The in vitro and in vivo effect of 1,25-dihydroxycholecalciferol (calcitriol) on tumor-derived endothelial cell (TDEC) and matrigel-derived endothelial cell (MDEC)

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Calcitriol (1,25-dihydroxycholecalciferol), the active form of vitamin D is known to have anti-tumor activity both in vitro and in vivo in a wide variety of malignant cell types. We demonstrated that calcitriol was not only anti-proliferative to tumor cells, but also to the endothelial cells that were isolated from tumors (TDEC) by flow cytometry, and that TDEC appeared to differ from mature endothelial cells isolated from normal tissues. To further examine the specificity of the anti-proliferative effect of calcitriol on TDEC, we isolated a pure population of normal endothelial cells from fibroblast growth factor (FGF) contained-matrigel plugs that were implanted subcutaneously into C3H mice (MDEC). We investigated the in vitro effect of calcitriol alone or in combination with dexamethasone (dex) on proliferation and apoptosis of TDEC and MDEC. Using the MTT assay, greater cell growth inhibition was seen with TDEC (50%) as compared to MDEC (25%) after 48 hours treatment of calcitriol and dex. Western blotting showed that both TDEC and MDEC have comparable vitamin D receptor (VDR) protein expression. However, calcitriol induced G₀/G₁ cell cycle arrest in TDEC, but not in MDEC. The induction of cell cycle arrest is further confirmed with a decrease p21 and an increase p27 expression in TDEC, but this was not observed in MDEC. Apoptotic and survival signaling pathway molecules were examined by Western blotting; calcitriol caused a significant decrease of phosphorylated-Erk and phosphorylated-Akt as well as an increase of PARP and pro-caspase 3 cleavage, in TDEC but not in MDEC. These effects were further validated using Annexin V staining and BrdU incorporation assay, in which more apoptotic cells and less S phase cells were observed in TDEC as compared to MDEC. The effect of calcitriol on TDEC and MDEC was further examined in vivo, in which squamous cell carcinoma (SCC) and matrigel plugs were inoculated into opposite flanks of the same mouse. The effect of calcitriol alone (total dose 1.875μg/mouse) and in combination with dex (total dose 0.036μg/mouse) on endothelial cells in the tumor and matrigel was evaluated by measuring mean vessel density (MVD) on CD31-stained histological specimens. Calcitriol caused a reduction of 40% MVD in tumor but not in matrigel, when compared to the control group. These results indicate that the anti-proliferative effect of combination treatment of calcitriol and dex is significantly greater in TDEC than in MDEC in vitro and in vivo, suggesting a differential effect of calcitriol in inhibition of tumor angiogenesis. Supported by NCI grants CA67267, CA95045 and CA85142.