European Journal of Pharmacology, 178 (1990) 247-250 Elsevier

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EJP 20588

Short communication

Inhibition of angiogenesis by vitamin D 3 analogues T s u t o m u O i k a w a 1, K e n j i H i r o t a n i 1,2, H i r o y u k i O g a s a w a r a 1,2, T a k a s h i K a t a y a m a 2, O s a m u N a k a m u r a 3, T a k a o Iwaguchi ~ a n d A k i y o s h i H i r a g u n 4 i Division of Cancer Therapeutics and 4 Oncology, The Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113, Japan, 2 Faculty of Pharmaceutical Sciences, Science University of Tokyo, Ichigaya Funagawara-cho, Shinjuku-ku, Tokyo 162, Japan and x Division of Neurosurgery, The Tokyo Metropolitan Komagome Hospital, Bunkyo-ku, Tokyo 113, Japan Received 9 January 1990, accepted 23 January 1990

The effects of vitamin D 3 and two analogues on embryonic angiogenesis were studied in 4.5-day-old chick embryo chorioallantoic membranes. The active metabolite of vitamin D3, la,25-dihydroxyvitamin D3, and a synthetic vitamin D 3 analogue, 22-oxa-la,25-dihydroxyvitamin D3, inhibited angiogenesis in a dose-dependent manner, the inhibition occurring in the picomolar range. In contrast, vitamin D 3 was not effective. The results suggest that these two vitamin D3 analogues might be promising anti-angiogenic agents for controlling the angiogenesis which occurs in several pathological conditions, including tumor development. Vitamin D 3 analogues; Embryonic angiogenesis; Chorioallantoic membrane; Anti-angiogenic activity

1. Introduction

Angiogenesis, the formation of new capillary blood vessels, plays an important role in a variety of normal physiological processes, such as in embryonic development and in the formation of the corpus luteum. In addition, aberrant angiogenesis occurs in several pathological processes, including the progressive growth of solid tumors and diabetic retinopathy. Thus, treatment with angiogenesis inhibitors might be a novel strategy for managing diseases accompanied by uncontrolled angiogenesis. It is well known that neovascularization involves the sequential actions of vascular endothelial cells, i.e., activation (migration and pro-

Correspondence to: T. Oikawa, Division of Cancer Therapeutics, The Tokyo Metropolitan Institute of Medical Science, Honkomagome 3-18-22, Bunkyo-ku, Tokyo 113, Japan.

liferation) and subsequent differentiation into vessel-forming quiescent cells (Folkman, 1985). We recently reported that herbimycin A, which causes the conversion of cells transformed with Rous sarcoma virus into normal phenotype cells (Uehara et al., 1986), had a strong anti-angiogenic effect, as assessed with the chick embryo chorioallantoic membrane assay system (Oikawa et al., 1989). Based on this finding, we speculated that agents capable of inducing the differentiation of malignant cells into normal phenotypes could also modify the activation/differentiation coupling of angiogenic endothelial cells, resulting in inhibition of angiogenesis. In a previous study we showed that several retinoids, which are well-known inducers of cell differentiation, markedly inhibited angiogenesis in chorioallantoic membranes (Oikawa et al., in press). The present study was conducted to determine the effects of vitamin D 3 and two analogues on embryonic angiogenesis because, like retinoids,

0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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vitamin D 3 analogues have recently been shown to have a differentiation-inducing action on several cell types (Suda et al., 1986).

2. Materials and methods

lc~,25-Dihydroxyvitamin D 3 (1,25(OH)2D3) and 22-oxa-l,25(OH)2D 3 were kindly provided by Chugai Pharmaceuticals (Tokyo, Japan). Vitamin D 3 was obtained from Sigma (St. Louis, MO). Ethylene-vinyl acetate copolymer (EV 40) was a generous gift from Mitsui-DuPont Polychemicals (Tokyo, Japan). Anti-angiogenic activity was determined by implanting an EV pellet containing the agent to be tested in a 4.5-day-old chorioallantoic membrane, as described previously (Oikawa et al., 1989; Oikawa et al., in press). Briefly, fertilized eggs were incubated in a humidified egg incubator at 37 ° C for 4.5 days. An EV pellet with or without the agent to be tested was implanted on a 4.5-dayold chorioallantoic membrane. The eggs, covered with teflon-coated metal cups, were incubated as described above. After a 2-day incubation, about 0.5 ml of a fat emulsion was injected into the chorioallantois. The anti-angiogenic response was assessed by measuring the avascular zone in the chorioallantoic membrane. Inhibition of angiogenesis was assessed as being present when the avascular zone exceeded 3 mm. Only the frequency was monitored; it was not indicated whether higher doses also yielded larger avascular zones. Data on the incidence of anti-angiogenic activity were analysed by means of Fisher's exact probability test, with P < 0.05 as the level of significance.

3. Results

The effects of vitamin D 3 and two analogues on embryonic angiogenesis were examined by placing EV pellets containing different doses of these agents on 4.5-day-old chorioallantoic membranes. The dose-response relationships for inhibition of embryonic angiogenesis are shown in fig. 1. Figure

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Fig. 1. Inhibitory effects of vitamin D 3 and two analogues on embryonic angiogenesis. EV pellets containing various doses of the agents were implanted in 4.5-day-old chorioallantoic membranes and then their anti-angiogenic effects were evaluated by measuring the avascular zones 2 days after implantation of the EV pellets. Vitamin D 3 (open triangles), 1,25(OH)2D 3 (open circles) and 22-oxa-l,25(OH)2D 3 (closed circles) were used. Points represent frequency (%) of an avascular zone showing anti-angiogenic activity. The number of membranes studied are indicated in parentheses. * P < 0.05 compared to control (i.e., empty) pellet-treated membranes (n = 35), which did not produce an avascular zone; * * P < 0.005 compared to the control; • * * P < 0.001 compared to the control.

2 shows the effects of the agents on angiogenesis 2 days after pellet implantation. Two vitamin D 3 analogues, i.e., 1,25(OH)2D 3 and 22-oxa-l,25(OH)2D 3, had potent anti-angiogenic effects. As compared with the effects of empty pellets in 35 control chorioallantoic membranes, the minimum doses required for the induction of significant anti-angiogenic activity (i.e., a minimum effective dose) were 10 ng (24 pmol) and 1 ng (2.4 pmol) per egg for 1,25(OH)2D 3 ( n = 1 4 ) and 22-oxa-l,25(OH)zD3 ( n = l l ) , respectively. The IDs0 values obtained for 1,25(OH)2D 3 and 22-oxa-l,25(OH)2D 3 were 140 and 40 ng per egg, respectively. In contrast, vitamin D 3, at doses of up to 1000 ng/egg, did not exhibit anti-angiogenic activity in any of the 45 chorioallantoic membranes used.

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Fig. 2. Effects of vitamin 0 3 and two analogues on angiogenesis in chorioallantoic membranes 2 days after EV pellet implantation. An appropriate volume of a fat emulsion was injected into the chorioallantois to show the vascular network better. The effects of 1,25(OH)2D3 (B, 100 ng per egg), 22-oxa-l,25(OH)2D 3 (C, 30 ng per egg) and vitamin D3 (D, 1000 ng per egg) are shown. Note the presence of an avascular zone (indicated by arrow heads) in chorioallantoic membranes treated with an EV pellet containing 1,25(OH)2D 3 or 322-oxa-l,25(OH)2D 3. Chorioallantoic membranes treated with empty (A) or vitamin D 3 (D) pellet show no disturbance of angiogenesis. Magnification ( × 2.4). Both 1 , 2 5 ( O H ) 2 D 3 (fig. 2B) a n d 22-oxa1,25(OH)2D 3 (fig. 2C) p r o d u c e d significant vascular zones in the c h o r i o a l l a n t o i c m e m b r a n e s . In contrast, e m p t y pellets (n = 35) d i d not affect the f o r m a t i o n of a new b l o o d vessel n e t w o r k in a n y of the control c h o r i o a l l a n t o i c m e m b r a n e s (fig. 2A), n o r d i d v i t a m i n D 3 pellets in a n y of the chorioallantoic m e m b r a n e s used over the dose range exa m i n e d (fig. 2D).

4. Discussion T h e p r e s e n t s t u d y d e m o n s t r a t e d that the active m e t a b o l i t e of v i t a m i n D 3, 1,25(OH)2D 3, which shows a d i f f e r e n t i a t i o n - i n d u c i n g effect on several cell types (Suda et al., 1986), h a d a strong inhibitory effect on e m b r y o n i c angiogenesis in chick embryo chorioallantoic membranes. The metabolite was active at a dose as low as 24 p m o l / e g g , with an IDs0 value of 340 p m o l / e g g . I n a d d i t i o n , a synthetic v i t a m i n D 3 analogue, 22-oxa-1,25(OH)zD3, which has a m o r e p o t e n t d i f f e r e n t i a t i o n - i n d u c ing activity than 1,25(OH)2D 3 ( A b e et al., 1987; A b e et al., 1989), was effective at doses as low as

2.4 p m o l / e g g , with an IDs0 value of 96 p m o l / e g g . Thus, the o r d e r of p o t e n c y of these two v i t a m i n D 3 analogues a p p e a r s to coincide with the o r d e r of their activity to induce cell differentiation, in agreement with o u r previous o b s e r v a t i o n for angiostatic retinoids ( O i k a w a et al., in press): the o r d e r of p o t e n c y was Ch 55, a synthetic retinoid (a m i n u m u m effective dose = 2.7 p m o l / e g g ; IDa0 = 22 p m o l / e g g ) > retinoic acid (a m i n i m u m effective dose = I70 p m o l / e g g ; IDs0 = 330 p m o l / e g g ) > r e t i n o l (a m i n i m u m effective d o s e = 3.5 n m o l / e g g ; IDs0 = 4.2 n m o l / e g g ) . W e have rep o r t e d previously that h e r b i m y c i n A dose d e p e n d e n t l y inhibits e m b r y o n i c angiogenesis, with a m i n i m u m effective dose = 170 p m o l / e g g a n d an IDs0 value = 260 p m o l / e g g ( O i k a w a et al., 1989; in press). C o n s i d e r i n g these d a t a , 22-oxa1 , 2 5 ( O H ) z D 3 seems to have a m o r e p o t e n t antiangiogenic effect than retinoic acid a n d h e r b i m y cin A, a n d exhibits roughly similar angiogenesisi n h i b i t o r y activity as C h 55, which is the most p o t e n t angiogenesis i n h i b i t o r identified so far (Oik a w a et al., in press). T h e p o t e n c y of 1 , 2 5 ( O H ) z D 3 a p p e a r s to be c o m p a r a b l e to that of retinoic acid or h e r b i m y c i n A.

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In contrast, at doses of up to 2.6 nmol/egg, vitamin D3, which on its own is unable to exert a differentiation-inducing effect, did not cause an avascular zone. Therefore, it is highly likely that the present results strengthen the validity of our idea that modulators of cell differentiation could also affect the activation/differentiation coupling of vascular endothelial cells during neovascularization, leading to inhibition of angiogenesis. Alternatively, it is possible that the embryonal chorioallantoic membranes used in this study could not provide sufficient hydroxylases to support the formation of the active vitamin D 3 metabolite with anti-angiogenic activity. At present, the mechanism by which the angiostatic vitamin D 3 analogues used in this study induce anti-angiogenic activity remains unknown. The biological effects of vitamin D 3 compounds are believed to be mediated by a mechanism involving an intracellular receptor for 1,25(OH)2D 3. The complementary DNA of this receptor has recently been isolated and characterized (McDonnell et al., 1987). However, the binding capacity of vitamin D 3 analogues to the 1,25(OH)zD 3 receptor has been found to not always correlate with the potency of their biological activity in several biological assay systems (Abe et al., 1987; Abe et al., 1989). Experiments with the human myeloid leukemia cell line, H L 60, showed that the receptor-binding potency of 22-oxa-1,25(OH)2D3 was one order of magnitude weaker than that of 1,25(OH)2D3, although the former exhibited a more potent differentiation-inducing activity toward the H L 60 cells than the latter (Abe et al., 1987). Similarly, in this study, 22-oxa-1,25(OH)2D 3 exhibited a more potent anti-angiogenic activity than 1,25(OH)2D 3. Both angiostatic vitamin D 3 analogues and retinoids exhibit several similar biological activities, such as inhibition of the induction of epidermal ornithine decarboxylase by 12-O-tetradecanoylphorbol-13-acetate, a known tumor promotor (Suda et al., 1986). This phorbol ester also shows angiogenic activity in chorioallantoic membranes (Morris et al., 1988). In addition, retinoic acid induces an increase in the number of 1,25(OH)zD 3 receptors (Petkovich et al., 1984). These observations indicate that these two angiostatic vitamins

could express anti-angiogenic activity through, at least in part, a shared mechanism of action. Although the mechanism by which vitamin D 3 analogues exhibit anti-angiogenic activity remains to be elucidated, like retinoids, these two vitamin D 3 analogues are promising angiogenesis inhibitors for use in the management of diseases accompanied by uncontrolled angiogenesis.

Acknowledgement This work was supported in part by The Mochida Memorial Foundation for Medical and Pharmaceutical Research.

References Abe, J., M. Morikawa, K. Miyamoto, S. Kaiho, M. Fukushima, C. Miyaura, E. Abe, T. Suda and Y. Nishii, 1987, Synthetic analogues of vitamin D 3 with an oxygen atom in the side chain skeleton, FEBS Lett. 226, 58. Abe, J., Y. Takita, T. Nakano, C. Miyaura, T. Suda and Y. Nishii, 1989, A synthetic analogue of vitamin D3, 22-oxala,25-dihydroxyvitamin D3, is a potent modulator of in vivo immunoregulating activity without inducing hypercalcemia in mice, Endocrinology 124, 2645. Folkman, J., 1985, Tumor angiogenesis, Adv. Cancer Res. 43, 175. McDonnell, D.P., D.J. Mangelsdore, J.W. Pike, M.R. Haussler and B.W. O'Malley, 1987, Molecular cloning of complementary DNA encoding the avian receptor for vitamin D, Science 235, 1214. Morris, P.B., T. Hida, PJ. Blackshear, G.K. Klintworth and J.L. Swain, 1988, Tumor-promoting phorbol esters induce angiogenesis in vivo, Am. J. Physiol. 254 (Cell Physiol. 23), C318. Oikawa, T., K. Hirotani, O. Nakamura, K. Shudo, A. Hiragun and T. Iwaguchi, A highly potent antiangiogenic activity of retinoids, Cancer Lett. (in press). Oikawa, T., K. Hirotani, M. Shimamura, H. Ashino-Fuse and T. Iwaguchi, 1989, Powerful antiangiogenic activity of herbimycin A (named angiostatic antibiotic), J. Antibiotics 42, 1202. Petkovich, P.M., J.N.M. Heersche, D.O. Tinker and G. Jones, 1984, Retinoic acid stimulates 1,25-dihydroxyvitamin 93 binding in rat osteosarcoma cells, J. Biol. Chem. 259, 8274. Suda, T., C. Miyaura, E. Abe and T. Kuroki, 1986, Modulation of cell differentiation, immune responses and tumor promotion by vitamin D compounds, in: Bone and Mineral Research/4, ed. W.A. Peck (Elsevier, Amsterdam) p. 1. Uehara, Y., M. Hori, T. Takeuchi and H. Umezawa, 1986, Phenotypic change from transformed to normal induced by benzoquinonoid ansamycins accompanies inactivation of p60 src in rat kidney cells infected with Rous sarcoma virus, Mol. Cell. Biol. 6, 2198.