Experimental Study On The Role Of VEGF Autocrine Loop In Leukemia Cells

Objective: To investigate the effects of anti-VEGF antibody on the proliferation and apoptosis of the leukemia cell line K562 when VEGF autocrine loop was blocked. To explore the possibility of inhibiting expression of VEGF in leukemia cells and observe whether the growth, apoptosis and tumorigenesis of cells could be affected after anti-VEGF hairpin ribozyme gene was introduced into the K.562 cells and its possible molecular mechanisms were then studied. To construct Short hairpin RNA (shRNA) eukaryotic expression vector specific to VEGF receptors Flt-1 and KDR and observe their silencing effects on Flt-1 gene and regulatory roles in the invasiveness of leukemia cells in vitro.Method: Firstly, the effect of gradient concentrations of 0.033 μg/ml~4.125μg/ml of anti-VEGF antibody on K562 cells was studied. The trypan blue exclusion assay and MTT assay were used to determine the growth curve and inhibited rate of K562 under different concentrations of anti-VEGF antibody. Expressions of MRP, TOPO Ⅱ and GST genes were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Agarose gel electrophoresis was performed to verify whether anti-VEGF antibody could induce apoptosis of K562 cells. Then, the recombinant eukaryotic expression plasmid(pcDNA3-RZ) including anti-VEGF hairpin ribozyme gene and the vector-alone were introduced into K562 cells by lipofectamine mediation and positive clones were screened by G418. Ribozyme gene in K562 cells was conformed by PCR. Fluorescent real time RT-PCR and western blotting were employed to detect the expression of VEGF mRNA and protein in leukemia cells. The proliferative ability of K562 cells was determined by MTT, methylcellulose semisolid clony forming assay and cell cycles analysis. Apoptosis of K562 cells was assayed by DNA agarose gel electrophoresis and Annexin V -FITC/PI double labeled flow cytometry. The tumorigenicity of transfected K562 cells transplanted in nude mice and microvasculardensity (MVD) in tumors were evaluated. Furthermore, a plasmid pEGFP-Cl containing human U6 snRNA promoter was ligated to a 19 bp reverse repeated motif of Flt-1 and KDR target sequence with 9 bp spacer, respectively. Enzyme digestion and DNA sequencing were used to exam whether the: recombinant plasmid Cl/U6/FltS and Cl/Ue/KDRS were correct. MMP-2 and MMP-9 mRNA were detected by RT-PCR after the recombinant plasmid Cl/U6/FltS2 transfected into K562 cells through liposome. The invasive ability of K562 cells was studied by Boyden chamber invasion assay before and after Flt-1 ShRNA transfection.,Results: The anti-VEGF antibody was able to inhibit growth and induce apoptosis of leukemia cells in a dose-dependent manner, and the growth inhibition rate was 13.2 + 6.3% when treated with antibody 0.033 u g/ml compared with 41.2±13.4% with dose of 0.825 u g/ml. Exposure to anti-VEGF antibody at 0.165 u g/ml for 72 hours, the K562 cell line exhibited typical DNA ladder observed by gel electrophoresis. The enhanced apoptosis rate appeared when antibody concentration reached 0.825 u g/ml. RT-PCR have showed a decreased level of MRP^ TOPOII genes and a relative constant expression of GST in leukemia cells. The pcDNA3-RZ and pcDNA3 had been transfected into the human leukemia cell line K562 and positive clones been screened by G418 for 2 weeks. Stable expression of the ribozyme gene in K562 cells was conformed by PCR. The level of VEGF mRNA and protein decreased dramatically in K562-RZ cells when compared with K562 or K562-PC(K562 cell transfected with empty vector) cells. The introduction of exogenous anti-VEGF ribozyme gene into K562 cells resulted in decreased levels of the proliferative ability and colony forming efficiency in K562 cells, higher Gl and lower S ratio in cell cycle distribution in comparison wilh the control groups. DNA electrophoresis and Annexin V/PI detection indicated typical DNA fragmentation and higher AnnexinV+ ratio in K562-RZ cells after treated with AS2O3 0.5uM for 3 days. Neoplasia formation test on nude mice showed that transfection of ribozyme caused less tumor growth after injection of leukemia cells, and ability of vessel formation reduced as well. Microvascular density of the tumor in K562-RZ groups was lower than that of the control groups (P<0.05). These results suggested that anti-VEGF hairpin ribozyme inhibited the over-proliferation of leukemia cells. The Flt-1 and KDR shRNA were successfullyinserted into eukaryotic expression vector pEGFP-Cl with U6 snRNA promoter and termination signal of RNA polymerase . Enzyme digestion by Pst I and Sail showed a 400bp DNA fragments. The sequences of shRNA in recombinant plasmid were same as that of designed fragments. Fit-1 gene was inhibited by plasmid-expressed shRNA after recombinant vetors C1/U6/FUS were transfectecl into K562 cells. The inhibitory effects of two different shRNA sequences targeting Flt-1 gene were 46.1% and 65.4%, respectively. The expression of MMP-2 and MMP-9 mRNA detected by RT-PCR was decreased, and the mean invasion percentage in C1/U6/FUS2 transfected K562 cells was lower than that in nontransfected cells.Conclusion: 1. Anti-VEGF antibody can inhibit growth, induce apoptosis and down-regulate the expression of MRP, TOPC) II genes of leukemia cells in vitro. 2. Transfection with anti-VEGF ribozyme gene can inhibit the proliferation of leukemia cells by delaying the progression Gi into S stage in cell cycles and induce cell apoptosis by down-regulating the VEGF gene expression. Neoplasia formation test on nude mice showed that transfection of ribozyme in K562 cells caused slower tumor growth and less vessel formation. 3. Eukargotic expression vectors of shRNA specific for Flt-1 and KDR were generated successfully. After Flt-1 shRNA were transfeced into K562 cells , the expression of MMP-2 and MMP-9 mRNA was decreased that may responsible for the lower invasive ability of transfected leukemia cells.