Role of vitamin D receptor in the antiproliferative effects of calcitriol in tumor-derived endothelial cells and tumor angiogenesis in vivo

Abstract

Calcitriol (1,25-dihydroxycholecalciferol), the major active form of vitamin D, is antiproliferative in tumor cells and tumor-derived endothelial cells (TDEC). These actions of calcitriol are mediated at least in part by vitamin D receptor (VDR), which is expressed in many tissues including endothelial cells. To investigate the role of VDR in calcitriol effects on tumor vasculature, we established TRAMP-2 tumors subcutaneously into either VDR wild-type (WT) or knockout (KO) mice. Within 30 days post-inoculation, tumors in KO mice were larger than those in WT (P < 0.001). TDEC from WT expressed VDR and were able to transactivate a reporter gene whereas TDEC from KO mice were not. Treatment with calcitriol resulted in growth inhibition in TDEC expressing VDR. However, TDEC from KO mice were relatively resistant, suggesting that calcitriol-mediated growth inhibition on TDEC is VDR-dependent. Further analysis of the TRAMP-C2 tumor sections revealed that the vessels in KO mice were enlarged and had less pericyte coverage compared with WT (P < 0.001). Contrast-enhanced magnetic resonance imaging showed an increase in vascular volume of TRAMP tumors grown in VDR KO mice compared with WT mice (P < 0.001) and FITC-dextran permeability assay suggested a higher extent of vascular leakage in tumors from KO mice. Using ELISA and Western blot analysis, there was an increase of hypoxia-inducible factor-1alpha, vascular endothelial growth factor, angiopoietin 1, and platelet-derived growth factor-BB levels observed in tumors from KO mice. These results indicate that calcitriol-mediated antiproliferative effects on TDEC are VDR-dependent and loss of VDR can lead to abnormal tumor angiogenesis.

Fig. 1. TDEC expressing VDR is more sensitive to growth inhibitory effects by calcitriol

A, Phase contrast pictures showing no significant obvious difference in cell morphology of TDEC isolated from VDR WT or KO mice. B, VDR protein expression (arrows) was observed in 48 hr calcitriol-treated TDEC isolated from VDR WT but not KO mice as shown by Western blot analysis. C, Transactivation activity of VDR is observed in TDEC expressing VDR (◆) but not in those from KO mice (○). TDEC were transfected with a constant dose of adenoviral 24-hydroxylase promoter luciferase reporter and adenoviral β-galactosidase expression vector for 3 hrs before treatment with vehicle (dotted lines) or 10 nM calcitriol (solid lines) for 48 hr. Luminescence data were normalized with β-galactosidase activities, and the empty adenoviral control vector showed minimal luciferase activity (data not shown). D, The effects of 48 hr treatment of 1–500 nM calcitriol on cell viability as measured by MTT assay. TDEC isolated from VDR WT (◆) but not those from KO mice (○) were responsive to the anti-proliferative effects of calcitriol. *, P < 0.001 (Student’s t test). RLU, relative luciferase unit. Representative of three independent experiments.

Fig. 2. Enlargement of tumor vessels in TRAMP tumors in VDR KO mice is associated with lower pericyte coverage

A, Representative of endothelial marker CD31-stained vessels (brown) found in tumors implanted in VDR WT and KO mice were shown. Magnification, ×100. B, Representative of endothelial marker CD31 (pink) and pericyte marker α-smooth muscle actin (brown) positive vessels found in tumors in both VDR WT and KO mice. Magnification, ×100.

Fig. 3. Difference in blood volume, vascular leakiness and tumor size of tumors in VDR WT and KO mice

A, Change in T1 relaxation rates (ΔR₁) over time of TRAMP tumors in VDR WT (◆) and KO (○) mice. Vascular volume and permeability values were calculated from ΔR₁ using linear regression analysis. Analysis of the slopes showed a slight, but not significant difference in permeability between the two groups. Significant differences were seen between the vascular volumes (Y-intercept) of tumors in VDR WT and KO mice (P < 0.001). B, Modified in vivo Evans blue dye assay showed a significant increase in Evans blue dye content in tumors from VDR KO compared to WT. Animals were given 0.2 ml 0.5% Evans blue intracardiacally under anesthesia for 5 min before sacrificied. The dye from tumors was extracted using formamide and readings were normalized with those from the livers. Data shown were from at least 3–5 animals per group.C, Confocal microscopy analysis shows a more extensive leakage of FITC-dextran from tumor vasculature in VDR KO than in WT animals. Animals were given 0.2 ml 2 µM FITC-dextran in saline intracardically before fixation with paraformaldehyde. Livers were used as a control for sufficient systemic delivery of the dye. Data shown were representative of three independent experiments. Scale bar, 200 µm. D, Tumors in VDR KO mice is larger than those in WT mice. 2 × 10⁶ of TRAMP cells were implanted subcutaneously into VDR WT (◆) and KO (○) mice. The growth of the tumors, as measured by the tumor size, was monitored over time. There were at least 4–6 animals per group, and the data shown is representative of two independent experiments.

Fig. 4. Abnormal vasculature in tumors from VDR KO mice is associated with increased HIF-1 alpha, VEGF, angiopoietin 1 and PDGF-BB expression

A–D, Tumor extracts were subjected to ELISA analyses for (A) HIF-1 alpha, (B) VEGF, (C) Ang 1 and (D) PDGF-BB and the quantification of the growth factors were normalized with total protein. Data shown were from at least 5 tumors per group and statistical analyses were performed using Student’s t test. E, Representative of 3 experiments showing increased protein expression of VEGF, Ang1 and PDGF-BB in 5 individual tumors from KO mice compared to WT using Western blot analysis. No significant change in the expression of the respective receptors in both groups. HIF1, hypoxia-induced factor; VEGF, vascular endothelial growth factor; ANG1, angiopoeitin-1; PDGF-BB, platelet-derived growth factor-BB; PDGFR, platelet-derived growth factor receptor.