Bevacizumab Targeted Therapy Of Ovarian Cancer And Its Inhibition On SKOV3 Cell Of VEGF Autocrine Pathway

1.Objective 1) Aim to find the relationship between the expression and clinical prognosis of VEGF and its receptor VEGFR1(Flt-1)、 VEGFR2(KDR) in epithelial ovarian carcinoma. 2) Aim to research the mechanism of VEGF autocrine phenomenon in epithelial ovarian cancer and the proliferation inhibition of Bevacizumab in SKOV3.2. Method 1) The research on the expression and prognostic significance of VEGF and its receptor VEGFR1、 VEGFR2 in epithelial ovarian carcinoma.(1)The difference in expressing of VEGF, VEGFR1 and VEGFR2 in normal tissues、 benign ovarian tumor and epithelial ovarian cancer.(2) The relationship of VEGF and VEGFR1, VEGFR2 in epithelial ovarian cancer tissue between the expression and clinical pathological parameters.(3) The correlation between the expression of VEGF and VEGFR1, VEGFR2.(4)The comparation on 5- year survival rates between VEGF and VEGFR1, VEGFR2 co- high expression than others. 2) The research on mechanism of VEGF autocrine phenomenon in EOC cell.(1) Comparison and screening of differentially expressed VEGF, VEGFR1(Flt-1), VEGFR2(KDR) in epithelial ovarian cancer cell lines.(2) The content of VEGF detected in serum culture medium of different times.(3)The proliferation of the selected cell line by adding different concentrations of rh VEGF.(4) Verification of the mechanism mainly combined by VEGFR2(KDR) in cell VEGF autocrine pathway. 3) The study on mechanism of which Bevacizumab inhibits proliferation in EOC cell.(1) Morphological changes of different concentrations of Bev in SKOV3 cells was observed by inverted microscope.(2) Detection the cells proliferation of different Bev concentrations by MTT.(3) Using the flow cytometry to monitor SKOV3 apoptosis with Annexin-FITC/PI staining.(4) Western Blot(WB) analysis of the expression of SKOV3 in Flt-1, KDR and p-KDR by adding different concentrations of Bev.(5) Selective inhibitor of KDR receptors, Ki8751, preliminary infer its downstream of ERK may be involved in SKOV3 cell’s VEGF autocrine pathway which the Bev directly inhibits.3. Results 1) The staining intensities of VEGF and its receptor VEGFR1, VEGFR2 in EOC tissue were significantly higher than that in benign ovarian tumors and normal ovary, which also reached a level of statistics significance in the aspects of FIGO stage and different ascites volumes. In the correlation analysis, VEGF and VEGFR2(r=0.316, p<0.05), VEGFR1 and VEGFR2(r=0.415, p<0.05) were highly associated. High co-expression of the three had a statistical significance in 5-year survival rate than others(p=0.044). 2) There are co-expression and relatively high expression of VEGF and its receptor Flt-1, KDR in human ovarian cancer cell line SKOV3(p<0.05). Different concentrations of rh VEGF can promote the proliferation of SKOV3, and showed dose dependence, which may be mediated by KDR(t=7.64; t=5.53; t=4.22; t=6.59, p<0.05;), not by Flt-1(t=2.05; t=2.34; t=1.32, p>0.05). While exogenous rh VEGF can stimulate VEGFR2(KDR)(t=5.81, *P<0.05) and p-VEGFR2(p-KDR)(t=12.43, *P<0.05) increasing expression in SKOV3 cell, suggesting that the VEGF autocrine mechanism in SKOV3 is mainly mediated by KDR, rather than Flt-1, and through p-KDR phosphorylation activating downstream signal transduction pathway. 3) Different concentrations of Bevacizumab could restrain cell reproduction, add apoptosis of SKOV3(*P<0.05); WB told us Bev produced effect on which may be media by the block of VEGF autocrine loop, namely reducing the corresponding receptor VEGFR2(KDR) /p-VEGFR2(p-KDR) expression(*P<0.05), and not by VEGFR1(Flt-1)(P>0.05). In addition, the expression of ERK and p-ERK in Bev, Ki8751(VEGFR2 inhibitor) and Bev+Ki8751 group are significantly higher than control one(*P<0.05), showing that Bevacizumab may inhibit VEGF autocrine by descreasing the ERK and p-ERK, the downstream signalling pathway of VEGFR2, to further inhibit the proliferation of SKOV3.4. Conclusion 1) The staining intensities of VEGF and its receptor VEGFR1, VEGFR2 in EOC tissue were significantly higher than that in benign ovarian tumors and normal ovary, which also reached a level of statistics significance in the aspects of FIGO stage and different ascites volumes. High and co-expression of VEGF, VEGFR1 and VEGFR2, indicating a poor prognosis, can be used as a guide to choose clinical targeted therapy of anti-angiogenesis drug(Bev) and to predict or evaluate the effectiveness of it. 2) There is VEGF autocrine mechanism in human ovarian cancer cell line SKOV3. rh VEGF can increase the proliferation of SKOV3, also active and up the expression of KDR, the surface receptors, and intracellular p-KDR, the downstream signal to further active autocrine loop. 3) Bevacizumab can reduce the expression of KDR and p-KDR by blocking SKOV3 cell VEGF autocrine loop, while Bevacizumab may inhibit cell autocrine function by indirect inhibition of expression of ERK and p-ERK, downstream signaling molecules of KDR, to demonstrate that Bev could inhibit SKOV3 cell’s proliferation through MAPK pathway inhibiting VEGF autocrine mechanism.