Effect of diindolylmethane supplementation on low-grade cervical cytological abnormalities: double-blind, randomised, controlled trial

This work was supported by a Cancer Research UK project grant (C8162/A4609 project costs) and a programme grant (C8162/A10406)

Induction of G1 and G2/M cell cycle arrests by the dietary compound 3,3'-diindolylmethane in HT-29 human colon cancer cells

<p>Abstract</p> <p>Background</p> <p>3,3'-Diindolylmethane (DIM), an indole derivative produced in the stomach after the consumption of broccoli and other cruciferous vegetables, has been demonstrated to exert anti-cancer effects in both <it>in vivo </it>and <it>in vitro </it>models. We have previously determined that DIM (0 – 30 μmol/L) inhibited the growth of HT-29 human colon cancer cells in a concentration-dependent fashion. In this study, we evaluated the effects of DIM on cell cycle progression in HT-29 cells.</p> <p>Methods</p> <p>HT-29 cells were cultured with various concentrations of DIM (0 – 30 μmol/L) and the DNA was stained with propidium iodide, followed by flow cytometric analysis. [³H]Thymidine incorporation assays, Western blot analyses, immunoprecipitation and <it>in vitro </it>kinase assays for cyclin-dependent kinase (CDK) and cell division cycle (CDC)2 were conducted.</p> <p>Results</p> <p>The percentages of cells in the G1 and G2/M phases were dose-dependently increased and the percentages of cells in S phase were reduced within 12 h in DIM-treated cells. DIM also reduced DNA synthesis in a dose-dependent fashion. DIM markedly reduced CDK2 activity and the levels of phosphorylated retinoblastoma proteins (Rb) and E2F-1, and also increased the levels of hypophosphorylated Rb. DIM reduced the protein levels of cyclin A, D1, and CDK4. DIM also increased the protein levels of CDK inhibitors, p21^CIP1/WAF1and p27^KIPI. In addition, DIM reduced the activity of CDC2 and the levels of CDC25C phosphatase and cyclin B1.</p> <p>Conclusion</p> <p>Here, we have demonstrated that DIM induces G1 and G2/M phase cell cycle arrest in HT-29 cells, and this effect may be mediated by reduced CDK activity.</p

3,3′-Diindolylmethane Induces G1 Arrest and Apoptosis in Human Acute T-Cell Lymphoblastic Leukemia Cells

Certain bioactive food components, including indole-3-carbinol (I3C) and 3,3′-diindolylmethane (DIM) from cruciferous vegetables, have been shown to target cellular pathways regulating carcinogenesis. Previously, our laboratory showed that dietary I3C is an effective transplacental chemopreventive agent in a dibenzo[def,p]chrysene (DBC)-dependent model of murine T-cell lymphoblastic lymphoma. The primary objective of the present study was to extend our chemoprevention studies in mice to an analogous human neoplasm in cell culture. Therefore, we tested the hypothesis that I3C or DIM may be chemotherapeutic in human T-cell acute lymphoblastic leukemia (T-ALL) cells. Treatment of the T-ALL cell lines CCRF-CEM, CCRF-HSB2, SUP-T1 and Jurkat with DIM in vitro significantly reduced cell proliferation and viability at concentrations 8- to 25-fold lower than the parent compound I3C. DIM (7.5 µM) arrested CEM and HSB2 cells at the G1 phase of the cell cycle and 15 µM DIM significantly increased the percentage of apoptotic cells in all T-ALL lines. In CEM cells, DIM reduced protein expression of cyclin dependent kinases 4 and 6 (CDK4, CDK6) and D-type cyclin 3 (CCND3); DIM also significantly altered expression of eight transcripts related to human apoptosis (BCL2L10, CD40LG, HRK, TNF, TNFRSF1A, TNFRSF25, TNFSF8, TRAF4). Similar anticancer effects of DIM were observed in vivo. Dietary exposure to 100 ppm DIM significantly decreased the rate of growth of human CEM xenografts in immunodeficient SCID mice, reduced final tumor size by 44% and increased the apoptotic index compared to control-fed mice. Taken together, our results demonstrate a potential for therapeutic application of DIM in T-ALL

Abstract P1-06-03: Chinese Herb Fructus Cornii Exhibits Preventive Efficacy in a Cell Culture Model for Hormone Responsive Breast Cancer

Abstract Background: Selective estrogen receptor modulators and combination of chemotherapeutic agents represent conventional therapeutic interventional strategies for estrogen receptor positive (ER+) clinical breast cancer. Herbal medicines have been extensively used in alternative and complementary medicine for cancer of various organ sites. Concerns of adverse toxicity or acquired tumor resistance from conventional chemo-endocrine therapy, rationalizes the use of herbal medicines independently or in combination with chemo-endocrine therapy. The present study has utilized a human cell culture model for ER+ breast cancer to assess potential preventive efficacy of a popular Chinese nutritional herb Fructus Cornii (FC). Material and Methods: The ER+ human mammary carcinoma derived MCF-7 cells represented the model. Status of cellular metabolism of estradiol (E2), anchorage dependent growth and anchorage independent colony formation provided the quantitative end point biomarkers for efficacy. Results: MCF-7 cells maintained in a serum depleted medium (E2&amp;lt;1 nM) retained E2 responsiveness as evidenced by growth modulation by E2, its metabolites, anti-estrogen Tamoxifen, and by the persistence of E2 metabolism via the C17-oxidation, C2-hydroxylation and C16α-hydroxylation pathways. Treatment of E2 stimulated MCF-7 cells with FC produced cytostatic growth arrest and reduction in the number of anchorage independent colonies in a dose dependent manner. Treatment of MCF-7 cells with FC at the maximum cytostatic concentration increased anti-mitogenic 2-hydroxyestrone (2-OHE1), and decreased pro-mitogenic 16α-hydroxyestrone (16α-OHE1) formation. Discussion: These data identify a mechanistic lead for the efficacy of FC and thereby, validate a human cell culture based approach for rapid prioritization of efficacious herbal medicinal products for prevention/therapy of ER+ clinical breast cancer. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-06-03.</jats:p

Comparison of plasma and urinary levels of 2-hydroxyestrogen and 16 alpha-hydroxyestrogen metabolites

A modified ELISA assay for measurement of the two estrogen metabolites 2-hydroxyestrone (2OHE1) and 16 alpha-hydroxyestrone (16 alpha OHE1) in plasma and serum has been developed. Previously, these have only been measured in urine. It is not known how well the measurements of these metabolites in urine and plasma are correlated. The goal of this study was to compare urinary and plasma levels of 2OHE1 and 16 alpha OHE1 and their ratios and to explore how they were affected by ethnicity, dietary and genetic factors, and medication use. Blood and urine samples were obtained from 511 nulliparous women, aged 17-35, from four ethnic groups during the same visit at the study center, on a random day of the menstrual cycle. The overall correlation between the 2OHE1/16 alpha OHE1 ratio in plasma and urine was fair (r(s) = 0.52; p < 0.0001). In general, the correlation between the 2OHE1/16 alpha OHE1 ratio in urine and plasma was higher among women not using oral contraceptives (OCs) (r(s) = 0.58; p < 0.0001) than among women currently using OCs (r(s) = 0.34; p < 0.0001). The correlation was highest for samples obtained during the mid-cycle in among non-OC users (r(s) = 0.83; p < 0.0001). Among non-OC users, the urinary 2OHE1/160 alpha OHE1 ratio was stable over the menstrual cycle while there was an increase in the plasma 2OHE1/16 alpha OHE1 ratio. The strongest factors predicting discordance between the urinary and plasma 2OHE1/16 alpha OHE1 ratios among non-OC users were a baseline urinary 20HE1/16 alpha OHE1 ratio in the three upper quartiles (p < 0.001), the menstrual cycle phase (p = 0.001), and the number of cups of coffee consumed per day (p = 0.006). Among current OC users, the strongest predictors of discordance between the urinary and plasma 2OHE1/16 alpha OHE1 ratios were a baseline urinary 2IHE1/16 alpha OHE1 ratio in the three lower quartiles (p < 0.001), being black (p = 0.001), and being Asian (p = 0.014). In conclusion, we found that the correlation between the two methods was fair and varied according to the baseline urinary 2OHE1/16 alpha OHE1 ratio, ethnic group, OC status, coffee consumption, and time of menstrual cycle when the samples were obtained. (C) 2005 Elsevier Inc. All rights reserved

Abstract P1-10-02: Comparative Preventive Efficacy of Select Chinese Herbs in Breast Carcinoma Derived Isogenic Cells with Modulated Estrogen Receptor Functions

Abstract Background: Human mammary carcinoma derived estrogen receptor positive (ER+) MCF-7 cells depend on 17β-estradiol (E2) for cellular proliferation in vitro and for tumor development in vivo. Traditionally, ER functions as a ligand activated nuclear transcription factor with E2 as its physiological ligand, and limited availability of E2 modulates ER functions. We have recently demonstrated preventive efficacy of selected Chinese nutritional herbs in this MCF-7 cell culture model. The present study screened additional Chinese nutritional herbs for their preferential preventive efficacy on MCF-7 cells with modulated ER functions. Methods: MCF-7 cells were maintained in medium supplemented with either 0.7% serum, &amp;lt;1 nM E2, or 0.7% serum+20 nM E2. This approach, while preserving the MCF-7 genotype, provides isogenic cells with either non-functional ER (ER-NF) or functional ER (ER-F) phenotype, and thereby, enables paired comparisons for the preventive efficacy of herbs on the two phenotypes. Anchorage dependent and anchorage independent growth, cell cycle progression, cellular apoptosis, and E2 metabolism represent the quantitative end points.Results: Long-term maintenance of MCF-7 cells in a serum-depleted culture medium (0.7% serum, &amp;lt; 1 nM E2) selects a proliferative sub-population of E2 responsive cells with transiently non-functional ER due to limited availability of E2. The growth inhibitory effects of non-fractionated aqueous extracts from 15 Chinese nutritional herbs were compared in MCF-7 cells selected for either the ER-NF or the ER-F phenotype. Comparative dose response experiments identified IC50 concentrations, and thereby, facilitated rank ordering for growth inhibition. Although all the herbs tested were effective in the present model, two herbs exhibited greater efficacy for the ER-NF phenotype, eight herbs exhibited greater efficacy for the ER-F phenotype, while five herbs exhibited equal efficacy for both the ER-NF and the ER-F phenotypes. Representative herbs at their respective maximum cytostatic concentrations from these three groups, inhibited anchorage independent growth, induced G1 arrest, induced apoptosis, and regulated E2 metabolism in favor of the anti-proliferative metabolites 2-hydroxyestrone and estriol. Conclusions: Preferential growth inhibition by Chinese nutritional herbs in isogenic ER-NF and ER-F phenotypes of MCF-7 cells and mechanistic efficacy of representative herbs identify possible leads towards their rapid prioritization for planning prevention trials on ER+ and ER− clinical breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-10-02.</jats:p

P3-09-04: Comparative Preventive Efficacy of Aqueous Extracts from Lycium Barbarum Bark and Fruit on Estrogen Receptor Positive Human Mammary Carcinoma MCF-7 Cells.

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 &amp; 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.</jats:p

Abstract P4-09-05: Preventive efficacy of the Chinese nutritional herb epimedium grandiflorum in a preclinical cell culture model for luminal A molecular subtype of breast cancer

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&amp;lt;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.</jats:p