The Study Of The Effect And Molecular Mechanism Of The Combination Of The Proteasome Inhibitor-Sorafenib And Homoharringtonine On MV4-11 Cell

BackgroundFLT3 gene mutations in acute myeloid leukemia(AML)accounts for about 35%of all types of leukemia,is one of the most common mutations in acute myeloid leukemia cells,of which FLT3/ITD in 25-30% patients with AML.It is characterized by high white blood cells,high early recurrence rates,and low survival rates at the time of initial diagnosis,and was identified as high-risk AML by the NCCN guidelines.Therefore,how to effectively control the progression of FLT3/ITD+AML disease,improve the cure rate,and clarify its specific pathogenesis,has been the focus of clinical and research workers.In drug treatment,sorafenib is a small molecule multikinase inhibitor and by competing with the FLT3 tyrosine kinase ATP binding sites and inhibit the activity of interference,signal transduction,proliferation and differentiation of leukemia cells.Sorafenib induced apoptosis of leukemia cells,but in clinical treatment,single use of sorafenib has not obtained remarkable curative effect.As Chinese traditional medicine,there are two important medical events affecting the status of HHT(Homoharringtonine)in the treatment of leukemia.On 2012 October,the new drug from Teva pharmaceuticals Inc.by harringtonine synthesized the Omacetaxine FDA approval,for the treatment of more than 2 kinds of tyrosine kinase inhibitors(TKI)resistant or intolerant of chronic myeloid leukemia(CML)patients.The biological China traditional medicine abroad and synthesis by American clinical researchers recognized and approved by America FDA in clinical treatment,there is no precedent,visible foreign scholars pay attention to the degree of harringtonine.In 2013 the clinical study published in the "The Lancet" magazine(impact factor: 44): summary of Chinese 17 research center in homoharringtonine as the coreconsisting of three induction chemotherapy(HAA,HAD,HAE)for the treatment of remission induction with newly diagnosed AML(609 cases of AML patients)confirmed the induction of HAA scheme,the remission rate of up to 73%,elaborated with HHT as the core for the curative effect of AML chemotherapy significantly,further demonstrates the importance of HHT in the treatment of leukemia.By the above two medical events,both in the basic experiment and in clinical treatment,HHT has become an important drug in the research of leukemia.Chinese traditional medicine has been a breakthrough in the "tradition".As the clinical workers of Hematology should recognize and review the Chinese traditional medicine-HHT.It is of great significance for the research and treatment of leukemia,both now and in the future.On the other hand,we are in the early clinical treatment of HAD or HAA scheme for FLT3/ITD was found in AML patients with positive first remission induction rate can reach about 70%.For the first time by far higher than the remission rate of IA,why HHT is such a significant effect? Where is the target and the way of action? With sorafenib targeting?FLT3/ITD positive AML research literature study found that there are two important genes is always accompanied by the progression of FLT3/ITD+AML,5BLOOD and 2 Leukemia literature have confirmed the significant role of Id1 and Mcl-1 gene in the occurrence and progression of FLT3/ITD+AML in.A large number of previous studies have confirmed that there are STAT5 specific binding sequences in the promoter of the two genes,and the gene can be trans activated by STAT5,so STAT5 is the key to the production of these two genes.Therefore,we think: in FLT3/ITD AML cells,STAT5 is an important gate of disease development,closed the gate of STAT5,Id1 and Mcl-1 gene will also be closed? Whether it will affect the rapid progress of the disease? Does the drug have an inhibitory effect on these genes?Some scholars have conducted a preliminary study on leukemia stem cells,found that the application of siRNA to silence STAT5 gene,can induce stem cell apoptosis,and inhibit the expression of Mcl-1 gene in AML cells,whether FLT3/ITD+ also exists in this signaling pathway? Worth studying.To sum up,the formation of our research aimed at FLT3/ITD +AML cells:1.The expression levels of STAT5,Id1 and Mcl-1 in FLT3/ITD +AML cells were analyzed;2.Si-RNA STAT5 was used to observe the apoptosis and the effects of Id1 and Mcl-1 gene on the molecular mechanism of FLT3/ITD +AML cells;3.HHT and sorafenibon the growth inhibition and apoptosis induction of FLT3/ITD cells,and the effects of STAT5,Id1 and Mcl-1;4.HHT and sorafenib two drugs on FLT3/ITD +AML cells,whether there is a synergistic inhibitory effect,and can improve the efficacy?5.Effect of HAD regimen and IA regimen in the treatment of FLT3/ITD +AML.Objective:1.To detect the expression of STAT5,Id1,Mcl-1 gene in MV4-11 cell line(FLT3/ITD+ acute myeloid leukemia cell line),K562 cell line(FLT3/ITD-acute myeloid leukemia cells)and normal human bone marrow cells;2.SiRNA-STAT5 on the proliferation and apoptosis of MV4-11 cells and the changes of mRNA and Mcl-1 levels and protein levels of Id1 and STAT5 were observed.3.Homoharringtonine and sorafenib alone and in combination on MV4-11 cell line,cell proliferation,cell apoptosis and the effect of FLT3/ITD,STAT5,Id1 and Mcl-1 gene expression,to investigate the molecular mechanism of the two drugs on MV4-11 cell line.4.Application of HAD and IA regimen for treatment of FLT3/ITD+ induced acute myeloid leukemia,the remission rate of the difference was observed by the two program,for patients with long-term overall survival(OS),progression free survival(PFS)of the difference.Methods:1.The expression of STAT5,Id1 and mRNA protein in normal human bone marrow cells,K562 cells and Mcl-1 cells were analyzed by RT-PCR and Western Blot.2.For STAT5 design 4 siRNA sequences,in 24 hours,48 hours to observe the transfection efficiency by using Western,Blot and RT-PCR screened sequencessilencing effect relatively significant;siRNA-STAT5 after transfection into MV4-11 cells,the cell proliferation was determined by MTT,Annexin V/ PI method for detection of apoptosis,expression of RT-PCR and Western Blot detection application Id1,Mcl-1 gene mRNA and protein.3.Respectively 1ng/ml,5ng/ml,10ng/ml concentration of group HHT and100nmol/l sorafenib in MV4-11 cells: proliferation of cells was detected by MTT;the apoptosis changes of Annexin cells was detected by V/PI assay;using RT-PC analysis of MV4-11 expression in cells STAT5,Id1,Mcl-1 gene mRNA;expression by STAT5,Id1,Mcl-1 protein was detected in MV4-11 cell Western in Blot.4.96 FLT3/ITD+ patients with acute myeloid leukemia(non APL),HAD regimen and IA regimen were selected for the first time,and the remission rate,OS and PFS were analyzed retrospectively.Results1.RT-PCR and Western Blot results confirmed that: FLT3,STAT5,Idl gene was highly expressed in the three cell,the lowest expression in normal control group,in MV4-11 cells was the highest among the three groups,FLT3,STAT5,Idl gene expression was statistically significant difference(P < 0.05);Mcl-1 gene in three cells has the expression,but the expression in MV4-11 cells was significantly increased,2 times higher than that of K562 cells,and the difference had statistical significance between the two groups(P < 0.01).2.Normal cultured MV4-11 cells and siRNA negative control group STAT5 mRNA expression remained at a high level,the expression of MV4-11 in mRNA cells transfected with siRNA-STAT5 of the other four groups were significantly decreased,and the normal cultured MV4-11 cells and siRNA negative control group had a significant difference(P < 0.05).However,siRNA-STAT5-2624 group had a significant inhibitory effect on the expression of STAT5 in mRNA,and the difference was statistically significant(P<0.05)between the two groups(siRNA-STAT5-2624),and then transfected into MV4-11 cells.3.After the silencing of STAT5 gene in 24 h,48h,detection of MV4-11 cells in different time periods of apoptosis,the results were not statistically significant(P >0.05).4.After silencing the STAT5 gene,detection of Id1,the expression of Mcl-1 gene,the results showed that: 48 hours after the Id1 2-CT value: 0.52(0.46-0.59),compared with the control group,the expression of Id1 mRNA was statistically significant difference(P < 0.05).Compared with the expression of Mcl-1 in the mRNA gene,the Mcl-1 value of 2-CT was 1.30(1.20-1.41)at the end of the last 48 hours.Compared with the control group,there was no significant difference in the expression of Mcl-1in the mRNA gene(P >0.05).5.HHT alone has a significant inhibitory effect on the growth of MV4-11 cells in a concentration and time dependent manner,and the effect is most significant at 72 hours,and the difference is significant(P < 0.05).Inhibition of cell growth effect was enhanced with SOR alone,the difference between groups was significant(P < 0.05);the combination of two drugs in 72 hours,the most significant effect,with HHT,compared with SOR alone,there were significant differences(P < 0.05).6.After using HHT alone,there was no significant change in 24 h concentration in each group,and there was no significant difference compared with the control group(P > 0.05);10ng/ml HHT was highest in the 72 hours of apoptosis,there was significant difference(P < 0.05);single SOR can effectively induce cell apoptosis(P <0.05);the two drug combination in 72 hours after the MV4-11 cells have an effect of cell apoptosis significantly(P < 0.05).7.Detection of FLT3,STAT5,Id1 gene expression changes confirmed mRNA:The FLT3,STAT5,Id1 mRNA expression levels were decreased with the increase of dose in 72 hours after HHT in group,there was significant difference between the two groups(P < 0.05).With sorafenib 24 h,48h,72 h,the expression level of FLT3,STAT5,Id1 and mRNA decreased with time,and there was significant difference between the groups(P <0.05).The combined effect of HHT and SOR two kinds of drugs,and the decrease of higher inhibitory effect on FLT3 and STAT5,Id1 gene mRNA with dose,and HHT alone and SOR,the two drug combination could significantly inhibit the expression of FLT3,STAT5,Id1 gene mRNA(P < 0.05).8.Mcl-1 gene of mRNA were different from the former three genes: 1ng/ml,5ng/ml with the effect of HHT on cells after 72 hours,no mRNA expression level of Mcl-1gene was significantly decreased with the dose of HHT increased,between the two groups compared with the control group had no significant difference(P > 0.05);10ng/ml HHT had significant inhibition on Mcl-1 gene the expression of mRNA,there was significant difference compared with the control group(P < 0.05).The expression level of Mcl-1 gene was decreased after using Sola Feeney 72h(26.13+ /-3.07),which was significantly different from that of the control group(P < 0.05).Two kinds of combined action of drugs after 72 h,and decreased increased inhibitory effect on Mcl-1 gene mRNA with dose,and HHT alone and SOR,the expression of the two drug combination could significantly inhibit the Mcl-1 gene of mRNA(P <0.05).9.Effects of drugs on the expression :Confirmed Western Blot detection with single HHT or sorafenib can inhibit FLT3 and STAT5,the expression level of Id1 protein.After 72 h with HHT and SOR,the expression of three proteins decreased significantly,and HHT alone and sorafenib were significantly different.10ng/ml HHT on the expression of Mcl-1 protein had no significant difference.Sorafenib decreased the expression of Mcl-1 protein;two drug combination can significantly inhibit the expression of Mcl-1 protein,and HHT alone and Sorafenib were significantly different.10.Clinical research: CR rate of IA was 53.4%(31/58,53.4%),and the CR rate of HAD was 78.9%(30/38,78.9%).In the treatment group for the first time CR rate compared with a significant difference(P = 0.02),HAD induced remission effect is stronger than the IA scheme.4 year overall survival of IA and HAD group(OS)had significant difference(P =0.008),respectively(27% + 7%)and(37% + 10%);disease progression free survival(PFS)compared with significant difference(P = 0.012),respectively(39% + 7%)and(62% + 10%),the HAD program is conducive to the long-term survival of the patients.ConclusionThis topic through the first three parts of the experiment confirmed that FLT3/ITD in +AML cells,the expression of FLT3,STAT5,Id1,Mcl-1 gene significantly increased,but in silencing STAT5 gene expression did not induce cell apoptosis and inhibit Mcl-1 gene,can significantly inhibit the expression of Id1 gene;that STAT5 is closely related with Id1.But the Mcl-1 gene may be the key site closely related to the apoptosis of MV4-11 cells,worthy of further study.HHT alone can not effectively induce cell apoptosis and can not effectively inhibit the expression of Mcl-1 gene.The proliferation and promotion of HHT combined with SOR can significantly inhibit MV4-11 cell apoptosis and expression of FLT3,STAT5,Id1.inhibition of Mcl-1 gene mRNA and protein levels,Two drugs more than single drug had significant difference,confirmed the association of synergistic effect of the two drugs,and explore new ideas for clinical treatment.The effect of HHT and the experiment of siRNA-STAT5 confirmed that the Mcl-1 gene may be closely related to the apoptosis of MV4-11 cells,which should be the key gene in the future FLT3/ITD +AML clinical and basic research.Clinical study of the last part verified the chemotherapy based on HHT(HAD)FLT3/ITD +AML for remission induction is better than the classical IA scheme,is conducive to the long-term survival of the patients,worthy of clinical samples to expand in-depth study.