Mechanism Of Autophagy As A Potential Therapeutic Target In Myeloma Proliferation,Invasion And Chemotherapy Resistance

Multiple myeloma(MM)is an incurable plasma cell tumor,but the causes of its poor prognosis remain unclear.Studies have found that autophagy plays an important role in the regulation of many tumors,including MM.Autophagy is a response of cells to hunger,toxic stimulation and chemotherapy,etc.Through the formation of autophagosomes,the lysosomal degradation of subcellular components and the recycling of intracellular components are an adaptive regulation of adverse environments.Autophagy is a double-edged sword with both good and bad aspects: on the one hand,autophagy is a way for cells to survive normally;On the other hand,excessive autophagy can promote autophagy death of cells.Therefore,this study took autophagy as a potential therapeutic target to study the mechanism of proliferation,invasion and chemotherapy resistance of MM cells.This study mainly includes the following two parts:Part ?: The mechanism of FTY720 induced autophagy activation and inhibition of multiple myelomaObjective: The S1P(sphingosin 1-phosphate)signaling pathway can activate autophagy in cancer cells.S1 P and its upstream precursor ceramide/sphingosin(CER/SPH)are metabolites of sphingolipids.In tumors,S1 P plays a major role in anti-apoptosis and promoting tumor growth,while CER and SPH play a role in promoting tumor cell apoptosis.The breaking of S1P/CER balance is one of the pathological mechanisms for the growth and survival of tumor cells(including MM cells).Fingolimod(FTY720)is an S1 P receptor antagonist that inhibits the activity of the S1 P signaling pathway.Therefore,the purpose of this study was to determine the effect of S1 P receptor antagonist FTY720 on the survival and apoptosis of MM cells.To clarify the regulatory mechanism of FTY720 on autophagy;To elucidate the mechanism of cell death induced by targeting the S1 P receptor in multiple myeloma.Methods: 1.The effects of FTY720 on survival rate and apoptosis of multiple myeloma patients and 4 myeloma cell lines were detected.Was collected in 5 cases of patients with multiple myeloma bone marrow fluid,extraction of bone marrow mononuclear cells,at the same time with myeloma cell line U266,LP-1,Nci-h929,RPMI8226 cell as the research object,by the method of CCK 8-FITC and Annexin V/PI double FTY720 flow cytometry detection dye concentrations(0,5,10,15,20 ?M)for patients with multiple myeloma cells and 4 kinds of myeloma cell survival and apoptosis rate.Rt-PCR was used to detect the expression of S1 PR in four myeloma cell lines,and the high-expression cell lines of S1 PR were selected for subsequent experiments.2 Test whether the cell death induced by FTY720 was caspase-3 dependent death,and test the survival of RPMI8226 cells treated with FTY720 in the presence or absence of caspase-3 inhibitor Z-vad-Fmk by cck-8 method.Western blot was used to detect the protein expression levels of apoptotic proteins including Mc L-1,Survivin,Bcl-2,Bak and Bax before and after FTY720 treatment.In order to investigate whether FTY 720 can induce autophagy in RPMI8226 cells,the expression of LC3-I to LC3-II in RPMI8226 cells after FTY 720 treatment was detected by western blot with anti-LC3 antibody,and the expression change of Beclin-1 after FTY720 treatment was detected by RT-PCR.To further clarify the role of autophagy in FTY720-induced cell death,we used CCK-8 and Annexin V-FITC/PI double-staining flow cytometry to detect the survival and apoptosis of RPMI8226 cells treated with FTY720 in the presence or absence of autophagy inhibitor Bafilomycin A1.To determine the relationship between apoptosis of RPMI8226 cells induced by FTY720 and autophagy and ROS,apoptosis of RPMI8226 cells was detected by Annexin V-FITC/PI double-staining flow cytometry after FTY720 alone or in combination with ROS inhibitor NAC or Tiron,and LC3-I to LC3-II transformation was detected by anti-lc3 antibody western blot.3 The effect of S1P5 R on the survival of RPMI8226 cells was detected.RPMI8226 cell line was detected by RT-PCR to detect the expression level differences of 5 S1PR(S1PR1,S1PR2,S1PR3,S1PR4,S1PR5),and the RPMI8226 cell line was selected with the highest S1P5 R background expression.Lentivirus interference S1P5R(si S1P5R)was used to detect S1P5 R gene interference in RPMI8226 cell line.RT-PCR was used to detect the expression of Rho B,ROCK1,Rac1 and CDC42 in the downstream signaling pathway.Expression of apoptotic and autophagy related proteins Akt,Survivin,PI3 K,m TOR,P53 and P62 were detected by RT-PCR and Western blot.Results: 1.FTY720 expression promoted the apoptosis of myeloma cells and 4myeloma cell lines.After the treatment of cells with different concentrations of FTY720,CCK-8 assay and flow cytometry apoptosis assay showed that FTY720 had a significant promoting effect on cell apoptosis,showing a concentration dependence,and the difference was statistically significant compared with the control group.RPMI8226 cell lines with the highest expression of S1 PR background were selected as follow-up experiments.2 RPMI8226 cells for FTY720 effects clearly whether the cause of death for caspase-3 dependence,we use 5 ?mol,FTY720 processing RPMI8226 cell CCK-8 testing,the results show that the cell survival rate is only 20%,and in the caspase inhibitor Z-3-VAD-FMK produce most of the action,the cell survival rate can increase to 40-60%,and a dose dependent,that can save FTY720 produce most cause cell death.To further clarify whether the observed FTY720 promoting myeloma cell death was mediated by apoptotic proteins,we examined the protein expression levels of apoptotic proteins Mcl-1,Survivin,Bcl-2,Bak and Bax before and after FTY720 treatment.The results showed that the expressions of anti-apoptotic proteins Mcl-1,Survivin and Bcl-2 were significantly decreased after treatment,while the expression of pro-apoptotic proteins Bak and Bax were not significantly changed.Thus,it was confirmed that FTY720 induces apoptosis by regulating the expression of anti-apoptotic protein.In order to investigate whether FTY 720 can induce autophagy in RPMI8226 cells,the expression of LC3-I to LC3-II in RPMI8226 cells was detected by western blot with anti-LC3 antibody,and the expression level of beclin-1 was detected by RT_PCR.The results showed that after FTY720 treatment,the amount of LC3-I to LC3-II was increased,and beclin-1 expression level was significantly increased in a dose-dependent manner.Thus,FTY720 can induce autophagy in RPMI8226 cells.In FTY720 effects induced in order to define the role of autophagy in the cell death,we examined the autophagy inhibitor Bafilomycin A1 exist or not exist when FTY720 effects of RPMI8226 cells survival situation,the result shows: the combined application of FTY720 and cell survival Bafilomycin A1 group was obviously higher than that of pure FTY720 effects of,shows that Bafilomycin A1 can save FTY720 effects of cell death caused by autophagy.In order to clarify the effect of ROS on apoptosis and autophagy induced by FTY720,RPMI8226 cells were treated with 15mol/L FTY720,the apoptosis rate of RPMI8226 cells was significantly increased compared with the blank control group.ROS inhibitor NAC or tir-on combined with FTY720 could reduce the apoptosis induced by FTY720.Combined use of ROS inhibitors after NAC or Tir-on and FTY720,LC3B-? expression is use FTY720 markedly reduced.Thus,FTY720 can induce cell apoptosis by activating ROS,and ROS inhibitors can inhibit the occurrence of autophagy.3Rt-PCR was used to detect the main expression of S1P5 receptor in RPMI8226 cells,and lentivirus interference was used to down-regulate RPMI8226 cells of S1P5 R.The results showed that compared with the control group,the survival rate of cells in the S1P5 R interference group was significantly reduced,with a statistical difference.RPMI8226 cells were treated with 0.1 mol S1 P,20 mol FTY720 and si S1P5 R,respectively,and rt-pcr was used to detect the expression of Rho B,ROCK1,Rac1 and CDC42 in the downstream signaling pathways.The results showed that the expressions of Rho B,ROCK1,Rac1 and CDC42 in the S1 P group increased,while the expressions of Rho B,ROCK1,Rac1 and CDC42 in the FTY720 group were opposite to those in the S1 P group.After S1P5 receptor was silenced,the expression of Rac1 and CDC42 decreased,but the expression of Rho B and Rock1 did not change,indicating that Rac1 and CDC42 may be the downstream signaling pathway of S1P5 R.After the S1P5 R gene knockdown of RPMI8226 cells,the expressions of apoptosis and autophagy related proteins Akt,Survivin,PI3 K,m TOR,P53 and P62 were detected by RT-PCR and Western blot.The results showed that the expressions of PI3 K,Akt and m TOR were significantly decreased at the m RNA level.The protein levels of Akt,Survivin and P62 were significantly decreased.This indicates that S1P5 can regulate the PI3K/AKT/m TOR signaling pathway,inhibit autophagy and up-regulate the expression of anti-apoptotic protein Survivin.Conclusion: 1.FTY720 can promote the death of myeloma cells and induce apoptosis.2.FTY720 can induce caspase-dependent cell death in RPMI8226 cells,which can induce apoptosis of RPMI8226 cells by regulating the expression of anti-apoptotic proteins,and induce autophagy,which promotes apoptosis.ROS is a co-regulatory pathway of apoptosis and autophagy induced by FTY720 in myeloma cells RPMI8226.3.S1P5 R is the main receptor of RPMI8226 cells,and Rac1 and CDC42 may be the downstream signaling pathways of S1P5 R.S1P5 can regulate the PI3K/AKT/m TOR signaling pathway,inhibit autophagy and up-regulate the expression of anti-apoptotic protein survivin.Part ?: study on the mechanism of centella asiatica saponins targeting autophagy to inhibit the proliferation and invasion of multiple myelomaObjective: there is growing evidence that molecules from plants may prove extremely beneficial in the development of chemotherapy for deadly cancer types.Chemotherapy for multiple myeloma has been poorly studied.Here,we examined the anticancer effects of Asiaticoside,a triterpenoid compound derived from plants,on the drug-resistant myeloma cell line KM3/BTZ.Materials and methods: cell activity was determined by cck-8,and autophagy was detected by transmission electron microscopy.ROS levels were determined by flow cytometry.Transwell method was used to detect cell migration and invasion.Protein expression was detected by Western blotting.Results: the results showed that saponins inhibited the growth of KM3/ BTZ cells and showed IC50 of 12 mol.It was further observed that the anticancer effect of saponins in centella centauri was due to the induction of autophagy and the increase of LC3-?expression.In addition,the expression of effector caspases in KM3/BTZ cells also changed.The saponins of asiatica asiatica can also promote the increase of ROS in KM3/BTZ cells and inhibit their migration and invasiveness by regulating the STAT3 signaling pathway.Conclusion: Centella saponins have been shown to be useful in the treatment of multiple myeloma.