What is Diindolylmethane

Diindolylmethane also called DIM or 3,3′-diindolylmethane, is  a bioactive compound found in cruciferous vegetables like broccoli, brussels sprouts, cauliflower, cabbage, kale, bok choy, radishes, turnips, and kohlrabi, that is being studied in the treatment of prostate cancer and in the prevention of cervical cancer and breast cancer ¹, ². Thomson and colleagues ³ previously reported, from a secondary analysis, a 52% reduction in breast cancer recurrence among women consuming more cruciferous vegetables, they hypothesized to be attributed to higher exposure to indole-3-carbinol (I3C) and the related dimer, DIM diindolylmethane ⁴.

Diindolylmethane has been shown to regulate immune responses and exhibit a broad range of biological activities in several disease models, including various cancers ⁵ and inflammatory diseases such as multiple sclerosis ⁶, colitis ⁷ and arthritis ⁸. Moreover, diindolylmethane has shown potential effects against infections with enteric viruses ⁹ and Staphylococcus aureus ¹⁰. The immunomodulatory properties of diindolylmethane are associated with its ability to regulate multiple receptors ¹¹ and signaling pathways, such as JNK, p38, NF-ĸB, AP-1, and FAK ¹², suggesting that the anti-inflammatory properties of diindolylmethane are associated with the regulation of T-cell differentiation and inhibition of Th1/Th17 cells ¹³.

Cruciferous vegetable consumption may confer a protective effect against cancer, possibly attributable to their glucosinolates ¹⁴. Glucobrassicin is a predominant glucosinolate ¹⁵, is converted upon chewing of cruciferous vegetables into indole-3-carbinol (I3C), which then undergoes acid condensation in the stomach, predominantly to DIM (3,3′-diindolylmethane) a compound with anti-cancer effects ¹⁶.

DIM is readily detected in the livers and feces of rodents fed indole-3-carbinol (I3C), whereas the parent I3C compound has not been detected in tissues of these rodents ¹⁷. Thus, the natural effects of I3C (indole-3-carbinol) are attributable to diindolylmethane (DIM), which show evidence of anti-cancer activities in vivo (animal) and in vitro (test tube) by reducing the growth of prostate, colon, and breast cancer cells ¹⁸. I3C and its derivatives also suppress cell propagation and induce apoptosis in colon cancer cells ¹⁹, as well as in other types of cancer cells including prostate cancer ²⁰, breast cancer ¹⁸, bladder cancer ²¹, pancreatic cancer ²² and liver cancer ²³. However, a number of studies have found that I3C actually promoted or enhanced the development of cancer when administered chronically after the carcinogen ²⁴. The cancer promoting effects of I3C is first reported in a trout model of liver cancer ²⁴. However, I3C also has been found to promote cancer of the thyroid, liver, colon, and uterus in rats ²⁵. Although the long-term effects of I3C supplementation on cancer risk in humans are not known, but the contradictory results of animal studies have led some to caution against the widespread use of I3C and DIM supplements in humans until their potential risks and benefits are better understood ²⁵.

Reed et al. ²⁶ in his extensive article of pharmacokinetic studies reported that women received oral doses of 400, 600, 800, 1,000, and 1,200 mg I3C (indole-3-carbinol) and these serial plasma samples were analyzed by high-performance liquid chromatography-mass spectrometry method for the detection and quantitation of the I3C and diindolylmethane (DIM). I3C itself is not detectable in plasma ²⁶. The only detectable I3C-derived product is DIM. High initial value, plasma DIM for all subjects, decreased to near or below the limit of quantitation within the 12 hour sampling period ²⁶. Physiologically based pharmacokinetic model is developed using plasma and tissue (brain, heart, liver, kidneys, and lungs) concentration data for DIM to compare the pharmacokinetic properties and biodistribution of pure crystalline and a novel formulation (BioResponse-DIM) of DIM after oral administration to mice ²⁷. I3C is hard to develop as a drug because it is highly unstable and can transform into many other derivatives such as DIM, 5,6,11,12,17,18-hexahydrocyclonona[1,2-b:4,5-b′:7,8-b′′] triindole (CTr), indolo [3,2-b] carbazole (ICZ), N-methoxyindole-3-carbinol (NI3C), two tetramers, one linear (LTET) and one cyclic (CTET), and 1-(3-hydroxymethyl) indolylmethane (HI-IM). Anderton et al. ¹⁷ reported oral doses of I3C to female CD-1 mice, and studied the disposition of I3C and its acid condensation products diindolylmethane (DIM), ICZ, LTr(1), and HI-IM in blood, liver, kidney, lung, heart, and brain. I3C was rapidly absorbed, distributed, and eliminated from plasma and tissues, falling below the limit of detection by 1 hour. The major acid condensation product of I3C, diindolylmethane (DIM), has proven very stable in acidic conditions during prolonged exposure to high temperature and humidity ²⁸. Another report BioResponse-DIM has proven very stable and it is not converted into other forms ²⁸. The tissue distribution of I3C has been determined in mice by radio-labelled I3C ²⁹. Both I3C and DIM have been detected in kidneys, lungs, heart, liver, plasma, and brain samples of mice treated with 250 mg/kg of I3C as early as 15 mins after administration ²⁹. These results suggest that I3C is rapidly absorbed and spread to a number of well-perfused tissues, where it is transformed to diindolylmethane (DIM) to perform its anticancer actions ²⁹. Based on these outcomes researchers suggested that one could exploit further development of diindolylmethane (DIM) as a potential therapeutic agent ².

Figure 1. Diindolylmethane (DIM)

Figure 2. Indole-3-carbinol (I3C)

[Source ² ]

Diindolylmethane benefits

For more than 25 years, the interdependence between nutrition and the development and progression of cancer have been recognized ³⁰. The key challenge is to identify the specific components responsible for contributing to this relationship ³⁰. In the United States, cancers of the lungs, colon, rectum, breast, and prostate account for almost half of the total cancer incidence ³⁰. Cohen et al. ³¹ examined the association of fruit and vegetable intake and prostate cancer risk among newly diagnosed men residing in the Seattle, WA, area. With each 10 g of cruciferous vegetables consumed per day, one could expect an 8% decrease in risk for colorectal cancer.

Several recent case-control studies in the US, Sweden, and China found that measures of cruciferous vegetable intake are significantly lower in women diagnosed with breast cancer than in cancer-free control groups ³². High intake of cruciferous vegetables has been associated with lower risk of lung and colorectal cancer in some epidemiological studies, but there is evidence that genetic polymorphisms may influence the effectiveness of cruciferous vegetables on human cancer risk ³³. Epidemiological studies show that consumption of large quantities of fruits and vegetables, particularly cruciferous vegetables, is associated with a reduced occurrence of cancer ³³.

Studies in various experimental models have shown that indole-3-carbinol (I3C) can alter the metabolism of carcinogens and provide protection against chemically induced carcinogenesis ³⁴. When administered before or at the same time as the carcinogen, oral I3C has been originated to inhibit the spreading out of cancer in a variety of animal models and tissues, including cancers of breast cancer ¹⁸, colon cancer ¹⁹, stomach cancer ³⁴, lung cancer ²⁹ and liver cancer ²⁴. However, a number of studies have found that I3C actually promoted or enhanced the development of cancer when administered chronically after the carcinogen ²⁴.

The cancer promoting effects of I3C is first reported in a trout model of liver cancer ²⁴. However, I3C also has been found to promote cancer of the thyroid, liver, colon, and uterus in rats ²⁵. Although the long-term effects of I3C supplementation on cancer risk in humans are not known, but the contradictory results of animal studies have led some to caution against the widespread use of I3C and DIM supplements in humans until their potential risks and benefits are better understood ²⁵.

Other uses of diindolylmethane supplements, such as weight loss and acne ³⁵ treatment, have not been studied in humans.

Anticancer effects

DIM inhibits proliferation of human breast cancer cells at concentrations achievable through oral supplementation with I3C (10–50 μM) ³⁶. Recently a report by Fan et al. ³⁷ demonstrated that DIM protects cancer cells and normal epithelial cells against reactive oxygen species (ROS) in a breast cancer type 1 susceptibility protein (BRCA1-)dependent manner. Moreover I3C inhibits DMBA initiated and TPA promoted mouse skin tumor formation ³⁸. I3C also exhibits inhibitory and preventive effects on prostate tumors in mice ²⁰. Other investigators reported that this activity of I3C is associated with its action as a nonspecific inducer of powerful cytochrome enzymes responsible for “Phase I” detoxification metabolism, the efficiency of I3C to inhibit prostate tumor growth ³⁴. The anticancer effect of I3C is well proven including the reduction of cervical intraepithelial neoplasia (CIN) and its progression to cervical cancer ³⁹. It is perceived that the diminished phosphatase and tensin homolog protein (PTEN) expression is observed during the progression from low-grade to high-grade cervical dysplasia in humans and in a mouse model for cervical cancer, the K14HPV16 transgenic mice promoted with estrogen ³⁹. PTEN could impede tumor progression by inhibiting proliferation and by increasing tumor cell apoptosis ³⁹. This is supported by the previous findings that I3C decreased proliferating cell nuclear antigen- (PCNA-)positive cells and increased TdT-mediated dUTP nick-end labeling- (TUNEL-)positive cells in abnormal cervical epithelium of HPV16 mice ⁴⁰.

Diindolylmethane estrogen blocker

Breast cancer survivors frequently self-prescribe bioactive dietary supplements with the intention to obtain survival benefit ⁴¹. Of these, DIM or diindolylmethane (3,3′-diindolylmethane), a stable in vivo metabolite of indole-3-carbinol (I3C) found in cruciferous vegetables ⁴², is among the most well-studied ⁴³. Thomson et al. ⁴⁴ previously reported, from a secondary analysis, a 52% reduction in breast cancer recurrence among women consuming more cruciferous vegetables, hypothesized to be attributed to higher exposure to indole-3-carbinol (I3C) and the related dimer, diindolylmethane ⁴⁵.

Accumulating evidence favors several anti-tumor actions of diindolylmethane, including favorable changes in estrogen metabolism toward 2-hydroxylation of estrogen metabolism ⁴⁶; increasing the ratio of 2-hydroxyestrone (2-OHE1) (anti-tumorigenic) to 16α-hydroxyestrone (16α-OHE1) (pro-tumorigenic) estrogens ⁴⁷, a shift associated with lower breast cancer risk ⁴⁸. Support of anti-estrogenic effect of diindolylmethane are evident in thyroid disease ⁴⁹, cell culture studies demonstrating diminished estrogen receptor alpha (ERα) levels ⁵⁰ and activation of estrogen receptor beta (ERβ) target genes ⁵¹.

This randomized, controlled trial of diindolylmethane supplementation ⁴⁶ provided as BioResponse-DIM, found that diindolylmethane modulates estrogen metabolism through increased 2-hydroxylation of estrogen metabolism and, ultimately, increasing the ratio of this “favorable” metabolite to the “punitive” 16α-hydroxylated metabolite confirming the results in a pilot clinical trial using one third the diindolylmethane dose in breast cancer survivors not on tamoxifen. The present results support prior observations in 3 patients taking tamoxifen where the 2-hydroxyestrone (2-OHE1)/16α-hydroxyestrone (16α-OHE1) ratio increased 126–229% with a daily dose of 100 mg diindolylmethane from BioResponse-DIM for 30 days ⁵². Mechanistically, 2-hydroxyestrone (2-OHE1) (anti-tumorigenic) has been shown to bind ERα with high affinity, but lacks the ability to induce transcriptional activity resulting in the reported anti-estrogenic/anti-growth effects ⁵³. Higher urinary 2-hydroxyestrone/16α-hydroxyestrone ratio has been associated with a lower risk of breast cancer in most ⁵⁴, but not all studies ⁵⁵. While a higher 2-hydroxyestrone/16α-hydroxyestrone ratio has been hypothesized as being anti-tumorigenic, direct evidence that manipulating 2-hydroxyestrone/16α-hydroxyestrone ratio prevents breast cancer or improves outcomes are lacking ⁵⁶.

In this study ⁵⁶, diindolylmethane did not affect serum estrogen levels, but increased sex hormone-binding globulin (SHBG), exposures which have been extensively studied in relation to breast cancer risk. In an analysis of worldwide data, the Endogenous Hormones and Breast Cancer Collaborative Group ⁵⁷ confirmed that breast cancer risk increased with higher total Estradiol (E2), free E2, non-SHBG-bound E2, Estrone (E1), E1 sulfate, and testosterone, despite some contrary findings ⁵⁸. As in this study, low levels and the high variations in measures may limit interpretation. Sex hormone-binding globulin (SHBG) has been inversely associated with breast cancer risk ⁵⁹ with recent evidence suggesting a strong inverse association with breast cancer specific mortality ⁵⁸. As noted by Duggan et al ⁶⁰, sex hormone-binding globulin has been shown to induce apoptosis and inhibit growth of breast cancer cells via a receptor-mediated binding, suggesting that the effects of diindolylmethane on sex hormone-binding globulin may be beneficial to breast cancer outcomes independent of serum hormone or tamoxifen metabolite levels.

In this study ⁵⁶, diindolylmethane lowered plasma levels of all three major Phase I tamoxifen metabolites. The clinical significance of lowered levels of tamoxifen metabolites remains unclear. Endoxifen and 4-OH tamoxifen, which exhibit higher activity for estrogen receptor at the tissue level, have been postulated as the active agents in tamoxifen therapy. While early work in the field failed to demonstrate a direct relationship between tamoxifen dose and tissue Ki67 levels as a biomarker of effects on proliferation, subsequent work suggested that low circulating endoxifen levels may limit the efficacy of tamoxifen. For example, in breast cancer survivors on tamoxifen circulating endoxifen levels > 5.6 ng/ml (upper four quintiles) were associated with a marginally significant lower risk of recurrence (26%) ⁶¹. Despite study limitations, this evidence has promoted inclusion of the 5.6 ng/ml endoxifen cut-point as a putative therapeutic threshold of tamoxifen ⁶¹. Lower doses of tamoxifen, including 1 and 5 mg daily, have demonstrated similar antitumor effects to therapeutic dosing and challenge the validity of the endoxifen cut-point for determining efficacy ⁶². Furthermore, diindolylmethane did not result in unfavorable change in breast density or sex hormone-binding globulin (SHBG), exposures which have been associated with tamoxifen efficacy ⁶³.

Apoptosis

Apoptosis or programmed cell death is a highly regulated process that involves activation of a series of molecular events, leading to cell death that is characterized by cellular morphological change, chromatin condensation, and apoptotic bodies which are associated with DNA cleavage into ladders ⁶⁴. The nuclear factor kappa B (NF-κB) signaling plays critical roles in regulating cell proliferation, survival, tumor invasion, metastasis, drug resistance, and stress response ⁶⁵. Banerjee et al ⁶⁶ confirmed that NF-κB activity is significantly upregulated by docetaxel, gemcitabine or oxaliplatin treatment and that the NF-κB inducing activity of these agents was completely abrogated in cells pretreated with DIM. They found that diindolylmethane or the formulated BR-DIM treatment, could restrict its nuclear localization and inactivate NF-κB DNA-binding activity in prostate ⁶⁷, breast ⁶⁸ and pancreatic cancer cells ⁶⁶, resulting in the inhibition of transcriptional downregulation of several NF-κB downstream genes causing inhibition of cell growth and inducing apoptotic cell death. Collectively, these results clearly suggest that diindolylmethane pretreatment, which inactivates NF-κB activity, along with other cellular effects of DIM, may contribute to enhanced cell growth inhibition and apoptosis with suboptimal doses of cytotoxic chemotherapeutic agents with minimal side effects.

I3C trigger the stress-induced MAP-kinases p38 and C-jun N-terminal kinase (JNK) in prostate cancer cells and to inhibit constitutively active STAT3, a transcription factor, in pancreatic cancer cells ⁶⁹. Irrespective of the cell type I3C suppressed NF-κB activation induced by various agents ⁶⁹. NF-κB inhibition correlated with suppression of inhibitor of kappa B kinase (IKK) and IκBα phosphorylation, ubiquitination, degradation with p65 phosphorylation, nuclear translocation, and acetylation ⁷⁰. I3C also downregulated NF-κB α regulated reporter gene transcription and gene products involved in cell proliferation, antiapoptosis, and invasion ⁷⁰. This led to the potentiation of apoptosis induced by cytokines and chemotherapeutic agents ⁷⁰. Collectively, the concerted effects on those proapoptotic components underlie the ability of I3C or diindolylmethane to induce mitochondrial dependent apoptosis in tumor cells ⁷⁰.

Regulation of redox status

Reactive oxygen species (ROS) including hydrogen peroxide (H2O2) can cause different combinations of apoptosis, necrosis, and autophagy in a cell line dependent and stimulus-dependent manner ⁷¹. The capacity of I3C to form adducts with electrophiles or free radicals appears too autonomous on their chemical reactivity hence the scavenging ability of I3C is compatible with the adduct formation ⁷². Arnao et al. ⁷² investigate the ability of I3C to trap a metastable synthetic-free radical and inhibition of carcinogenesis. This induction may be produced by I3C itself and/or I3C derived polymerization products such as diindolylmethane and others ⁷³.

According to Benabadji et al. ⁷⁴, they reported that diindolylmethane and 6-methoxy-diindolylmethane in DPPH model, their IC50, were 50% and 40% smaller than that of vitamin E, due to their hydrogen-donating ability with the presence of two N–H groups as an H-donating group necessary to react with free radical and slightly less potent than the standard phenolic antioxidant BHA in β-carotene model with IC50 4% and 9% smaller for diindolylmethane and 6-methoxy-diindolylmethane.

Cell cycle arrest

Cell cycle arrest is defined as the halt of the cell cycle. I3C is reported to inhibit cyclin dependent kinase 2 (CDK2) kinase activities in MCF-7 cells through selective alterations in cyclin E composition, size distribution, and subcellular localization of the CDK2 protein complex ⁷⁵. Cell-cycle arrest involves the upregulation of the CDK inhibitors p21 WAF1 and p27 KIP1 and the concurrent downregulation of cyclin D1, cyclin E, and CDKs 2, 4, and 6 attributable to the effect of I3C and diindolylmethane on regulating SP1-promoter binding activity [19]. Inhibition of CDK4/6 cyclin D1 and CDK2 cyclin E activities led to decreased Rb phosphorylations, which cause the Rb protein to bind to the E2F transcription factor ⁷⁶. This E2F sequestration blocks the transcription of S phase genes resulting in G1 arrest; also the involvement of p53 and cell cycle arrest in the I3C-mediated effect has been studied in various cancer cells ⁷⁷.

Treatment of I3C betters the expression of p53 (Ser 15) and CDKIs such as p21 and p27, while cyclin D1 expression is suppressed and cyclin E is not altered ⁷⁸. Collectively, the data for the cell cycle analysis, the Plk-1 assay, and the western blot of p53 and CDKIs explain that I3C augments the expression of p53 and CDKIs and that I3C induced cell cycle arrest at G0/G1 in A549 cells ⁷⁸. In addition, cotreatment with I3C and wortmannin prevents both phosphorylation of p53 at Ser 15 and p21 expression. Hence it is clear that A549 cell arrest by I3C is involved in the PI3 K and p53 signal pathways ⁷⁸.

Angiogenesis

Angiogenesis is the physiological process through which new blood vessels form from preexisting vessels. This is distinct from vasculogenesis, which is the de novo formation of endothelial cells from mesoderm cell precursors ⁷⁹. Dysregulated angiogenesis that consists of the unbalanced production of pro- and antiangiogenic factors is linked to a number of pathological situations ⁸⁰. For example, the overexpression of angiogenic factors, including vascular endothelial growth factor (VEGF), IL-6, and matrix metalloproteinases (MMP-9) is closely associated with the development of cancers and metastasis [21]. The effect of I3C on LPS-activated macrophage-induced tube formation and its associated factors in endothelial EAhy926 cells are investigated ⁸⁰. LPS significantly enhanced the capillary-like structure of endothelial cells cocultured with macrophages, but no such effect was observed in single-cultured ECs ⁸⁰. I3C, on the other hand, suppressed such enhancement in concert with decreased secretions of VEGF, NO, IL-6, and MMPs ⁸⁰. The results obtained from cultivating endothelial cells with conditioned medium collected from macrophages suggested that both endothelial cells and macrophages were inactivated by I3C ⁸⁰.

Anti-inflammatory effect

The effect of diindolylmethane on inflammatory responses and its molecular mechanisms of diindolylmethane have been examined using lipopolysaccharide (LPS) stimulated RAW264.7 murine macrophages ⁸¹. Diindolylmethane inhibits LPS-induced increases in protein levels of inducible nitric oxide synthase (iNOS), which are accompanied by decreased iNOS mRNA levels and transcriptional activity ⁸¹. In addition, diindolylmethane suppresses LPS-induced NF-κB transcriptional and DNA-binding activity, translocation of p65 (RelA) to the nucleus, and degradation of inhibitor of kappa B alpha (IκBα) ⁸¹. Also the results of RT-PCR analysis exposed that LPS augmented the steady-state levels of proinflammatory cytokines such as TNF-α, IL-1β, PLA2, and interleukin-6 (IL-6) transcripts, which are substantially suppressed by diindolylmethane treatment ⁸¹. Diindolylmethane pretreatment significantly repressed LPS-induced phosphorylation of SAPK/JNK, whereas the phosphorylation of other MAPK family proteins (p38 or ERK-1/2) is unaltered by diindolylmethane pretreatment ⁸². Consequently, the use of certain anti-inflammatory agents, such as indole-3-carbinol (I3C) and 3,3’-diindolylmethane (DIM), could be beneficial for the treatment of autoimmune diseases ⁶.

Detoxification

Detoxification is the physiological or medicinal removal of toxic substances from living organisms ⁸³. Both the Phase I and Phase II detoxification centers in the liver and the intestinal epithelial cells can be accelerated by some minor exogenous agent like I3C ⁸³. Many researchers indicate that the ability of cruciferous vegetables to motivate Phase I and Phase II detoxification, particularly their I3C content, is a primary factor in which these nutrients are related to reduce cancer risk in humans ⁸⁴. Animals are exposed to or injected with carcinogens; the animals receiving the cruciferous vegetables or the I3C in their food supply have a significantly lower tumor incidence than the animals fed the same diet, but without cruciferous vegetables or I3C fortification ⁸⁴.

Diindolylmethane dosage

Due to limited research, proper dosages for DIM or diindolylmethane are unknown. In human research, doses typically range from 108–900 mg per day — though these studies were only related to treatments for cancer and prostate enlargement ⁸⁵, ⁸⁶.

However, a study in 24 healthy people ⁸⁷ found that although diindolylmethane doses of up to 200 mg were well tolerated and didn’t cause side effects, one person experienced nausea, headache, and vomiting after taking a 300-mg dose, suggesting that higher doses may be associated with adverse side effects. Therefore, it’s best to talk to your healthcare provider to obtain personalized dosage recommendations based on your intended use.

Diindolylmethane side effects

Due to a lack of research in humans, little is known about the long-term safety and side effects of diindolylmethane supplements. Current human research doesn’t show diindolylmethane supplements to be toxic or have serious side effects. The most common side effects include darkening of the urine, an increase in bowel movements, headaches, and gas ⁸⁵. Less common side effects include nausea, vomiting, diarrhea, and skin rash ⁸⁸.

As diindolylmethane supplements interact with estrogen levels, they may affect people with hormone-sensitive cancers or who are on hormone therapies. Such individuals should steer clear of diindolylmethane supplements unless under the supervision of a medical professional. No matter your medical history, it’s important to consult your healthcare provider before taking diindolylmethane supplement.

Diindolylmethane summary

Diindolylmethane is a compound found in cruciferous (Brassica) vegetables. These vegetables include cabbage, cauliflower, Brussels sprouts, kale, turnips, and broccoli ⁸⁹. Limited studies suggest that diindolylmethane supplements may help reduce prostate enlargement, prostatic intraepithelial neoplasia (PIN) ⁸⁶ and protect against certain cancers. However, diindolylmethane’s effectiveness for other hormone-related conditions hasn’t been widely studied.

DIM supplements have not been shown to cause serious side effects, though more safety research is needed. People on hormone therapies or with certain hormone-related cancers should avoid diindolylmethane supplement.

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