Study On Effect Of Vascular Endothelial Growth Factor(VEGF) Antisense Oligonucleotide (ASODN)on Human Leukemic Cell Line HL60

Objective:VEGF is a kind of multifunction glycoprotein .It's a specific mitogen for endothelial cells and a vascular growth factor .Overexpression of VEGF is correlated with the malignancy of solid tumors,Increased serum VEGF was found in leukemia patients.The recent study of leukemic cells in vitro shows that leukemic cells can secrete endogenous VEGF and express VEGF receptors(VEGFR),VEGF can not only promote endothelial cells to proliferate and secrete other cytokines but is involved in bone marrow angiogenesis,which can contribute to indirectly leukemic cells proliferation.In addition,VEGF binding to its special receptor can stimulate leukemic cells to proliferate and decrease the sensibility of chemotherapy and radiotherapy.Little is known about mechanism of this action.some scholars support that VEGF can directly promote leukemic cells to proliferate, other believes that VEGF has anti-apoptotic effects.Antisense oligonucleotide technology plays an important role in the tumor gene therapy.And it can directly block aberrant intrinsic signal pathway against tumor growth.There is no report on the relations between VEGF ASODN and leukemic cells apoptosis so far.VP16 induces leukemic cells apoptosis through inhibition of topoisomeraseI .Could VEGF enhance VP16-induced apoptosis in HL60 cells? In this study,We investigate expression of VEGF mRNA after VEGF ASODN transferred into HL60 cells.to clarify the relations between VEGF ASODN and leukemic cells apoptosis and whether combination of VEGF and VP16 enhanced VP16-induce apoptosis in leukumic cells or not.Materials and methods:l.Leukemic cell line HL60 is provided by Department of histology and Embryology of ZhengZhou University.2.Cells are divided into three groups:VEGF ASODN group,VEGF missense oligonucleotide (VEGF MSODN) group,non-transfection group.First,We transferred phosphorothiate VEGF ASODN into human leukemic cells in vitro with cation poly mediated method.Then the inhibitory rate of cell proliferation is assayed by MTT after 24h and 48h incubation.Finnally optimal concentration and time was found.3.0ptimal concentration of VEGF ASODN is transfected into HL60 cells.At 48h we measured the level of expression of VEGFmRNA by RT-PCR.4.Detection of cell apoptosis:Apoptotic body by morphology,DNA ladder formation of cell apoptosis by agarose gel electrophoresis,Annexin V FITC(+)/PI(-)(±) analysis by FCM.5.The rate of cell apoptosis is determined by FCM Annexin V FITC/PI after combination of VEGF ASODN with VP16.6.The results were analyzied with ANOVA analysis by SPSS 1 O.O.We established the standard of statistical significance as a ^0.05.Results:l.There is statistic significance between The inhibitory rate of cells proliferation of ASODN group and that of control groups under the same condition (VEGF ASODN:24h-1.62%, 28.65%, 39.64%; 48h-37.51%, 52.40%, 67.29%;VEGF MSODN: 24h-0.46%, 2.14%, 2.20%; 48h-1.76%, 2.93%, 3.52% ) (P<0.05) .The inhibitory rate of cells proliferation of each concentration of ASODN has statistical significance in different time(24h or 48h) (5/*mol/L VEGF ASODN group 1.62 %,37.51 %, lO^mol/L VEGF ASODN group 28.65 %, 52.40%, 20/Mmol/L VEGF ASODN group 39.64%, 67.29%) (P<0.05) .48h, 20/anol/L VEGF ASODN is the optimal time and concentrationg among all groups.2.The relative expression of VEGF mRNA of each group(ASODN group, MSODN group and non-transfection group)is 1.11 + 0.03, 1.08+0.02, 0+0.01 by RT-PCR after 20/amol/L VEGF ODN transfected into HL60 cells for 48h incubation. Significant difference between ASODN and control groups was found (P<0.05 ) .3.1nspections of cell apoptosis after 20[amol/L VEGF ASODN transfected into HL60 cells for 48h incubation,In ASODN group The number of clusters of cell is decreased,Cells apoptosis morphologies,such as cells shrinkingmore gtanula in cytoplasm, nuclear contracting and many fragments of cell were fbundJn control groups, However, cells is plump and clear,and grow healthily.The results of agarose gel electrophoresis showed that DNA ladder was found in ASODN group.But there is one strand of DNA in control groups.The rate of cell apoptosis is 19.46% in ASODN group by FCM Annexin V FlTC/PiBut only 3.67%^ 1.89% in MSODN and non-transfection groups/There is a significant difference among ASODN group and control groups (P<0.05) .4.The rates of HL60 cells apoptosis in combination of VEGF ASODN with VP16(5 u g /L) group is 20.87% or 23.28% after 24h or 48h incubtion,which is higher significantly than 4.25% or 4.70% in VP16(5u g /L) alone group( (P<0.05) . The rates of HL60 cells apoptosis in combination VEGF ASODN with VP16(10u g /L) group is 25.59% or 30.07% after 24h or 48h incubtion,which is higher significantly than that in VP16(10 Hg/L) alone group (P<0.05) .The rates of HL60 cells apoptosis in combination of VEGF ASODN with VP16(20 P g /L) group is 36.47% or 41.72% after 24h or 48h incubation,which is higher significantly than that in VP16(20u g /L) alone group (P<0.05) .The data suggests that combination of VEGF ASODN with VP16 has high rate of cells apoptosis in comparision with that of VP16 alone.The difference between the rates cells apoptosis combination of VEGF ASODN with VP16 at 24h and that of 48h (24h: 5 u g/L VP16 + VEGF ASODN group 20.87%, 48h: 5 u g/LVP16 + VEGF ASODN group 23.28%; 24h: 10 u g/L VP16+VEGF ASODN group 25.59%, 48h: 10 u g/L VP16+VEGF ASODN group 30.07%; 24h:20n g/L VP16+VEGF ASODN group36.47%, 48h:20ug /L VP16+VEGF ASODN group 41.72%) has statistical significance (P<0.05 ) . Significant difference of the rate cell apoptosis was found among groups of different concentrationgof VP16/VEGF ASODN (24h: 5 u g/LVP16/VEGF ASODN group 20.87%, 10 u g/LVP16/VEGF ASODN group 25.59%, 20 u g/LVP16/VEGF ASODN group 36.47%; 48h: 5 u g/LVP16/VEGF ASODN group 23.28%, 10 u g/L VP16/VEGF ASODN group 30.07 %, 20 u g/L VP16/VEGF ASODN group 41.72%) at the same time (P<0.05) (P<0.05) .ConJusionsl.VEGF ASODN can down-regulate expression of VEGF mRNA of HL60 cells.2.VEGF ASODN can induce the apoptosis and inhibit the proliferation of HL60 cells.3.VEGF ASODN can enhance VP16-induced apoptosis of HL60 cells.