Prevention of breast cancer in the rat with 9-cis-retinoic acid as a single agent and in combination with tamoxifen.

We show that 9-cis-retinoic acid (9cRA) is a potent inhibitor of mammary carcinogenesis induced by N-nitroso-N-methylurea in Sprague-Dawley rats. Rats were first treated with a single dose of N-nitroso-N-methylurea (50 mg/kg body weight) and then fed non-toxic levels of 9cRA (120 or 60 mg/kg of diet). 9cRA was highly effective in reducing tumor incidence, average number of tumors per rat, and average tumor burden, as well as extending tumor latency. The combination of 9cRA with low levels of tamoxifen (TAM; fed at either 1.0 or 0.5 mg/kg of diet) was particularly effective; addition of 9cRA to a TAM regimen doubled the number of animals that were tumor-free at autopsy and significantly diminished tumor number and tumor burden. For suppression of carcinogenesis in vivo, 9cRA was much more potent than all-trans-retinoic acid, both as a single agent or in combination with TAM, although both retinoids had equivalent inhibitory effects on DNA synthesis in cultured human breast cancer cell lines. Both 9cRA and all-trans-retinoic acid induce the expression of the adhesion molecule, E-cadherin, in the SK-BR-3 cell line. We suggest that clinical evaluation of the combination of 9cRA and TAM, either for chemoprevention or for adjuvant therapy, should be considered.

1 alpha,25-Dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalciferol (Ro24-5531), a new deltanoid (vitamin D analogue) for prevention of breast cancer in the rat.

We have used the vitamin D analogue, 1 alpha,25-dihydroxy-16-ene-23-yne-26,27-hexafluorocholecalcifero l (Ro24-5531), for inhibition of mammary carcinogenesis induced by N-nitroso-N-methylurea (NMU) in Sprague-Dawley rats. Rats were first treated with a single dose of either 15 or 50 mg/kg body weight NMU and then fed Ro24-5531 (2.5 or 1.25 nmol/kg of diet) for 5-7 months. Ro24-5531 significantly extended tumor latency and lessened tumor incidence as well as tumor number in rats treated with the lower dose of NMU. In rats treated with the higher dose of NMU, Ro24-5531 was fed in combination with tamoxifen; in these experiments, Ro24-5531 significantly enhanced the ability of tamoxifen to reduce total tumor burden, as well as to increase the probability that an animal would be tumor free at the end of the experiment. In vitro, Ro24-5531 was 10-100 times more potent than 1,25-dihydroxyvitamin D3 for inhibition of proliferation of human breast cancer cell lines as well as primary cultures of cells from 2 patients with acute myelogenous leukemia. When fed chronically, Ro24-5531 did not elevate serum calcium in the present studies. We propose the new term, "deltanoids," for the set of molecules composed of vitamin D and its synthetic analogues, in a manner similar to the naming of "retinoids" for the corresponding set of molecules related to vitamin A.

N-(4-Hydroxyphenyl)retinamide, a new retinoid for prevention of breast cancer in the rat.

The synethesis of a new retinoid, N-(4-hydroxyphenyl)-all-trans-retinamide, which has useful biological properties, is described. This retinoid was more potent than retinyl acetate in reversing keratinization caused by retinoid deficiency in tracheal organ culture. It was markedly less toxic than retinyl acetate when fed p.o. to rats over 2-week or 6-month periods. It was an effective agent for inhibition of the development of breast cancer induced in rats by N-nitroso-N-methylurea, although it was not as potent as retinyl acetate in this regard. However, the lesser toxicity of 4-hydroxyphenylretinamide makes it a superior agent for prevention of breast cancer. High-pressure liquid chromatographic analyses of liver and breast extracts from rats treated for 6 months with retinoids show the pharmacokinetic basis for the superiority of 4-hydroxyphenylretinamide; this retinoid and its metabolites were found in high concentrations in breast tissue, without any measurable accumulation in the liver or evident liver toxicity. In contrast, chronic feeding of retinyl acetate caused marked deposition of retinyl esters in the liver and severe hepatotoxicity. Whole mounts of rat mammary glands, made after chronic feeding of 4-hydroxyphenylretinamide, showed that it had a marked antiproliferative effect on mammary epithelium.

Localization of transforming growth factor-beta isotypes in lesions of the human breast.

Transforming growth factor-beta s (TGF-beta) comprise a highly conserved family of multifunctional cell regulatory peptides that may play a role in a variety of pathologic processes. To date, five TGF-beta isotypes have been identified, three of these in mammalian systems. A number of cultured human breast carcinoma cell lines produce biologically inactive latent TGF-beta and are growth inhibited by activated TGF-beta; TGF-beta production is estrogen-influenced in some of these cell lines. To investigate the potential role of the TGF-beta isotypes in human breast disease, we localized TGF-beta s 1, 2, and 3 immunohistochemically in normal breast, fibrocystic change, epithelial hyperplasia, sclerosing adenosis, fibroadenoma, cystosarcoma phyllodes, and several carcinoma variants. Transforming growth factor-beta s 1, 2, and 3 were identified intracellularly in most active mammary epithelia, regardless of the lesion, including carcinoma; the associated stroma contained little or no intracellular TGF-beta. An antibody that recognizes an extracellular conformation of TGF-beta stained normal intralobular stroma and, more extensively, the stroma of active fibroadenomas and low-grade phyllodes tumors and the desmoplastic stroma of carcinomas. The results indicate the potential for paracrine and autocrine regulation of the mammary gland by TGF-beta and suggest an association between TGF-beta and abnormal stromal proliferations. Altered expression of TGF-beta s 1, 2, and 3 at the protein level in mammary epithelia appears not to be a major feature of most breast lesions, raising the possibility that altered cellular response to the TGF-beta already present may play a role in the development of breast disease.

Antibodies to transforming growth factor-beta 2 peptides: specific detection of TGF-beta 2 in immunoassays.

Polyclonal antibodies have been raised to synthetic peptides corresponding to several regions of transforming growth factor-beta 2 (TGF-beta 2). All antisera were tested for their ability to react with either the native or reduced forms of both TGF-beta 1 and TGF-beta 2 in enzyme-linked immunosorbent assays, Western blots, and immunoprecipitation assays. On Western blots, antisera raised to a peptide corresponding to residues 50-75 of TGF-beta 2 specifically detected 5 ng TGF-beta 2, while antisera raised to regions 1-30 and 79-108 cross-reacted with TGF-beta 1. Anti-P 50-75(2) also localized TGF-beta 2 in murine placenta in immunohistochemical studies. In immunoprecipitation assays with either iodinated TGF-beta s or with media conditioned by cells labeled with [35S]cysteine, both anti-P 50-75(2) and anti-P 79-108(2) specifically immunoprecipitated TGF-beta 2 under reducing conditions only, while anti-P 79-108(2) also reacted with TGF-beta 1. None of the TGF-beta 2 peptide antibodies was able to block receptor binding of either TGF-beta 1 or 2. Analysis of the cross-reactivity patterns of these peptide antibodies suggests conformational differences between TGF-beta 1 and TGF-beta 2.