| Recent researches show that various kinds of leukemia all highly express the vascular endothelial growth factor(VEGF),and the expression level of VEGF in the leukemic patients at the developing and late period is even higher.Restraining the proliferation of the leukemic cells and releasing VEGF may probably inhibit the growth and metastasis of tumor,and improve the prognosis.So far,survivin is the strongest apoptosis inhibitor.It also is detected to the excessive expression of survivin protein ever detected in the proterozoic leukemic cells of the ALL,AML and the CML at the accelerating period and blast crisis.Diallyl disulfide(DADS)is a valid lipid soluble composition in the garlic and may inhibit the leukemic cells.This experiment investigate the effect of DADS on the expression of VEGF mRNA and the secretion of VEGF proteins during the proliferation of human leukemic cell line HL-60,and to explore the inhibition mechanism in HL-60 cells.This experiment also determines whether the expression of survivin protein is down-regulated in HL-60 cells at the same time,and hopes to provide evidence for anti-tumor experiments of the clinical application of DADS. PART ONE Effect of diallyl disulfide on the expression and secretion of VEGF in human leukemic cell line HL-60Objective To investigate the inhibitive effect of DADS on the proliferation of human leukemic cell line HL-60 and the expression of vascular endothelial growth factor(VEGF)mRNA and secretion of VEGF protein in HL-60 cellsMethods MTT was used to test the effect of DADS on the growth of HL-60 cells;Semi-quantitative RT-PCR and ELISA were used to determine the expression of VEGF mRNA and secretion of VEGF protein in HL-60 cells treated by DADS.Results DADS significantly inhibited the proliferation of IlL-60 cells and the inhibiting effect was dose-dependent(r＞0.9,P＜0.01)and time-dependent(r＞0.7,p＜0.01).The expression of VEGF mRNA and secretion of VEGF protein could be down-regulated by 0.625,1.25,and 2.5μg/mL DADS-treated HL-60 cells for 48 and 72 hours and the effect was dose-dependent(r＞0.9,P＜0.01).The differences in the DADS-treated HL-60 cell groups and the control group were statistically significant(P＜0.01).There were also statistically significant differences among the three DADS-treated HL-60 cell groups(P＜0.01).Conclusion DADS can effectively inhibit the proliferation of human leukemic cell line HL-60.Mechanism of the anti-leukemia effect of DADS might be to reduce the expression of VEGF mRNA and secretion of VEGF protein in HL-60 cells.PART TWO Effect of diallyl disulfide on the proliferation of human leukemic cell line HL-60 and its mechanismObjective To investigate the effect of DADS on the proliferation of human leukemic HL-60 cell line and the correlation between the expression of survivin protein and the cell cycleMethod MTT was used to test the growth of HL-60 cells treated by DADS;SP-immunohistochemical and flow cytometry were used to determine the expression of survivin protein in HL-60 cells and the cell cycle arrest treated by DADS.Results DADS significantly inhibited the proliferation of HL-60 cells and the inhibiting effect was both dose-dependent and time-dependent(r＞0.9,P＜0.01).The inhibition fraction of HL-60 cells rose from 8.94%to 70.66%with DADS increasing from 0.625μg/mL for 24 hours to 20.0μg/mL for 72 hours.Survivin was highly expressed in blank control group and DMSO solvent group.Their SII was 1125 and 1126 respectively and the difference was not statistically significant(P＞0.05).The expression of survivin protein could be down-regulated by 5, 10,and 20 mg/L DADS-treated HL-60 cells for 24,48,and 72 hours and the effect was both time-dependent and dose-dependent(r＞0.9,P＜0.01). The SII of HL-60 cells treated by 20 mg/L DADS for 72 hours was 119, which was the lowest level in the test.The differences in the DADS-treated HL-60 cells groups and the control group were all statistically significant(P＜0.01),and there were statistically significant differences among the three DADS-treated HL-60 cells groups(P＜0.01). G2/M phase arrest which was accompanied with the down-regulation of survivin protein could be found in DADS interference in a dose- and time- dependent manner(r＞0.9,P＜0.01).After being treated with 20μg/mL DADS for 72 hours,the percentage of cells in G2/M phase rose to the highest level of 66.56%.Conclusion DADS can effectively inhibit the proliferation of human leukemia cell line HL-60,down-regulate the level of survivin protein and bring about G2/M phase arrest.In conclusion,DADS can effectively inhibit the proliferation of human leukemia cell line HL-60,down-regulate the level of survivin protein and bring about G2/M phase arrest.The mechanism of the anti-leukemia effect of DADS might be to reduce the expression of VEGF mRNA and secretion of VEGF protein in HL-60 cells.
|
PDF Full Text Request