| Objective:This study was designed to investigate the effect of Zhebei Huangqin decoction on proliferation、apoptosis and chemosensitivity in human-derived leukemia cell lines Kasumi-1.Wip1-p38MAPK-p53 signaling pathway and autophagy effect were analyzed.Exploring the molecular mechanism of Zhebei Huangqin decoction-mediated anti-leukemia activity.Method:1.The proliferation of Kasumi-1 exposure to chemotherapy were examined using MTS assay.HL-60 cells that are more sensitive to chemotherapy were used as control.Kasumi-1 cells were intervened with different concentrations of daunorubicin or cytarabine,the inhibition rate of cell proliferation in each Kasumi-1 cells was determined by measuring the absorbance at OD=490nm.Susceptibility to chemotherapy was determined by the method above.2.Kasumi-1 cells were intervened with different concentrations of the alcoholic extracts of Zhebei Huangqin decoction,the inhibition rate of cell proliferation in each Kasumi-1 cells was detected by MTS assay.3.Kasumi-1 cells were divided into three groups,including blank control group,chemotherapy group,and a combined group.The chemothapy group was intervened with daunorubicin and cytarabine.The combined group was intervened with the alcoholic extracts of Zhebei Huangqin decoction,as well as daunorubicin and cytarabine.The inhibition rate of cell proliferation in each group was detected by MTS assay.Apoptosis analysis using annexin V/PI double staining was determined by flow cytometry.4.Wip1、p38MAPK、p53 protein and mRNA levels were analyzed by real-time PCR and western blot,respectively.5.This study further examined effects of Zhebei Huangqin decoction on autophagy,focusing on the changes in LC3 protein by using Western blot assay.Result:1.Kasumi-1 cells were intervened with 0.01 ug/ml、0.02 ug/ml、0.05ug/ml、0.1 ug/ml、0.5 ug/ml、1 ug/ml、2 ug/ml、5 ug/ml daunorubicin,respectively.The inhibition rate of cell proliferation was-7.21±5.5、-6.47±5.84、0.02±4.60、0.97±8.78、47.6±2.04、62.67±1.00、62.52±1.41、56.94±1.12,respectively.Human-derived leukemia cell lines HL-60 was used as control,HL-60 cells were intervened with daunorubicin that with 0.01 ug/ml-1 ug/ml concentration above,respectively.The inhibition rate of cell proliferation was 6.46±5.54、23.94±3.26、58.47±1.73、63.07±2.15、73.25±0.3、73.25±0.16,respectively.The IC50 of daunorubicin to Kasumi-1 and HL-60 were 2.833ug/ml and 0.092 ug/ml,respectively.The multiple resistance of Kasumi-1 to daunorubicin was 30.79.Daunorubicin 0.1ug/ml was selected for follow-up experiment.Kasumi-1 cells were intervened with 0.1mg/ml、0.5 mg/ml、1 mg/ml、2 mg/ml、4 mg/ml、8 mg/ml、10 mg/ml、20 mg/ml cytarabine,respectively.The inhibition rate of cell proliferation was17.65±9.34、22.09±10.16、27.64±6.10、37.13±6.05、46.06±4.61、64.86±1.23、69.55±1.35、77.92±0.52,respectively.HL-60 cells were intervened with 0.01 mg/ml、0.02 mg/ml、0.05 mg/ml、0.1 mg/ml、0.5 mg/ml、1 mg/ml、2 mg/ml、5 mg/ml cytarabine,respectively.The inhibition rate ofcell proliferation was28.51±1.92、45.55±5.81、63.36±4.56、68.64±7.03、70.92±2.18、72.99±2.32、64.79±2.32、74.42±1.16,respectively.The IC50 of cytarabine to Kasumi-1 and HL-60 were 7.820 mg/ml and 0.070 mg/ml,respectively.The multiple resistance of Kasumi-1 to daunorubicin was 111.71.Daunorubicin 2mg/ml was selected for follow-up experiment.2.Kasumi-1 cells were intervened with 25mg/ml、50mg/ml、100 mg/ml、200 mg/ml、400 mg/ml、800 mg/ml、1600 mg/ml the alcoholic extracts of Zhebei Huangqin decoction.respectively.The inhibition rate of cell proliferation was 15.17±2.57、17.59±2.62、24.72±1.84、24.99±3.79、23.74±2.25、25.44±1.45、21.27±0.66,respectively.The alcoholic extracts of Zhebei Huangqin decoction 200ug/ml was selected for follow-up experiment.3.Kasumi-1 cells were divided into three groups,the intervention drug of chemotherapy group was daunorubicin 0.1ug/ml+cytarabine 2mg/ml,the intervention drug of combined group was Zhebei Huangqin Decoction 200ug/ml+daunorubicin 0.1ug/ml+cytarabine 2mg/ml,and the blank group had no drug intervention.After 48 hours of drug intervention,the proliferation inhibition rate was 29.00±2.85 in the chemotherapy group and 62.52±2.13 in the combined group,the difference was statistically significant(P<0.05).After 24 hours of drug intervention,Annexin V/PI double staining and flow cytometry were used to detect the apoptotic rate.The early apoptotic rate of blank group,chemotherapy group and combined group were 4.14%,10.4%and 23.5%,the late apoptotic rate was 1.7%,3.37%and 7.48%,and the overall apoptotic rate was 5.84%,13.77%and 30.98%,respectively.The alcoholic extracts of Zhebei Huangqin decoction can partially reverse the chemoresistance of Kasumi-1 cells and promote apoptosis.4.The ethanol extract of Zhebei Huangqin Decoction significantly decreased the mRNA expression of wip1,p38 and p53 in Kasumi-1 cells.Real-time RCR showed that the relative expression of wip1 mRNA in blank group,chemotherapy group and combination group was 1、73.52773±1.27401、6.587956±6.5439,respectively.And the relative expression of p38 mRNA was 1、26.65348±26.60724、0.005633±0.000147,respectively.And the relative expression of p53 mRNA were 1、127.2598±14.27375、10.51457±10.37919,respectively.The differences were statistically significant(P<0.05).The relative expression levels of wip1,p38 and p53 mRNA in the chemotherapy group were significantly higher than those in the blank group.The combination of Zhebei Huangqin Decoction and chemotherapy could significantly reduce the expression of these genes.Western blot showed that the expression of p38MAPK protein in the combination group was significantly lower than that in the chemotherapy group,while the expression of wipl and p53 protein was not detected.5.Zhebei Huangqin decoction can promote the autophagy of Kasumi-1 cells.Western blot results showed that the autophagy protein LC3 of Kasumi-1 was significantly decreased in daunorubicin or cytarabine group,but increased in combined group.Conclusion:Zhebei Huangqin Decoction reverses drug resistance of myeloid leukemia cells by regulating wip1-p38MAPK-p53 signaling pathway. |
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