Estrogens stimulate the proliferation of human cholangiocarcinoma by inducing the expression and secretion of vascular endothelial growth factor

Background: Estrogens may induce the proliferation of neoplastic cells by activating neo-angiogenesis. Aim: To evaluate the effect of estrogens on the expression of vascular endothelial growth factor (VEGF) and related receptors (VEGF-R) in human cholangiocarcinoma and the role played by VEGF in mediating the proliferative effects of estrogens. Methods: Seven biopsies of intra-hepatic cholangiocarcinoma and the HuH-28 cell lines were investigated. Cell proliferation was measured by both PCNA Western blot and MTS proliferation assay. Results: By immunohistochemistry, biopsies of human cholangiocarcinoma stained positively for VEGF-A and VEGF-C and related receptors. HuH-28 cells expressed VEGF-A, -C, and VEGFR-1, -2, -3 and, their protein level was enhanced by 17β-estradiol in association with the stimulation of cell proliferation. 17β-Estradiol-stimulated proliferation of HuH-28 cells was blocked by 70% by VEGF-TRAP, a receptor-based VEGF inhibitor. 17β-Estradiol induced the secretion of VEGF in the supernatant of HuH-28 cells. The stimulatory effect of 17β-estradiol on the protein expression of VEGF-A, VEGF-C and VEGFR-1, -2, -3 was blocked by antagonists of ER (Ici182,780) or insulin-like growth factor 1-receptor (αIR3). Conclusions: With the limitations of experiments performed in a cell line, our study indicates that VEGF plays a major role in mediating the proliferative effects of estrogens on human cholangiocarcinoma. © 2008 Editrice Gastroenterologica Italiana S.r.l.