Nitin T. Telang

The Role of Estrogen in Mammary Carcinogenesisa

The in vivo and in vitro studies conducted to examine whether E2 functions as an initiator or a promoter in mammary carcinogenesis can be summarized as follows: (1) Clinical and animal studies in vivo have shown a positive correlation of up-regulation of E2 C16 alpha-hydroxylation with either the presence of or the risk for breast cancer, suggesting that this metabolic alteration may represent an early-occurring event in the multistep process of tumorigenesis. (2) The mammary tissue, target for carcinogenesis, exhibits cancer risk-dependent alteration in E2 metabolism in the rodent and human mammary explant culture model, indicating that E2 metabolites may directly influence the mammary epithelium. (3) The 16 alpha-hydroxylated metabolite of E2, 16 alpha-OHE1, induces genotoxic DNA damage and aberrant hyperproliferation similar to that induced by chemical carcinogens in the rodent cell culture model. In preinitiated or fully transformed rodent or human cells, 16 alpha-OHE1 promotes the expression of transformed phenotype. (4) The initiator-mediated perturbation of E2 C16 alpha-hydroxylation in rodent and human mammary explant cultures is modulated by naturally occurring dietary constituents that are known to modulate rodent mammary tumorigenesis. (5) The observed effect of E2 on mammary tumorigenesis may be due in part to the generation of 16 alpha-OHE1, which functions as a weak initiator or a potent promoter of tumorigenic transformation in mammary epithelial cells. (6) The reaction of 16 alpha-OHE1 with the transcription factor ER is unique in that it can be irreversible and leads to aberrant gene expression.

Indole‐3‐carbinol

The results show that all of the carcinogens, oncogenes, and tumor-associated viruses that we have studied profoundly affect the extent of 2- and 16 alpha-hydroxylation in a prorisk direction. All of the dietary and biological responses associated with increased cancer risk decrease 2-hydroxylation and increase 16 alpha-hydroxylation. Remarkably, although PAHs are reported to induce P450-1A1, we have found them to decrease 2-hydroxylation. Finally, using indole-3-carbinol to induce 2-hydroxylation results in the chemoprevention of mammary tumors in rodents and recurrences of laryngeal papillomas in humans. Also correlating with these studies in HPV is the decrease in the C-2/C-16 alpha metabolite ratio observed in women with CIN relative to control subjects. The greatest decrease was observed in women with the most severe form, CIN3 (Figure 23). These findings are under further investigation.

Experimental down-regulation of intermediate biomarkers of carcinogenesis in mouse mammary epithelial cells

SummaryThe polycyclic aromatic hydrocarbon 7,12-dimethylbenz(a)anthracene (DMBA) is a metabolism-dependent procarcinogen whose tumorigenicity is modified by dietary and endocrine manipulationsin vivo. DMBA initiates molecular and cellular alterations in the mammary tissue, while dietary components and estrogens affect the post-initiational phase of tumorigenic transformation. The mechanism(s) responsible for modulation of tumorigenic transformation remain unclear. This study examines the effects of selected tumor suppressing agents and estradiol (E2) metabolites onin vitro DMBA carcinogenesis utilizing a newly established mouse mammary epithelial cell line C57/MG. Alteration in DNA repair synthesis, metabolism of E2 via the C2- and C16α-hydroxylation pathways, and acquisition of anchorage-independent growth were utilized as molecular, endocrine, and cellular biomarkers to quantitate the cellular transformation by DMBA and its modulation by tumor suppressing agents and E2 metabolites. A single 24 hr exposure of 0.78 µM DMBA to C57/MG cells resulted in a 193.9% increase in DNA repair synthesis and a 73.1% decrease in C2/C16α hydroxylation of E2. The DMBA treated C57/MG cells also exhibited increased anchorage-independencein vitro prior to tumorigenesisin vivo. A simultaneous treatment of cells with DMBA and with the highest non-cytotoxic doses of the tumor suppressing agents 5 µM N-(4-hydroxyphenyl) retinamide (HPR), 50 µM indole-3-carbinol (I3C), or 1 µM tamoxifen (TAM) resulted in a 35.6% to 63.9% decrease in DNA repair synthesis, a 23.8% to 1347.6% increase in C2/C16α hydroxylation of E2, and a 53.8% to 72.4% decrease in anchorage-independent growth. The E2 metabolites at the highest non-cytotoxic doses of 0.76 µM estrone (E1), 0.69 µM 2-hydroxyestrone (2-OHE1), and 0.66 µM 2-methoxyestrone (2-MeOHE1) suppressed DMBA-induced DNA repair synthesis by 56.0% to 68.8%. These tumor suppressing agents and E2 metabolites also effectively suppressed post-initiational, anchorage-independent growth by 24.9% to 72.4%. These results indicate that DMBA induces cellular transformation in part by causing DNA damage, altering C2/C16α hydroxylation in favor of C16α-hydroxylation, and inducing anchorage-independent growth prior to tumor development. Effective downregulation of these genotoxic, endocrine and proliferative end points by prototypic tumor suppressing agents and by E2 metabolites generated via the C2-hydroxylation pathway suggest that these agents may influence mammary tumorigenesis by inhibiting early occurring initiational and/or post initiational events.

Chemoprevention of mammary tumor virus-induced and chemical carcinogen-induced rodent mammary tumors by natural plant products

SummaryThe natural plant products turmeric, β-carotene, catechin, and betel leaf extract were evaluated for their antitumor effects on mammary tumorigenesis in murine mammary tumor expressing C3H (Jax) mice and in Wistar rats treated with the chemical carcinogen 7-12-dimethylbenz(a)anthracene (DMBA). Administration of turmeric through the diet and of β-carotene, catechin, and betel leaf extract through the drinking water to virgin female C3H mice resulted in decreased tumor incidence and tumor burden. Administering 5% turmeric in the diet from 2 months of age showed suppression of mammary tumor virus-related reverse transcriptase activity and of preneoplastic changes in the mammary glands. Furthermore, feeding turmeric from 6 months of age resulted in a 100% inhibition of mammary tumors. In the DMBA model of rat mammary tumorigenesis, administration of turmeric, catechin, and betel leaf extract resulted in decreased tumor burden and tumor incidence, and a delay in the onset of mammary tumors.

ESTROGEN METABOLITES AS BIOREACTIVE MODULATORS OF TUMOR INITIATORS AND PROMOTERS

A role for estrogens in the initiation and promotion of breast and endometrial cancer has been clear for more than 100 years since Beatson demonstrated that oophorectomy induced remissions in breast cancer (1). The details of these responses are still not clear and are under further study.

Chemoprevention of Mammary Preneoplasia

The major findings of this study can be summarized as follows: (1) the c-myc oncogene-transfected and MTV-expressing mammary epithelial cells exhibit aberrant hyperproliferation in vitro preceding tumorigenesis in vivo; (2) upregulation of aberrant hyperproliferation (i.e., anchorage-independent growth) in initiated cells represents a cellular marker for preneoplastic transformation; (3) the tea polyphenol EGCG differentially downregulates aberrant hyperproliferation in myc oncogene- and MTV-initiated cells; (4) the present in vitro model provides an efficient assay for chemoprevention of mammary preneoplasia by naturally occurring compounds.

Alteratioon in proliferative and endocrine responsiveness of human mammary carcinoma cells by prototypic tumor-suppressing agents

The experiments performed in this study were designed to establish that (1) acquisition of anchorage-independent growth, a biological characteristic of tumorigenically transformed phenotype, can be modulated by prototypic tumor-suppressing agents, and (2) modulation of growth is influenced by the metabolic competence of the cells to biotransform estradiol, MCF-7 human breast carcinoma cells exhibited linear cell proliferative kinetics with a 41-hour population doubling time, and a 15% colony-forming efficiency in 0.33% agar. Indole-3-carbinol (13C), a naturally occurring tumor-suppressive agent; tamoxifen (TAM), an antiestrogenic agent; and 4-hydroxytamoxifen (4-OHTAM), a metabolite of TAM, demonstrated 73.7%, 72.5%, and 89.9% suppression in anchorage-independent growth of MCF-7 cells, respectively. At the metabolic level, 13C and 4-OHTAM induced 2.3-fold (P < 0.0001) and 1.3-fold increase (P = 0.001) relative to their own controls in the extent of 2-hydroxylation of estradiol. The results indicate that growth inhibition by 13C, TAM, and 4-OHTAM may in part be due to altered estradiol metabolism in MCF-7 cells. Thus, anchorage-independent growth and altered biotransformation of estradiol may constitute useful cellular and endocrine markers to evaluate the biological response of chemosuppressive agents.

Genotoxic Damage and Aberrant Proliferation in Mouse Mammary Epithelial Cells

Publisher Summary This chapter discusses genotoxic damage and aberrant proliferation in the mouse mammary epithelial cells. Conversion to the fully transformed tumorigenic phenotype is the end result of a series of molecular, metabolic, and cellular events leading to the neoplastic state. This series of events has been reported in vivo as well as in vitro during mammary carcinogenesis. In addition to intermediate biomarkers, such as deregulated expression of oncogenes, mutagenic changes, and altered proliferation, changes in estrogen metabolism may be a biomarker for target tissue susceptibility to tumorigenic changes. Increases in DNA repair have been extensively validated as a marker for genotoxic DNA damage. Using hydroxyurea to suppress replicative DNA synthesis, one can readily measure DNA repair activity. 16α-OHE1 can directly interact with cellular DNA and thereby function as an initiator. The hyperproliferation of the epithelial component has been reported to occur in tissues at increased risk for tumors or following exposure to carcinogens and is implicated as an intermediate biomarker preceding transformation. The ability to acquire anchorage-independent growth has been correlated with treatment with various initiators of tumorigenic transformation and may be a cellular marker for tumor genesis. Preliminary studies have shown that inhibiting the formation of 16α-OHE1 by treatment with indole-3-carbinol decreases the incidence of tumors in susceptible mice.

In Vitro and In Vivo Modulation of Growth Regulation in the Human Breast Cancer Cell Line MCF-7 by Estradiol Metabolites.

BackgroundThe natural estrogen 17β-estradiol (E2) functions as a potent tumor promoter during tumorigenic transformation of the mammary gland. From amongst the various pathways of E2 metabolism upregulation of C16α-hydroxylation of E2 has been associated with carcinogenesis. In the present studyin vitro andin vivo experiments were performed on estrogen receptor positive human breast cancer MCF-7 cells to examine whether the natural estrogen E2 and its metabolites 16α-hydroxyestrone (16α-OHEi) and 2-hydroxyestrone (2-OHE1) function as modulators of tumor cell growth.MethodsAn anchorage-independent growth assay was used forin vitro study by counting the number of tri-dimensional colonies formed by MCF-7 cells suspended in 0.33% agar.In vivo experiments examined the effect of implanting metabolite material pellets into female nude mice.ResultsIn the anchorage-independent growth assay (AIG), continuous 14-day exposure to E2 and to 16α-OHE1 at 200 ng/ml induced a 59.4% and a 105.9% increase (P=0.001) respectively in the number of colonies of MCF-7 cells. Identical treatment with 2-OHE1, however, failed to increase AIG relative to that seen in the solvent treated control cultures. In thein vivo tumorigenicity assay, treatment of nude mice with 1.5 mg E2 or 16α-OHE1 resulted in a 335.4% and a 384.1% increase (P<0.0002) in tumor growth, while identical treatment with 2-OHE1 failed to exhibit any increase relative to the control group.ConclusionsThese results suggest that the 16α- and 2-hydroxylated metabolites of E2 may directly affectin vitro growth of MCF-7 cells via an autocrine mechanism andin vivo growth via paracrine mechanisms. Thus, E2-mediated growth regulation in MCF-7 cells may in part be due to distinct effects of specific E2 metabolites on the breast cancer cells.

Negative Growth Regulation of Oncogene-Transformed Mammary Epithelial Cells by Tumor Inhibitors

Deregulated expression of Ras and myc oncogenes confers neoplastic transformation in non-tumorigenic mammary epithelial cells. Down-regulation of perturbed biomarkers prior to tumorigenesis may provide a means of effective chemoprevention. In vitro experiments were designed to i) identify specific molecular, endocrine and cellular biomarkers as quantitative end points for preneoplastic transformation, and ii) utilize these end points to evaluate chemopreventive efficacy of selected naturally-occurring and synthetic tumor inhibitors. Stable Ras and myc transfectants exhibited persistent expression of oncogene specific mRNA transcripts, altered estradiol biotransformation and enhanced anchorage-independent growth in vitro prior to tumorigenesis in vivo. Treatment of the transfectants with omega-3-fatty acid, indole-3-carbinol and tamoxifen individually suppressed the perturbed molecular, endocrine and cellular biomarkers in vitro. Thus, suppressed oncogene expression and altered estrogen metabolism may be important determinants for antiproliferative mechanisms in mammary tumor inhibition, providing useful end points for chemopreventive intervention of preneoplasia.