Calcitriol reduces thrombospondin-1 and increases vascular endothelial growth factor in breast cancer cells: Implications for tumor angiogenesis

Calcitriol, a potent antineoplastic vitamin D metabolite, inhibits proliferation, induces apoptosis and slows the growth of tumors. Calcitriol also may exert either antiangiogenic or proangiogenic effects depending on the tissue. Vascular endothelial growth factor (VEGF) and thrombospondin-1 (Tsp-1) are key factors involved in promoting and inhibiting angiogenesis, respectively. The effects of calcitriol on Tsp-1 have not been studied in the mammary gland, while VEGF regulation is not clear, since opposite outcomes have been demonstrated. Therefore, the present study was undertaken to investigate the effects of calcitriol on VEGF and Tsp-1 expression in primary breast tumor-derived cells and a panel of established breast cancer cell lines. In vivo studies in athymic mice were also performed in order to gain further insight into the biological effects of calcitriol on angiogenesis. Real time-PCR and ELISA analyses showed that calcitriol stimulated VEGF mRNA expression and protein secretion while elicited the opposite effect on Tsp-1 in 7 out of 8 cell lines studied, independently of the cell phenotype (P < 0.05 in n = 5). In vivo, calcitriol significantly inhibited the relative tumoral volume after 4 weeks of treatment; however, serum VEGF was higher in calcitriol-treated animals compared to controls (P < 0.05). The integrated fluorescence intensity analysis of CD31, a vessel marker, showed that xenografted breast cancer cells developed tumors with similar vascular density regardless of the treatment. Nevertheless, larger necrotic areas were observed in the tumors of calcitriol-treated mice compared to controls. Since the antineoplastic activity of calcitriol has been consistently demonstrated in several studies including this one, our results suggest that the antitumoral effect of calcitriol in vivo involve different mechanisms not necessarily related to the inhibition of tumor vascularization. Overall, our findings indicate that calcitriol can impact the angiogenic process in breast cancer by regulating VEGF and Tsp-1 expression. This article is part of a Special Issue entitled '16th Vitamin D Workshop'. (C) 2013 Elsevier Ltd. All rights reserved.