PREVENTION OF PRENEOPLASTIC MAMMARY LESION DEVELOPMENT BY A NOVEL VITAMIN-D ANALOG, 1-ALPHA-HYDROXYVITAMIN D-5

Background: the form of vitamin D (vitamin D-3) in fortified milk and the provitamin D produced by the body undergo metabolic activation to a biologically active form, 1 alpha,25-dihydroxyvitamin D-3 [1 alpha,2 5(OH)(2)D-3]. This compound can induce cell differentiation and can pr event proliferation of cancer cells. However, because 1 alpha,25(OH)(2 )D-3 is hypercalcemic (effective in increasing serum calcium level), i t is not suitable for use in cancer prevention or cancer therapy trial s. Purpose: We synthesized a vitamin D-5 series analogue, 1-alpha-hydr oxy, 24-ethyl-cholecalciferol, or 1 alpha-hydroxyvitamin D-5 and evalu ated its chemopreventive activity in carcinogen-treated mammary glands in organ culture experiments. Methods: The analogue 1 alpha(OH)D-5 wa s synthesized from sitosterol acetate and was characterized by nuclear magnetic resonance. Its purity mas evaluated by high-pressure liquid chromatography, The calcemic activities of vitamin D-3 and D-5 analogu es were determined in vitamin D-deficient Sprague-Dawley rats. Mammary glands of BALB/c mice were placed in organ culture and treated with t he carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) to induce preneopl astic lesions. Vitamin D analogues were added to the culture medium at four different concentrations, and formation of mammary lesions was e valuated, The effects of 1 alpha(OH)D-5 and 1 alpha,25(OH)(2)D-3 on th e expression of vitamin D receptors (VDRs) and transforming growth fac tor-beta 1 (TGF-beta 1) were studied by immunohistochemistry. Statisti cal significance was determined by the chi-squared test. All reported P values were two-sided. Results: 1 alpha,25(OH)(2)D-3 was fourfold mo re calcemic than 1 alpha(OH)D-5 at a dose of 0.042 mu g/kg per day in rats. Both 1 alpha,25(OH)(2)D-3 and 1 alpha(OH)D-5 inhibited the devel opment of DMBA-induced preneoplastic lesions in mouse mammary glands c ompared with untreated glands. The effect of the vitamin D-3 analogue was observed at a much lower concentration (0.01 mu M). Treatment with 1 alpha(OH)D-5 resulted in a dose-related (0.01-10.0 mu M) inhibition without any toxicity, whereas the vitamin D-3 analogue was highly pot ent but toxic at concentrations of 1.0 mu M or higher. Normal mouse ma mmary glands poorly express VDR and TGF-beta 1; incubation with 1 alph a(OH)D-5 or 1 alpha 25(OH)(2)D-3 dramatically induced their expression . Conclusions: This is the first report showing the possibility of che moprevention by a vitamin D-5 series compound. We conclude that 1 alph a(OH)D-5 is less calcemic than 1 alpha,25(OH)(2)D-3. It is nontoxic at a wide range of concentrations, but it is potent in inhibiting the de velopment of preneoplastic lesions in mammary glands in organ culture. In addition, we show for the first time the induction of TGF-beta 1 i n normal mammary tissues by a chemopreventive agent. Implications: 1 a lpha(OH)D-5 is a good candidate for in vivo chemoprevention studies. I t may mediate its action by inducing expression of VDR and of TGF-beta 1, as is seen in other systems.