Prevention of preneoplastic mammary lesion development by a novel vitamin D analogue, 1alpha-hydroxyvitamin D5.
BACKGROUND
The form of vitamin D (vitamin D3) in fortified milk and the provitamin D produced by the body undergo metabolic activation to a biologically active form, 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. This compound can induce cell differentiation and can prevent proliferation of cancer cells. However, because 1alpha,25(OH)2D3 is hypercalcemic (effective in increasing serum calcium level), it is not suitable for use in cancer prevention or cancer therapy trials.


OBJECTIVE
We synthesized a vitamin D5 series analogue, 1alpha-hydroxy, 24-ethyl-cholecalciferol, or 1alpha-hydroxyvitamin D5 [1alpha(OH)D5], and evaluated its chemopreventive activity in carcinogen-treated mammary glands in organ culture experiments.


METHODS
The analogue 1alpha(OH)D5 was synthesized from sitosterol acetate and was characterized by nuclear magnetic resonance. Its purity was evaluated by high-pressure liquid chromatography. The calcemic activities of vitamin D3 and D5 analogues were determined in vitamin D-deficient Sprague-Dawley rats. Mammary glands of BALB/c mice were placed in organ culture and treated with the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) to induce preneoplastic lesions. Vitamin D analogues were added to the culture medium at four different concentrations, and formation of mammary lesions was evaluated. The effects of 1alpha(OH)D5 and 1alpha,25(OH)2D3 on the expression of vitamin D receptors (VDRs) and transforming growth factor-beta1 (TGF-beta1) were studied by immunohistochemistry. Statistical significance was determined by the chi-squared test. All reported P values were two-sided.


RESULTS
1alpha,25(OH)2D3 was fourfold more calcemic than 1alpha(OH)D5 at a dose of 0.042 microg/kg per day in rats. Both 1alpha,25(OH)2D3 and 1alpha(OH)D5 inhibited the development of DMBA-induced preneoplastic lesions in mouse mammary glands compared with untreated glands. The effect of the vitamin D3 analogue was observed at a much lower concentration (0.01 microM). Treatment with 1alpha(OH)D5 resulted in a dose-related (0.01-10.0 microM) inhibition without any toxicity, whereas the vitamin D3 analogue was highly potent but toxic at concentrations of 1.0 microM or higher. Normal mouse mammary glands poorly express VDR and TGF-beta1; incubation with 1alpha(OH)D5 or 1alpha,25(OH)2D3 dramatically induced their expression.


CONCLUSIONS
This is the first report showing the possibility of chemoprevention by a vitamin D5 series compound. We conclude that 1alpha(OH)D5 is less calcemic than 1alpha,25(OH)2D3. It is nontoxic at a wide range of concentrations, but it is potent in inhibiting the development of preneoplastic lesions in mammary glands in organ culture. In addition, we show for the first time the induction of TGF-beta1 in normal mammary tissues by a chemopreventive agent.


CONCLUSIONS
1alpha(OH)D5 is a good candidate for in vivo chemoprevention studies. It may mediate its action by inducing expression of VDR and of TGF-beta1, as is seen in other systems.
Mehta
R G
RG
Department of Surgical Oncology, College of Medicine, University of Illinois, Chicago 60612, USA.
Moriarty
R M
RM
Mehta
R R
RR
Penmasta
R
R
Lazzaro
G
G
Constantinou
A
A
Guo
L
L
D015127:9,10-Dimethyl-1,2-benzanthracene; D000818:Animals; D002117:Calcitriol / Q000494:pharmacology*; D002118:Calcium / Q000097:blood; D004305:Dose-Response Relationship, Drug; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006887:Hydroxycholecalciferols / Q000494:pharmacology*; D008297:Male; D008325:Mammary Neoplasms, Experimental / Q000139:chemically induced / Q000737:chemistry* / Q000517:prevention & control*; D051379:Mice; D008807:Mice, Inbred BALB C; D009924:Organ Culture Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley; D018167:Receptors, Calcitriol / Q000096:biosynthesis / Q000187:drug effects* / Q000235:genetics; D016212:Transforming Growth Factor beta / Q000096:biosynthesis / Q000187:drug effects* / Q000235:genetics; D014807:Vitamin D / Q000031:analogs & derivatives*
C103958:1-hydroxyvitamin D5; D006887:Hydroxycholecalciferols; D018167:Receptors, Calcitriol; D016212:Transforming Growth Factor beta; D014807:Vitamin D; D015127:9,10-Dimethyl-1,2-benzanthracene; D002117:Calcitriol; D002118:Calcium