Cytostatic effects of 3,3'-diindolylmethane in human endometrial cancer cells result from an estrogen receptor-mediated increase in transforming growth factor-alpha expression.

3,3'-Diindolylmethane (DIM), a major in vivo product of indole-3-carbinol (I3C), is a promising anticancer agent derived from vegetables of the Brassica genus including broccoli, Brussels sprouts and cabbage. We report here that DIM has a potent cytostatic effect in cultured human Ishikawa endometrial cancer cells. A combination of northern blot and quantitative PCR analyses revealed that DIM induced the level of TGF-alpha transcripts by approximately 4-fold within 24 h of indole treatment. DIM also induced a 4-fold increase in the activity of the estrogen response marker, alkaline phosphatase (AP). Co-treatment of cells with the estrogen receptor (ER) antagonist ICI, or with the inhibitor of PKA-mediated activation of the ER, H89, ablated the DIM induction of both TGF-alpha expression and AP activity. Furthermore, DIM increased the maximum stimulatory effect of estrogen on TGF-alpha expression. Co-treatment with the protein synthesis inhibitor, cycloheximide, abolished the inductive effects of DIM, indicating differences in the mechanistic requirements of DIM and estrogen. DIM treatment also stimulated levels of secreted TGF-alpha protein by >10-fold. The ectopic addition of TGF-alpha inhibited the growth of Ishikawa cells, whereas incubation with a TGF-alpha antibody partially reversed the growth inhibitory effects of DIM. Taken together, these results extend our previous findings of the ligand independent estrogen receptor agonist activity of DIM, and uncover an essential role for the stimulation in TGF-alpha expression and the TGF-alpha activated signal transduction pathway in the potent cytostatic effects of DIM in endometrial cancer cells.

Indole-3-carbinol inhibits CDK6 expression in human MCF-7 breast cancer cells by disrupting Sp1 transcription factor interactions with a composite element in the CDK6 gene promoter.

Indole-3-carbinol (I3C), a compound naturally occurring in Brassica vegetables, can induce a G(1) cell cycle arrest of human MCF-7 breast cancer cells that is accompanied by the selective inhibition of cyclin-dependent kinase 6 (CDK6) expression. Reverse transcriptase-polymerase chain reaction analysis of CDK6 mRNA decay rates revealed that I3C had no effect on CDK6 transcript stability. We report the first identification and functional characterization of the CDK6 promoter in order to determine whether I3C inhibits CDK6 transcription. In MCF-7 cells stably transfected with CDK6 promoter-linked luciferase reporter plasmids, I3C inhibited CDK6 promoter activity in an I3C-specific response that was not a consequence of the growth-arrested state of the cells. Deletion analysis revealed a 167-base pair I3C-responsive region of the CDK6 promoter between -805 and -638. Site-specific mutations within this region revealed that both Sp1 and Ets-like sites, which are spaced 5 base pairs apart, were necessary for I3C responsiveness in the context of the CDK6 promoter. Electrophoretic mobility shift analysis of protein-DNA complexes formed with nuclear proteins isolated from I3C-treated and -untreated cells, in combination with supershift assays using Sp1 antibodies, demonstrated that the Sp1-binding site in the CDK6 promoter forms a specific I3C-responsive DNA-protein complex that contains the Sp1 transcription factor. Taken together, our results suggest that I3C down-regulates CDK6 transcription by targeting Sp1 at a composite DNA site in the CDK6 promoter.

N-methoxyindole-3-carbinol is a more efficient inducer of cytochrome P-450 1A1 in cultured cells than indol-3-carbinol.

The well-documented reduction of cancer risk by high dietary cruciferous vegetable intake may in part be caused by modulation of cytochrome P-450 (CYP) expression and activity by indoles. The purpose of the present experiments was to study the mechanism of CYP 1A1 induction by N-methoxyindole-3-carbinol (NI3C) in cultured cells and to compare the CYP-inducing potential of NI3C and indole-3-carbinol (I3C) administered to rats. NI3C induced 7-ethoxyresorufin-O-deethylase (EROD) activity in Hepa-1c1c7 cells in a concentration-dependent manner with 10-fold higher efficiency than I3C. Inasmuch as NI3C induced binding of the aryl hydrocarbon receptor (AhR) to the dioxin-responsive element and induced expression of endogenous CYP 1A1 mRNA and an AhR-responsive chloramphenicol acetyl transferase construct, we conclude that NI3C can activate the AhR. Besides the induction of CYP 1A1, we observed an inhibition of EROD activity, with a concentration causing 50% inhibition of 6 microM. Oral administration of NI3C at 570 mumol/kg body wt to male Wistar rats increased the hepatic CYP 1A1 and 1A2 protein levels, as well as the EROD and 7-methoxyresorufin O-demethylase activities at 8 and 24 hours after administration, but the responses were less pronounced than after administration of I3C at 570 mumol/kg body wt. Furthermore, NI3C did not induce hepatic 7-pentoxyresorufin O-depentylase activity, as I3C did. Ascorbigen, another indolylic compound formed during degradation of glucobrassicin in the presence of ascorbic acid, was tested in the same animal model, and ascorbigen only weakly induced hepatic CYP 1A1 and 1A2, but not CYP 2B1/2. In conclusion, NI3C is a more efficient inducer of CYP 1A1 in cultured cells than I3C but is less active when administered to rodents.

Ligand-independent activation of estrogen receptor function by 3, 3'-diindolylmethane in human breast cancer cells.

3,3'-Diindolylmethane (DIM), a major in vivo product of acid-catalyzed oligomerization of indole-3-carbinol (I3C), is a promising anticancer agent present in vegetables of the Brassica genus. We investigated the effects of DIM on estrogen-regulated events in human breast cancer cells and found that DIM was a promoter-specific activator of estrogen receptor (ER) function in the absence of 17beta-estradiol (E(2)). DIM weakly inhibited the E(2)-induced proliferation of ER-containing MCF-7 cells and induced proliferation of these cells in the absence of steroid, by approximately 60% of the E(2) response. DIM had little effect on proliferation of ER-deficient MDA-MB-231 cells, suggesting that it is not generally toxic at these concentrations. Although DIM did not bind to the ER in this concentration range, as shown by a competitive ER binding assay, it activated the ER to a DNA-binding species. DIM increased the level of transcripts for the endogenous pS2 gene and activated the estrogen-responsive pERE-vit-CAT and pS2-tk-CAT reporter plasmids in transiently transfected MCF-7 cells. In contrast, DIM failed to activate transcription of the simple E(2)- and diethylstilbesterol-responsive reporter construct pATC2. The estrogen antagonist ICI 182780 (7alpha-[9-[(4,4,5,5, 5-pentafluoropentyl)sulfonyl]nonyl]-estra-1,3,5(10)-triene-3, 17beta-diol) was effective against DIM-induced transcriptional activity of the pERE-vit-CAT reporter, which further supports the hypothesis that DIM is acting through the ER. We demonstrated that ligand-independent activation of the ER in MCF-7 cells could be produced following treatment with the D1 dopamine receptor agonist SKF-82958 [(+/-)6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4, 5-tetrahydro-1H-3-benzazepinehydrobromide]. We also demonstrated that the agonist effects of SKF-82958 and DIM, but not of E(2), could be blocked by co-treatment with the protein kinase A (PKA) inhibitor H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide). These results have uncovered a promoter-specific, ligand-independent activation of ER signaling for DIM that may require activation by PKA, and suggest that this major I3C product may be a selective activator of ER function.

The major cyclic trimeric product of indole-3-carbinol is a strong agonist of the estrogen receptor signaling pathway.

Indole-3-carbinol (I3C), a component of Brassica vegetables, is under study as a preventive agent of cancers of the breast and other organs. Following ingestion, I3C is converted to a series of oligomeric products that presumably are responsible for the in vivo effects of I3C. We report the effects of the major trimeric product, 5,6,11,12,17,18-hexahydrocyclonona[1,2-b:4,5-b':7,8-b' ']triindole (CTr), on the estrogen receptor (ER) signaling pathways. Tumor-promoting effects of high doses of I3C may be due to activation of aryl hydrocarbon receptor (AhR)-mediated pathways; therefore, we also examined the effects of CTr on AhR activated processes. We observed that CTr is a strong agonist of ER function. CTr stimulated the proliferation of estrogen-responsive MCF-7 cells to a level similar to that produced by estradiol (E(2)) but did not affect the growth of the estrogen-independent cell line, MDA-MD-231. CTr displaced E(2) in competitive-binding studies and activated ER-binding to an estrogen responsive DNA element in gel mobility shift assays with EC(50)s of about 0.1 microM. CTr activated transcription of an E(2)-responsive endogenous gene and exogenous reporter genes in transfected MCF-7 cells, also with high potency. CTr failed to activate AhR-mediated pathways, consistent with the low-binding affinity of CTr for the AhR reported previously. Comparisons of the conformational characteristics of CTr with other ER ligands indicated a remarkable similarity with tamoxifen, a selective ER antagonist used as a breast cancer therapeutic agent and suggest an excellent fit of CTr into the ligand-binding site of the ER.

Modulation of cytochrome P4501A1 activity by ascorbigen in murine hepatoma cells.

Modulation of cytochrome P4501A1 (CYP1A1) activity is a mechanism whereby indoles present in cruciferous vegetables could affect the metabolism of xenobiotics. Ascorbigen (ASG) is the predominant indole formed during the degradation of glucobrassicin, although the mechanism by which ASG modulates CYP1A1 activity is not known. The major focus of this study was to examine the mechanism of CYP induction by ASG using a murine hepatoma-derived cell line (Hepa 1c1c7). ASG was shown to induce the activity of 7-ethoxyresorufin O-deethylase, a marker for CYP1A1, in a concentration-responsive manner with a maximum induction at 700 microM. Maximum ASG induction after 24-hr treatment was 7% of maximal CYP1Al activity induced by the well-known potent CYP1A1 inducer, indolo[3,2-b]carbazole (ICZ) (1 microM), and the EC50 values differed by 2-fold. The CYP1A1 activity increased continuously up to 72 hr, where ASG showed an induction efficiency in the same range as for the positive control (1 microM ICZ) after 24 hr, whereas the CYP1A1 protein level, measured by Western blot analysis, was maximally induced after 24 hr. ASG significantly inhibited CYP1A1 activity in whole cells at concentrations above 1 microM. ASG increased the chloramphenicol acetyl transferase (CAT) activity via a CAT reporter construct containing a dioxin-responsive element in Hepa 1c1c7 cells, indicating involvement of the aryl hydrocarbon receptor. ASG was shown to be transformed into ICZ, or a compound with the same chromatographic mobility as ICZ, in the medium. Taken together, the results indicate that ASG inhibits CYP1A1 activity at low concentrations, but induces the same activity at higher concentrations.

Cytostatic and antiestrogenic effects of 2-(indol-3-ylmethyl)-3,3'-diindolylmethane, a major in vivo product of dietary indole-3-carbinol.

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.

Lipoxin A4: a new class of ligand for the Ah receptor.

The Ah receptor is a ligand-activated transcription factor that mediates many of the biological actions of a large class of environmental compounds. Support for a role of the Ah receptor in normal physiology also has been reported, but an endogenous regulating ligand has not been identified. We have examined candidate endogenous lipophilic substances and report here the ability of the arachidonic acid metabolite, lipoxin A4, to bind to and activate the Ah receptor in Hepa-1 cells. Lipoxin A4 produced a concentration-dependent response in a DRE-driven CAT reporter construct, with a greater than 10-fold increase in CAT activity at 0. 3 microM. Lipoxin A4 transformed the Ah receptor to an active DRE-binding form in a concentration-dependent manner as indicated by gel mobility shift analysis. Results of Ah receptor competitive binding experiments indicated that at a concentration of 100 nM, lipoxin A4 produced a half-maximum displacement (EC50) of [3H]TCDD binding. Results of Northern blot analyses indicated a transient increase in mRNA levels of the Ah receptor-responsive gene CYP1A1, which peaked at 4 h, consistent with the kinetics observed for lipoxin A4-induced CYP1A1 enzyme activity. Further, lipoxin A4 was found to be a competitive inhibitor for the CYP1A1 enzyme, with a calculated Ki = 1.1 microM. These results establish lipoxin A4 as a new class of Ah receptor ligand, one that differs dramatically from classical Ah receptor ligands.

Indole-3-carbinol and tamoxifen cooperate to arrest the cell cycle of MCF-7 human breast cancer cells.

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.

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.

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.

Inhibition of 2-amino-3-methylimidazo[4,5-f]quinoline-DNA adducts by indole-3-carbinol: dose-response studies in the rat colon.

Indole-3-carbinol (I3C) inhibits the formation of colonic aberrant crypt foci and DNA adducts in rats given heterocyclic amine colon carcinogens, such as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Mechanism studies indicate that I3C induces cytochromes P4501A1 and 1A2 (CYP1A1 and CYP1A2), isozymes that respectively metabolize IQ via ring hydroxylation or activate the carcinogen by N-hydroxylation. The present study examined the dose-response for induction of CYP1A1 versus CYP1A2 by I3C, and compared the profiles of induction with the dose-response for inhibition of IQ-DNA adducts in the colon of the F344 rat. Dietary equivalent doses of I3C in the range 100-1000 p.p.m. increased in a dose-related manner both ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) activities in the liver and colonic mucosa, and Western blots showed a corresponding induction of CYP1A1 and CYP1A2 proteins. However, dietary equivalent doses of I3C in the range 10-25 p.p.m. (i) reduced hepatic EROD and MROD activities and CYP1A protein levels compared with controls, (ii) increased the ratio of CYP1A2 versus CYP1A1, and (iii) activated IQ to a more potent mutagen when liver microsomes from rats given I3C were used for metabolic activation in the Salmonella assay. Rats given a single oral dose of I3C shortly before administering IQ (5 mg/kg body wt, p.o.) exhibited dose-related inhibition of colonic IQ-DNA adducts in the range 25-100 p.p.m. I3C, reaching 95% inhibition at doses > or = 100 p.p.m. I3C, but IQ-DNA adducts were elevated slightly at the lowest I3C dose as compared with the controls. The possible significance of the low versus high dose effects of I3C are discussed in the context of human dietary exposures to I3C and the reported chemopreventive mechanisms of I3C in vivo.

Mechanisms of indole-3-carbinol (I3C) anticarcinogenesis: inhibition of aflatoxin B1-DNA adduction and mutagenesis by I3C acid condensation products.

Possible inhibitory mechanisms of indole-3-carbinol (I3C) against aflatoxin B1 (AFB1), a potent hepatocarcinogen, were examined in rainbow trout. In the Salmonella assay using a trout post-mitochondrial activation system, I3C itself was not an antimutagen against AFB1. The study also evaluated: the antimutagenic ability of I3C oligomers; an acid reaction mixture (RXM) of I3C, generated at low pH to simulate I3C products formed under acidic conditions of the stomach; 3,3-diindolylmethane (I33'), the major derivative of I3C found in trout liver; and 5,6,11,12,17,18- hexahydrocyclononal [1,2-b:4,5-b':7,8-b"]triindole , the cyclic trimer of I3C (CT), a derivative of I3C in liver and one of the major components of RXM. Concentrations of 3.5 microM and greater of I33', CT or RXM showed about 80% inhibition compared with the control. Higher concentrations (70 microM) of the various I3C oligomers also inhibited (to a maximum of 55%) mutagenesis of synthetic AFB1-8,9-epoxide added to the Salmonella assay, in the absence of activating enzymes. I33' inhibited total microsome catalysed AFB1-DNA binding in vitro in an apparently non-competitive manner (Kis = 27.6 +/- 9.4 microM, Kii = 37.5 +/- 32.2 microM). These results suggest that the anticarcinogenic effect of I3C against AFB1 in rainbow trout, and perhaps other species, is due in part to inhibition of AFB1 bioactivation enzymes and to scavenging of the activated AFB1-8,9-epoxide.

The anticarcinogen 3,3'-diindolylmethane is an inhibitor of cytochrome P-450.

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.

Regulation of hepatic cytochrome P4501A by indole-3-carbinol: transient induction with continuous feeding in rainbow trout.

This study investigated the kinetics of hepatic cytochrome P-4501A (CYP1A) induction in rainbow trout by indole-3-carbinol (I3C), a natural tumour modulator from cruciferous vegetables, and its low pH reaction products 3,3'-diindolylmethane (I33'), 5,6,11,12,17,18-hexahydrocyclononal[1,2-b:4,5-b':7,8-b"]triindo le cyclic trimer (CT), and the unresolved I3C acid reaction mixture (RXM). RXM, CT and I33' were potent inducers of total embryonic CYP1A following direct microinjection, and of fingerling hepatic CYP1A following ip exposure, whereas I3C itself produced only a transient and relatively weak induction. It is also reported for the first time that dietary I3C induced hepatic CYP1A and its associated ethoxyresorufin O-deethylase (EROD) activity in trout but, again, the induction was weak and transient even with continuous I3C feeding. Mechanism studies and mixed exposures with the Ah agonist beta-naphthoflavone indicated that transient induction by I3C was not due to diet ageing, but appears to involve inactivation of the Ah inductive pathway and irreversible inactivation of CYP1A-mediated EROD activity by I3C-derived metabolites. Thus, I3C derivatives exhibit dual capacities for CYP1A induction and inhibition in trout.

Modulation of glutathione S-transferase activity and isozyme pattern in liver and small intestine of rats fed goitrin- and T3-supplemented diets.

Goitrin is a potent goitrogen that has been shown to induce glutathione S-transferase (GST) activity and to increase aflatoxin detoxification. In the present study with rats, dietary goitrin (200 mg/kg diet) produced a hypothyroid state and significantly increased levels of hepatic GSSG (1.4-fold), GST protein (1.4-fold) and GST activity against chlorodinitrobenzene (CDNB) (1.7-fold). Cotreatment with dietary triiodothyronine (T3) reversed these effects in a dose-related manner. Intestinal GST activities against CDNB and epoxynitrophenoxypropane did not change with goitrin or T3 treatment. HPLC analysis showed that, in the liver, goitrin treatment increased the levels of GST-1b and -7 by 3.5- and 5-fold, respectively, and decreased the level of GST-3 by 50%. Cotreatment with T3 returned levels of GST-7 and -3 to control levels but only partially reduced the level of GST-1b. In the small intestine, goitrin increased the level of GST-1b by 28% and decreased the level of GST-7 by 34% compared with those of controls; thyroid hormone treatment produced no additional effect on GST in this organ. Selenium deficiency altered thyroid hormone status but significantly affected the level only of hepatic GST-3, which was reduced by 30% compared with that of controls. These results indicate that a modified thyroid hormonal status plays an important role in the GST-inducing effects of goitrin. A possible mechanism of thyroid-dependent GST induction by goitrin is discussed.

Indolo[3,2-b]carbazole: a dietary-derived factor that exhibits both antiestrogenic and estrogenic activity.

BACKGROUND: Indole-3-carbinol (I3C) and related compounds have been identified in vegetables of the Brassica genus. I3C and its acid-derived condensation product, indolo[3,2-b]carbazole (ICZ), bind to the aryl hydrocarbon (Ah) receptor and induce CYP1A1/1A2 gene expression in both in vivo and in vitro models. I3C also inhibits mammary tumor development in rodent models. PURPOSE: The major focus of this study was to investigate the induction of CYP1A1-dependent activity and antiestrogenic effects of ICZ in the MCF-7 human breast cancer cell line and determine if induction of CYP1A1 is required for observed antiestrogenic responses. METHODS: The induction of CYP1A1 in MCF-7 cells was determined by measuring time- and concentration-dependent changes in ethoxyresorufin O-deethylase (EROD) activity in response to ICZ treatment. The effects of ICZ on occupied nuclear estrogen receptor (ER) levels and inhibition of estrogen (17 beta-estradiol [E2])-induced cell proliferation, [3H]thymidine uptake, secretion of the 52-kd protein, and nuclear progesterone receptor (PR) levels were also measured. Chloramphenicol acetyl transferase (CAT) activity was assayed in MCF-7 cells transiently transfected with an estrogen-responsive vit-CAT plasmid. Competitive binding to rat cytosolic ER was also examined. RESULTS: ICZ (> or = 10 nM) induced CYP1A1 in MCF-7 human breast cancer cells. This compound also elicited a diverse spectrum of antiestrogenic responses, including inhibition of E2-induced cell proliferation, [3H]thymidine uptake, occupied nuclear PR binding, and CAT activity in cells transfected with the estrogen-responsive vit-CAT plasmid. In nuclear extracts from ICZ-treated cells, there was a decrease in ER levels and binding to an estrogen-responsive element in a gel shift assay. I3C also decreased nuclear ER binding in MCF-7 cells. ICZ bound with low affinity to the ER and exhibited weak estrogen-like activity. CONCLUSIONS: Like other Ah receptor agonists, ICZ is antiestrogenic in human breast cancer cells, and this activity is consistent with the inhibitory activity of I3C on mammary tumor formation in rodents. ICZ-induced antiestrogenic responses can be observed at times or concentrations in which EROD activity is unchanged, indicating an interaction between the Ah receptor and ER-mediated endocrine pathways that is independent of P450-induced hormone metabolism. ICZ also is a weak estrogen in MCF-7 cells and binds to the ER. IMPLICATIONS: The current focus on the role of dietary and environmental estrogens in human disease should take into account the possible contra-active effects of Ah receptor agonists such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), ICZ, I3C, and related compounds that exhibit antiestrogenic activity.

Anticarcinogenic activity of indole-3-carbinol acid products: ultrasensitive bioassay by trout embryo microinjection.

The relative contribution of indole-3-carbinol (I3C) and its acid condensation products to the anticarcinogenic activity of this crucifer phytochemical has been studied using trout embryo microinjection. I3C was treated with 0.07 N HCl to give a reaction mixture (RXM) comprising < 0.5% parent compound and over 20 products, the most prevalent being the dimer 3,3'-diindolylmethane (I33') and a related cyclic trimer (CT). RXM, I33' or CT was injected into embryos with [3H]aflatoxin B1 (AFB1) and total embryonic DNA was isolated 1, 3, or 10 days postinjection. Compared with controls given AFB1 alone, I3C failed to inhibit carcinogen-DNA binding at any time point. In contrast I33', CT, and RXM inhibited AFB1-DNA binding by an average of 37, 51, and 65%, respectively. Coinjection of AFB1 and 350 microM I3C, RXM, or I33' into trout embryos reduced AFB1-induced hepatocarcinogenesis after 1 year from 43.4% in positive controls to 36.0, 12.2 (P < 0.05), and 24.6% (P < 0.05), respectively. No tumor data were obtained in the AFB1 plus CT group due to poor survival of the embryos posthatching. These results indicate that acid condensation products, not the parent compound, represent the anticarcinogenic species in trout and that their formation in the stomach is a likely prerequisite for I3C anticarcinogenesis.

Organ-selective induction of cytochrome P-450-dependent activities by indole-3-carbinol-derived products: influence on covalent binding of benzo[a]pyrene to hepatic and pulmonary DNA in the rat.

Indole-3-carbinol (I3C) is a dietary modulator of carcinogenesis that can reduce the level of carcinogen binding to DNA. I3C-derived products are potent inducers of certain cytochrome P-450(CYP)-dependent enzyme activities. To investigate whether the protective effects of I3C against carcinogen damage to DNA are associated with increased activities of CYP1A1 enzymes, we examined the relationship of I3C-mediated organ-specific CYP enzyme induction with total levels of benzo[a]pyrene (BP) binding to hepatic and pulmonary DNA of rats. Oral intubation (PO) of I3C (500 mumol/kg body wt.) in 10% DMSO in corn oil produced after 20 h, increases in ethoxyresorufin O-deethylase (EROD) activities (associated with CYP1A1 isozyme) of 700-fold, 245-fold and 36-fold in small intestine, lungs and liver, respectively, compared with activities in untreated controls. Hepatic aryl hydrocarbon hydroxylase (AHH) activity was increased 4-fold under these conditions. Pentoxyresorufin O-depentylase (PROD) activity (associated with CYP2B isoenzyme) was increased 6-fold in the liver but was unaffected in lung and small intestine. Intraperitoneal injection (IP) of I3C (500 mumol/kg body wt.) produced no significant change in EROD or PROD activities in lung, liver, or small intestine. PO administration of the acid reaction mixture (RXM) of I3C increased hepatic AHH activity (5-fold) and EROD activities in small intestine (650-fold), lung (100-fold) and liver (18-fold). IP administration of RXM (equivalent to 500 mumol I3C/kg body wt.) significantly increased only EROD activity in lung and liver, but did not affect EROD activity in small intestine, AHH activity in liver, or PROD activity in any of the organs examined. Twenty hours after inducer treatment, half of the rats were treated PO with 0.2 mumol [3H]BP in corn oil. Analysis of tissues 5 h after BP administration indicated that compared with untreated controls, administration of I3C and RXM by either route reduced by 30-50% the level of BP binding to hepatic DNA, an effect that was not correlated to CYP1A1 enzyme induction in any of the organs examined. However, PO administration of I3C and RXM produced a 50-70% decrease in carcinogen binding to pulmonary DNA, while IP administration of inducers had no effect on DNA binding in this organ. These results with the lung are consistent with an increased presystemic clearance of BP in the intestine and are discussed in terms of the role of induction of intestinal CYP1A1 activity in the decreased lymphatic and venous transport of unmetabolized BP to the lung.

Oligomerization of indole-3-carbinol in aqueous acid.

Indole-3-carbinol [I3C, also called 3-(hydroxymethyl)indole] is a naturally occurring modulator of carcinogenesis with a biological activity that is at least partially dependent on its conversion to active substances in acidic media. We compared the identities of the major oligomeric products of I3C produced under conditions approximating those found in gastric juice with the reported identities of products of 3-substituted indoles produced under enzymatic and other nonenzymatic conditions. After a 10-min treatment in aqueous HCl solution, I3C was converted in 18% yield to a mixture of acetonitrile-soluble products, the major components of which (as determined by HPLC) were diindol-3-ylmethane (5.9%), 5,6,11,12,17,18-hexahydrocyclononal[1,2-b:4,5-b':7,8-b"]triindo le (2.0%), and [2-(indol-3-ylmethyl)indol-3-yl]indol-3-ylmethane (5.9%). Tentative assignments were made for 3,3-bis(indol-3-ylmethyl)indolenine (0.59%), a symmetrical cyclic tetramer (0.64%), and a linear tetramer (1.1%). Indolo[3,2-b]carbazole (ICZ) was formed slowly in aqueous acidic solutions in low yields (2.0 ppm) which increased to greater than 90 ppm following addition of an organic solvent [tetrahydrofuran (THF) or dimethylformamide (DMF)] to a neutralized solution. Relative yields of trimers vs dimer increased with decreasing pH and with decreasing starting concentration of I3C. Evidence is presented that ICZ formation may not involve radical intermediates as is characteristic of photodynamic processes. A mechanistic rationale is presented for the formation of the identified products.

Aromatic hydrocarbon responsiveness-receptor agonists generated from indole-3-carbinol in vitro and in vivo: comparisons with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Indole-3-carbinol (I3C) is a secondary plant metabolite produced in vegetables of the Brassica genus, including cabbage, cauliflower, and brussels sprouts. I3C is both an anti-initiator and a promoter of carcinogenesis. Consumption of I3C by humans and rodents can lead to marked increases in activities of cytochrome P-450-dependent monooxygenases and in a variety of phase II drug-metabolizing enzymes. We have reported previously that the enzyme-inducing activity of I3C is mediated through a mechanism requiring exposure of the compound to the low-pH environment of the stomach. We report here the aromatic hydrocarbon responsiveness-receptor Kd values (22 nM-90 nM), determined with C57BL/6J mouse liver cytosol and the in vitro- and in vivo-molar yields (0.1-6%) of the major acid condensation products of I3C. We also show that indolo[3,2-b]carbazole (ICZ) is produced from I3C in yields on the order of 0.01% in vitro and, after oral intubation, in vivo. ICZ has a Kd of 190 pM for aromatic hydrocarbon responsiveness-receptor binding and an EC50 of 269 nM for induction of cytochrome P4501A1, as measured by ethoxyresorufin O-deethylase activity in murine hepatoma Hepa 1c1c7 cells. The binding affinity of ICZ is only a factor of 3.7 x 10(-2) lower than that of the highly toxic environmental contaminant and cancer promoter 2,3,7,8-tetrachlorodibenzo-p-dioxin. ICZ and related condensation products appear responsible for the enzyme-inducing effects of dietary I3C.