Study On Mechanisms Of The Apoptosis In Human Multiple Myeloma RPMI8226 Cells Induced By Diosgenin

Multiple myeloma is a hemopoietic system malignancy that is characterized by the malignant proliferation of plasma cells.The majority of patients have clinicle symptom such as renal insufficiencebone distruction,anemia,immunoldeficiency and so on.Seriously,MM is still unable to cure and the incidence of MM is the second highest in the hematological malignancies.The mechanisms of multiple myeloma is also unclear now,but the occurrence of tumor is closely related to the imbalance of proliferation and apoptosis,and there are three main apoptoic pathway: mitochondrial pathway,death receptor pathway and endoplasmic reticulum pathway.In addition,induction of apoptosis is one of the important mechanisms of anti-tumor drugs.Now chemotherapy and autologous stem-cell transplantations have improved the clinical symptoms of patients and slowed down the progress of the disease,the application of proteasome inhibitor bortezomib and other targeted drug also have achieved good results,but they inevitably lead to drug resisitance and recurrence and the side effects of chemotherapy,seriously affecting the quality of life of patients.So to development new drugs that can improve outcomes and overcome resistance is still of great significance.Diosgenin is an aglycone of steroidal saponins,witch is extracted from Dioscorea species.Morden pharmacological studies show that diosgenin have positive effect on relaxing blood vessels,lowering blood fat,regulating immunity and resisting tumor,especially the anti-tumor effect has get more and more attention.There are researches report that diosgenin exhibits anti-proliferation and promote apoptosis activities on a vriaty of cancer cells in vitro including human breast cancer Hela cells,melanoma A375-S cells,and human chronic myeloid leukemia K562 cells and so on.However,though reaerches show that Fas/FasL,cytochrome C,p53,Caspase,and nuclear factor kappa B(NF-?B)were involved in the apoptosis induced by diosgenin,whether diosgenin has an inhibitory effect on MM cells,has not been reported.Therefore we intend to use multiple myeloma RPMI8226 cells as the research object to observe whether Dio has an effect on apoptosis of RPMI8226 cells and to explore its mechanisms.Objective:To investigation the effect of diosgenin on apoptosis of multiple myeloma RPMI8226 cells,then reveal the mechanism of apoptosis of RPMI8226 cells induced by diosgenin,and provide experimental basis for the further development and application of diosgenin.Methods:Multiple myeloma RPMI8226 cells were treated with different concentrations of diosgenin in vitro.The cell proliferation was assessed by CCK-8 assay at different concentration and different time to screen the effective inhibitory concentration.With 10nmol/L bortezomib as the positive control,the cell apoptosis was detected by flow cytometry;the changes of Bax,Bcl-2,Caspase-3,Caspase-9,Cyt-C mRNA expression were analyzed by RT-PCR;the changes of Bax,Bcl-2,Caspase-3,Caspase-9,Cyt-C protein expression were evaluated by Western Blot.Results:1 Inhibitory action of diosgenin on the proliferation of RPMI8226 cells assessed by CCK-81.1 After treatment with diosgenin for 24 h,compared with the blank group,the Dio groups display an inhibitory effect on the cells(P<0.05),and the inhibition rates of 5,10,20,30,40,50 ?mol/LDio groups are respectively 4.96%,11.95%,24.09%,36.59%,41.76%,48.96%.1.2 After treatment with diosgenin for 48 h,compared with the blank group,the Dio groups display an inhibitory effect on the cells(P<0.05),and the inhibition rates of 5,10,20,30,40,50 ?mol/LDio groups are respectively 6.42%,11.27%,26.02%,49.29%,67.04%,86.50%.CCK-8 cell proliferation test tesrults suggest that Dio can inhibite the proliferation of RPMI8226 cells in a time-does-dependent manner.According to the inhibition rate of diosgenin,the drug concentration was determined to be 10,20,30?mol/L.2 RPMI8226 cell apoptosis rate detected by FCMCompared with the blank group,the apoptotic ratio of BTZ group was obviously improved(P<0.05),in 10?mol/L,20?mol/L and 30?mol/L Dio groups the apoptotic ratio were also higher than that in blank group(P<0.05),and had dose-dependent.The apoptotic ratio of 20?mol/L and 30?mol/L Dio groups were significantly increased compared with BTZ group(P<0.05),and had dose-dependent.3 Expression of Bax,Bcl-2,Caspase-3,Caspase-9,Cty-C mRNA detected by RT-PCRCompared with the blank group,in BTZ group Bax/Bcl-2,Caspase-9,Cty-C mRNA increased(P<0.05),but Caspase-3 mRNA have no obviously change(P>0.05);the ratio of Bax/Bcl-2 mRNA and the expression of Cty-C mRNA were increased in 10?mol/L compared with the blank group;20?mol/L and 30?mol/L Dio groups(P<0.05),and had dose-dependent;Caspase-3,Caspase-9 mRNA were higher in 20?mol/L and 30?mol/L Dio groups than that in blank group(P < 0.05),and had dose-dependent.Compared with BTZ group,in 10?mol/L,20?mol/L and 30?mol/L Dio groups the Caspase-3 mRNA obviously increased(P < 0.05),and had dose-dependent;Caspase-9 mRNA increased in 20?mol/L and 30?mol/L Dio groups,and had dose-dependent;Cty-C mRNA showed no difference between10?mol/L,20?mol/L,30?mol/L Dio groups and BTZ group(P>0.05);the ratio of Bax/Bcl-2 mRNA increased significantly in BTZ group compared with 10?mol/L,20?mol/L and 30?mol/L Dio groups(P<0.05).4 Expression of Bax,Bcl-2,Caspase-3,Caspase-9,Cty-C protein detected by Western BlotCompared with the blank group,Bax/Bcl-2,Caspase-3,Caspase-9,Cty-C protein increased(P < 0.05),Bax/Bcl-2,Caspase-3,Cty-C protein were significantly higher in 10?mol/L,20?mol/L and 30?mol/L Dio groups than blank group(P<0.05),and had dose-dependent;Caspase-9 protein was higher in 30?mol/L Dio groups than blank group(P<0.05),and had dose-dependent.Compared with BTZ group,the protein expression of Bax/Bcl-2,Caspase-3 were much higher in 20?mol/L and 30?mol/L Dio groups(P<0.05),and had dose-dependent;the protein expression of Caspase-9,Cty-C had enhanced in 30?mol/L Dio groups compared with BTZ group(P < 0.05),and had dose-dependent.Conclusion:1.Diosgenin can inhibite the proliferation of human multiple myeloma RPMI8226 cells and induce apoptosis of the cells.2.Diosgenin can induce apoptosis of RPMI8226 cells by activating mitochondrial-mediated endogenous apoptotic pathway in a Caspase-dependent manner.