Effects Of Chidamide Combined With Sorafenib On The Proliferation And Apoptosis Of MV-411 Cells And Its Related Mechanism

Objective:To explore the effects of single-agent and two-drug combination of chidamide(CS055),sorafenib(Sorafenib)on FLT3-ITD-positive acute myeloid leukemia cell line MV-411 cell proliferation inhibition and apoptosis promotion.At the same time,observe whether CS055 can synergize or enhance the effect of sorafenib and possible mechanism,explore new methods for the treatment of FLT3-ITD-positive acute myeloid leukemia and provide a theoretical basis for its clinical application.Methods:1.Thiazole blue(MTT)reduction method was used to detect the inhibitory effect of sorafenib(50nmol / L),chidamide(0.5?mol /L)single drug and two-drug combination on the proliferation of MV-411 cells2.Flow cytometry(FCM)was used to detect the effects of sorafenib(50nmol/ L),chidamide(0.5?mol / L)single drug and the combination of two drugs on the apoptosis of MV-411 cells.3.Real-time fluorescence quantitative polymerase chain reaction(RT-PCR)was used to detect FLT3 ? ?-catenin,C-myc mRNA expression after sorafenib(50nmol / L),chidamide(0.5?mol / L)single drug and two-drug combination group intervention in MV-411 cells.4.Western blot was used to detect FLT3,?-catenin,C-myc and Ac-H3 proteins expression after sorafenib(50nmol / L),chidamide(0.5?mol / L)single drug and two-drug combination group intervention in MV-411 cells.Results:1.MTT assay results: after intervention of different concentrations of sorafenib and chidamide in cell lines for 24 h,48h,and 72 h,respectively,the growth of MV-411 cell lines was significantly inhibited.With the increase of the concentration of sorafenib and chidamide and the increase of the intervention time,the cell survival rate decreased compared with the blank control group.The inhibition rate increased,showing a dose and time dependence.The IC50 values of sorafenib on MV-411 cells at 24,48,and 72 h were 101.28,79.07,and 63.33 nmol / L.The IC50 values of chidamide on MV-411 cells at 24,48,and 72 h were 4.28?3.87 and 1.41?mol / L,compared with the blank control group,the difference was statistically significant(p<0.05).When 50 nmol / L sorafenib and 0.5?mol / L chidamide were applied to MV-411 cell line for 72 h,the two-drug combination group had a significantly higher cell proliferation inhibitory effect than the blank control group,and also higher than in the single-drug group,according to the calculation of King's formula,there is a synergy between sorafenib and chidamide(q = 1.6);The difference between the two-drug combination group and the single-drug group was statistically significant.(p<0.05).2.The results of FCM showed that the MTT results showed that sorafenib and chidamide both had the strongest inhibitory effect at 72 h,so 72 h was selected as the time for flow detection of cell apoptosis.The results showed that the blank control group had a low apoptosis rate,and both the single-drug group and the combined drug group showed obvious apoptosis.The blank control group,sorafenib single-druggroup,chidamide group,and two-drug combination group had apoptosis rates of(2.53±0.60)%?(13.75±0.76)%?(25.63±0.67)%?(44.98±0.82)% at 72 hours,respectively.The rate was significantly higher than the single drug group and the blank control group,the difference was statistically significant(p <0.05).3.RT-PCR results showed that after using sorafenib(50nmol / L),chidamide(0.5?mol / L),and two drugs to intervene in the MV-411 cell line for 72 h,the expression levels of FLT3??-catenin and C-myc mRNA were significantly lower than that of the blank control group,and the difference was statistically significant(all p <0.05).The expression of the two-drug combination group was more down-regulated,which was lower than that of the single-drug group and the blank control group,and the difference was statistically significant(all p <0.05).4.Western Blot protein determination results showed that after using sorafenib(50nmol / L),chidamide(0.5?mol / L)and two drugs to intervene in the leukemia MV-411 cell line for 72 h,the expression levels of FLT3??-catenin and C-myc protein were significantly lower than that of the blank control group,the difference was statistically significant(all p <0.05),and the down-regulation of the two-drug combination group was significantly lower than that of the single drug group and the blank control group,the difference was statistically significant(all p <0.05).The expression of Ac-H3 in the MV-411 cell line of the blank control group was low,but after 72 hours of sorafenib(50nmol / L),chidamide(0.5?mol / L)and two drugs combined intervention,the expression of Ac-H3 protein Up-regulated,the expression of Ac-H3 protein in each single-drug group was significantly higher than that in the blank control group,the difference was statistically significant(all p <0.05),and the expression of the two-drug combination group was significantly higher than that in the single-drug group and the blank control group,the difference was statistically significant(all p <0.05).Conclusions:1.Sorafenib and chidamide acted on MV-411 cell line as a single agent,which had a proliferation-inhibiting effect on cell growth and was time-dose dependent.2.Sorafenib combined with chidamide can act on MV-411 cell line,which can synergistically inhibit cell proliferation and induce cell apoptosis.3.After sorafenib combined with chidamide acted on the MV-411 cell line,the FLT3,?-catenin,C-myc gene and protein expression of the cells decreased,and the Ac-H3 protein expression level increased.4.Sorafenib combined with chidamide can synergistically inhibit cell proliferation and induce apoptosis in MV-411 cell lines,which may be related to the synergistic inhibition of FLT3,?-catenin and C-myc genes and proteins,and is also related to the up-regulation of Ac-H3 protein expression.