Huaier Reverses The Resistance Of Human Acute T-Lymphocytic Leukemia CEM-C1 Cells To Glucocorticoids

Objective: To investigate whether Huaier can reverse the glucocorticoid(GC)resistance of dexamethasone(DEX)-resistant human acute T-lymphocytic leukemia(T-ALL)cell line CEM-C1 and its possible mechanism.Methods:1.Cell culture: In vitro cell culture technology was used to culture human T-ALL cell CEM-C1 and CEM-C7 cell lines.2.Experimental grouping:(1)CEM-C1,CEM-C7 cells were divided into6 groups according to the intervention concentration of Huaier(50,100,200,400,600,800 ?g/ml);(2)CEM-C1 and CEM-C7 cells were divided into 6groups according to the concentration of dexamethasone(DEX)(12.5,25,50,100,200 ?g / ml);and different concentrations of DEX(12.5,25,50,100,200?g/ml)combined with Huaier(100?g/ml)to divide CEM-C1,CEM-C7 cells into 6 groups respectively;(3)CEM-C1 and sensitive Whether the cell line CEM-C7 was treated with Huaier optimized concentration(0,200,600?g/ml)and divided into 3 groups respectively;(4)CEM-C1 cells were divided into Control group,AZD1208 group and Huaier group and Huaier+AZD1208group.3.Cell counting kit-8(CCK8):(1)Detecting the change of the proliferation of two kinds of cells after treatment with different concentrationsof Huaier ear for 24 h,48h,72h;(2)Detecting the combination and treatment of DEX with different concentrations Changes in cell proliferation of Huaier in each group;(3)Determining the optimal intervention concentration and duration of Huaier;determining the IC50 of Huaier to the two cells and the sensitivity to DEX.4.Quantitative real-time PCR(qRT-PCR): Detecting CEM-C1 and CEM-C7 cells and the expression of PIM3 gene in CEM-C1 cells after Huaier interfering with different concentrations(0,200,600 ?g/ml).5.Western Blot:(1)Detection of PIM3 and MDR1 protein expression in CEM-C1 and CEM-C7 cells;(2)Detection of different concentrations(0,200,600 ?g/ml)of Huaier intervention in CEM-C1 cells expression of PIM3 and MDR1 proteins;(3)Detecting the expression of PIM3 and MDR1 proteins in each group of cells after treatment of CEM-C1 cells with the PIM3 inhibitor AZD1208(5?M).6.Detection of apoptosis:(1)Flow cytometry(FCM)was used to detect the apoptosis of two ALL cell lines after 48 h of intervention with different concentrations of Huaier;(2)Hoechst 33258 stain was used to detect different concentrations of Huaier Apoptosis of two ALL cell lines after intervention for48 hours.7.Cell cycle detection: The cell cycle of two ALL cell lines after 48 h intervention with different concentrations of Huaier was detected.Results:1.Huaier inhibits ALL cell proliferation:(1)Huaier inhibits CEM-C1,CEM-C7 cell proliferation in a dose-dependent manner,and the effect is most obvious at 48h;(2)IC50 is 313.4?g/ml and 757.5?g/ml of CEM-C1 and CEM-C7 cells respectively;(3)Experiments show that when the concentration of Huaier is less than 100?g/ml,there is no obvious toxic effect,that is,the inhibition rate of cell proliferation is <10%,and 100?g/ml is the intervention concentration when combined.2.Reversal effect of Huaier on drug resistance of CEM-C1 cells:(1)IC50of CEM-C1 cells treated with different concentrations of DEX for 48 h was98.89 ?g/ml,while IC50 of CEM-C7 cells was 0.16 ?g/ml,calculated according to the formula,the resistance multiple is 618.06 times;(2)the IC50 of DEX + Huaier for CEM-C1 cells is 73.75?g/ml,the resistance multiple is1.34 times,and there is no IC50 value for CEM-C7 cells significant effects;(3)Compared with DEX alone,Huaier+DEX inhibition rate increased significantly,and both Coefficient of Drug Interaction Index(CDI)were <1.3.Huaier promoted the apoptosis of ALL cells:(1)FCM detection with the increase of Huaier's concentration,the apoptosis rate increased significantly(P<0.05);(5)Hoechst 33258 detected Huaier to promote ALL in a dose-dependent manner apoptosis.4.The cell cycle arrest of Huaier in CEM-C1 and CEM-C7 cell lines was not significant(P> 0.05).5.Effect of Huaier on the expression of PIM3 in CEM-C1 cells:(1)Compared with CEM-C7 cells,the expression of PIM3-mRNA and protein in CEM-C1 cells increased significantly(P<0.05);(2)with The concentration ofHuaier the expression of PIM3-mRNA and protein in CEM-C1 cells was increased(P<0.05).6.Effect of Huaier on MDR1 expression in CEM-C1 cells:(1)As the concentration of Huaier increased,the expression of MDR1 protein decreased in CEM-C1 cells;(2)in CEM-C1 cells,compared with the Control group,the expression of MDR1 protein in the AZD1208 group was reduced(P <0.05);(2)in CEM-C1 cells,compared with the Control group,the expression of MDR1 protein was reduced in AZD1208 + Huaie group(P <0.05).Conclusion:1.Huaier can inhibit the proliferation of CEM-C1 and CEM-C7 cells and promote their apoptosis;2.Huaier reverses the resistance of CEM-C1 cells to GC to a certain extent,and the mechanism may be related to the down-regulation of PIM3.