Chidamide Sensitizes Acute Myeloid Leukemia Cell Line Kasumi-1 To Chemotherapy By Inhibit EZH2/Hedgehog Pathway

Background and ObjectivesAcute myeloid leukemia(AML)is the most prevalent malignant tumors with shorter survival and nearly most of patients will eventually relapse or change into refractory acute myeloid leukemia,One of the major causes of failure in the treatment is drug resistance.In recent years,it has been found that the induction of multidrug resistance phenotype of tumors is related to the regulation of epigenetics,Chidemide(CS055)is one of the histone deacetylase inhibitor(HDACi),it had reported that CS055 could cause chromatin remodeling by inhibiting HDAC to increase the acetylation of histones and decrease the methylation of histones,thereby altering the expression of multiple signaling pathway genes,and thus recovering the sensitivity of drug-resistant cells to drugs.Our previous study showed that AML cell line Kasumi-1 was resistant to adriamycin(ADM)and cytarabine.Silenting the EZH2 gene can significantly increase the drug sensitivity in Kasumi-1 cells by inhibit the Hedgehog pathway.The aim of this study is to investigate whether CS055 can increase the sensitivity of Kasumi-1 cells to chemotherapeutic drugs in vivo and in vitro,and to study its mechanism of reversing drug resistance.Materials and Methods1.CS055 effect the proliferation and apoptosis of Kasumi-1 cell:Using the cell counting kit-8(CCK-8)to detect the proliferation inhibition rate after treatment with various concentration and different time of CS055.Using the flow cytometric analysis the percentage of apoptotic cells in Kasumi-1 cells,after treatment with various concentration of CS055 for 48h.2.CS055 can increase the drug sensitivity in Kasumi-1 cells:Using the cell counting kit-8(CCK-8)to detect the proliferation inhibition rate after treatment with various concentration ADM and CS055 coupled with various concentration ADM,and according to IC50 values calculated the reversal fold.Using the flow cytometric analysis the percentage of apoptotic cells in Kasumi-1 cells,after treatment with CS055,ADM and CS055 coupled with ADM for 24h.3.CS055 downregulate EZH2/Hedgehog pathway:Western blotting analysis of the expression level of proteins:EZH2、Gli-1、Smo、DNMT3A、Acetyl-H3、H3K27me3,after treatment with CS055 for 48h on Kasumi-1 cells.4.1n vivo studies:Approximately 1×107 Kasumi-1 or Si-EZH2 Kasumi-1 cells were injected into the right posterior flank of 6 weeks old BALB/c nude mice subcutaneously.When the tumor size was about 150-200 mm3,the mice which bearing Kasumi-1 neoplasms were randomly divided into 4 groups:Kasumi-1 cells + vehicle;Kasumi-1 cells + ADM;Kasumi-1 cells + CS055;Kasumi-1 cells+ADM + CS055;Mice bearing Si-EZH2 Kasumi-1 neoplasms were randomly divided into 2 groups:Si-EZH2 Kasumi-1 cells + vehicle;Si-EZH2 Kasumi-1 cells + ADM.Calculation of tumor volume is V=0.5×longestxshortest2.After ten days of drug treatment,the mice were sacrificed and the tumors were weighting and then fixed in 10%neutralized formalin overnight.5.Histopathological and immunohistochemical staining examinations:Immunohistochemical(IHC)examined the EZH2、Gli-1、Smo、p-AKT、MRP1 protein expression of the tumor tissues.6.Statistical analysis:Statistical analysis was performed by SPSS 20.0.Using Student’ s t-test,one-way analysis of variance(ANOVA)followed by the Bonferroni test when equal variances were assumed,while Dunnett’s T3 test were used when equal variances were not assumed.P values<0.05 were regarded to be significantly different.Results1.Effect on proliferation and apoptosis in Kasumi-1 Cells treat with CS055:The cell counting kit-8 cytotoxicity experiment results indicated that various concentration and different time CS055 could significantly inhibit the Kasumi-1 cells proliferation,inhibition ratio was elevated accompanied with time extension and dose increase,it was a time-dose dependent.Apoptosis was detected by flow cytometry,various concentration CS055 could remarkably induce apoptosis,in a dose-dependent mode,the ANOVA stastical analysis indicated that CS055 induced significantly Kasumi-1 cell apoptosis,compared with CON group,there was significantly stastical differences(F=88.09,P=0.000).2.Effect on drug resistance in Kasumi-1 Cells treat with CS055:CCK8 experiment results indicated CS055 can increase the inhibitory effect of ADM on Kasumi-1 cells.The IC50 of 1umol/L CS055 combined with ADM was 0.301±0.082μg/ml,which was significantly lower than that of ADM single drug group(2.421±0.045μg/ml),and the reversal drug resistance was 8.1 times.Apoptosis results showed that CS055 significantly increased the apoptotic rate of Kasumi-1 cells,The total apoptosis rate of Kasumi-1 cells treat with CS055 coupled with ADM was 43.1%which was significantly higher than the blank group,CS055 group and ADM group.3.CS055 down-regulated the activity of EZH2/Hedgehog pathway:Western blot results showed that the expression of acetylated histone-3 protein in Kasumi-1 cells treated with CS055 for 48h was significantly higher than that of control group,and the expression level of EZH2,Smo,Gli-1,DNMT3A and H3K27me3 was significantly lower than that control.4.1n vivo study:The results showed that tumors grew rapidly and the tumor weight in Kasumi-1 CS055 alone group,Si-EZH2 Kasumi-1 ADM alone group,Kasumi-1 CS055 combined with ADM group were significantly lower than Kasumi-1 control group,Kasumi-1 ADM group and Si-EZH2 Kasumi-1 Control group(P<0.001).And the growth rate and tumor weight of Kasumi-1 CS055 combined with ADM group was lower than that of Kasumi-1 CS055 alone group(P<0.05).5.Immunohistochemistry:Immunohistochemistry examination showed that the expressions of EZH2,Smo,Gli-1,p-AKT,MRP1 in Kasumi-1 tumor tissues were high,and they were decreased after treatment with CS055.At the same time,the expression of Smo,Gli-1,p-AKT,MRP1 protein was also decreased after knock-down EZH2 in Kasumi-1 cells.Conclusion1.CS055 had a significant inhibitory effect on proliferation and apoptosis of Kasumi-1 cells in a time-dose-dependent manner.2.CS055 could increase the proliferation inhibition and apoptosis effect of ADM on Kasumi-1 cells.3.CS055 could reduse the expression of EZH2 in Kasumi-1 cells to inhibit histone methylation and DNA methylation,and then down-regulate Hedgehog signaling pathway and the downstream p-AKT/MRP1 to reverse the resistance of Kasumi-1 cells to chemotherapeutic drugs.