VEGF111b, a C-terminal splice variant of VEGF-A and induced by mitomycin C, inhibits ovarian cancer growth

Xiuli Li, Fang Gu, Chenguang Niu, Yuanfen Wang, Zhongyu Liu, Na Li, Bing Pan, Dan He, Jian Kong, Shaobo Zhang, Xu Wang, Yuanqing Yao, Lemin Zheng

Alternative splicing of VEGF-A gives rise to two families - the pro-angiogenic VEGFxxx family and the anti-angiogenic VEGFxxxb family that differ by only six amino acids at their C-terminal end. The first verified and widely reported VEGFxxxb family member is VEGF165b, and here VEGF165b is a positive control. VEGF111b mRNA was detected in ovarian cancer cell lines SKOV3 and OVCAR3 by RT-PCR. Western blot was used to detect VEGF111b and VEGF165b protein in the CMs and lysates of OVCAR3 cells. MTT and colony formation assay were used to detect the short-term and long-term proliferation inhibition ability of ovarian cancer cells with VEGF111b overexpression. Cell-cycle analysis was performed to further characterize VEGF111b inhibition effects. VEGF111b signaling on ovarian cancer cells were determined by western blot. The expression levels of Ki67, PCNA, CD31 and VEGF in VEGF111b overexpression xenograft model were detected by immunohistochemistry. Under the effect of mitomycin C, we identify a new member of VEGFxxxb family-VEGF111b in ovarian cancer cell lines. SKOV3 and OVCAR cells were transfected with empty lentivirus, VEGF111b or VEGF165b lentivirus. VEGF111b and VEGF165b overexpression inhibits proliferation of the ovarian cancer cells, but inhibition effect of VEGF111b is slightly less efficient than VEGF165b. Cell cycle analysis was further used to elucidate the mechanism involved in the inhibition effect. Further, we detected the expression of VEGF-R2 in SKOV3 and OVCAR3 cells, and shown that VEGF111b might bind to conventional VEGF-R2 with the results of reducing VEGF-R2 tyrosine phosphorylation and downstream signaling to have anti-tumor effects. In vivo VEGF111b overexpression inhibits ovarian cancer growth in xenograft mice. Our results show that VEGF111b, as a new member of VEGFxxxb family, with similar properties to VEGF165b, plays potent anti-tumor effect in vitro and in vivo that can target the VEGF-R2 and its signaling pathway to inhibit ovarian tumor growth. This also opens a new avenue for treating ovarian cancer.