The Role And Mechanism Of Cardamonin In Inhibiting The Malignant Biological Behavior Of IL-6 Induced Multiple Myeloma U266 Cells

Objective:Study on the inhibition of IL-6 induced malignant biological behavior of Human multiple Myeloma U266 cells by cardamonin and its mechanism.To provide an objective experimental basis for the treatment of multiple myeloma with cardamonin.It is expected that the application of Chinese medicine extract and chemotherapy together can reduce the complications of multiple myeloma and improve the disease remission rate and the total survival time of the patients.Methods:? Culture the multiple myeloma U266 cells,U266 cells were exposed to different concentrations of IL-6(10 ng/ml,20 ng/ml,40 ng/ml)at different time points,detected the proliferation of myeloma cells by CCK8,the best concentration of IL-6 was selected.Detected STAT3 phosphorylation and expression of STAT3 downstream Bcl-2,VEGF,cyclin D1 protein by Western blot.? Divided into control group,IL-6 group,IL-6+12.5 ? M cardamonin group,IL-6+25.0 ? M cardamonin group,IL-6+50.0 ? M cardamonin group,U266 cells were treated at different time points.Detected the proliferation of myeloma cells by CCK8,detected apoptosis rate of myeloma cells by Annexin V-FITC/PI double staining flow cytometry.Western blot assay was used to detect the expression of STAT3 and STAT3 downstream gene Bcl-2,VEGF,cyclin D1.Results:1.The results of CCK-8 method showed that:?IL-6 can promote the increment of U266 cells,the proliferation rate of U266 cells treated with different concentrations of IL-6 was significantly higher than that of the control group(P<0.01).The value added of 10 ng/ml IL-6 was 2.53%,7.15%and 11.17%,respectively,after the treatment of U266 24 h,48 h,72 h.The value added of 20 ng/ml IL-6,U266 24h,48h and 72h,was 12.13%,15.01%,17.09%,respectively.After treated with 40 ng/ml IL-6 for 24h,48 h and 72 h,the cell proliferation rate was 13.32%,15.46%,17.54%,respectively.The optimum concentration is 20 ng/ml.?Cardamonin inhibited the proliferation of U266 cells induced by IL-6 in a time-dependent and dose-dependent manner.After treated with IL-6+12.5 ? M cardamonin for 24h,48 h,72h.the inhibition rates were 15.63%,44.05%and 49.47%,respectively.After treated with IL-6+25.0 ? M for 24h,48h and 72h,the inhibition rates were 20.75%,57.72%,69.15%,respectively.After treated with IL-6 +50.0? M for 24h,48 h and 72 h,the inhibition rates were 21.49%?68.63%?76.15%.2.The apoptosis rate of myeloma cells was detected by Annexin V-FITC/PI double dye flow cytometry:?Wardamonin promoted apoptosis of multiple myeloma cells in a dose-dependent manner.The apoptosis rate of control group was 5.01%,the apoptosis rate of U266 cells treated with 12.5 ? M cardamonin for 24 h was 16.87%,the apoptosis rate was 35.74%after treatment of 25.0 ?M cardamonin for 24 hours.The apoptosis rate was 59.63%after treatment with 50?M cardamonin for 24 hours.Compared with the control group,the apoptosis rate of the above treatment was significantly higher than that of the control group(p<0.01).?IL-6 can reduce apoptosis rate,but cardamonin could antagonize the apoptosis of IL-6 and increase the rate of apoptosis.The apoptosis rate of control group was 4.93%,the apoptosis rate in IL-6 group was 3.78%.the apoptosis rate of IL-6+12.5 ? M cardamonin group was 12.67%.the apoptosis rate of IL-6+25.0 ? M cardamonin group was 31.35%,the apoptosis rate increased to 51.64%of IL-6+50.0,M cardamonin group.There was significant difference in apoptosis rate between the additive group and the control group(p<0.01).There was a significant difference compared with the IL-6 group(p<0.01).3.Western blot detection results show:IL-6 could activate STAT3,and promote the expression of Bel-2,VEGF,cyclin D1,the downstream gene product of STAT3.Cardamomin inhibited STAT3 activation induced by IL-6 and down-regulated the expression of downstream STAT3 gene products in U266 cells.Conclusion:Cardamonin inhibits the malignant biological behavior of multiple myeloma U266 cells induced by IL-6,the mechanism may be that cardamonin regulates the IL-6/STAT3 pathway,down-regulation the expression of VEGF,Bel-2,Cyclin D1 in multiple myeloma cell line,thus inhibit the proliferation of multiple myeloma cell and promot cell apoptosis.