Previous work has shown that the efficacy of cancer prevention by selenium-enriched garlic (Se-garlic) is primarily dependent on the action of selenium. An aqueous extract containing 43 micro Se/ml was prepared from lyophilized Se-garlic powder by the Soxhlet method. The activity of this Se-garlic extract was evaluated in a transformed mammary epithelial cell culture model for its effect on cell morphology, cell growth, cell cycle progression and the induction of single and double stranded breaks in DNA. Comparisons were also made with a similarly prepared extract from regular garlic, Se-methylselenocysteine (a major water-soluble seleno-amino acid identified in Se-garlic) and selenite (used for fertilizing Se-garlic). In contrast to the regular garlic extract which produced little or no modulation of the above parameters, treatment with the Se-garlic extract resulted in growth inhibition, GI phase cell cycle arrest and apoptotic DNA double strand breaks in the absence of DNA single strand breaks. This pattern of cellular responses was duplicated with exposure to Se-methylselenocysteine. Selenite, on the other hand, induced cell cycle blockage in the S/G2-M phase, and a marked increase in DNA single strand breaks (a measure of genotoxicity) in addition to growth suppression. The chemopreventive efficacy of the two garlic extracts was also investigated in the rat methylnitrosourea mammary tumor model. Both extracts were supplemented in the diet for 1 month immediately following carcinogen administration. Significant cancer protection was observed with treatment by the Se-garlic extract (at 3 p.p.m. Se in the diet), while little benefit was noted with treatment by the regular garlic extract. Based on the above in vitro and in vivo findings, it is hypothesized that the Se-garlic extract, in part via the action of Se-methylselenocysteine, is able to inhibit tumorigenesis by suppressing the proliferation and reducing the survival of the early transformed cells. Furthermore, the data also support the concept that the modulation of certain in vitro markers may be of value in predicting the effectiveness of novel forms of selenium for cancer prevention.

Experimentally induced models of breast carcinogenesis in the rat are widely used for studying the biology of breast cancer and for developing and evaluating cancer prevention and control strategies. However, very little is known about gene expression changes that are associated with experimentally induced mammary carcinogenesis. This paper reports the identification, by differential display of mRNA and molecular cloning, of seven cDNA fragments of gene transcripts overexpressed in mammary carcinomas induced by 1-methyl-1nitrosourea. These genes included the rat homologues of human galectin-7 gene, the human/mouse melanoma inhibitory activity/bovine chondrocyte-derived retinoic acid sensitive protein gene, the mouse stearoyl-CoA desaturase-2 gene, and the mouse endo B cytokeratin/human cytokeratin-18 gene. Although each of these genes has been implicated in some aspect of carcinogenesis in other organs, this paper is the first report of their overexpression in chemically induced mammary carcinomas. Two previously uncharacterized gene transcripts were also identified. A comparison of the expression levels of several genes in mammary carcinomas with those in the normal mammary gland tissue of virgin rats, mid-stage pregnant rats, and of day 1 postpartum lactating dams indicated that the overexpression of several genes observed in mammary carcinomas could not be accounted for by either a difference in the mammary epithelial content between mammary carcinoma and normal mammary tissue or by mammary epithelium-specific proliferation associated with pregnancy. Several genes were also overexpressed in rat mammary carcinomas induced by 7,12-dimethylbenz[a]anthracene but not in azoxymethane-induced rat colon adenocarcinomas. The genes identified in this study may therefore represent mammary carcinoma-specific molecular markers that may be helpful in investigations of mammary carcinogenesis and its prevention.

We used acute selenium (Se) treatments (i.e., daily single oral gavage of 2 mg Se per kilogram of body weight for 3 days) of female Sprague-Dawley rats bearing 1-methyl-1-nitrosourea -induced mammary carcinomas to increase the probability of detecting in vivo apoptosis and the associated gene/protein changes in the cancerous epithelial cells. The results show that whereas control carcinomas doubled in volume in 3 days, Se-methylselenocysteine and selenite treatments regressed approximately half of the carcinomas, accompanied by a 3-to 4-fold increase of morphologically observable apoptosis and f40% inhibition of 5-bromo-2 ¶-deoxyuridine index of the cancerous epithelial cells. The mRNA levels of growth arrest-DNA damage inducible 34 (gadd34), gadd45, and gadd153 genes were, contrary to expectation, not higher in the Se-treated carcinomas than in the gavage or diet restriction control groups. The gadd34 and gadd153 proteins were localized in the nonepithelial cells and not induced in the cancer epithelial cells of the Se-treated carcinomas. On the other hand, both Se forms decreased the expression of cyclin D1 and increased levels of P27Kip1 and c-Jun NH 2 -terminal kinase activation in a majority of the mammary carcinomas. Furthermore, the lack of induction of gadd genes in vivo by methylseleninic acid was confirmed in a human prostate xenograft model in athymic nude mice. In summary, these experiments showed the induction of cancer epithelial cell apoptosis and inhibition of cell proliferation by Se in vivo through the potential involvement of cyclin D1, P27Kip1, and c-Jun NH 2 -terminal kinase pathways. They cast doubt on the three gadd genes as mediators of Se action in vivo. [Mol Cancer Ther 2009;8(3):682 -91]

Our earlier work has shown a selective promotional effect on the genesis of mammary carcinomas bearing a wild-type, but not mutant, Ha-ras codon 12 in a 1-methyl-1-nitrosourea (MNU)-induced carcinogenesis model by high-fat diets (Nutr Cancer 23, 283-290, 1995). To test the hypothesis that activation of the mitogen-activated protein kinase (MAPK) pathway is associated with this promotional effect, we compared the in vivo MAPK phosphorylation state of carcinomas from rats consuming a low-fat (5% corn oil, modified AIN-93G) with that from rats consuming a high-fat (25% corn oil) diet. Specifically, 21-day-old female Sprague-Dawley rats were given an intraperitoneal injection of MNU and one week later were randomized to the two diets for six weeks. The number of mammary carcinomas per rat was 68% greater in the high-fat group, and Ha-ras mutation was rare in this short-term model. The levels of the phosphorylated MAPK2 (active) and of proliferating cell nuclear antigen (PCNA) were significantly higher in carcinomas from the high-fat group, and the two parameters were substantially correlated (r2 = 0.43, p < 0.01). The expression level of c-Raf was fourfold higher in the high-fat group but was only modestly associated with MAPK activation (r2 = 0.35, p < 0.05). The levels of the total MAPK1 and MAPK2, guanosine triphosphatase-activating protein, Ha-Ras, and MAPK kinase did not change. These results suggest that an upregulation of c-Raf expression by high fat may in part account for the in vivo MAPK activation, which in turn may enhance cell proliferation and mammary carcinogenesis.