Effects Of Low-dose Triptolide Combined With Sorafenib On Proliferation And Apoptosis Of MV411 Cells And Study On STAT5 Related Pathway

Objective:To investigate the inhibition of proliferation and inducing apoptosis of low-dose triptolide and sorafenib alone or in combination on FLT3-ITD positive acute myeloid leukemia cell line MV411,and the study of the STAT5 related pathway.To provide an experimental basis for finding effective methods for treating FLT3-ITD positive AML.Methods:1.Cell Counting Kit-8（CCK-8）method was used to determine the proliferation inhibition effect of different concentrations of TPL and sorafenib monotherapy-treated MV411 cell line;2.The proliferation inhibition of the group(low-dose TPL(5 nmol/L,closed to IC₂₀),sorafenib(3μmol/L,closed to IC₅₀)and their combination（TPL 5 nmol/L+Sorafenib 3μmol/L）)was detected by CCK-8;3.The apoptosis of the MV411 cell line treated by the group（low-dose TPL（5 nmol/L）,sorafenib（3μmol/L）and their combination（TPL 5 nmol/L+Sorafenib 3μmol/L））was detected by flow cytometry（FCM）;4.The expression of FLT3 and STAT5 mRNA in MV411 cells treated by the group（low-dose TPL（5 nmol/L）,sorafenib（3μmol/L）and their combination（TPL 5 nmol/L+Sorafenib 3μmol/L））was detected by real-time fluorescence quantitative polymerase chain reaction（RT-PCR）;5.The expression of FLT3,STAT5,and P-STAT5 proteins in the MV411treated by the group（low-dose TPL（5 nmol/L）,sorafenib（3μmol/L）and their combination（TPL 5 nmol/L+Sorafenib 3μmol/L））was detected by Western blot.Results:1.CCK-8 test results showed that TPL and sorafenib alone have obvious proliferation inhibition effect on MV411 cells.With the increase of drug concentration,cell survival rate decreases,inhibition rate increases,and in concentration-dependent.At 48h,the IC₂₀0 of TPL in MV411 cells was 5.161 nmol/L,and the IC₅₀0 of sorafenib was 3.193μmol/L.2.CCK-8 test results showed that at 48h,TPL 5nmol/L(closed to IC₂₀),sorafenib3μmol/L(closed to IC₅₀)and their combination on MV4-11 cell lines showed significant proliferation inhibition.The inhibition rate of the two drug combination group was higher than that of the single drug group,and the combination of the two drugs had synergistic effect.3.The results of flow cytometry showed that the average apoptotic rate of the control group was（4.67±1.15）%、the average apoptotic rate of 5 nmol/L TPL group was（9.94±1.38%）、the average apoptotic rate of 3μmol/L sorafenib group was（31.05±1.69）%and the average apoptotic rate of the two drugs combination group（TPL 5nmol/L+Sorafenib 3μmol/L）was（50.61±2.08%）after 48 hours of different drugs acting on MV411 cells.The apoptotic rate of the combination group on MV411 cells was greater than that of the single drug group.4.RT-PCR results showed that TPL（5 nmol/L）,sorafenib（3μmol/L）single drug group and combined group（TPL 5 nmol/L+Sorafenib 3μmol/L）after 48h treatment with MV411 cells,the expression levels of FLT3 and STAT5 mRNA in the combination group were significantly lower than those in the control group,and lower than that in the single drug group.5.Western Blot results showed that TPL（5 nmol/L）,sorafenib（3μmol/L）and two drugs（TPL 5 nmol/L+3μmol/L）were applied to MV411 cells for 48 h.The expression levels of FLT3,STAT5 and its phosphorylated active form P-STAT5 protein in the combination group were significantly lower than those in the control group,and significantly lower than the sorafenib（3μmol/L）and TPL（5 nmol/L）groups alone.Conclusions:1.TPL and sorafenib alone inhibited the proliferation of MV411 cells in a concentration-dependent manner.2.Low-dose TPL combined with sorafenib has synergistic inhibition and synergistic induction of apoptosis in MV411 cells.3.After low-dose TPL combined with sorafenib in MV411 cells,the expression levels of FLT3,STAT5 mRNA and FLT3,STAT5,and P-STAT5 protein in the combination group were lower than those in the single drug treatment group.4.The inhibitory effect of low-dose TPL combined with sorafenib on proliferation of MV411 cells may be related to the synergistic inhibition of FLT3 by both drugs and down-regulation of STAT5 and P-STAT5 expression.