.The proportion of degranulation was increased in CD107a+primary NK cells.When NK-92 cells and primary MM cells were treated according to E:T=1:1,the proportion of apoptosis of primary MM cells increased,and the proportion of CD 107a+NK-92 cells expression increased.All the differences were statistically significant.(4)The concentrations of IFN-γ and TNF-α in the supernatant of NK-92 cells in TWS119 group were significantly higher than those in DMSO group.After adding RPMI1226,the concentrations of IFN-y and TNF-α in the two groups increased.In the supernatant of primary NK cells,the concentrations of IFN-y and TNF-α in DMSO,2.5μM and 5μM group showed an increasing trend.After stimulation with target cell(RPMI1226),the concentrations of IFN-γ and TNF-α in the three groups all increased,and the concentration of 5μM group was the highest.(5)Compared with TWS 119,the expression of NKG2D in DMSO group increased from 28.23±2.6%to 50.3±6.4%,and the expression of Tim-3 receptor decreased from 72.7±6.6%to 44.3±3.1%.There were significant differences between the two groups.(6)The expression of RAB27A mRNA in TWS 119 group was higher than that in DMSO group,and the experimental results were statistically significant.(7)Compared with DMSO group,TWS 119 treatment increased the mRNA expression and supernatant concentration of IFN-y and TNF-α,while PTL or MSAB treatment decreased the mRNA expression and supernatant concentration of IFN-y and TNF-α.The combined effects of TWS119+PTL or TWS119+MSAB neutralized the inhibitory effects of PTL or MSAB.The protein expression levels of β-catenin,p-NF-κB and c-Rel increased in TWS 119 group,which were reduced by PTL or MSAB treatment to DMSO group,and the increased protein expression levels were offset by TWS119+PTL or TWS119+MSAB combination treatment.(8)Co-IP analysis showed that β-catenin and NF-κB had a colocalization relationship.LSCM showed that β-catenin(green fluorescence)and NF-κB(red fluorescence)were located outside the nucleus and had a colocalization relationship(R=0.77).In TWS119 group,green fluorescence and red fluorescence were located in the nucleus,and the fluorescence intensity and colocalization relationship were enhanced(R=0.92).(9)NOG mouse model was established by subcutaneous injection of RPMI8226-luciferase,compared with control group(con),adoptive transfer of DMSO-NK and TWS119-NK cells had an inhibitory effect on tumor,and TWS119-NK had a stronger inhibitory effect,a smaller tumor size,and significantly prolonged the survival time of mice.Conclusions(1)In NK-92,YTS and primary NK cells,mRNA and protein levels of GSK-3β were the highest in NK-92 cells.GSK-3β inhibitor TWS119 could increase the expression of β-catenin.(2)TWS119 at a concentration of 5μM did not affect the activity of primary NK cells.The cellular activity of NK-92 cells was not affected by TWS119 at a concentration of 20μM.(3)TWS119 can significantly enhance the cytotoxic activity of NK-92 or primary NK cells against MM cell lines or primary MM cells and CD 107a degranification level.(4)TWS119 could change the surface receptors of NK-92 cells,up-regulate the expression of activating receptor NKG2D and down-regulate the expression of inhibitory receptor Tim-3.(5)Mechanism studies showed that TWS119 treatment significantly increased the secretion concentration of IFN-γ and TNF-α supernatant and mRNA expression of NK-92 cells,and up-regulated the expression of RAB27A,a key gene for degranulation of NK cells,and induced the colocalization of β-catenin and NF-κB in the nucleus of NK cells.The GSK-3β/β-catenin/NF-κB signaling pathway regulated the cytokines IFN-γ,TNF-α and RAB27A.(6)Adoptive transfer of TWS119-treated NK cells significantly delayed the tumor growth rate,reduced tumor burden and prolonged the survival time of MM bearing mice. |
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