| BackgroundDaunorubicin is an anthracycline antibiotics, as the basic drug of clinic chemotherapyscheme because of its damage DNA, inhibit the topological isomerase Ⅱ (Topo Ⅱ) role.Butwith strong side effects, and happened resistance and recurrence easyly, make its applicationsubject to limit, the mechanisms of resistance may is NF-kB signal path abnormal activation andcause cells resistant gene expression increasing. PTEN gene is tumor-suppressor gene, is alsoregulatory factors of the start apoptosis. By inhibiting PIP3/AKT signal transduction pathways,reduce NF-kB molecular activation and play inhibiting tumor effect. PTEN gene exists differentdegree of loss and lower expression in leukemia, and its relate to promoter methylation, which isone important mechanism of the blood system disease occur. Decitabine is the methylationinhibitor, which it have certain curative effect for drug resistance and recurrence of leukemiapatients. DAC single drug has been successfully used in a variety of malignant blood diseases,but less in the side of combination drugs. We used different concentrations of DNR, DAC andthe two drug combination treatment HL-60cells by MTT method and flow cytometry to detectapoptosis rate,FQ-PCR detected the expression level of PTENmRNA, MSP detect themethylation status of PTENmRNA, which to provide the more theory basis for the clinicalcombine medication in the future.Methods1.HL-60Cells’s proliferation effect was analyzed by MTT assay;2.HL-60Cell’ s apoptosis rate was examined by floe cytometry(FCM);3.The expression of PTENmRNA level was detected by FQ-PCR;4.The methylation status of PTENmRNA by methylation specific PCR(MSP).Result1. MTT result displayed that after different concentrations and time points of decitabineand daunorubicin,cells have a certain effect of inhibition. The same group with time in theinhibition rate increased with the extension,72h time point reached a peak inhibition, difference was statistically significant (P <0.001); The same time with different concentration betweengroup compared and between the two drug exist interaction effects (P <0.001), difference wasstatistically significant; The inhibition effect of combined treatment group is more obvious (P <0.001).2. FCM displayed that the drug treatment group apoptosis phenomenon can be seen exceptthe control group have no significant apoptosis by72h training (Figure3A). Along with the DNRand DAC each single medicine group increase of concentration, HL-60apoptosis rate willincrease gradually（P <0.001）. Apoptosis rate reached24.17%(Figure3B)after1.0μmol/L DNReffect72h and8.5%(Figure3C) after5.0μmol/L DAC achieves,after the combination group,apoptosis rate increase significantly increased to36.5%（F=30.199，P<0.001）(Figure3D).3. FQ-PCR test results displayed, HL-60cells had a low expression of PTENmRNA. Withthe increase of DAC drug concentration, its expression increased.4. MSP technology testing results showed that: Promoter methylation state of the controlgroup in HL-60cells PTEN genes, after low concentration of DAC intervention, PTEN genepromoter methylation levels dropped, at the same time appear methylation and thenon-methylation strip.When drug concentration for5.0μmol/L, PTEN genes be foundedcompletely to methylation (figure5).Conclusion1. DAC ally DNR have a synergistic effect on HL-60cell proliferation and induction ofapoptosis;2. HL-60cells PTENmRNA low expression involved with gene promoter regions of theCpG island methylation;3. DAC can increase HL-60cell lines of sensitivity to DNR, its mechanism may relate torecovery or increase the expression of PTENmRNA relevant. |
PDF Full Text Request