Indole 3 Carbinol
Description
The naturally occurring chemical indole-3- carbinol (I3C) is a promising anticancer agent shown previously to induce a G1 cell cycle arrest of human breast cancer cell lines, independent of estrogen receptor signaling.
Combinations of I3C and the antiestrogen tamoxifen cooperate to inhibit the growth of the estrogen-dependent human MCF-7 breast cancer cell line more effectively than either agent alone.
Indole-3-carbinol (I3C) (2) is produced endogenously from naturally occurring glucosinolates contained in a wide variety of plant food substances including members of the family Cruciferae, and particularly members of the genus Brassica, whenever they are crushed or cooked. It may be derived from cruciferous vegetables such as broccoli, cabbage, cauliflower, Brussels sprouts and bok-choi.
Method of Action
Hundreds of research studies conducted over the last 25 years have clearly shown that indole-3-carbinol (I3C), a substance found in cruciferous vegetables, has a powerful cancer fighting ability. Some of these studies have shown that I3C itself is very unstable and under the acid conditions found in the stomach breaks down into a number of metabolites largely responsible for the effectiveness at fighting cancer. According to the studies, diindolylmethane (DIM) is the most active and effective metabolite of I3C. Animal studies in which stomach acid conditions were bypassed indicate little effectiveness of I3C by itself.
In recent years there has been considerable interest in the effects of estrogen exposure on hormone related diseases including breast, uterine and prostate cancer. Excessive estrogen, whether endogenous (from within the body) or exogenous (originating outside the body), is being studied in both men and women.
Estrogen is a name used to describe the family of hormones consisting of Estrone (E1), Estradiol (E2) and Estriol (E3). Estrogens are converted from testosterone and androstenedione by way of the aromatase enzyme. Aromatase activity is present in many tissues including the liver, fat cells and skin. Estrogens are also synthesized in the ovaries. Estradiol, the most predominant estrogen hormone, is converted to estrone and finally to estriol. Estrogens are bound to a protein known as sex hormone binding globulin (SHBG). Binding the hormone to a protein (SHBG) supplies a circulating pool of the estrogens, which most likely provides a safeguard against sudden variations in the plasma level. Changes in SHBG due to normal aging or thyroid dysfunction can affect estrogen metabolism.
Downstream metabolites of estrogen have been the principal focus of current hormone research. Estrogen is metabolized primarily in the liver using the Phase 1 hydroxylation enzymes and Phase II methylation, glucuronidation and sulfation pathways. The cytochrome P-450 enzymes are responsible for the hydroxylation of estradiol and estrone in the Phase I pathway. There are two crucial positions on the estrogen molecule where hydroxylation takes place, either at the 2 carbon (C-2) site or at the 16 carbon (C-position, yielding 2-hydroxyestrone (2-OH) and 16 -hydroxyestrone (16 -OH) respectively. Hydroxylation at the 4-carbon (C-4) position has a slight involvement, yielding 4-hydroxyestrone (4-OH). The C-2 metabolite, 2-OH, presents very weak estrogenic action and is often referred to as the good estrogen, while the 16 -OH and 4-OH metabolites have significant estrogenic activity implicating them as tumor promoting metabolites. There is a considerable body of evidence relating the 2-hydroxyestrone (2-OH) and 16 -hydroxyestrone (16 -OH) ratio to an increase risk of breast cancer. Women with elevated levels of 2-hydroxyestrone (2-OH) had a lower risk of breast cancer, while women with elevated 16 -hydroxyestrone showed increased risk.
Estrogen, through its binding to the estrogen receptor (ER), plays an important role in breast cancer cell proliferation and tumor development. Various researchers suggest that Indole 3 Carbinol (I3C) exhibits potent antitumor activity by regulating estrogen activity and metabolism. One method of action for I3C is its ability to increase levels of 2-hydroxyestrone (2-OH), the so-called good estrogen, and decrease 4-hydroxyestrone (4-OH), and 16 -hydroxyestrone (16 -OH), potential carcinogenic estrogen metabolites. Another probable scenario is that I3C and its metabolite diindolylmethane (DIM) inhibit estrogen-induced proliferation of MCF-7 human breast cancer cells. Further substantiation comes from Tiwari et al, who disclosed that I3C inhibits the growth of estrogen-responsive cell line MCF-7 while at the same time up regulates 2-hydroxyestrone (2-OH). Meng et al found that I3C inhibits cell adhesion, up-regulates a tumor-suppressing gene, known as PTEN and limited metastasis. The protective effects of I3C on human breast cancer may also be due to its ability to induce cellular apoptosis. This course of action may be critical in limiting tumor growth.
Therapeutic Approaches
Indole 3 Carbinol (I3C) and breast cancer
Estrogen, through its binding to the estrogen receptor (ER), plays an important role in breast cancer cell proliferation and tumor development. Various researchers suggest that Indole 3 Carbinol (I3C) exhibits potent antitumor activity by regulating estrogen activity and metabolism (11). One method of action for I3C is its ability to increase levels of 2-hydroxyestrone (2-OH), the so-called good estrogen, and decrease 4-hydroxyestrone (4-OH), and 16 -hydroxyestrone (16 -OH), potential carcinogenic estrogen metabolites. Another probable scenario is that I3C and its metabolite diindolylmethane (DIM) inhibit estrogen-induced proliferation of MCF-7 human breast cancer cells. Further substantiation comes from Tiwari et al, who disclosed that I3C inhibits the growth of estrogen-responsive cell line MCF-7 while at the same time up regulates 2-hydroxyestrone (2-OH). Meng et al found that I3C inhibits cell adhesion, up-regulates a tumor-suppressing gene, known as PTEN and limited metastasis. The protective effects of I3C on human breast cancer may also be due to its ability to induce cellular apoptosis. This course of action may be critical in limiting tumor growth.
Indole 3 Carbinol (I3C) and tamoxifen
The anti-estrogen drug tamoxifen is commonly used to treat breast cancer. Studies combining I3C with tamoxifen have demonstrated positive results. The combination works together to inhibit the growth of the estrogen-dependent human MCF-7 breast cancer cell line more effectively than either agent alone.
Post-menopausal Estrogen Replacement Therapy
There is still considerable controversy about the increased risk of breast cancer with post-menopausal estrogen administration. Serum levels of estradiol produced by commonly prescribed doses of postmenopausal estrogen therapy are equal to those of the follicular phase of the menstrual cycle. However, these levels may be near the threshold of breast cancer promoting effects when combined with genetics and lifestyle factors. According to Zumoff et al, the risk of breast cancer with postmenopausal estrogen replacement can be reduced by various methods, including the use of I3C. These researchers suggest that women on estrogen replacement therapy will have some protection against tumor development by decreasing 16 -hydroxyestrone (16 -OH) levels with I3C supplementation.
Endometrial and cervical tumors
Human papillomavirus (HPV) infects the genital tract and is generally acknowledged to be a contributing mediator of cervical cancer. However, HPV infection alone is not enough to bring on cervical cancer. Additional factors, such as hormones, are thought to contribute to the development and/or progression of this disease. 16 -hydroxyestrone (16 -OH) appears to be necessary for the development of abnormal cell growth as well as the death of healthy cells.(20) Oral supplementation of I3C has been found to reduce tumor development of the endometrium and cervix. The positive effects were due to the improvement in the 2/16 alpha-hydroxyestrone ratios.
Estrogens and men
Testosterone or dihydrotestosterone (DHT) in combination with estradiol seems to be implicated in the widespread and uncontrolled division of prostate cells that result in benign prostatic hypertrophy (BPH). Correlations were found between prostate-specific antigen (PSA) levels, testosterone, epitestosterone and estradiol, while the estrogen metabolites have been associated with prostate tumor development. From their research, Chimni et al concluded that I3C inhibited the growth of prostate cancer cells. I3C appears to suppress tumor-promoting agents such as cyclin-dependent kinase 6 (CDK6) and Retinoblastoma (Rb) protein in prostate cells.
Exogenous Estrogens
People all around the world are being exposed to a multitude of foreign chemicals known as xenobiotics. Xenobiotics (Xeno is from Greek meaning stranger) are any compounds foreign to the body. These compounds can be found in environmental pollutants, pesticides, drugs and food additives. As of 1996, more than 200,000 manufactured environmental chemicals exist. The liver is the primary organ involved in the detoxification and elimination of xenobiotics. Many of these foreign compounds exhibit hormone-like characteristics. Exposure to these exogenous estrogens may directly or indirectly modify estrogen metabolism. If they are not properly detoxified and eliminated, serious health problems may present themselves. Earlier research illustrates that some organochlorine-based pesticides raised the production of 16 alpha-hydroxyestrone relative to 2-hydroxyestrone in MCF-7 breast cancer cells. As previously discussed, I3C has been shown to modify this ratio. Tobacco smoke also contains several exogenous carcinogens including nitrosamine 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Human and animal studies reveal that I3C may inhibit lung tumor formation by enhancing the liver clearance of NNK.
By measuring the urinary estrogen metabolite ratio of 2-hydroxyestrone to 16 alpha- hydroxyestrone, optimum dosing of I3C was determined. In a study by Wong et al, it was suggested that 300-400 mg of I3C daily was the minimum effective dose. In conclusion, I3C has many benefits, including the ability to modulate 2/16 alpha-hydroxyestrone ratios and provide antimutagenic and anticarcinogenic properties against a variety of carcinogens.
Nutritional Sciences, Inc. gratefully acknowledges Rx Vitamins, Inc. for providing excellent research on the subject of Indole 3 Carbinol. Rx Vitamins has long been the leader in nutritional supplementation in the pharmacy environment, in part due to the extensive and well-written research conducted on each of its products.
Toxicity Factors
There is no toxicity reported at this time.
Abstracts
Propoxur is a widely used dithiocarbamate pesticide. In the present set of investigations, mutagenicity of propoxur (in formulation) was studied using the micronucleus assay in bone marrow of Swiss mice. Single intraperitoneal (i.p.) administration of 25 mg/kg body weight dose of propoxur, which is a maximum tolerated dose (MTD), significantly induced the micronucleus formation in bone marrow cells after a 24- and 48-hr exposure. A half and a quarter of the MTD (12.5 and 6.25 mg/kg) were found ineffective to induce the micronuclei formation after 24- and 48-hr time periods by the i.p. route. However, the PCE:NCE ratio was inhibited significantly with all the dose levels at both time periods. Oral administration of propoxur at different dose levels also induced micronuclei formation. A single application of 50 and 25 mg/kg dose levels of propoxur, which are MTD and 50% of MTD, also significantly induced micronuclei formation after 24- and 48-hr time periods in bone marrow cells of Swiss mice as compared with solvent control group, whereas a 12.5 mg/kg dose of propoxur was ineffective in inducing micronuclei formation. Single application of indole-3-carbinol (I3C), a glucobrassicin derivative present in cruciferous vegetables, significantly inhibited the propoxur-induced micronuclei formation when it was given at the dose level of 500 mg/kg body weight 48 hr before the single application of propoxur. Therefore, it seems that propoxur is mutagenic in the above test systems and I3C inhibited the mutagenicity of propoxur significantly.
Agrawal RC Mehrotra Nk. Assessment of mutagenic potential of propoxur and its modulation by indole-3-carbinol.Food Chem Toxicol (1997 Oct-Nov) 35(10-11):1081-4
Indole-3-carbinol (I3C) is a glucobrassicin derivative isolated from cruciferous vegetables. In this study, the protective effect of 13C is reported against cyclophosphamide (CP)-induced micronuclei formation in mouse bone marrow cells. The three test doses, namely 500, 250 and 125 mg/kg body weight of 13C provided protection when given 48 hr prior to the single ip administration of cyclophosphamide (50 mg/kg). The efficacy of the test doses of 13C was also evaluated using a lower dose of CP (25 mg/kg body weight). A significant inhibition in micronuclei formation was noticed with 13C at 250 and 125 mg/kg body weight dose. 13C could not induce micronuclei formation at the test doses 500 and 250 mg/kg body weight. 13C, therefore seems to have a preventive potential against CP-induced micronuclei formation in Swiss mouse bone marrow cells.
Agrawal RC Kumar S.Prevention of cyclophosphamide-induced micronucleus formation in mouse bone marrow by indole-3-carbinol.Food Chem Toxicol (1998 Nov) 36(11):975-7
In an effort to understand the mechanism by which dietary indoles inhibit chemically initiated tumorigenesis in experimental animals, we have investigated the potency of 3-substituted and 1,3- disubstituted indoles on the induction of intestinal and hepatic cytochrome P-448-dependent monooxygenases in the rat. Oral intubation with indole-3-carbinol (13C), 1-methoxyindole-3-carbinol (N13C), 1- methoxyindole-3-carboxaldehyde (NCHO), and 3,3'-diindolylmethane (133') at 31 mumol/animal led to significant increases in hepatic ethoxyresorufin O-deethylase activity (EROD; 15, 7, 6, and 5-fold over control, respectively), while intubation with indole (IND), 3- methylindole (3MI), indole-3-carboxaldehyde (13CHO), and indole-3- acetonitrile (IAN) did not increase this monooxygenase activity over control levels. For the eight indoles tested, there was a strong relationship between instability in acidic solution, as indicated by the generation of insoluble products, and capacity to induce hepatic EROD. Further experiments indicated that 13C did not induce hepatic EROD when dosed ip (thus bypassing the acidity of the stomach). Acid treatment of 13C generated a reaction mixture (RXM) that induced EROD after ip or po dosing. Chromatographic fractionation of the RXM indicated that there exist at least four different 13C acid- condensation products in the RXM with the ability to induce EROD. The results presented strongly support the hypothesis that dietary indoles influence the levels of monooxygenase activities via a series of acid-condensation products generated upon introduction of the indole into the acidic environment of the stomach.
Bradfield CA Bjeldanes LF Structure-activity relationships of dietary indoles: a proposed mechanism of action as modifiers of xenobiotic metabolism. J Toxicol Environ Health (1987) 21(3):311-23
Previous studies from this laboratory have suggested that 2- hydroxyestrone is protective against breast cancer, whereas the other principal metabolite, 16 alpha-hydroxyestrone, and the lesser metabolite quantitatively, 4-hydroxyestrone, are potent carcinogens. Attempts to directly decrease the formation of the 16-hydroxylated metabolite were either unsuccessful or required such high levels of the therapeutic agent as to be impractical. On the other hand the concentration of the protective metabolite, 2-hydroxyestrone, proved to be readily modulated by a variety of agents, both in the direction of increased protection and the opposite direction, increased risk by a variety of agents and activities. We have focussed our attention on indole-3-carbinol, a compound found in cruciferous vegetables, and its further metabolites in the body, diindolylmethane (DIM) and indolylcarbazole (ICZ), because of its relative safety and multifaceted activities. It has been shown that it induces CyP4501A1, increasing 2-hydroxylation of estrogens, leading to the protective 2- OHE1, and also decreases CyP1B1 sharply, inhibiting 4-hydroxylation of estradiol, thereby decreasing the formation of the carcinogenic 4- OHE1. In addition to these indirect effects as a result of altered estrogen metabolism, indole-3-carbinol has been shown to have direct effects on apoptosis and cyclin D, resulting in blockage of the cell cycle. In addition to its antitumor activity in animals, it has also been shown to be effective against HPV-mediated tumors in human patients. All of these responses make the study of its behavior as a therapeutic agent of considerable interest.
Bradlow HL Sepkovic DW Telang NT Osborne MP. Multifunctional aspects of the action of indole-3-carbinol as an antitumor agent. Ann NY Acad Sci (1999) 889:204-13
Indole-3-carbinol (I3C) is produced endogenously from naturally occurring glucosinolates contained in a wide variety of plant food substances including members of the family Cruciferae, and particularly members of the genus Brassica, whenever they are crushed or cooked. The acid environment of the gut very facilely converts it into a range of polyaromatic indolic compounds, e.g., which appear to be responsible for many of the physiological effects observed following the ingestion of these foods. 3- (Methoxymethyl)indole is formed with great ease whenever 2 contacts methylating agents, including methanol, and it is often found as a contaminant of 2. This contamination is often not recognized or easily removed because of the great similarities of the two in melting points and solubilities. However, their biological properties are essentially identical. These so-called chemopreventive compounds are important because of their enzyme induction and suppression, mutagenic, carcinogenic and, particularly, antimutagenic and anticarcinogenic properties. The natural occurrence, formation, preparation, identification, separation, quantification, chemical transformations and general toxicological properties of these substances are critically reviewed in detail in this paper of 146 references, the first of two parts. At the present time it appears that these have considerable potential as natural prophylactic anticancer agents against certain common neoplasms, especially inasmuch modern diets are increasingly deficient in these vegetable-derived substances.
Broadbent TA Broadbent HS. The chemistry and pharmacology of indole-3-carbinol (indole-3- methanol) and 3 (methoxymethyl)indole. Curr Med Chem (1998) 5(5):337-52
Xenobiotic estrogens are external compounds with estrogenic activity that may thereby affect the risk of breast cancer. This paper describes a mechanism by which xeno-estrogens may affect the development of breast cancer. Estradiol metabolism proceeds by hydroxylation at one of two mutually exclusive sites at C-2 and C-16 alpha. The catechol pathway yields the weakly estrogenic 2- hydroxyestrone (2-OHE1), which inhibits breast cell proliferation. In contrast, the alternative pathway yields the genotoxic 16 alpha- hydroxyestrone (16 alpha-OHE1), which enhances breast cell growth, increases unscheduled DNA synthesis, and oncogene and virus expression, and increases anchorage-independent growth. Using a radiometric assay that measures the relative formation of 16 alpha- OHE1 versus 2-OHE1 from specifically tritiated estradiol in (ER+) MCF- 7 cells, we compared the ratio of 16 alpha-OHE1/2-OHE1 observed after treatment with the known rodent carcinogen 7,12- dimethylbenz[a]anthracene (DMBA) with the ratios after treatment with DDT, atrazine, gamma-benzene hexachloride, kepone, coplanar PCBs, endosulfans I and II, linoleic and eicosapentenoic acids, and indole- 3-carbinol (I3C). These pesticides significantly increase the ratio of 16 alpha-OHE1/2-OHE1 metabolites to values comparable to or greater than those observed after DMBA. In contrast, the antitumor agent I3C increased 2-OHE1 formation and yielded ratios that are 1/3 of those found in unexposed control cells and 1/10th of those found in DMBA-treated cells. Thus the ratio of 16 alpha-OHE1/2-OHE1 may provide a marker for the risk of breast cancer. Assays of this ratio, which can be measured in spot urines, may prove useful for a variety of in vitro and in vivo studies bearing on breast cancer risk.
Broadbent TA Broadbent HS. The chemistry and pharmacology of indole-3-carbinol (indole-3- methanol) and 3 (methoxymethyl)indole. Curr Med Chem (1998) 5(5):337-52
Indole-3-carbinol (I3C) (2) is produced endogenously from naturally occurring glucosinolates contained in a wide variety of plant food substances including members of the family Cruciferae, and particularly members of the genus Brassica, whenever they are crushed or cooked. The acid environment of the gut very facilely converts it into a range of polyaromatic indolic compounds, e.g. (3, 4, 5), which appear to be responsible for many of the physiological effects observed following the ingestion of these foods. These so-called chemopreventive compounds are important because of their enzyme induction and suppression, mutagenic, carcinogenic and, particularly, antimutagenic and anticarcinogenic properties against a variety of classes of carcinogens. These properties as well as other miscellaneous properties of these substances are critically reviewed in detail in this paper of >170 references, the second of two parts. At the present time it appears that I3C and its congeners have considerable potential as natural prophylactic anticancer agents against certain common neoplasms, especially inasmuch modern diets are increasingly deficient in these vegetable-derived substances. A short general assessment of the substantial potential of the title compounds concludes the review.
Broadbent TA Broadbent HS. The chemistry and pharmacology of indole-3-carbinol (indole-3-methanol) and 3-(methoxymethyl)indole Curr Med Chem (1998 Dec) 5(6):469-91
Under acidic conditions, indole-3-carbinol (13C) is converted to a series of oligomeric products thought to be responsible for the biological effects of dietary 13C. Chromatographic separation of the crude acid mixture of 13C, guided by cell proliferation assay in human MCF-7 cells, resulted in the isolation of 2-(indol-3-ylmethyl)- 3,3'-diindolylmethane (LTr-1) as a major antiproliferative component. LTr-1 inhibited the growth of both estrogen-dependent (MCF-7) and - independent (MDA-MB-231) breast cancer cells by approximately 60% at a non-lethal concentration of 25 microM. LTr-1 had no apparent effect on the proliferation of MCF-7 cells in the absence of estrogen. LTr-1 was a weak ligand for the estrogen receptor (ER) (IC50 70 microM) and efficiently inhibited the estradiol (E2)-induced binding of the ER to its cognate DNA responsive element. The antagonist effects of LTr-1 also were exhibited in assays of endogenous pS2 gene expression and in cells transiently transfected with an estrogen-responsive reporter construct (pERE-vit-CAT). LTr-1 activated both binding of the aryl hydrocarbon (Ah) receptor to its cognate DNA responsive element and expression of the Ah receptor-responsive gene CYP1A1. LTr-1 was a competitive inhibitor of CYP1A1-dependent ethoxyresorufin-O- deethylase (EROD) activity. In summary, these results demonstrated that LTr-1, a major in vivo product of I3C, could inhibit the proliferation of both estrogen-dependent and -independent breast tumor cells and that LTr-1 is an antagonist of estrogen receptor function and a weak agonist of Ah receptor function.
Chang YC Riby J Chang GH Peng BC Firestone G Bjeldanes LF. Cytostatic and antiestrogenic effects of 2-(indol-3-ylmethyl)-3,3'-diindolylmethane, a major in vivo product of dietary indole-3-carbinol.Biochem Pharmacol (1999 Sep 1) 58(5):825-34
Indole-3-carbinol (I3C) is a major component of Brassica vegetables, and diindolylmethane (DIM) is the major acid-catalyzed condensation product derived from I3C. Both compounds competitively bind to the aryl hydrocarbon (Ah) receptor with relatively low affinity. In Ah- responsive T47D human breast cancer cells, I3C and DIM did not induce significantly CYP1A1-dependent ethoxyresorufin O-deethylase (EROD) activity or CYP1A1 mRNA levels at concentrations as high as 125 or 31 microM, respectively. A 1 nM concentration of 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) induced EROD activity in these cells, and cotreatment with TCDD plus different concentrations of I3C (1-125 microM) or DIM (1-31 microM) resulted in a > 90% decrease in the induced response at the highest concentration of I3C or DIM. I3C or DIM also partially inhibited (< 50%) induction of CYP1A1 mRNA levels by TCDD and reporter gene activity, using an Ah-responsive plasmid construct in transient transfection assays. In T47D cells cotreated with 5 nM [3H]TCDD alone or in combination with 250 microM I3C or 31 microM DIM, there was a 37 and 73% decrease, respectively, in formation of the nuclear Ah receptor. The more effective inhibition of induced EROD activity by I3C and DIM was due to in vitro inhibition of enzyme activity. Thus, both I3C and DIM are partial Ah receptor antagonists in the T47D human breast cancer cell line.
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 (1996 Apr 26) 51(8):1069-76
Phytochemicals such as indole-3-carbinol (I3C) and sulforaphane are components of cruciferous vegetables which exhibit antitumorigenic activity associated with altered carcinogen metabolism and detoxification. Diindolylmethane (DIM) is a major acid-catalyzed metabolite of I3C formed in the gut that binds to the aryl hydrocarbon receptor (AhR) and treatment of MCF-7 human breast cancer cells with 10-50 microM DIM resulted in rapid formation of the nuclear AhR complex and induction of CYP1A1 gene expression was observed at concentrations >50 microM. Previous studies have demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a high affinity AhR ligand, inhibits 17beta-estradiol (E2)-induced responses in MCF-7 cells and growth of E2-dependent 7,12-dimethylbenzanthracene (DMBA)-induced mammary tumors in female Sprague-Dawley rats. Results of this study show that like TCDD, DIM inhibits E2-induced proliferation of MCF-7 cells, reporter gene activity in cells transiently transfected with an E2-responsive plasmid (containing a frog vitellogenin A2 gene promoter insert) and down-regulates the nuclear estrogen receptor. Moreover, DIM (5 mg/kg every other day) also inhibits DMBA-induced mammary tumor growth in Sprague-Dawley rats and this was not accompanied by induction of hepatic CYP1A1- dependent activity. Thus, DIM represents a new class of relatively non-toxic AhR-based antiestrogens that inhibit E2-dependent tumor growth in rodents and current studies are focused on development of analogs for clinical treatment of breast cancer.
Chen I McDougal A Wang F Safe S. Aryl hydrocarbon receptor-mediated antiestrogenic and antitumorigenic activity of diindolylmethane. Carcinogenesis (1998 Sep) 19(9):1631-9
Indole-3-carbinol (I3C) is a major component of Brassica vegetables and diindolylmethane (DIM) is an acid-catalyzed dimeric condensation product derived from I3C. Both compounds competitively bind to the aryl hydrocarbon (Ah) receptor with relatively low affinity. In Ah- responsive T47D human breast cancer cells, I3C and DIM did not significantly induce CYP1A1-dependent ethoxyresorufin O-deethylase (EROD) activity or CYP1A1 mRNA levels at concentrations as high as 125 or 31 uM, respectively. One nM 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD) induced EROD activity in these cells and cotreatment with TCDD plus different concentrations of I3C (1 to 125 uM) or DIM (1 to 31 uM) resulted in greater than 90% decrease in the induced response at the highest concentration of I3C or DIM. I3C or DIM also partially inhibited (greater than 50%) induction of CYP1A1 mRNA levels by TCDD and reporter gene activity using an Ah-responsive plasmid construct in transient transfection assays. In T47D cells cotreated with 5 nM [3H]TCDD alone or in combination with 250 AM I3C or 31 uM DIM, there was a 37 and 73% decrease, respectively, in formation of the nuclear Ah receptor. The more effective inhibition of induced EROD activity by I3C and DIM was due to in vitro inhibition of enzyme activity. Thus, both I3C and DIM are partial Ah receptor antagonists in the T47D human breast cancer cell line.
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 (Meeting abstract).Fundam Appl Toxicol (201996) 30(1):183
A major obstacle to successful chemotherapy is the development of multidrug resistance (MDR) by cancer cells. MDR is characterized by enhanced cellular efflux of many structurally and functionally diverse compounds, including many anticancer drugs, due to overexpression of the MDR-1 gene product, P-glycoprotein. We hypothesized that the phytochemical, indole-3-carbinol (I3C), and some of its acid-condensation derivatives may inhibit P-glycoprotein- mediated transport due to their aromatic and nitrogen components, thus increasing the accumulation and efficacy of anticancer drugs and acting as a dietary adjuvant to conventional chemotherapy. I3C was subjected to acid conditions similar to those occurring in the stomach following ingestion and three acid-condensation products; a dimer, a noncyclic trimer, and a cyclic trimer were isolated and purified by high-performance liquid chromatography. The ability of I3C and its acid-condensation derivatives to reverse MDR was investigated using murine B16 melanoma cells that were transfected with the human MDR-1 gene (B16/hMDR-1 cells) and were cross-resistant to vinblastine and doxorubicin. The I3C acid-condensation product mixture, but not I3C, sensitized B16/hMDR-1 transfectants to the toxicity of vinblastine and doxorubicin. All three I3C acid- condensation products also increased the accumulation of the P- glycoprotein substrate, doxorubicin, in B16/hMDR-1 transfectants to levels comparable to parental B16 cells. The I3C acid-condensation product mixture competed with azidopine for binding to P- glycoprotein, suggesting that the observed MDR-reversing effect of the acid-condensation products was due to direct interaction with P- glycoprotein. The ability of p.o. administered I3C to reverse MDR was also tested in vivo. The resistance of B16/hMDR-1 transfectants to vinblastine and doxorubicin was preserved after i.p. injection and growth in nude mice. Tumor mass in mice that were provided with 333 or 500 mg/kg mouse/day I3C in their diet and injected s.c. with the anticancer drugs doxorubicin or vinblastine was significantly reduced as compared to tumor mass in mice provided with standard diet and injected with these anticancer drugs or mice provided with 500 mg/kg mouse/day I3C and not injected with anticancer compound. The concentrations of I3C used had no effect on survival or the general appearance and behavior of the mice. Collectively, these results indicate that ingestion of the common dietary constituent I3C results in its conversion to acid-condensation derivatives that sensitized MDR tumors to chemotherapeutic drugs without eliciting direct toxicity to the host.
Christensen JG LeBlanc GA. Reversal of multidrug resistance in vivo by dietary administration of the phytochemical indole-3-carbinol.Cancer Res (1996 Feb 1) 56(3):574-81
The current options for treating breast cancer are limited to excision surgery, general chemotherapy, radiation therapy, and, in a minority of breast cancers that rely on estrogen for their growth, antiestrogen therapy. The naturally occurring chemical indole-3- carbinol (I3C), found in vegetables of the Brassica genus, is a promising anticancer agent that we have shown previously to induce a G1 cell cycle arrest of human breast cancer cell lines, independent of estrogen receptor signaling. Combinations of I3C and the antiestrogen tamoxifen cooperate to inhibit the growth of the estrogen-dependent human MCF-7 breast cancer cell line more effectively than either agent alone. This more stringent growth arrest was demonstrated by a decrease in adherent and anchorage- independent growth, reduced DNA synthesis, and a shift into the G1 phase of the cell cycle. A combination of I3C and tamoxifen also caused a more pronounced decrease in cyclin-dependent kinase (CDK) 2- specific enzymatic activity than either compound alone but had no effect on CDK2 protein expression. Importantly, treatment with I3C and tamoxifen ablated expression of the phosphorylated retinoblastoma protein (Rb), an endogenous substrate for the G1 CDKs, whereas either agent alone only partially inhibited endogenous Rb phosphorylation. Several lines of evidence suggest that I3C works through a mechanism distinct from tamoxifen. I3C failed to compete with estrogen for estrogen receptor binding, and it specifically down-regulated the expression of CDK6. These results demonstrate that I3C and tamoxifen work through different signal transduction pathways to suppress the growth of human breast cancer cells and may, therefore, represent a potential combinatorial therapy for estrogen-responsive breast cancer.
Cover CM Hsieh SJ Cram EJ Hong C Riby JE Bjeldanes LF Firestone GL. Indole-3-carbinol and tamoxifen cooperate to arrest the cell cycle of MCF-7 human breast cancer cells.
In: Cancer Res (1999 Mar 15) 59(6):1244-51
Indole-3-carbinol (I3C), a naturally occurring component of Brassica vegetables such as cabbage, broccoli, and Brussels sprouts, has been shown to reduce the incidence of spontaneous and carcinogen-induced mammary tumors. Treatment of cultured human MCF7 breast cancer cells with I3C reversibly suppresses the incorporation of [3H]thymidine without affecting cell viability or estrogen receptor (ER) responsiveness. Flow cytometry of propidium iodide-stained cells revealed that I3C induces a G1 cell cycle arrest. Concurrent with the I3C-induced growth inhibition, Northern blot and Western blot analyses demonstrated that I3C selectively abolished the expression of cyclin-dependent kinase 6 (CDK6) in a dose- and time-dependent manner. Furthermore, I3C inhibited the endogenous retinoblastoma protein phosphorylation and CDK6 phosphorylation of retinoblastoma in vitro to the same extent. After the MCF7 cells reached their maximal growth arrest, the levels of the p21 and p27 CDK inhibitors increased by 50%. The antiestrogen tamoxifen also suppressed MCF7 cell DNA synthesis but had no effect on CDK6 expression, while a combination of I3C and tamoxifen inhibited MCF7 cell growth more stringently than either agent alone. The I3C-mediated cell cycle arrest and repression of CDK6 production were also observed in estrogen receptor-deficient MDA-MB-231 human breast cancer cells, which demonstrates that this indole can suppress the growth of mammary tumor cells independent of estrogen receptor signaling. Thus, our observations have uncovered a previously undefined antiproliferative pathway for I3C that implicates CDK6 as a target for cell cycle control in human breast cancer cells. Moreover, our results establish for the first time that CDK6 gene expression can be inhibited in response to an extracellular antiproliferative signal.
Cover CM Hsieh SJ Tran SH Hallden G Kim GS Bjeldanes LF Firestone GL. Indole-3-carbinol inhibits the expression of cyclin-dependent kinase- 6 and induces a G1 cell cycle arrest of human breast cancer cells independent of estrogen receptor signaling.J Biol Chem (1998 Feb 13) 273(7):3838-47
The potency of indole-3-carbinol (I3C) to form condensation products under acidic aqueous conditions was studied. After identifying a known dimer, 3,3'-diindolylmethane (DIM), we elucidated the structures of two trimers also found in acid reaction mixtures: 5,6,11,12,17,18-hexahydrocyclonona[1,2-b:4,5-b':7,8-b"]tri-indole (CTI), and 2,3-bis[3-indolylmethyl] indole (BII). The formation of these indole oligomers was shown to be pH dependent. The highest amounts of DIM and BII were formed in aqueous solutions having a pH value ranging from 4 to 5. No CTI could be detected at pH values above 4.5. In rats that received an oral dose of I3C we could detect DIM and BII in gastric contents, stomach tissue, small intestine and liver. No CTI could be detected in vivo after oral exposure to I3C. In in-vitro experiments, using rat hepatocytes, the cytochrome P- 450IA1 apoprotein level, 7-ethoxyresorufin O-deethylation activity (EROD) and DT-diaphorase activity (DTD) were markedly enhanced by DIM and CTI as well as BII.
De Kruif CA Marsman JW Venekamp JC Falke HE Noordhoek J Blaauboer BJ Wortelboer HM. Structure elucidation of acid reaction products of indole-3-carbinol: detection in vivo and enzyme induction in vitro. Chem Biol Interact (1991) 80(3):303-15
Glucosinolates hydrolysis products are attracting increasing attention since many studies have suggested that they may be involved in the anticarcinogenic property of cruciferous vegetables. In this study, we show that diindolylmethane (DIM) and sulforaphane, produced during the hydrolysis of glucobrassicin and glucoraphanin, respectively, exert a dose-dependent cytotoxicity on human colon adenocarcinoma HT29 cells. Moreover, these products are able to inhibit quiescent cells to re-enter the cell cycle. Interestingly, our results clearly show that low doses of DIM and sulforaphane, although very effective on undifferentiated intestinal HT29 cells, do not affect the viability of the differentiated CaCo2 cells. The reversibility of their effects has also been tested and is discussed.
Gamet-Payrastre L Lumeau S Gasc N Cassar G Rollin P Tulliez J. Selective cytostatic and cytotoxic effects of glucosinolates hydrolysis products on human colon cancer cells in vitro.Anticancer Drugs (1998 Feb) 9(2):141-8
3,3'-Diindolylmethane is a dimer of indole-3-carbinol formed both in vivo and in vitro. In this study, human cancer cells MCF-7 (with wild- type p53), T47-D (mutant p53), and Saos-2 (deficient in p53 gene), were used to examine the anticancer activities of 3,3'- diindolylmethane. The dose-dependent growth inhibitory effect was found in all these cell lines. Exposure of the cells to 50 microM solution of 3,3'-diindolylmethane for 48 h, apoptosis (programmed cell death) was evidenced by the characteristic morphology of cell nuclei under fluorescence microscope and the DNA "ladder" in agarose gel electrophoresis. The percentage of apoptotic cells in each cell line was found to be 12% for MCF-7, 14% for T47D and 13% for Saos2 cells. Exposure of MCF-7 cells to 100 microM 3,3'-diindolylmethane for 24 h, 19% of apoptotic cells were detected by flow cytometry analysis. The lowest dose required for induction of apoptosis in MCF- 7 cells was found to be 10 microM after 72 h incubation. Western blot showed that wild-type p53 protein was unchanged after MCF-7 cells had been exposed to 50 microM 3,3'-diindolylmethane for 8 h. This study provides evidences that 3,3'-diindolylmethane induces apoptosis in human cancer cells and that the induction of apoptosis is independent of p53 pathway.
Ge X Yannai S Rennert G Gruener N Fares FA. 3,3'-Diindolylmethane induces apoptosis in human cancer cells.Biochem Biophys Res Commun (1996 Nov 1) 228(1):153-8
The anticancer activity of indole-3-carbinol and the possible mechanisms involved were explored in human breast cancer cell lines MCF-7 and T47D. Treatment with indole-3-carbinol suppressed the growth of MCF-7 and T47D cells. MCF-7 cells were more sensitive to indole-3-carbinol than T47D cells. The growth suppression caused by indole-3-carbinol was found to be partially involved in its ability to induce apoptosis (programmed cell death) in MCF-7 cells. Western blot analysis demonstrated that wild-type p53 was not induced after treatment of MCF-7 cells with indole-3-carbinol. Northern blot analysis showed that treatment of MCF-7 cells with indole-3-carbinol did not affect the expression of bax gene (one of the death genes). In the tissue culture medium, indole-3-carbinol was found to be partially converted to 3,3'-diindolylmethane. The experiments indicated that indole-3-carbinol suppressed MCF-7 cell growth in part by induction of apoptosis which was independent of p53 and bax expression and that the effect caused by indole-3-carbinol was partially due to its conversion to a more potent compound, 3,3'- diindolylmethane, in vitro.
Ge X Fares FA Yannai S. Induction of apoptosis in MCF-7 cells by indol-3-carbinol is independent of p53 and bax. Anticancer Res (1999 Jul-Aug) 19(4B):3199-203
The chemopreventive properties of dietary indole-3-carbinol (I3C) were evaluated by assessing its effect on DNA adduct formation and metabolism of the dietary carcinogen, 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine (PhIP), and the induction of cytochromes P450 1A1 and -1A2 in female F344 rats. In experiment 1, animals on I3C diets (0, 0.02% or 0.1%, w/w) were treated by gavage with 1mg/kg/day of PhIP for 23 days. On days 2, 9, 16 and 23, their 24-hr urine was collected and unmetabolized PhIP was measured by GC/MS. On day 24, the animals were sacrificed, and DNA from pancreas, spleen, white blood cells (WBCs), lung, colon, kidney, mammary epithelial cells, caecum, heart, small intestine, liver and stomach was isolated for determination of PhIP-DNA adduct levels by (32)P-postlabelling assays. Except in the mammary gland, I3C diets significantly inhibited PhIP-DNA adduct formation in WBCs and in all organs, ranging from 34.7 to 56.7% with the 0.02% I3C diet to 68.4 to 95.3% with the 0.1% I3C diet. I3C diets also significantly decreased the concentration of urinary unmetabolized PhIP to 29.5-38.4% (0.02% I3C) and 12.8-17.8% (0.1% I3C) of values obtained with the I3C-free diet. In experiment 2, animals were either treated by intubation of I3C at 100 or 200mg/kg for 2 consecutive days or given an I3C-containing diet (0.02% or 0.1%, w/w) for 2 weeks. The expression and activity of cytochromes P450 1A1 and -1A2 were studied by Northern blots, Western blots, and in vitro enzyme determinations. Both the expression and activity of these cytochromes were induced by all of the I3C treatments. It is concluded that, in the female F344 rat, dietary I3C inhibits PhIP-DNA adduct formation and accelerates PhIP metabolism, probably through induction of cytochromes P450 1A1 and -1A2. The chemopreventive properties of I3C in PhIP-induced carcinogenesis are probably mediated through enhancement of PhIP detoxification pathways.
He YH Friesen MD Ruch RJ Schut HA.Indole-3-carbinol as a chemopreventive agent in 2-amino-1-methyl-6- phenylimidazo[4,5-b]pyridine (PhIP) carcinogenesis: inhibition of PhIP-DNA adduct formation, acceleration of PhIP metabolism, and induction of cytochrome P450 in female F344 rats.Food Chem Toxicol (2000 Jan) 38(1):15-23
Indole-3-carbinol(I3C), a naturally-occurring inhibitor of experimental carcinogenesis, was evaluated for its inhibitory effect on DNA-adduct formation of 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine(PhIP), a dietary mutagen, in female F344 rats. Four-week old rats (4/group) were maintained on AIN-76A diet without or with I3C (0.02%, 0.1%,w/w) for 58 days. PhIP(0.04%,w/w) was added to the diet from days 15 through 42. Animals were killed on days 43 and 58. DNA isolated from mammary epithelial cells(MEC), colon, liver and white blood cells(WBC) was analyzed for PhIP-DNA adducts by 32P- postlabeling assays. On day 43, the adduct levels of the groups receiving 0.1% I3C in the diet decreased in MEC (91.5%), colon (56.2%), liver (69.2%) and WBC (82.3%). On day 58, DNA adduct formation was inhibited in colon (81.3%-82.2%) in both groups receiving I3C, and in liver (46.7%) only in the animals receiving 0.1% I3C. When incorporated in the diet after exposure to dietary PhIP (0.04% for 2 weeks), I3C (0.1%) had no effect on the rate of removal of PhIP-DNA adducts over the next 28 days. It is concluded that dietary I3C inhibits PhIP-DNA adduct formation in a dose dependent manner in the female F344 rat but does not affect adduct removal.
He YH Schut HA. Indole-3-carbinol inhibits DNA adduct formation of the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in female F344 rats (Meeting abstract).Proc Annu Meet Am Assoc Cancer Res (1997) 38:A2487
Mice that express transgenes for human papillomavirus type 16 under a keratin 14 promoter (K14-HPV16 mice) develop cervical cancer when they are given 17beta-estradiol chronically. We asked whether the antiestrogenic phytochemical indole-3-carbinol (I3C), found in cruciferous vegetables, administered at physiological doses, would prevent the cervical-vaginal cancer that is promoted in these mice by high doses of estrogen. We compared mice that were fed a control diet with those that were fed a diet supplemented with 2000 ppm I3C. In the group fed the control diet, at a dose of estradiol of 0.125 mg per 60-day release, 19 of 25 transgenic mice developed cervical- vaginal cancer within 6 months, and the remainder had dysplasia. Only 2 mice of 24 in the group fed the I3C supplemented diet developed cancer, and the remainder had dysplasia or hyperplasia. I3C reduced dysplasia in the nontransgenic mice. Similar results were obtained at a higher dose of estradiol (0.250 mg per 60-day release), and I3C helped to prevent morbidity associated with retention of fluid in the bladder that frequently occurred with the higher estradiol dose. Additionally, I3C appeared to reduce skin cancer in transgenic mice. These data indicate that I3C is a useful preventive for cervical- vaginal cancer and, possibly, other cancers with a papillomavirus component.
Jin L Qi M Chen DZ Anderson A Yang GY Arbeit JM Auborn KJ.Indole-3-carbinol prevents cervical cancer in human papilloma virus type 16 (HPV16) transgenic mice.Cancer Res (1999 Aug 15) 59(16):3991-7
Aberrant proliferation and modulated apoptosis leading to impaired cellular homeostasis represent crucial early events in the multi-step carcinogenic process. Regulation of these perturbed biomarkers may predict efficacious prevention of cancer development. Present experiments on non-cancerous human mammary epithelial 184-B5 cells were designed to examine whether i) exposure to suspect environmental human carcinogen Benzo (a) pyrene (BP) alters the status of cell proliferation and apoptosis and ii) BP-induced alterations are modulated in response to select natural phytochemicals that inhibit rodent mammary tumorigenesis. Flow cytometric analysis, cellular immunoreactivity to proliferation specific and apoptosis specific gene products and anchorage-dependent colony formation represented quantitative endpoints. Cruciferous glucosinolate indole-3-carbinol (I3C), tea polyphenol (-) epigallo catechin gallate (EGCC) and soy isoflavone genistein (GEN) represented the chemopreventive test compounds. A single 24 h exposure to 39 lM BP resulted in a 50% decrease (P=0.02) in the ratio of quiescent (Q=G0) to proliferative (P=S + M) population in part due to increase in aberrantly proliferative cells. The BP-initiated cells also exhibited an 87.8% inhibition (P=0. 0001) in confluency-associated apoptosis and a concomitant decrease in cellular immunoreactivity to wild-type p53. Simultaneous treatment of cultures with BP + I3C, BP + EGCG and BP + GEN resulted in a 1.8- to 3.4-fold increase (P<0.01) in Q/P ratio and 1.8- to 6. 9-fold increase (P=0.001) in sub G0 (apoptotic) population. The induction of apoptosis was accompanied by enhanced p53 immunoreactivity (P<0.01). In long-term (21 day) experiments, BP treatment induced a 145.3% increase (P=0.001) in anchorage-dependent colony formation. This aberrant proliferation was inhibited by 44.2% to 65.3% (P=0.01) in the presence of the three phytochemicals. Thus, BP-induced aberrant proliferation is inhibited by the natural phytochemicals in part due to regulation of cell cycle progression and induction of p53 dependent apoptosis.
Katdare M Osborne MP Telang NT.Inhibition of aberrant proliferation and induction of apoptosis in pre-neoplastic human mammary epithelial cells by natural phytochemicals.Oncol Rep (1998 Mar-Apr) 5(2):311-5
3IC, which is found at high levels in vegetables of the Brassica genus, was administered ig (600, 150 or 38 mg/Kg BW/day) to 53 day old rats initiated 3 days previously with N-methyl-N-nitrosourea (MNU). Inhibition of tumor multiplicity was approx 35% at the two higher doses while the lowest dose of 3IC had no effect. In contrast 3IC caused a dose dependent increase in the levels of CYP 1A1 and CYP 1A2 in the liver. 3IC or its metabolites indole carbazole (ICZ) or a dimeric condensation product (DIM) could not be detected in mammary tissue. A dose dependent increase in ICZ in both liver and feces as well as relatively high levels of DIM in feces (0.05-1.0 mg/gram) was observed over the dose range of 3IC examined. Thus, 3IC is an effective chemopreventive even when administered following MNU- initiation.
Lubet RA Beebe L Bjeldanes L Bradlow LH Kelloff GJ Steele VE Eto I Grubbs CJ. Chemopreventive effects of indole-3-carbinol (3IC) on MNU induced mammary cancer in Sprague Dawley rats (Meeting abstract).Proc Annu Meet Am Assoc Cancer Res (201996) 37:1890
Estrogen, via its binding to the estrogen receptor (ER), plays an important role in breast cancer cell proliferation and tumor development. Indole-3-carbinol (I3C), a compound occurring naturally in cruciferous vegetables, exhibits a potent antitumor activity via its regulation of estrogen activity and metabolism. This study was designed to determine the effect of I3C on the potential to inhibit the ER-alpha. Using a reporter gene driven by the estrogen receptor, I3C (10-125 micromol/L) significantly repressed the 17ss-estradiol (E2)-activated ER-alpha signaling in a dose-dependent manner. I3C and breast cancer susceptibility gene 1 (BRCA1) synergistically inhibited transcriptional activity of ER-alpha. Moreover, I3C down-regulated the expression of the estrogen-responsive genes, pS2 and cathepsin-D, and up-regulated BRCA1. The inhibitory effects of I3C did not contribute to its cytotoxic effects because these activities were observed at less than toxic concentrations. These results further suggest that antitumor activities of I3C are associated not only with its regulation of estrogen activity and metabolism, but also its modulation of ER transcription activity.
Meng Q Yuan F Goldberg ID Rosen EM Auborn K Fan S. Indole-3-carbinol is a negative regulator of estrogen receptor-alpha signaling in human tumor cells.J Nutr (2000 Dec) 130(12):2927-31
Indole-3-carbinol (I3C) is a promising phytochemical agent in chemoprevention of breast cancer. Our present study is the first description of I3C that significantly inhibits the cell adhesion, spreading and invasion associated with an up-regulation of PTEN (a tumor suppressor gene) and E-cadherin (a regulator of cell-cell adhesion) expression in T47-D human breast cancer cells. Therefore, I3C exhibits anti-cancer activities by suppressing breast tumor cell growth and metastatic spread. Metastatic breast cancer is a devastating problem, clinical application of I3C as a potent chemopreventive agent may be helpful in limiting breast cancer invasion and metastasis.
Meng Q Goldberg ID Rosen EM Fan S.Inhibitory effects of Indole-3-carbinol on invasion and migration in human breast cancer cells.Breast Cancer Res Treat (2000 Sep) 63(2):147-52
The oxidative metabolism of estrogens in humans is mediated primarily by cytochrome P450, many isoenzymes of which are inducible by dietary and pharmacologic agents. One major pathway, 2- hydroxylation, is induced by dietary indole-3-carbinol (I3C), which is present in cruciferous vegetables (e.g., cabbage and broccoli). PURPOSE: Because the pool of available estrogen substrates for all pathways is limited, we hypothesized that increased 2-hydroxylation of estrogens would lead to decreased activity in competing metabolic pathways. METHODS: Urine samples were collected from subjects before and after oral ingestion of I3C (6-7 mg/kg per day). In the first study, seven men received I3C for 1 week; in the second study, 10 women received I3C for 2 months. A profile of 13 estrogens was measured in each sample by gas chromatography-mass spectrometry. RESULTS: In both men and women, I3C significantly increased the urinary excretion of C-2 estrogens. The urinary concentrations of nearly all other estrogen metabolites, including levels of estradiol, estrone, estriol, and 16alpha-hydroxyestrone, were lower after I3C treatment. CONCLUSIONS: These findings support the hypothesis that I3C-induced estrogen 2-hydroxylation results in decreased concentrations of several metabolites known to activate the estrogen receptor. This effect may lower estrogenic stimulation in women. IMPLICATIONS: I3C may have chemopreventive activity against breast cancer in humans, although the long-term effects of higher catechol estrogen levels in women require further investigation.
Michnovicz JJ Adlercreutz H Bradlow HL. Changes in levels of urinary estrogen metabolites after oral indole-3-carbinol treatment in humans. J Natl Cancer Inst (1997 May 21) 89(10):718-23
Diindolylmethane (DIM) is an acid-catalyzed condensation product of indole-3-carbinol, a constituent of cruciferous vegetables, and is formed in the stomach. DIM alters estrogen metabolism and inhibits carcinogen-induced mammary tumor growth in rodents. DIM is a weak agonist for the aryl hydrocarbon (Ah) receptor and blocks the effects of estrogens via inhibitory Ah receptor-estrogen receptor cross-talk. DIM and various structural analogs were examined in H295R cells for effects on 3 cytochrome P450 (CYP) enzymes involved in estrogen synthesis and/or metabolism: CYP1A1, CYP1B1, and CYP19 (aromatase). Aromatase activity was measured by conversion of 1 beta-(3)H- androstenedione to estrone and (3)H(2)O. H295R cells were exposed to the test chemicals dissolved in dimethyl sulfoxide for 24 h prior to analyses. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) (0--30 nM) and DIM (0--10 microM) induced ethoxyresorufin-O-deethylase (EROD) activity, as a measure of CYP1A1 and possibly 1B1 activity, with EC(50) values of about 0.3 nM and 3 microM, respectively. DIM, but not TCDD, induced aromatase activity with an apparently maximal 2- fold increase at 10 microM; higher concentrations of DIM and many of its analogs were cytotoxic. TCDD (30 nM) significantly increased CYP1A1 and 1B1 mRNA levels, but had no effect on mRNA for CYP19. DIM (3 microM) significantly increased mRNA levels for all three CYPS: DIM analogs with substitutions on the 5 and 5' position (3 microM) induced aromatase and EROD activity, together with mRNA levels of CYP1A1, 1B1, and 19; analogs that were substituted on the central carbon of the methane group showed little or no inductive activity toward the CYPS: In conclusion, DIM and several of its analogs appear to induce CYPs via multiple yet distinct pathways in H295R human adrenocortical carcinoma cells.
Sanderson JT Slobbe L Lansbergen GW Safe S van den Berg M.2,3,7,8-Tetrachlorodibenzo-p-dioxin and diindolylmethanes differentially induce cytochrome P450 1A1, 1B1, and 19 in H295R human adrenocortical carcinoma cells. Toxicol Sci (2001 May) 61(1):40-8
Dietary indole-3-carbinol inhibits carcinogenesis in rodents and trout. Several mechanisms of inhibition may exist. We reported previously that 3,3'-diindolylmethane, an in vivo derivative of indole-3-carbinol, is a potent noncompetitive inhibitor of trout cytochrome P450 (CYP) 1A-dependent ethoxyresorufin O-deethylase with Ki values in the low micromolar range. We now report a similar potent inhibition by 3,3'-diindolylmethane of rat and human CYP1A1, human CYP1A2, and rat CYP2B1 using various CYP-specific or preferential activity assays. 3,3'-Diindolylmethane also inhibited in vitro CYP- mediated metabolism of the ubiquitous food contaminant and potent hepatocarcinogen, aflatoxin B1. There was no inhibition of cytochrome c reductase. In addition, we found 3,3'-diindolylmethane to be a substrate for rat hepatic microsomal monooxygenase(s) and tentatively identified a monohydroxylated metabolite. These observations indicate that 3,3'-diindolylmethane can inhibit the catalytic activities of a range of CYP isoforms from lower and higher vertebrates in vitro. This broadly based inhibition of CYP-mediated activation of procarcinogens may be an indole-3-carbinol anticarcinogenic mechanism applicable to all species, including humans.
Stresser DM Bjeldanes LF Bailey GS Williams DE. The anticarcinogen 3,3'-diindolylmethane is an inhibitor of cytochrome P-450. J Biochem Toxicol (1995 Aug) 10(4):191-201
There are striking associations between certain components of the human diet and reduced incidence of experimental cancer. Thus, implementing specific changes in diet may be an effective means to control human cancer, a disease responsible for about 25% of all deaths in the United States. 'Chemoprevention' is a cancer control strategy entailing the deliberate administration of chemicals in order to prevent the occurrence of the disease. The micronutrient indole-3-carbinol (I3C) is a common dietary constituent shown to modulate tumor incidence in animals and is under consideration as a possible chemopreventive agent. Central to the implementation of this strategy is understanding the mechanisms by which 'chemopreventives' exert their effects. Evidence is presented here that dietary I3C may inhibit tumor formation in the rat by a number of mechanisms, including induction of important carcinogen detoxifying enzymes such as cytochromes P-450 (CYP) and the glutathione S-transferases. For comparison, beta-naphthoflavone, a synthetic flavonoid, was found to be a less potent inducer at doses approximately equally anticarcinogenic. The major role of CYP enzymes is to form or expose functional groups on lipophilic chemicals which enter the organism, thereby serving to enhance their excretion. Some lipophilic chemicals are acted on by CYPs to 'bioactivate' them into carcinogenic forms. Evidence is also presented that I3C metabolites and BNF will inhibit this enzymatic process, thus suggesting another mechanism of protection. Unlike I3C metabolites, BNF was found to enhance bioactivation under certain conditions in vitro. It is shown that concentrations of I3C metabolites that can inhibit CYP in vitro are present in vivo following an anticarcinogenic I3C dose. Aflatoxin B1 was used as a model carcinogen throughout this study both because of its extreme potency and because it is a common contaminant of human foodstuffs, particularly in lesser developed countries.
Stresser DM.Mechanisms of inhibition of chemical carcinogenesis by indole-3-carbinol in the rat. Diss Abstr Int [B] (201995) 55(8):3268
I3C occurs in cruciferous vegetables and has chemopreventive activity in laboratory animals. This activity relates to its ability to influence cytochromes P450 that metabolize carcinogens, but the effects of I3C on human carcinogen metabolism have not been reported. NNK is a tobacco-specific lung carcinogen believed to play a role in lung cancer induction in smokers. Its lung tumorigenicity is inhibited by I3C in the mouse; I3C increases hepatic clearance of NNK resulting in lower bioavailability to lung (Morse et al, Cancer Res; 50:2613, 1990). We studied I3C effects on NNK metabolism in 13 females (mean age, 35.2 ± 11.0 yr, range 24-60) who received 400 mg/day I3C orally for 5 consecutive days. The women maintained constant smoking (confirmed by cotinine). Urine was collected before and after treatment and analyzed for two metabolites of NNK: 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronide (NNAL-Gluc). Levels of NNAL decreased significantly as a result of treatment (mean ± S.D., -0.27 ± 0.09 pmol/mg creatinine, p=0.016) as did total levels of NNAL plus NNAL-Gluc (- 0.43 ± 0.16 pmol/mg creatinine, p=0.023). The NNAL-Gluc/NNAL ratio increased, 1.1 ± 0.5, p=0.046. These results suggest that I3C increases hepatic clearance of NNK, probably via cytochrome P450 induction. The results are similar to those observed in mice protected against NNK pulmonary tumorigenesis by I3C.
Taioli E Garbers SV Bradlow LH Carmella SG Hecht SS. Effects of indole-3-carbinol (I3C) on the metabolism of the tobacco- specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK) in smokers (Meeting abstract).Proc Annu Meet Am Assoc Cancer Res (1997) 38:1413
The natural estrogen 17-beta-estradiol (E2) influences the mammary cell proliferation, morphogenesis, and neoplastic transformation in vivo. The role of epithelial-stromal interaction in the cellular effects of E2 is equivocal. Experiments were designed using an in vitro model developed from mammary explants and cell culture to examine whether: (i) stromal component affects cellular metabolism of E2 and (ii) naturally-occurring agents modulate E2 metabolism. Metabolic conversion of E2 via C2- and C16-alpha-hydroxylation pathways was monitored by a radiometric assay, and cellular proliferation was measured by 3H-thymidine uptake or by anchorage- independent growth. In the explants of terminal duct lobular units (TDLU) containing inter-lobular and inter-ductal stroma, the chemical carcinogen benzo(alpha)pyrene (BP) induced 164.8% increase in E2 C16- alpha-hydroxylation (p=0.006) and 51.9% increase in 3H-thymidine uptake (p=0.002) BP treatment also exhibited a 77.6% decrease (p less than 0.0001) in E2 C2/C16-alpha-hydroxylation ratio, which was abrogated (p less than 0.0001) in the presence of eicosapentaenoic acid (EPA) or indole-3-carbinol (I3C). In the cultures of human mammary carcinoma MCF-7 cells EPA and I3C enhanced E2 C2/C16-alpha- hydroxylation ratio (p less than 0.0001) and inhibited anchorage- independent growth (p=0.001) In TDLU and MCF-7 cultures the extent of E2 C2-hydroxylation was down-regulated (p=0.005) by human adipocyte- conditioned medium (h-ACM) obtained from obese subjects. Thus, BP- and h-ACM-induced alteration of E2 metabolism and its modulation by EPA and I3C in TDLU suggests a paracrine role of stroma in human mammary carcinogenesis. This in vitro model may provide a system to assess the effects of naturally-occurring chemopreventive agents on human mammary cell transformation.
Telang NT.In vitro modulation of human mammary carcinogenesis: a model for chemoprevention (Meeting abstract). Proc Annu Meet Am Assoc Cancer Res (201995) 36:A3509
Aberrant proliferation is an early-occurring intermediate event in carcinogenesis whose inhibition may represent preventive intervention. Indole-3-carbinol (I3C), a glucosinolate metabolite from cruciferous vegetables, inhibits organ site carcinogenesis in rodent models. Clinically relevant biochemical and cellular mechanisms for the anticarcinogenic effects of I3C, however, remain unclear. Experiments were conducted on reduction mammoplasty derived 184-B5 cells initiated with chemical carcinogen (184-B5/BP) or with oncogene (184-B5/HER), and on mammary-carcinoma-derived MDA-MD-231 cells to examine whether (i) I3C inhibits aberrant proliferation in initiated and transformed cells, and (ii) inhibition of aberrant proliferation is associated with altered cell-cycle progression, estradiol (E2) metabolism, and apoptosis. Aberrant proliferation in 184-B5/BP, 184-B5/HER, and MDA-MB-231 cells was evident by a 55%-56% decrease in the ratio of quiescent (Q = G0) to proliferative (P = S + M) phase of the cell cycle, a 72%-90% decrease in apoptosis, and a 76%-106% increase in anchorage-dependent growth. These cells also exhibited a 88%-90% decrease in the ratio of C2 to C16alpha- hydroxylation products of E2. Treatment of 184-B5/BP, 184-B5/HER, and MDA-MB-231 cells to cytostatic dose of 50 microM I3C resulted in an 137%-210% increase in Q/P I3C ratio, a 4- to 18-fold increase in E2 metabolite ratio, a 2-fold increase in cellular apoptosis, and a 54%- 61% inhibition of growth. The preventive efficacy of I3C on human mammary carcinogenesis may be due in part to its ability to regulate cell-cycle progression, increase the formation of antiproliferative E2 metabolite, and induce cellular apoptosis.
Telang NT Katdare M Bradlow HL Osborne MP Fishman J.Inhibition of proliferation and modulation of estradiol metabolism: novel mechanisms for breast cancer prevention by the phytochemical indole-3-carbinol.Proc Soc Exp Biol Med (1997 Nov) 216(2):246-52
Sixty women at increased risk for breast cancer were enrolled in a placebo-controlled, double-blind dose-ranging chemoprevention study of indole-3-carbinol (I3C). Fifty-seven of these women with a mean age of 47 years (range 22-74) completed the study. Each woman took a placebo capsule or an I3C capsule daily for a total of 4 weeks; none of the women experienced any significant toxicity effects. The urinary estrogen metabolite ratio of 2-hydroxyestrone to 16 alpha- hydroxyestrone, as determined by an ELISA assay, served as the surrogate endpoint biomarker (SEB). Perturbation in the levels of SEB from baseline was comparable among women in the control (C) group and the 50, 100, and 200 mg low-dose (LD) group. Similarly, it was comparable among women in the 300 and 400 mg high-dose (HD) group. Regression analysis showed that peak relative change of SEB for women in the HD group was significantly greater than that for women in the C and LD groups by an amount that was inversely related to baseline ratio; the difference at the median baseline ratio was 0.48 with 95% confidence interval (0.30, 0.67). No other factors, such as age and menopausal status, were found to be significant in the regression analysis. The results in this study suggest that I3C at a minimum effective dose schedule of 300 mg per day is a promising chemopreventive agent for breast cancer prevention. A larger study to validate these results and to identify an optimal effective dose schedule of I3C for long-term breast cancer chemoprevention will be necessary.
Wong GY Bradlow L Sepkovic D Mehl S Mailman J Osborne MP. Dose-ranging study of indole-3-carbinol for breast cancer prevention. J Cell Biochem Suppl (1997) 28-29:111-6
Groups of male Wistar rats were fed semi-synthetic diets containing 0, 200 or 500 mg indole-3-carbinol (13C)/kg for 2, 7, 14 or 28 days. After 2 days, P-450 activities were already induced, but the isoenzyme pattern induced was different in the liver and the small intestine. Hepatic P4501A1, P4501A2 and P4502B1 apoprotein levels were dose-relatedly enhanced, whereas in the small intestine induced levels of P4502B1 and P4501A1 were detected but P4501A2 was not induced. Pentoxy- and ethoxyresorufin dealkylation (PROD and EROD) were dose-relatedly enhanced in the liver (5- and 7-fold, respectively, in the higher dose group) as well as in the small intestine (8- and 13-fold, respectively, at 500 mg 13C/kg diet). Testosterone 16 alpha- and 16 beta-hydroxylation in the small intestine were enhanced (6-9-fold) from day 2 onwards, but in the liver these activities were only slightly enhanced from day 7 onwards. Thus, the major forms induced in the liver appear to be P4501A1, P4501A2, P4502B1 and, to a lesser extent, P4503A, whereas in the small intestine all of the effects that were found are associated with only one cytochrome P-450, P4502B1. After 2 days I3C (500 mg/kg) induced glutathione S-transferase in the liver (1.3-fold) and small intestine (1.5-fold). Hepatic glucuronyl transferase (GT1) was induced (about 1.6-fold) after 7, 14 and 28 days. DT-diaphorase was induced in the liver (2.7-fold) and small intestine (1.5-fold) after 14 days of exposure to 500 mg I3C/kg diet. Treatment of rat hepatocytes with indole-3-acetonitrile and 3,3'-diindolylmethane, but not I3C and indole-3-carboxaldehyde, enhanced EROD activity and halved testosterone 16 alpha- and 2 alpha-hydroxylation. All four indoles slightly induced glutathione S-transferase in cultured hepatocytes. Thus, the in vitro studies suggest that the in vivo effects of I3C have to be attributed to indole-condensation products, such as 3,3'-diindolylmethane, but not to I3C itself.
Wortelboer HM van der Linden EC de Kruif CA Noordhoek J Blaauboer BJ van Bladeren PJ Falke HE. Effects of indole-3-carbinol on biotransformation enzymes in the rat: in vivo changes in liver and small intestinal mucosa in comparison with primary hepatocyte cultures.Food Chem Toxicol (1992 Jul) 30(7):589-99
Cervical cancer constitutes the second most common cancer in women. Estrogen promotes development of cervical cancer in cells infected with high risk human papillomaviruses (HPVs). We asked whether the phytochemical indole-3-carbinol (I3C) has anti-estrogenic activities in cervical cells with the goal of preventing cancer in HPV infected cells. MATERIALS AND METHODS: Using the cervical cancer cell line CaSki, we evaluated expression of HPV and cytochrome p450 (CYP) enzymes by Northern, RNase protection or quantitative RT-PCR. I3C binding to estrogen receptor was measured by competition with estradiol. Estrogen metabolites were measured by gas chromarography- mass spectrometry (GC-MS). RESULTS: Estradiol increased expression of HPV oncogenes whereas I3C and the estrogen metabolite 2- hydroxyestrone (2-OHE) abrogated the estrogen-increased expression of HPV oncogenes. Both I3C and 2-OHE competed with estradiol for estrogen receptor binding. I3C enhanced gene expression of CYP enzymes responsible for 2-hydroxylation of estrogen, and induced the formation of 2-OHE. CONCLUSION: I3C has anti-estrogenic activities which should prevent cancer in cervical cells.
Yuan F Chen DZ Liu K Sepkovic DW Bradlow HL Auborn K.Anti-estrogenic activities of indole-3-carbinol in cervical cells implication for prevention of cervical cancer. Anticancer Res (1999 May-Jun) 19(3A):1673-80
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