The Luminal A subtype of breast cancer expresses the estrogen receptor (ER)-α and progesterone receptor (PR), but not the human epidermal growth factor receptor (HER)-2 oncogene. This subtype of breast cancer responds to endocrine therapy involving the use of selective estrogen receptor modulators and/or inhibitors of estrogen biosynthesis. However, these therapeutic agents are frequently associated with long-term systemic toxicity and acquired tumor resistance, emphasizing the need to identify non-toxic alternative treatments for chemo-endocrine therapy responsive breast cancer. The present study utilized the human mammary carcinoma-derived, ER+/PR+/HER-2- MCF-7 cell line as a model of the Luminal A subtype of breast cancer to examine the growth inhibitory effect of the Chinese nutritional herb Epimedium grandiflorum (EG) and determine the mechanisms underlying this effect. MCF-7 cells maintained in a serum-depleted culture medium retained their ability to grow in response to 17β-estradiol (E2). Treatment of the MCF-7 cells with EG resulted in dose-dependent inhibition of E2-promoted growth. Mechanistically, EG inhibited E2-promoted cell cycle progression through G1 stage arrest and modulated the cellular metabolism of E2, increasing the formation of the anti-proliferative metabolites 2-hydroxyestrone and estriol. Long-term treatment of MCF-7 cells with EG inhibited E2-promoted anchorage independent growth, a surrogate in vitro biomarker of tumorigenesis. In conclusion, the results of the present study demonstrate the growth inhibitory effects of EG on MCF-7 cells and identified clinically relevant mechanistic leads for its anti-tumorigenic efficacy.

In estrogen receptor (ER)+ MCF-7 cells, ER represents a ligand-activated transcription factor, and 17β-estradiol (E2) represents its physiological ligand. Maintenance of the human breast carcinoma-derived MCF-7 cells with 0.7% serum selected a proliferative sub-population of E2-responsive cells with transiently non-functional ER due to limited availability of E2. Culture of MCF-7 cells in the presence of either 0.7% serum, <1 nM E2 or 0.7% serum + 20 nM E2 selected isogenic cells with either non-functional ER (ER-NF) or functional ER (ER-F) phenotype. The two phenotypes responded to the growth-promoting effects of E2 and to the growth-inhibitory effects of the selective ER modulator tamoxifen, indicating retention of E2 responsiveness. Comparative dose-response experiments with Chinese nutritional herbs on ER-NF and ER-F cells identified the inhibitory concentration (IC)50 values for these herbs, while the IC50 ratios for the ER-NF:ER-F phenotypes facilitated their rank ordering in terms of efficacy. Out of the 11 efficacious herbs tested, five herbs exhibited ER-F > ER-NF inhibitory activity, four exhibited ER-F = ER-NF inhibitory activity and two exhibited ER-NF > ER-F inhibitory activity. Extracts from representative herbs, Lycium barbarum bark, Epimedium grandiflorum and Cornus officinalis, from each of the three groups inhibited anchorage-independent growth, induced G1 or G2/M arrest and/or apoptosis, and generated anti-proliferative E2 metabolites. The differential growth inhibition in ER-NF and ER-F phenotypes, together with the mechanistic efficacy of representative herbs, identified potential leads for their efficacy on ER+ and/or ER- breast cancer.

Hormonal exposures are known to influence breast cancer risk among women with a BRCA1 mutation. Thus, dietary factors that increase the 2-hydroxyestrone (OHE):16α-OHE ratio, a biomarker inversely related to breast cancer development, may also influence cancer risk. We conducted a dietary intervention study to evaluate the ability of 300 mg/day of 3,3′-diindolylmethane (DIM) to increase the urinary 2:16α-OHE ratio in 20 women with a BRCA1 mutation. BRCA1 mutation carriers (n = 15) were assigned to receive 300 mg/day of Rx Balance BioREsponse DIM for 4–6 weeks (intervention group) and five BRCA1 mutation carriers did not take DIM (control group). The urinary 2:16α-OHE ratio was assessed at baseline and after 4–6 weeks by immunoassay. There was no significant effect of DIM on the 2:16α-OHE ratio (2.4 at baseline vs. 3.0 after the intervention, P = 0.35). A short dietary intervention with DIM did not significantly increase the 2:16α-OHE ratio in female BRCA1 mutation carriers. Larger studies investigating the effect of dietary or lifestyle interventions on circulating hormone levels in these high-risk women are warranted.

Abstract While there is a large volume of literature describing a role for obesity as a risk factor for breast cancer and many other cancers, in the main a causal relationship has not been established. If the study is limited to breast cancer risk, it has been suggested that the increase in sex steroid formation that occurs in postmenopausal women plays a role. Obesity is known to be associated with chronic low grade inflammation, but no reason for this association has been offered in the past. The gut microbiome, while known to be enormous, has not in the past been considered as a metabolic role player in the body. This is now recognized to be the case. Recent studies have found the obesity is correlated with an alteration in the gut microbiome. In obese individual there is a change in the relative proportions of the two major classes of bacteria – bacteroides and firmacutes – with the latter dominant in obesity and resulting in the formation of increased amounts of metabolic endotoxins like deoxycholic acid and lipopolysaccharides (LPS). Obese individuals show a decrease in the concentration of Akkermansia muciniphila in the mucus that lines the intestinal wall, resulting in thinner mucus and a weakened intestinal lining and permitting metabolic endotoxins formed by other bacterial flora like LPS to enter the blood steam and cause the chronic inflammation associated with obesity. The change in the microbiome profile results in increases in bacterial strains that are more efficient at generating energy, leading to increased obesity. In mice, it has been shown that introducing gut bacterial flora from the cecum of obese mice into germ-free mice results in increased obesity with lesser food consumption while the reverse, introducing bacterial flora from lean mice results in a loss in weight. This raises the attractive possibility that manipulating the gut microbiome could facilitate weight loss or prevent obesity in humans.

Abstract Background: The Luminal A molecular sub-type of clinical breast cancer expresses estrogen receptor (ER) and progesterone receptor (PR), but lacks the expression of HER-2 oncogene. This cancer sub-type responds to endocrine therapy involving the use of selective estrogen receptor modulators or inhibitors of estrogen biosynthesis. Conventional therapeutic options are frequently associated with acquired tumor resistance and systemic toxicity, and therefore, emphasize a need for identification of promising new non-toxic agents for secondary prevention. Nutritional substances that do not incur long-term toxicity represent ideal candidates. Nutritional herbs have been used in traditional Chinese medicine for a variety of health related issues, including prevention of breast cancer. Methods: The present study utilized the human mammary carcinoma derived ER+/PR+/HER-2- MCF-7 cells as a model for the Luminal A breast cancer to examine the preventive efficacy of non-fractionated aqueous extract from Epimedium grandiflorum (EG), a popular Chinese nutritional herb. Anchorage dependent growth, cell cycle progression, cellular metabolism of 17b-estradiol (E2), and anchorage independent colony formation represented the quantitative end point biomarkers for preventive efficacy. Results: Maintenance of MCF-7 cells in a chemically defined serum depleted culture medium (serum 0.7%, E2<1 nM) retained their cellular growth promoting response to the physiologically relevant concentration range of 1 nM to 20 nM E2, exhibiting a 10.3% to a 91.9% increase in the viable cell number, respectively. A 7 day treatment of MCF-7 cells with EG resulted in a dose dependent inhibition of E2 promoted growth (EG IC50: 0.49%). At its maximum cytostatic concentration, EG inhibited cell cycle progression via G1 arrest, resulting in a 1.6 fold increase in the G1:S+G2/M ratio, and modulated the cellular metabolism of E2 in favor of formation of anti-proliferative metabolites 2-hydroxyestrone (2-OHE1) and estriol (E3), exhibiting a 5.1 and a 7.6 fold increase, respectively. In addition, EG produced a favorable 3.9 fold increase in the 2-OHE1: 16α-OHE1 ratio, an endocrine biomarker of breast cancer risk. A 21 day treatment of MCF-7 cells with EG produced a dose dependent inhibition in anchorage independent growth (EG IC50: 0.49%; IC90: 1.03%). Conclusions: These data demonstrate the growth inhibitory effects of EG and identify clinically relevant mechanistic leads for its preventive efficacy. The present approach promises to facilitate identification of new efficacious herbs for the secondary prevention of the Luminal A subtype of clinical breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-09-05.

Background: The effects of 3,3'-diindolylmethane (DIM) together with the Gardasil vaccine on cervical histology were evaluated using the K14-HPV16-transgenic mouse model. The possibility that DIM could enhance the efficacy of this preventive vaccine in this model was explored. Materials and Methods: Transgenic mice were given 1000 mg/kg of DIM in the diet for 28 weeks. The mice were injected with Gardasil Quadrivalent HPV vaccine. Some mice were sacrificed at 28 weeks. Other groups were removed from the DIM diet after 28 weeks to a diet with no DIM for either 4 or 8 weeks. Results: Cervical histology indicated that a high percentage of transgenic mice fed DIM and vaccinated with Gardasil manifested normal cervical epitheliums at 4 weeks after DIM discontinuation. Conclusion: Vaccination pre-supplemented with DIM may provide with a window of protection of at least four weeks in this transgenic model. However, extrapolation to the effect in humans is beyond the limited scope of the histological data presented here.

Chemo-endocrine therapy for estrogen receptor positive (ER+) breast cancer exhibits acquired tumor resistance. Herbal medicines provide integrative support for breast cancer patients. Present study compared the efficacy of aqueous extracts from Lycium barbarum bark (LBB) and Lycium barbarum fruit (LBF) on ER+ MCF-7 cells. Cellular growth and 17ß-estradiol (E2) metabolism quantified the efficacy. MCF-7 cells maintained in serum depleted medium+ E2 exhibited increased anchorage-dependent and anchorage-independent growth. LBB exhibited greater potency than LBF (95% reduction in IC50). LBB produced a 6.8-fold increase, 40% decrease, and a 3.7-fold increase in 2-hydroxyestrone (2-OHE1), 16α-hydroxyestrone (16α-OHE1), and estriol (E3) formation. The corresponding values for LBF were 3.9, 33, and 10.5. LBB produced a16.3-fold and a twofold increase in 2-OHE1:16α-OHE1 and E3:16α-OHE1 ratios, whereas LBF produced a sixfold and a 2.9-fold increase. The efficacy of LBB is due to increased 2-OHE1 formation, whereas that of LBF is due to accelerated conversion of 16α-OHE1 to E3. Specific growth inhibitory profiles of LBB and LBF may be due to their distinct chemical composition and their complementary actions on E2 metabolism. This study validates a mechanistic approach to identify efficacious herbal extracts for clinical ER+ breast cancer.

Selective estrogen receptor modulators and a combination of mechanistically distinct chemotherapeutic agents represent conventional therapeutic interventions for estrogen receptor-positive (ER+) clinical breast cancer. Long-term treatment with these agents is associated with acquired tumor resistance and other adverse side effects that impact on patient compliance. Herbal medicines are being widely used in complementary and alternative medicine. However, long-term safety and efficacy of the use of herbal medicines, as well as their interaction with conventional endocrine and chemotherapeutic drug regimens remain largely unknown. The present study utilized a human cell culture model for ER+ clinical breast cancer to examine the potential therapeutic efficacy of an aqueous extract prepared from the fruit of popular Chinese herb Cornus officinalis (CO), also known as Fructus cornii. The human mammary carcinoma-derived MCF-7 cell line represented the model. Status of anchorage-independent growth and cellular metabolism of 17β-estradiol (E₂) represented the quantitative end-point biomarkers for efficacy. MCF-7 cells adapted for growth in serum-depleted medium (0.7% serum, <1 nM E₂) retained their endocrine responsiveness as evidenced by growth promotion by physiological levels of E₂, and growth inhibition by the selective ER modulator tamoxifen at the clinically achievable concentrations. Treatment of MCF-7 cells with CO resulted in inhibition of E₂-stimulated growth in a dose-dependent manner. Similarly, CO treatment also produced a dose-dependent progressive reduction in the number of anchorage-independent colonies, indicating effective reduction of the carcinogenic risk. Treatment of MCF-7 cells with CO at a maximally effective cytostatic concentration resulted in a 5.1-fold increase in the formation of the anti-prolifertive E₂ metabolite 2-hydoxyestrone (2-OHE₁), a 63.6% decrease in the formation of the pro-mitogenic metabolite 16α-hydroxestrone (16-αOHE₁) and a 9.1% decrease in the formation of mitogenically inert metabolite estrone (E₃). These alterations led to a 14.5-fold increase in the 2-OHE₁:16α-OHE₁, and a 3.3-fold increase in the E₃:16α-OHE1 ratios. These data validate a rapid cell culture-based mechanistic approach to prioritize efficacious herbal medicinal products for long-term animal studies and future clinical trials on ER+ clinical breast cancer.

BACKGROUND:Studies have shown associations of diabetes and endogenous hormones with exposure to a wide variety of organochlorines. We have previously reported positive associations of polychlorinated biphenyls (PCBs) and inverse associations of selected steroid hormones with diabetes in postmenopausal women previously employed in a capacitor manufacturing plant.METHODS:This paper examines associations of PCBs with diabetes and endogenous hormones in 63 men previously employed at the same plant who in 1996 underwent surveys of their exposure and medical history and collection of bloods and urine for measurements of PCBs, lipids, liver function, hematologic markers and endogenous hormones.RESULTS:PCB exposure was positively associated with diabetes and age and inversely associated with thyroid stimulating hormone and triiodothyronine-uptake. History of diabetes was significantly related to total PCBs and all PCB functional groupings, but not to quarters worked and job score, after control for potential confounders. None of the exposures were related to insulin resistance (HOMA-IR) in non-diabetic men.CONCLUSIONS:Associations of PCBs with specific endogenous hormones differ in some respects from previous findings in postmenopausal women employed at the capacitor plant. Results from this study, however, do confirm previous reports relating PCB exposure to diabetes and suggest that these associations are not mediated by measured endogenous hormones.

UNLABELLED While cervical cancer incidence and mortality rates have declined in the United States, this cancer represents a worldwide threat. Human papilloma viral infection causes cervical neoplasia (CIN). 3,3'-Diindolylmethane (DIM) prevents or inhibits the progression from cervical dysplasia to cancer. The aim of this study is to determine the most effective dose of DIM given continuously in food, that significantly increases serum interferon gamma levels (IFN-γ) in the K14-HPV16 transgenic mouse model for cervical cancer. MATERIALS AND METHODS Five doses of DIM in food were administered to the mouse model for 20 weeks. Serum Interferon gamma (IFN-γ) levels and estrogen metabolite levels were quantified. RESULTS At 1000 ppm DIM, serum IFN-γ concentrations were significantly increased (p<0.0396). The estrogen metabolites were unchanged. IFN-γ concentrations in CIN free mice and the percentage of CIN free transgenic mice were well correlated (r=0.88). DISCUSSION Significant increases in IFN-γ serum concentrations that correlate with the percentage of CIN free mice in each group indicate that 1000 ppm of DIM in food may be the most effective dose for future studies. These results may eventually lead to new and effective vaccination strategies in women already infected with the human papilloma virus.

BACKGROUND The incidence of thyroid cancer is four to five times higher in women than in men, suggesting a role for estrogen (E₂) in the pathogenesis of thyroid proliferative disease (TPD) that comprises cancer and goiter. The objective of this study was to investigate the antiestrogenic activity of 3,3'-diindolylmethane (DIM), a bioactive compound derived from cruciferous vegetables, in patients with TPD. METHODS In this limited phase I clinical trial study, patients found to have TPD were administered 300 mg of DIM per day for 14 days. Patients subsequently underwent a total or partial thyroidectomy, and tissue, urine, and serum samples were collected. Pre- and post-DIM serum and urine samples were analyzed for DIM levels as well as estrogen metabolites. DIM levels were also determined in thyroid tissue samples. RESULTS DIM was detectable in thyroid tissue, serum, and urine of patients after 14 days of supplementation. Urine analyses revealed that DIM modulated estrogen metabolism in patients with TPD. There was an increase in the ratio of 2-hydroxyestrones (C-2) to 16α-hydroxyestrone (C-16), consistent with antiestrogenic activity that results in more of C-2 product compared with C-16. CONCLUSION Our data suggest that DIM enhances estrogen metabolism in TPD patients and can potentially serve as an antiestrogenic dietary supplement to help reduce the risk of developing TPD. The fact that DIM is detected in thyroid tissue implicates that it can manifest its antiestrogenic activity in situ to modulate TPD.

The human papilloma virus is the major cause of cervical cancer. Viral infection initiates cervical intraepithelial neoplasia, which progresses through several stages to cervical cancer. The objective of this study is to identify the minimum effective dose of diindolylmethane that prevents the progression from cervical dysplasia to carcinoma in situ. We document cervical histology in K14-HPV16 mice receiving different doses of diindolylmethane. Urinary diindolylmethane concentrations are reported. Diindolylmethane could enhance the efficacy of human papilloma virus vaccines, creating a new therapeutic use for these vaccines in women already infected with the virus. Five doses (0–2,500 ppm) of diindolylmethane were incorporated into each mouse diet. The reproductive tract was serially sectioned and urine was obtained for analysis of urinary diindolylmethane. The results indicate that 62% of mice receiving 1,000 ppm diindolylmethane remained dysplasia-free after 20 weeks compared with 16% of mice receiving no diindolylmethane and 18% receiving 500 ppm; 1,000 ppm of 3,3′-diindolylmethane in the diet completely suppressed the development of cervical cancer. Urinary diindolylmethane levels increased significantly as diindolylmethane in food increased. These findings imply usefulness for diindolylmethane in the search to prevent cervical cancer when used in combination with prophylactic or therapeutic vaccines. Cancer Prev Res; 4(6); 890–6. ©2011 AACR.

There is an increasing body of literature showing associations of organochlorine exposure with risk of diabetes and insulin resistance. Some studies suggest that associations differ by gender and that diabetes risk, in turn, may be affected by endogenous steroid hormones. This report examines the relationships of serum PCBs and endogenous hormones with history of diabetes in a cohort of persons previously employed at a capacitor manufacturing plant. A total of 118 women were post-menopausal with complete data, of whom 93 were not using steroid hormones in 1996, at the time of examination, which included a survey of exposure and medical history, height, weight and collection of blood and urine for measurements of lipids, liver function, hematologic markers and endogenous hormones. This analysis examines relationships of serum polychlorinated biphenyls (PCBs), work exposure and endogenous hormones with self-reported history of diabetes after control for potential confounders. All PCB exposure groups were significantly related to history of diabetes, but not to insulin resistance as measured by the homeostatic model assessment of insulin resistance (HOMA-IR) in non-diabetics. Diabetes was also independently and inversely associated with follicle stimulating hormone (FSH), dehydroepiandrosterone sulfate (DHEAS) and triiodothyronine (T3) uptake. HOMA-IR was positively associated with body mass index (BMI) and C-reactive protein (CRP) and inversely associated with sex hormone binding globulin (SHBG) and T3 uptake after control for PCB exposure. Possible biologic mechanisms are discussed. This study confirms previous reports relating PCB exposure to diabetes and suggests possible hormonal pathways deserving further exploration.

The role of body fat as a risk factor for breast cancer has been well established. A decrease in the urinary 2/16α-hydroxyestrone ratio has also been shown to be a risk marker for breast cancer. These two observations are connected by the fact that obese women have decreased levels of 2-hydroxyestrone. To test the hypothesis that fat depots secrete factors that inhibit 2-hydroxylation, the effect of substances released into the media from adipocytes incubated in Krebs-Ringer buffer, on estrogen metabolism by MCF-7 cells in minimum essential medium eagle (MEM) plus adipocyte-conditioned media (ACM) was studied. The 1:1 ACM-MEM culture system resulted in a substantial and highly significant decrease in 2-hydroxylation of estradiol. This inhibition was partially reversed by the addition of indole-3-carbinol, a potent inducer of 2-hydroxylation of estradiol. Centrifugal sizing showed that the active 2-hydroxylation inhibitor in the medium had a molecular weight of about 30 kDa. These results suggest a mechanism for the decrease in 2-hydroxylation of estradiol that is observed in obese women and the increase in 2-hydroxylation observed in women with depleted fat depots.

Abstract Background: Chemotherapy, selective estrogen receptor modulators and aromatase inhibitors represent major treatment options for estrogen receptor positive (ER+) clinical breast cancer. These modalities frequently exhibit acquired tumor resistance and adverse systemic toxicity. Natural herbs are increasingly being used in the integrative support of breast cancer patients undergoing conventional treatment modalities and for prevention purposes. We previously reported favorable findings for the fruit of Lycium barbarum (LB), known commonly as goji berry, in the prevention for ER+ breast cancer (Nutrition & Cancer 61:408–414, 2009). The present study compares the preventive efficacy of non-fractionated aqueous extracts from the bark of LB (LBB) and the fruit of LB (LBF) on a cell culture model for ER + breast cancer. Material and Methods: The ER+ human mammary carcinoma derived MCF-7 cells represented the model. Anchorage dependent growth, anchorage independent colony formation and cellular metabolism of 17β-estradiol (E2) represented the mechanistic bases for efficacy. Results: MCF-7 cells grown in serum depleted medium retained their responsiveness to E2, exhibiting E2 stimulated promotion of anchorage dependent growth and anchorage independent colonies. LBB was much more potent than LBF as evidenced by a 95% reduction of IC50 from 0.38% for LBF to 0.02% for LBB, and a 95% reduction of maximum cytostatic concentration (MCC) from 1% for LBF to 0.05% for LBB. At their respective MCC, LBB treatment and LBF treatment produced a 93% and an 89% decrease, respectively, in the number of anchorage independent colonies. Also, LBB produced a 6.8 fold increase in 2-hydroxyestrone (2-OHE1), a 40% decrease in 16a-hydroxyestrone (16a-OHE1) and a 3.7 fold increase in estriol (E3) formation. The corresponding values for LBF were 3.9, 33 and 10.5. LBB treatment resulted in a 16.3 fold increase in 2-OHE1:16a-OHE1 ratio and a 2 fold increase in E3:16a-OHE1 ratio, while LBF treatment produced a 6.0 fold increase and a 2.9 fold increase, respectively, in these endocrine biomarkers. These data suggest that the preventive efficacy of LBB may predominantly be due to up-regulation of the anti-proliferative 2-OHE1 formation, while accelerated conversion of the pro-mitogenic 16α-OHE1 to the mitogenically inert E3 is the case with LBF. Discussion: Growth inhibitory profiles of LBB and LBF may in part be due to their distinct chemical composition and their complementary actions on E2 metabolism. The superior sensitivity of LBB relative to LBF, together with its non-toxic nature, suggests the feasibility of its use in the prevention of human breast cancer. A prevention trial to assess its efficacy and to establish relevant dose schedule is warranted. This study validates a mechanism based approach to identify and prioritize efficacious herbal extracts for the prevention of ER+ breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-09-04.

UNLABELLEDIndole-3-carbinol (I3C) when given orally is converted to diindolylmethane (DIM) and other oligomers catalyzed by stomach acid. This suggests that DIM is the predominant active agent and that I3C is a precursor, 'pro-drug' in vivo. However, in cell culture studies carried out in neutral solutions, I3C has been considered fully active.MATERIALS AND METHODSThe stability of I3C in cell culture media was studied.RESULTSIn the 8 different cell culture media tested, greater than 50% dimerization of I3C into DIM occurred in 24 hours. At 48 hour, greater than 60% conversion was found. When neutral synthetic cerebrospinal fluid (CSF) or peritoneal fluid (PF) was studied, a large peak, tentitively identified as I3C's linear trimer (LTR) conversion product by mass spectra, and two smaller peaks, were seen. When CSF or PF was diluted 1:1 with media, the formation of these additional peaks was diminished.CONCLUSIONBecause of the greater biologic potency of DIM when studied in parallel with I3C in vitro, this extent of dimerization shows that DIM rather than I3C is the active agent in cell culture studies.