The Mechanism And The Role Of Autophagy In Multiple Myeloma Induced By NVP-BEZ235

AIM:The PI3K/Akt/mTOR pathway is constitutively activated in human myeloma cell lines and in freshly isolated plasmocytes from patients with MM.mTOR signalling pathway has been designed as an attractive anti-tumor target in multiple myeloma.NVP-BEZ235,a novel,dual class Ⅰ PI3K/mTOR inhibitor,is an imidazoquinoline derivative.NVP-BEZ235 binds the ATP-binding clefts of PI3K and mTOR kinase,thereby inhibiting their activities.Increasing evidence shows that NVP-BEZ235 is able to effectively and specifically reverse the hyperactivation of the PI3K/mTOR pathway,not only resulting in potent antiproliferative and antitumor activities in a broad range of cancer cell lines and experimental tumors but also autophagy.We aimed to investigate(1)the inhibitory effects and(2)autophagy of NVP-BEZ235 on multiple myeloma and its relationships with cell death and apoptosis,(3)the mechanism of autophagy induced by NVP-BEZ235 in MM cells.We aim to explore the role of autophagy in NVP-BEZ235 treatment of multiple myeloma for the theoretical foundation of NVP-BEZ235s’ clinical application.Methods:1.The study of the autophagy,apoptosis,cell viability induced by NVP-BEZ235 on MM cell lines:Effects of NVP-BEZ235 on viability of U266、KM3 and RPMI8226 MM cells.Human myeloma cell lines U266,KM3 and RPMI8226 were treated with NVP-BEZ235 at different concentrations(0,100nM、200nM、300nM 和 400nM)for 0,12,24,48 and 72 h.Cell viability was detected by MTT assay.NVP-BEZ235 induces ultrastructural features of autophagy.KM3 cells were treated with NVP-BEZ235 for 12 h and processed for electron microscopy.Acridine orange was used to stain AVOs in untreated or BEZ235(50,100nM)-treated U266,KM3 and RPMI8226 cells for 12 h.The cells were visualized under a red filter fluorescence microscope.Autophagy bubble ratio were detected by flow cytometry.Effects of NVP-BEZ235 on the expression of LC3II and Atg5 in MM cells.Cells were treated with 50nM and 100nM NVP-BEZ235 for 12 h and LC3Ⅱ and Atg5 expressions in U266,KM3 and RPMI8226 cells was tested using Western blot analysis.2.The role of autophagy in multiple myeloma cells inhibition and apoptosis induced by NVP-BEZ235:NVP-BEZ235 in combination with CQ,3-MA and BafA1 exerts enhanced effects on inhibiting the viability of U266>KM3 and RPMI8226 MM cells.Human myeloma cell lines U266,KM3 and RPMI8226 were treated with NVP-BEZ235 in combination with CQ,3-MA and BafAl.Cell viability was detected by MTT assay.NVP-BEZ235(12.5nM,25nM,50nM,100nM)in combination with CQ(5nM,10nM,20nM,40nM)at different concentrations for48h.Morphological and numerary changes of MM U266、KM3 and RPMI8226cells colonies formation by CQ or NVP-BEZ235 alone for 21 days by methylcellulose elonogenic assay in microscopy.NVP-BEZ23 5 alone or in combination with A,G5-siRNA exerts enhanced effects on inhibiting the viability of U266 MM cells.Cell viability was detected by MTT assay.Morphological and numerary changes of U266cells colonies formation by SiAtg5 or NVP-BEZ235 alone for 14 days by methylcellulose clonogenic assay in microscopy.Apoptotic cell death was revealed by Hoechst 33258 staining with increasing BEZ235(0,100,200 and 300nmol/L)treatment for 48h.Apoptotic cells exhibited highly condensed and fragmented nuclei morphology.Apoptotic cell death was revealed by Annexin-V-FITC/PI staining with increasing BEZ235(100,200 and 300nmol/L)treatment for 48h.Apoptotic cells exhibited highly condensed and fragmented nuclei morphology.Chloroquine enhanced the apoptosis induced by NVP-BEZ23 5 in U266、KM3、RPMI8226 cell lines.Apoptotic cell death was revealed by Annexin-V-FITC/PI staining with chloroquine、NVP-BEZ235 treatment for 48h.SiAtg5 enhanced the apoptosis induced by NVP-BEZ235 in U266 cell lines.Apoptotic cell death was revealed by Annexin-V-FITC/PI staining with SiAtg5、NVP-BEZ235 treatment for 48h.3.The mechanism of the role of autophagy in multiple myeloma cells inhibition induced by NVP-BEZ235.Effects of NVP-BEZ235 on the expression of mTOR2-Akt pathway proteins and FOXO3a in myeloma cells.Cells were treated with 50nM and 1OOnM NVP-BEZ235 for 12 h and mTOR,p-mTOR(ser 2481),AKT,p-AKT,FOXO3a and p-FOXO3a(Thr32)protein expressions in U266 cells was tested using Western blot analysis.Effects of NVP-BEZ235 and/or siFOXO3a on the expression of BNIP proteins in MM cells.Effects of NVP-BEZ235 on the expression of BNIP proteins in MM cells.Effects of NVP-BEZ235 on the expression of BNIP3 and LCⅡ proteins transfected by siBNIP.Cells were treated with 100nM NVP·BEZ235 for 12 h and BNIP3 and LCⅡ protein expressions in U266 cells was tested using Western blot analysis.4.NVP-BEZ235 induced autophagy in MM cells selectively.Acridine orange was used to stain AV0s in untreated or BEZ235(50,100nM)-treated THP-1 and HL60 cells for 12 h.The cells were visualized under a red filter fluorescencemicroscope.Autophagy bubble ratio were detected by flow cytometry.Effects of NVP-BEZ235 on the expression of LC3II and Atg5 in THP-1 cells.Cells were treated with 50nM and 100nM NVP-BEZ235 for 12 h and LC3Ⅱ and Atg5 expressions in THP-1 and HL60 cells was tested using Western blot analysis.5.The study of the autophagy,cell viability induced by NVP-BEZ235 on primary CD138+myeloma cells:Primary CD138+myeloma cells in bone marrow cell sorting.Effects of NVP-BEZ235 on viability of primary myeloma cells.primary myeloma cells were treated with NVP-BEZ235 at different concentrations for different times.Cell viability was detected by MTT assay.Patients’ MM cells were treated with NVP-BEZ235 for 12 h and processed for electron microscopy.The relative expression levels of ATG5、FOXO3a and BNIP3 genes in CD138+ cells before and after NVP-BEZ235 treatment measured by qRT-PCR.Bone marrow mononuclear cell separation in patients with multiple myeloma.Effects of NVP-BEZ235 on the expression of LC3-Ⅱ/Ⅰ,ATG5,FOXO3a,p-FOXO3a and BNIP3 proteins in patients’ MM cells was tested using Western blot analysis.6.The study of the autophagy,tumor proliferation inhibition induced by NVP-BEZ235 on u266 myeloma cells tumor-burdened nude mice:To establish animal model of u266 myeloma cellstumor-burdened nude mice.Divide group.Compare the general condition,animal body weight and size of tumors.Calculate the tumor volumeinhibition rate.To verify whether NVP-BEZ235 myeloma cell may induce autophagy in tumor-burdened nude mice by transmission electron microscopy(sem).7.Statistical analysis.Data are expressed as mean±SEM.Statistical analyses involved use of SPSS vl9.0.Data were processed with analysis of variance of repeated measurement,one-way analysis of variance,Dunnett’sT3 and LSD test.P<0.05 was considered statistically significant.Results1.The NVP-BEZ235 treatment of U266、KM3 and RPMI8226 cells reduced cell viability in a dose-and time-dependent manner.Autophagy bubbles with double membrane were oberserved s in myeloma cells treated with NVP-BEZ235.Acridine orange stain and flow cytometry was used to measure the autophagy levels in untreated or BEZ235-treated myeloma cells.The result revealed autophagy cell ratio is higher in NVP-BEZ235 group than the control groupThe treatment of myeloma cells with NVP-BEZ235 affected the expression of light chain 3(LC3)and Atg5 proteins involved in the process of cellular autophagy.Hoeschst33258 stain and the flow cytometric analysis revealed that,the NVP-BEZ235 treatment increased the rate of apoptosis in myeloma cells.2.The autophagy inhibitors 3-MA,CQ and BafA1 were used to examine the role of autophagy in NVP-BEZ235-induced cell damage in myeloma cells.The combination NVP-BEZ235+3-MA showed a higher growth inhibition in myeloma cells than NVP-BEZ235 alone.A similar result was observed in myeloma treated with the combination NVP-BEZ235+CQ and NVP-BEZ235=BafA1.We also assessed the level of cell colony forming ability in myeloma cells treated with the combination NVP-BEZ235alone and NVP-BEZ235+CQ.The combination NVP-BEZ235+CQ showed a lower cell colony forming ability in myeloma cells than NVP-BEZ235 alone.Similarly,The combination of NVP-BEZ235 and siAtg5 transfected showed a higher growth inhibition in myeloma cells and a lower cell colony forming ability in myeloma cells than NVP-BEZ235 alone.The autophagy inhibitors 3-MA,CQ and BafA1 were used to examine the role of autophagy in NVP-BEZ235-induced apoptosis in myeloma cells.The combination NVP-BEZ235+CQ showed a higher growth inhibition in myeloma cells than NVP-BEZ235 alone.Similarly,The combination of NVP-BEZ235 and siAtg5 transfected showed a higher growth inhibition in myeloma cells and a lower cell colony forming ability in myeloma cells than NVP-BEZ235 alone.3.Acridine orange stain and flow cytometry was used to measure the autophagy levels in untreated or BEZ235(50,100nM)-treated THP-1 and HL60 cells.LC3II and Atg5 expressions was tested using Western blot analysis.in THP-1 and HL60 cells.There is no proof of autophagy occurs in THP-1 and HL60 cells treated with NVP-BEZ235.4.NVP-BEZ235 group had a significantly higher FOXO3a,BNIP protein level and lower p-mTOR(ser 2481),p-AKT,p-FOXO3a(Thr32)protein level than control group.After the treatment of NVP-BEZ235 andior siFOXO3a,the BNIP3 protein level of siFOXO3a decreased in siFOXO3a group,while increased in NVP-BEZ235 group than the control group.After the treatment of NVP-BEZ235 and/or si BNIP3,the BNIP3 and LCⅡ protein level NVP-BEZ235 group than the control group.5.The NVP-BEZ235 treatment of primary CD 138+myeloma cells reduced cell viability in a dose-and time-dependent manner.Autophagy bubbles with double membrane were oberserved in primary myeloma cells treated with NVP-BEZ235 under the electron microscope.The relative expression levels of ATG5、FOXO3a and BNIP3 genes in CD 138+cells from patients were significantly higher with NVP-BEZ235 than control treatment by qRT-PCR.The expression of LC3-Ⅱ/Ⅰ,ATG5,FOXO3a,and BNIP3 proteins in patients,MM cells were significantly higher with NVP-BEZ235 treatment than the control group.The expression of.p-FOXO3a proteins in patients’ MM cells were significantly lower with NVP-BEZ235 than control treatment.6.We established the animal model of u266 myeloma cells tumor-burdened nude miee.There was no statistical difference of the animal body weight in NVP一BEZ235 group and the control group.The size of the tumors decreased in NVP-BEZ235 group than the control group,Tumor growth inhibition rate is(83.33±6.50)%.Autophagy bubbles with double membrane were oberserved in primary myeloma cells in vivo in myeloma mice treated with NVP-BEZ235.Conclusion1.NVP-BEZ235 inhibits multiple myeloma cell line cells proliferation.NVP-BEZ235 induce autophagy in multiple myeloma cells.NVP-BEZ235 selectively induce autophagy in multiple myeloma cell lines.There is no proof of autophagy occurs in THP-1 and HL60 cells treated with NVP-BEZ235.The role of autophagy play an important role induced by NVP-BEZ235 in the cell death of myeloma cell lines,the combination NVP-BEZ235 autophagy inhibitors/siAtg5 transfected showed a higher growth inhibition in myeloma cells than NVP-BEZ235 alone.NVP-BEZ235 induce multiple myeloma cells apoptosis.The role of autophagy play an important role induced by NVP-BEZ235 in the apoptosis of myeloma cell lines,the combination NVP-BEZ235 autophagy inhibitors/siAtg5 transfected showed a higher apoptosis rate in myeloma cells than NVP-BEZ235 alone.(7)The mechanisms of autophagyinduced by NVP-BEZ235.NVP-BEZ235 induced autophagy through the mTOR2-Akt-FOXO3a-BNIP3 pathway of in multiple myeloma cells.2.NVP-BEZ235 inhibits primary myeloma cells proliferation.NVP-BEZ235 induce autophagy in primary myeloma cells.The mechanism is involve the mTOR2-Akt-FOXO3a-BNIP3 pathway.3.We establish animal model of myeloma nude mice.NVP-BEZ235 treatment decreased the size of the tumors in MM mice.Autophagy occurs in vivo in myeloma mice treated with NVP-BEZ235.