Effect Of Zhebei Huangqin Decoction On Chemosensitivity Of Acute Myeloid Leukemia

Objective1.To observe the effect of Compound Fritillaria Scutellaria Decoction (CFSD)extracted by water,alcohol and acid water on acute myeloid leukemia HL60, HL60/ADR cell proliferation and chemotherapeutic drug sensitivity.2.To investigate the expression level of Wipl in peripheral blood neutrophils and mononuclear cells of refractory acute myeloid leukemia(RAML).3.To observe the effect of CFSD extracted by alcohol on acute leukemia HL60 and HL60/ADR cell apoptosis and its correlation with Wipl expression.4.To investigate the correlation between Wipl expression and chemosensitizing effect of CFSD extracted by alcohol in acute leukemia HL60 and HL60/ADR cell lines.Method1.MTS assay was carried out to detect the inhibitory rate and IC50 value of CFSD extracted by water,alcohol and acid water combined or not combined with ADR in acute myeloid leukemia HL60 and HL60/ADR cell lines.2.Peripheral blood neutrophils and mononuclear cells of 5 RAML patients(refractory group),3 chemotherapy-sensitive AML patients(CR group),3 healthy adults(normal control group) were isolated respectively.Total RNA was extracted from neutrophils and mononuclear cells of blood sample from 8 patients and 3 healthy adults.The Real-time PCR method was performed to detect the expression levels of Wipl.3.Annexin/PI double-staining method was used to detect cell apoptosis induced by alcohol extracted CFSD in HL60 and HL60/ADR cell lines. The Real-time PCR method was performed to detect the expression levels of Wipl.4.The Real-time PCR method was performed to detect the expression levels of Wip1 in HL60 and HL60/ADR cell lines. And changes of Wip1mRNA expression were detected in HL60 and HL60/ADR cells treated with ADR combined with noncytotoxic dose(< IC10) of CFSD extracted by alcohol. HL60 cell transfection with plasmid DNA(hWIPl-FLAG-pCMV-Neo-Bam and pCMV-Neo-Bam) was performed using Lipofectamine3000 Transfection Reagent. The IC50 values of ADR in HL60 cells transfected with plasmid hWIPl-FLAG-pCMV-Neo-Bam and pCMV-Neo-Bam were detected by MTS method.Result1.The IC50 value of CFSD extracted by alcohol(141.06± 11.29ug/ml)was higher compared with the CFSD extracted by water(177.28 ±4.00 ug/ml)and acid water (217.66 ±16.78 ug/ml)in HL60 cells,respectively, P< 0.05. In HL60/ADR cells,the IC50 values of CFSD extracted by water and alcohol were higher than 200ug/ml,the IC50 value of CFSD extracted by acid water was higher than 400ug/ml.The inhibition rates of 200 ug/ml CFSD extracted by water,alcohol and acid water was(27.28±4.69)%,(37.67±3.43)%,(25.39± 4.98)%,the inhibition rate of CFSD extracted by alcohol was higher compared with the CFSD extracted by water and acid water,respectively,P< 0.05.Noncytotoxic dose (< IC10) of CFSD extracted by water and alcohol could reduce the IC50 value of ADR in HL60 cells,the potentiation folds were 1.60 and 2.00,respectively.Noncytotoxic dose (< IC10) of CFSD extracted by alcohol could reduce the IC50 value of ADR in HL60/ADR cells,the reversal index was 1.50.2.Real-time PCR results showed that,WiplmRNA expression level of refractory group,CR group and normal control group in peripheral blood neutrophils were 0.0969±0.0684,0.0083±0.0078,0.0116(0.0021,0.0118). WiplmRNA expression level of refractory group was higher compared with the normal control group and the CR group,respectively,P<0.05. WiplmRNA expression level of CR group had no significant difference compared with the normal control group, P>0.05. WiplmRNA expression level of normal control group,CR group and refractory group in peripheral blood mononuclear cells had no significant difference,P>0.05.3.The early and late apoptosis rates in HL60 cells were increased after treated with CFSD extracted by alcohol,P<0.05. The early apoptosis rates in HL60/ADR cells were increased afer treated with CFSD extracted by alcohol, P<0.05; however, the late apoptosis rates in HL60/ADR cells did not change significantly, P>0.05.After treated with alcohol extracted CFSD,Wip1mRNA expression was down-regulated in HL60 cells, but had no significant change in HL60/ADR cells.4.Wip1mRNA expression level in HL60/ADR cells were lower than in HL60 cells.WiplmRNA expression level in HL60 cells treated with ADR combined with einoncytotoxic dose(< IC10) of CFSD extracted by alcohol was higher compared with the ADR group.WiplmRNA expression level in HL60/ADR cells treated with ADR combined with einoncytotoxic dose(< IC10) of CFSD extracted by alcohol was lower compared with the ADR group.The the IC50 value of ADR in HL60 cells transfected with plasmid hWIPl-FLAG-pCMV-Neo-Bam was 0.35±0.09ug/ml, the the IC50 value of ADR in HL60 cells transfected with plasmid pCMV-Neo-Bam was 0.36±0.17ug/ml, showing no significant differences between two groups,P>0.05.Conclusion1.CFSD extracted by water,alcohol and acid water inhibited HL60 and HL60/ADR cell proliferation at different degree,the inhibitory effect of ethanol extract was the strongest.The inhibitory effect of CFSD in HL60 cells were stronger than in HL60/ADR cells.Noncytotoxic dose (<IC10) of CFSD extracted by alcohol could increase the sensitivity of chemotherapeutics of HL60 cells,and partly revese the drug resistance of HL60/ADR cells.2.The expression level of Wipl in peripheral blood neutrophils of RAML patients was up-regulated in this research.Wipl maybe highly expressed in peripheral blood neutrophils of RAML.The expression level of Wipl in acute myeloid leukemia remains to be further studied.3.CFSD extracted by alcohol could promote cell apoptosis of HL60 and HL60/ADR cells. But the correlation brtween this apoptosis-inducing effect and Wipl expression needs further research.4.The Result in this research showed no correlation between Wipl expression and chemosensitizing effect of CFSD extracted by ethanol in HL60 and HL60/ADR cell lines.