Breast Cancer Research and Treatment

, Volume 66, Issue 2, pp 147–157 | Cite as

Methyl‐substituted diindolylmethanes as inhibitors of estrogen‐induced growth of T47D cells and mammary tumors in rats

  • Andrew McDougal
  • Mona Sethi Gupta
  • Derek Morrow
  • Kavita Ramamoorthy
  • Jeong‐Eun Lee
  • Stephen H. Safe


Diindolylmethane (DIM) is formed by acid catalyzed dimerization of the phytochemical indole‐3‐carbinol, and both compounds inhibit formation and/or growth of mammary tumors in rodents. In this study, we have investigated the aryl hydrocarbon receptor (AhR) agonist activity and inhibitory AhR‐estrogen receptor crosstalk induced by the following methyl‐substituted DIMs: 1,1′‐dimethyl‐, 2,2′‐dimethyl‐, 5,5′‐dimethyl‐, 6,6′‐dimethyl‐, and 7,7′‐dimethylDIM and 1,1′,2,2′‐tetramethylDIM. The six compounds bound to the rat cytosolic AhR in a transformation assay but, at concentrations <10μM, exhibited minimal to non‐detectable AhR agonist or antagonist activities associated with CYP1A1 induction. In contrast, the methyl‐substituted DIMs inhibited estrogen‐induced T47D human breast cancer cell growth and the four most active compounds (1,1′‐, 2,2′‐, 5,5′‐dimethylDIM and 1,1′,2,2′‐tetramethylDIM) inhibited one or more estrogen‐induced responses in the 21‐day‐old female B6C3F1 mice at a dose of 100mg/kg/day (X3). Induction of hepatic CYP1A1‐dependent activity was not observed at this high dose. The antitumorigenic activity of these compounds was examined in 7,12‐dimethylbenz[a]anthracene‐induced rat mammary tumor model in which the DIM analogs were orally administered (by gavage in corn oil) at a dose of 1mg/kg/day (X10). 1,1′‐DimethylDIM, 5,5′‐dimethylDIM and 1,1′,2,2′‐tetramethylDIM significantly inhibited mammary tumor growth, and this was not accompanied by changes in organ/body weights or histopathology. These studies demonstrate that methyl‐substituted DIMs are selective AhR modulators (SAhRMs) with potential for clinical treatment of breast cancer.

antitumorigenic selective AhR modulators substituted diindolylmethanes 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Sondik EJ: Breast cancer trends. Incidence, mortality, and survival. Cancer 74: 995-999, 1994Google Scholar
  2. 2.
    Hulka BS, Liu ET, Lininger RA: Steroid hormones and risk of breast cancer. Cancer 74: 1111-1124, 1994Google Scholar
  3. 3.
    Hulka BS: Epidemiologic analysis of breast and gynecologic cancers. In: Aldaz M, Gould MN, McLachlan J, Slaga TJ (eds) Etiology of Breast and Gynecological Cancers. Wiley-Liss, 1997, pp 17-29Google Scholar
  4. 4.
    Jordan VC: Molecular mechanisms of antiestrogen action in breast cancer. Breast Cancer Res Treat 31: 41-52, 1994Google Scholar
  5. 5.
    McDonnell DP: The molecular pharmacology of SERMs. TEM 10: 301-311, 1999Google Scholar
  6. 6.
    Smith CL, O'Malley BW: Evolving concepts of selective estrogen receptor action: from basic science to clinical applications. TEM 10: 299-300, 1999Google Scholar
  7. 7.
    Jordan VC: Targeted antiestrogens to prevent breast cancer. TEM 10: 312-317, 1999Google Scholar
  8. 8.
    Moon RC, Mehta RG, Detrisac CJ: Retinoids as chemopreventive agents for breast cancer. Cancer Detect Prev 16: 73-79, 1992Google Scholar
  9. 9.
    Bollag W: Experimental basis of cancer combination chemotheraphy with retinoids, cytokines, 1,25-dihydroxyvitamin D3, and analogs. J Cell Biochem 56: 427-435, 1994Google Scholar
  10. 10.
    Bischoff ED, Heyman RA, Lamph WW: Effect of the retinoid X receptor-selective ligand LGD1069 on mammary carcinoma after tamoxifen failure. J Natl Cancer Inst 91: 2118-2118, 1999Google Scholar
  11. 11.
    Suh N, Wang Y, Williams CR, Risingsong R, Gilmer T, Willson TM, Sport MB: A new ligand for the peroxisome proliferator-activated receptor-g (PPAR-g), GW7845, inhibits rat mammary carcinogenesis. Cancer Res 59: 5671-5673, 1999Google Scholar
  12. 12.
    Safe S: 2,3,7,8-Tetrachlorodibenzo-p-diox in (TCDD) and related environmental antiestrogens: characterization and mechanism of action. In: Naz RK (ed) Endocrine Disruptors. CRC Press, Boca Raton, FL, 1999, pp 187-221Google Scholar
  13. 13.
    Safe S, Qin C, McDougal A: Development of selective aryl hydrocarbon receptor modulators (SARMs) for treatment of breast cancer. Expert Opin Invest Drugs 8: 1385-1396, 1999Google Scholar
  14. 14.
    Zacharewski T, Safe S: Antiestrogenic activity of TCDD and related compounds. In: Korach KS (ed) Reproductive and Developmental Toxicology. Marcel Dekker, New York, 1998, pp 431-448Google Scholar
  15. 15.
    Porter W, Safe S: Estrogenic and antiestrogenic compounds. In: Puga A, Wallace KB (eds) Molecular Biology Approaches to Toxicology. Techbooks, Fairfax, VA, 1998, pp 267-283Google Scholar
  16. 16.
    Ema M, Sogawa K, Watanabe N, Chujoh Y, Matsushita N, Gotoh O, Funae Y, Fujii-Kuriyama Y: cDNA cloning and structure of the putative Ah receptor. Biochem Biophys Res Comm 184: 246-253, 1992Google Scholar
  17. 17.
    Burbach KM, Poland AB, Bradfield CA: Cloning of the Ah-receptor cDNA reveals a distinctive ligand-activated transcription factor. Proc Natl Acad Sci USA 89: 8185-8189, 1992Google Scholar
  18. 18.
    Hoffman EC, Reyes H, Chu F-F, Sander F, Conley LH, Brooks BA, Hankinson O: Cloning of a factor required for activity of the Ah (dioxin) receptor. Science 252: 954-958, 1991Google Scholar
  19. 19.
    Wilson CL, Safe S: Mechanisms of ligand-induced aryl hydrocarbon receptor-mediated biochemical and toxic responses. Toxicol Pathol 26: 657-671, 1998Google Scholar
  20. 20.
    Probst MR, Hankinson O: Interactions of ARNT with AhR and HIF-1a. In: Puga A, Wallace KB (eds) Molecular Biology of the Toxic Response. Taylor and Francis, Philadelphia, 1999, pp 377-392Google Scholar
  21. 21.
    Whitlock JP, Okino ST, Dong L, Ko HP, Clarke-Katzenberg R, Ma Q, Li H: Induction of cytochrome P4501A1: a model for analyzing mammalian gene transcription. FASEB J 10: 809-818, 1996Google Scholar
  22. 22.
    Bjeldanes LF, Kim JY, Grose KR, Bartholomew JC, Bradfield CA: Aromatic hydrocarbon responsiveness-receptor agonists generated from indole-3-carbinol in vitro and in vivo-comparisons with 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin. Proc Natl Acad Sci USA 88: 9543-9547, 1991Google Scholar
  23. 23.
    Chen I, Safe S, Bjeldanes L: Indole-3-carbinol and diindolylmethane as aryl hydrocarbon (Ah) receptor agonists and antagonists in T47D human breast cancer cells. Biochem Pharmacol 51: 1069-1076, 1996Google Scholar
  24. 24.
    Casper RF, Quesne M, Rogers IM, Shirota T, Jolivet A, Milgrom E, Savouret JF: Resveratrol has antagonist activity on the aryl hydrocarbon receptor: implications for prevention of dioxin toxicity. Mol Pharmacol 56: 784-790, 1999Google Scholar
  25. 25.
    Seidel SD, Li V, Winter GM, Rogers WJ, Martinez EI, Denison MS: Ah receptor-based chemical screening bioassays: application and limitations for the detection of Ah receptor agonsists. Toxicol Sci 55: 107-115, 2000Google Scholar
  26. 26.
    Gasiewicz TA, Kende AS, Rucci G, Whitney B, Willey JJ: Analysis of structural requirements for Ah receptor antagonist activity: ellipticines, flavones, and related compounds. Biochem Pharmacol 52: 1787-1803, 1996Google Scholar
  27. 27.
    Gradelet S, Astorg P, Pineau T, Canivenc MC, Siess MH, Leclerc J, Lesca P: Ah receptor-dependent CYP1A induction by two carotenoids, canthaxanthin and b-apo-80-carotenal, with no affinity for the TCDD binding site. Biochem Pharmacol 54: 307-315, 1997Google Scholar
  28. 28.
    Jellinck PH, Forkert PG, Riddick DS, Okey AB, Michnovicz JJ, Bradlow HL: Ah receptor binding properties of indole carbinols and induction of hepatic estradiol hydroxylation. Biochem Pharmacol 43: 1129-1136, 1993Google Scholar
  29. 29.
    Denison MS, Seidel SD, Rogers WJ, Ziccardi M, Winter GM, Heath-Pagliuso S: Natural and synthetic ligands for the Ah receptor. In: Puga A, Kendall RJ (eds) Molecular Biology Approaches to Toxicology. Taylor and Francis, London, 1998, pp 3-33Google Scholar
  30. 30.
    McDougal A, Wilson C, Safe S: Inhibition of 7, 12-dimethylbenz[a]anthracene-induced rat mammary tumor growth by aryl hydrocarbon receptor agonists. Cancer Lett 120: 53-63, 1997Google Scholar
  31. 31.
    Chen I, McDougal A, Wang F, Safe S: Aryl hydrocarbon receptor-mediated antiestrogenic and antitumorigenic activity of diindolylmethane. Carcinogenesis 19: 1631-1639, 1998Google Scholar
  32. 32.
    McDougal A, Sethi-Gupta M, Ramamoorthy K, Sun G, Safe S: Inhibition of carcinogen-induced rat mammary tumor growth and other estrogen-dependent responses by symmetrical dihalo-substituted analogs of diindolylmethane. Cancer Letts 151: 169-179, 2000Google Scholar
  33. 33.
    Romkes M, Piskorska-Pliszczynska J, Keys B, Safe S, Fujita T: Quantitative structure-activity relationships: analysis of interactions of 2,3,7,8-tetrachlorodibenzo-p-dioxon and 2-substituted analogues with rat, mouse, guinea pig and hamster cytosolic receptor. Cancer Res 47: 5108-5111, 1987Google Scholar
  34. 34.
    Safe S: Modulation of gene expression and endocrine response pathways by 2,3,7,8-tetrachlorodibenzo-p-diox in and related compounds. Pharmacol Therap 67: 247-281, 1995Google Scholar
  35. 35.
    Romkes M, Safe S: Comparative activities of 2,3,7,8-tetrachlorodibenzo-p-dioxin and progesterone on antiestrogens in the female rat uterus. Toxicol Appl Pharmacol 92: 368-380, 1988Google Scholar
  36. 36.
    Romkes M, Piskorska-Pliszczynska J, Safe S: Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on hepatic and uterine estrogen receptor levels in rats. Toxicol Appl Pharmacol 87: 306-314, 1987Google Scholar
  37. 37.
    Astroff B, Safe S: Comparative antiestrogenic activities of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 6-methyl-1,3,8-trichlorodibenzofuran in the female rat. Toxicol Appl Pharmacol 95: 435-443, 1988Google Scholar
  38. 38.
    Chang Y-C, Riby J, Chang GH-F, Peng B-C, Firestone G, Bjeldanes LF: Cytostatic and antiestrogenic effects of 2-(indol-3-ylmethyl)-3,30-diindolylmethane, a major in vivo product of dietary indole-3-carbinol. Biochem Pharmacol 58: 825-834, 1999Google Scholar
  39. 39.
    Riby JE, Feng C, Chang Y-C, Schaldach CM, Firestone GL, Bjeldanes LF: The major tricyclic trimeric product of indole-3-carbinol is a strong agonist of the estrogen receptor signaling pathway. Biochemistry 39: 910-918, 2000Google Scholar

Copyright information

© Kluwer Academic Publishers 2001

Authors and Affiliations

  • Andrew McDougal
    • 1
  • Mona Sethi Gupta
    • 1
  • Derek Morrow
    • 1
  • Kavita Ramamoorthy
    • 1
  • Jeong‐Eun Lee
    • 1
  • Stephen H. Safe
    • 1
  1. 1.Department of Veterinary Physiology & PharmacologyTexas A&M UniversityCollege StationUSA

Personalised recommendations