Diindolylmethane Research Articles

Rapid measurement of indole levels in Brassica vegetables using one millilitre binary organic extraction solvent and capillary electrophoresis-UV analysis.

Brassica vegetables contain high levels of indole compounds which have been found to provide health benefits, especially as cancer-preventive agents. An efficient and rapid method using solvent extraction with capillary electrophoresis (CE) and ultraviolet (UV) detection was developed for the determination of four major indoles from four types of Brassica vegetables. Freeze-dried samples of four Brassica vegetables, i.e. broccoli, cauliflower, Chinese cabbage and cabbage, were selected. Hence, 1 mL of the binary solvent dimethylformamide (DMF)-methanol, 4:1 (v/v), was used for sample extraction. The extracts were diluted with the running buffer and directly analysed using CE with UV detection of four indole compounds. The binary solvent DMF-methanol, 4:1 (v/v) was selected from studies of the extraction efficiency of standard indoles spiked in ivy gourd (as the negative control sample) and using diphenylamine as the internal standard. Recovery was 80(±10)-120(±3)% for the four indoles: indole-3-carbinol (I3C), indole-3-acetonitrile (I3A), indole-3-acetic acid (IAA), and 3,3'-diindolylmethane (DIM). For direct analysis suitable dilution of the extract with the running buffer was required. The linear range of the quantitation is 0.75-25.0 μg/mL, limit of detection (LOD) of 0.14-0.52 μg/mL and r2 > 0.998. The amount of indole in the Brassica vegetables are in the order I3C > > IAA, I3A > DIM. A rapid method for extraction and quantitation of four indoles in four Brassica vegetables using CE with UV detection was developed. It has the potential as an efficient technique for generating data for use in agricultural and nutritional studies.

3,3'-Diindolylmethane and its derivatives: nature-inspired strategies tackling drug resistant tumors by regulation of signal transduction, transcription factors and microRNAs.

Indoles of cruciferous vegetables are promising anti-tumor agents. Studies with indole-3-carbinol and its dimeric product, 3,3’-diindolylmethane (DIM), suggest that these compounds have the ability to deregulate multiple cellular signaling pathways that are essential for tumor growth and spread. These natural compounds are also effective modulators of transcription factors and non-coding RNAs. These effects explain their ability to inhibit tumor spread and to overcome drug resistance. In this work, pertinent literature on the effects of DIM and its synthetic derivatives on resistant tumors and resistance mechanisms in tumors is highlighted.

3,3'-Diindolylmethane Promotes BDNF and Antioxidant Enzyme Formation via TrkB/Akt Pathway Activation for Neuroprotection against Oxidative Stress-Induced Apoptosis in Hippocampal Neuronal Cells.

3,3′-Diindolylmethane (DIM), a metabolite of indole-3-carbinol present in Brassicaceae vegetables, possesses various health-promoting effects. Nonetheless, the effect of DIM on neurodegenerative diseases has not been elucidated clearly. In this study, we hypothesized DIM may protect neuronal cells against oxidative stress-induced apoptosis by promoting the formation of brain-derived neurotrophic factor (BDNF) and antioxidant enzymes through stabilizing the activation of the tropomyosin-related kinase receptor B (TrkB) cascade and we investigated the effect of DIM on oxidative stress-mediated neurodegenerative models. DIM protected neuronal cells against oxidative stress-induced apoptosis by regulating the expression of apoptosis-related proteins in glutamate-treated HT-22 cells. Additionally, DIM improved the expression of BDNF and antioxidant enzymes, such as heme oxygenase-1, glutamate-cysteine ligase catalytic subunit, and NAD(P)H quinine oxidoreductase-1, by promoting the activation of the TrkB/protein kinase B (Akt) pathway in the cells. Consistent with in vitro studies, DIM attenuated memory impairment by protecting hippocampal neuronal cells against oxidative damage in scopolamine-treated mice. Conclusionally, DIM exerted neuroprotective and antioxidant actions through the activation of both BDNF production and antioxidant enzyme formation in accordance with the TrkB/Akt pathway in neuronal cells. Such an effect of DIM may provide information for the application of DIM in the prevention of and therapy for neurodegenerative diseases.

Amelioration of Hyperglycemia-Induced Nephropathy by 3,3'-Diindolylmethane in Diabetic Mice.

Type 1 diabetes mellitus (insulin-dependent diabetes) is characterized by hyperglycemia caused by an insulin deficiency. Diabetic nephropathy is a major complication of hyperglycemia. 3,3′-diindolylmethane (DIM)-a natural compound produced from indole-3-carbinol, found in cruciferous vegetables-enhances glucose uptake by increasing the activation of the insulin signaling pathway in 3T3-L1 adipocytes. In this study, we investigated whether DIM could improve insulin-dependent diabetes and nephropathy in streptozotocin (STZ)-induced diabetic mice. In mice, STZ induced hyperglycemia, hunger, thirst, and abnormally increased kidney weight and serum creatinine, which is a renal functional parameter. DIM decreased STZ-increased high blood glucose levels and food and water intake in diabetic mice. DIM also improved diabetic nephropathy by inhibiting the expression of PKC-α, the marker of albuminuria, and TGF-β1, an indicator of renal hypertrophy, in diabetic mice. Our findings suggest that DIM may ameliorate hyperglycemia and diabetic nephropathy through the inhibition of PKC-α and TGF-β1 signaling.

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans.

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a nonmammalian animal model that is widely used as an assay system due to its short lifespan, transparency, cost-effectiveness, and lack of animal ethics issues. In this study, a method was developed to investigate the effects of different bacteria and 3,3'-diindolylmethane (DIM) on the intestinal permeability of C. elegans with a high-throughput image analysis system. The worms were infected with different gut bacteria or cotreated with DIM for 48 h and fed with fluorescein isothiocyanate (FITC)-dextran overnight. Then, the intestinal permeability was examined by comparing the fluorescence images and the fluorescence intensity inside the worm bodies. This method may also have the potential to identify probiotic and pathogenic intestinal bacteria that affect intestinal permeability in the animal model and is effective for examining the effects of harmful or health-promoting chemicals on intestinal permeability and intestinal health. However, this protocol also has some considerable limitations at the genetic level, especially for determining which genes are altered to control illness, because this method is mostly used for phenotypic determination. In addition, this method is limited to determining exactly which pathogenic substrates cause inflammation or increase the permeability of the worms' intestines during infection. Therefore, further in-depth studies, including investigation of the molecular genetic mechanism using mutant bacteria and nematodes as well as chemical component analysis of bacteria, are required to fully evaluate the function of bacteria and chemicals in determining intestinal permeability.

Anti-Cancer Effects of 3, 3'-Diindolylmethane on Human Hepatocellular Carcinoma Cells Is Enhanced by Calcium Ionophore: The Role of Cytosolic Ca2+ and p38 MAPK.

Purpose: 3,3′-Diindolylmethane (DIM), derived from indole-3-carbinol (I3C) in the Brassica species of cruciferous vegetables, has anticancer effects, but its exact underlying mechanism of action is unknown. We explored the roles of cytosolic free calcium ([Ca2+]i) and p38 MAPK in the anti-cancer effects of DIM in human hepatocellular carcinoma cells. Methods: Cell proliferation was measured with a Cell Counting Kit-8 (CCK-8) and the clonogenic formation assay. Cell apoptosis was examined by flow cytometric analysis and Hoechst dye staining. Cleaved-caspase3, cleaved-PARP, Bax, total, and phosphorylated p38 MAPK were assayed by western blotting. [Ca2+]i was measured with Fluo-3/AM by fluorescence microscopy. A23187, a calcium ionophore, was used to increase [Ca2+]i levels. Results: DIM inhibited cell proliferation in both SMMC-7721 and HepG2 cells in a concentration- and time-dependent manner. DIM also enhanced phosphorylation of p38 MAPK (p-p38), which was attenuated by SB203580. The proliferation inhibition and apoptosis induction by DIM were also blunted. In addition, DIM increased [Ca2+]i in HCC cells, and this effect was inhibited by the calcium chelator, BAPTA-AM, resulting in reduced p-p38 MAPK activation and apoptosis in DIM-treated cells, though the proliferation inhibition by DIM was unchanged. However, the DIM-induced cell proliferation inhibition and apoptosis were significantly enhanced by A23187, a selective calcium ionophore, which was attributed to exaggerated p-p38 MAPK. Conclusions: The calcium ionophore enhanced DIM-induced anti-cancer effects in hepatocellular carcinoma cells, secondary to [Ca2+]i-dependent activation of p38 MAPK. Treatment with a combination of DIM and calcium ionophore may offer a new approach to enhance the chemotherapeutic efficacy in liver cancer.

Preparation and Characterization of Whey Protein Isolate-DIM Nanoparticles.

3,3’-Diindolylmethane (DIM) is a bioactive compound found in Cruciferous vegetables that possesses health benefits such as antioxidant, anticancer, and anti-inflammatory effects. However, hydrophobicity and photolabile limit its pharmaceutical applications. This study aims to prepare and characterize DIM-encapsulated whey protein isolate (WPI) nanoparticles mixed at different ratios of WPI and DIM using the combined heating–ultrasound method. Results showed that all the samples showed adequate physicochemical characteristics: The mean particle size of the nanoparticles could be controlled down to 96–157 nm depending on the DIM to WPI ratio used in the preparation with a low polydispersity index (<0.5), higher negative values of zeta potential (>−40 mV) as well as with greater encapsulation efficiency (>82%). Flow behavior indices showed the shear-thinning Non-Newtonian or pseudoplastic (n < 1) behavior of the nanoparticles. The thermal properties were characterized by differential scanning calorimetry (DSC), which showed that DIM was successfully entrapped in WPI nanoparticles. The secondary structure of WPI was changed after DIM incorporation; electrostatic interaction and hydrogen bonding were major facilitating forces for nanoparticles formation, confirmed by Fourier Transform Infrared Spectroscopy (FT-IR). Transmission electron microscopy (TEM) micrographs showed that all the samples had a smooth surface and spherical structure. The wall material (WPI) and encapsulation method provide effective protection to DIM against UV light and a broad range of physiologically relevant pH’s (2.5, 3.5, 4.5, 5.5, and 7). In conclusion, whey protein isolate (WPI)-based nanoparticles are a promising approach to encapsulate DIM and overcome its physicochemical limitations with improved stability.

3,3'-Diindolylmethane Improves Intestinal Permeability Dysfunction in Cultured Human Intestinal Cells and the Model Animal Caenorhabditis elegans.

3,3'-Diindolylmethane (DIM), a digestive metabolite originating from cruciferous vegetables, has dietary potential for the treatment of various human intestinal diseases. Although intestinal permeability dysfunction is closely related to the initiation and progression of human intestinal inflammatory diseases (IBDs), the effect of DIM on intestinal permeability is unclear. We evaluated the effect of DIM on the intestinal permeability of human intestinal cell monolayers and the animal model Caenorhabditis elegans, which were treated with IL-1β and Pseudomonas aeruginosa, respectively, to mimic IBD conditions. DIM substantially restored the intestinal permeability of differentiated Caco-2 cells by enhancing the expression of tight junction proteins (including occludin and ZO-1). Compared to the IL-1β single treatment (551.0 ± 49.0 Ω·cm2), DIM (10 μM) significantly increased the transepithelial electrical resistance (TEER) of Caco-2 cell monolayers (919.0 ± 66.4 Ω·cm2, p < 0.001). DIM also ameliorated the impaired intestinal permeability and extended the lifespan of C. elegans fed P. aeruginosa. The mean lifespan of DIM-treated worms (10.8 ± 1.3 days) was higher than that of control-treated worms (9.7 ± 1.1 days, p < 0.01). Thus, DIM is a potential nutraceutical candidate for the treatment of leaky gut syndrome by improving intestinal permeability.

Abstract 3739: Characterization of the effects of DIM (3,3’-Diindolylmethane) on irradiated tumors and anti-tumor immunity

Abstract The overall goal of this project is to determine the efficacy of DIM (3,3’-Diindolylmethane) to protect normal tissues during radiotherapy but not enhance the survival of irradiated tumors. We tested expression change of p53-downstream genes in the absence and presence of DIM after radiation exposure using hTERT-immortalized human mammary epithelial cells. Radiation-induced gene inductions for both p21 and Wip1 were found attenuated in cells pretreated with various concentrations of DIM compared to the vehicle control. To reveal DIM’s effects on the global transcriptome of irradiated cells, we performed whole genome gene expression microarray analysis. Pathway analysis results showed that the differentially expressed genes are involved in cell cycle, p53 network, DDR, and related pathways. Gene regulation pattern at the later post-radiation time point in the presence of DIM shows similarity with that of early time point with no DIM, which suggests that DIM treatment delays responses of cell-cycle-related genes. We monitored tumor growth after localized radiation in tumor xenograft models of MDA-MB-231 and HCC1937 human breast carcinoma cell lines using immunocompromised NSG mice. The fractionated regimen consists of a total dose of 24 Gy in four fractions (6 Gy per fraction given every other day) with administration of DIM or vehicle at one hour prior to each fraction. DIM showed no significant effect on the growth of irradiated tumors, i.e. DIM did not affect the treatment efficacy of radiation on tumors. To investigate effects of DIM on tumor microenvironment, we treated tumors with fractioned radiation in an E0771 syngeneic tumor model. DIM treatment enhanced tumor infiltration of lymphocytes population. These studies support the goal of developing DIM as a clinical radioprotector in order to improve the therapeutic index by allowing higher doses of radiation to improve local tumor control by reducing late dose-limiting normal tissue toxicity by radiation. Citation Format: Renxiang Chen, Lijun Li, Lorreta Yun-Tien Lin, Albert J. Fornace, Heng-Hong Li. Characterization of the effects of DIM (3,3’-Diindolylmethane) on irradiated tumors and anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3739.

Abstract 16: 3,3'-diindolylmethane induces cytoglobin expression and synergizes with poly ADP ribose polymerase inhibitor PJ34 to inhibit triple negative breast cancer cell growth

Abstract Triple negative breast cancer (TNBC), characterized by tumors that lack expression of the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), carries a poor prognosis. While recently approved poly ADP ribose polymerase (PARP) inhibitor olaparib shows favorable activity in patients with TNBC harboring tumors deficient in DNA repair enzyme BRCA1, patients with TNBC possessing BRCA1-proficient tumors are largely unresponsive to olaparib. Emerging evidence suggests that small molecules that activate aryl hydrocarbon receptor (AhR) signaling have the capacity to confer anticancer actions. 3,3′-diindolylmethane (DIM), an AhR ligand, is the major metabolite of indole-3-carbinol (I3C) found in cruciferous vegetables such as broccoli. Patients often consume natural product-derived AhR ligands while undergoing chemotherapy. It is therefore crucial to enhance our understanding of nutraceutical-pharmaceutical interactions. We previously demonstrated the ability of synthetic AhR ligands to induce the expression of putative tumor suppressor cytoglobin (CYGB) and inhibit Akt signaling in breast cancer cells. Since PARP inhibitor PJ34 has been shown to synergize with Akt inhibitors we hypothesize that DIM induces CYGB expression and synergizes with PJ34 to inhibit TNBC cell growth. Using the Alamar Blue assay, we found that DIM substantially enhanced the sensitivity of TNBC BRCA1 proficient cells to PJ34, though knockdown of CYGB did not significantly influence the responsiveness of cells to DIM. Chromatin immunoprecipitation followed by next-generation sequencing data revealed that DIM promoted the binding of AhR to the CYGB promoter in breast cancer cells. Finally, DIM induced CYGB gene expression in TNBC cells as determined by quantitative PCR. Taken together, our data suggest that DIM upregulates CYGB in TNBC cells and synergizes with PJ34 to confer anticancer actions in TNBC. Our data provide a rationale for incorporating the use of natural product-derived AhR ligands as a strategy to enhance PARP inhibitor efficacy in patients with TNBC that have BRCA1-proficient tumors. Citation Format: Jonathan V. Wooten, Nicole Mavingire, Leah Rowland, Jason Matthews, Eileen Brantley. 3,3'-diindolylmethane induces cytoglobin expression and synergizes with poly ADP ribose polymerase inhibitor PJ34 to inhibit triple negative breast cancer cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 16.

Abstract 1304: Aryl hydrocarbon receptor ligands 5F 203 and 3,3'-Diindolylmethane disrupt mammospheres derived from MCF-7 cells and induce tumor suppressor miR125b-2 expression

Abstract While anti-estrogen tamoxifen (Tam) effectively treats many patients with estrogen receptor positive (ER+) tumors, up to 40% experience relapse following resistance. Breast cancer stem cells (CSCs) within tumors greatly contribute to tamoxifen resistance (TamR) and exhibit unique molecular signatures that drive metastasis and promote relapse. Tumor suppressor miRNAs aid in suppressing breast cancer progression. We have previously shown that aryl hydrocarbon receptor (AhR)-ligand Aminoflavone disrupts the formation of spheres and inhibits the expression of putative stemness marker α6-integrin and α6-integrin-src-Akt signaling. We hypothesize that two AhR ligands, 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) and 3,3'-Diindolylmethane (DIM), exhibit anticancer properties in MCF-7 breast cancer cells by targeting the CSC population in an AhR-dependent fashion. We found that DIM and 5F 203 disrupted mammospheres derived from MCF-7 cells but demonstrated a reduced capacity to do so in mammospheres derived from AHR100 cells (MCF-7 variants that are AhR-unresponsive). Wound healing and colony forming assays respectively revealed that 5F 203 and DIM also decreased cell migration and cell proliferation in MCF-7 cells and to a much lesser extent in AhR100 cells. 5F 203 and DIM induced miR125b-2 expression and suppressed the expression of stemness-regulating genes such as α6-integrin, a predicted miR125b-2 target. The reduction in stemness-gene expression in MCF-7 cells was attenuated following pretreatment with AhR antagonist CH223191. These data suggest that AhR ligands such as DIM and 5F 203 confer their anticancer actions including those against breast CSCs in an AhR-dependent manner. Our data is expected to provide a rationale for the development of anticancer AhR ligands designed to combat ER+ breast cancer and decrease the risk of relapse. Citation Format: Eileen Brantley, Nicole Mavingire, Jonathan Wooten, Petreena Campbell. Aryl hydrocarbon receptor ligands 5F 203 and 3,3'-Diindolylmethane disrupt mammospheres derived from MCF-7 cells and induce tumor suppressor miR125b-2 expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1304.

Interactions between β-Lactoglobulin and 3,3'-Diindolylmethane in Model System.

The compound 3,3′-diindolylmethane (DIM) has a broad spectrum of anticancer activities. However, low stability and bioavailability limit its application. Elucidating interactions between DIM and β-lactoglobulin (β-LG) may be useful for fabricating whey protein-based protecting systems. Interaction with DIM increased the diameter and absolute zeta potential value of β-LG. UV-absorption spectra suggested that there was a complex of DIM and β-LG. β-LG showed enhanced fluorescence intensity by complexing with DIM with a binding constant of 6.7 × 105 M−1. Upon interaction with DIM, β-LG was decreased in secondary structure content of helix and turn while increased in β-sheet and unordered. FT-IR spectra and molecular docking results indicated the roles of hydrophobic interaction and hydrogen bond for the formation of DIM and β-LG nanocomplexes. Data suggested that β-LG may be a good vehicle for making a protein-based DIM protection and delivery system due to the tight binding of DIM to β-LG.

3,3′-Diindolylmethane nanoencapsulation improves its antinociceptive action: Physicochemical and behavioral studies

This study aimed to characterize the physicochemical properties of 3,3′-diindolylmethane (DIM)-loaded nanocapsules (NCs) as well as the antinociceptive effect using distinct animal models (hot plate test, formalin-induced nociception and complete Freud’s adjuvant induced paw inflammation). The DIM-loaded NCs (composed by primula oil and ethylcellulose) were characterized using differential scanning calorimetry, thermogravimetric analysis, Fourier-transformed infrared spectroscopy, X-ray diffractometry and scanning electron microscopy. The physicochemical characterization demonstrated that DIM could be molecularly dispersed into the NCs, whose size was nanometric with a spherical shape. An improvement in DIM thermal stability was achieved by its encapsulation and there were no interactions among the formula components. For the nociceptive evaluation, male adult Swiss mice were pretreated with the NCs or free DIM by the intragastric route at the dose of 10 mg/Kg (time-response curve), 5 or 2.5 mg/Kg (dose-response curve). The behavioral tests were performed over an experimental period of 0.5–8 h. Both free and nanoencapsulated DIM reduced the mechanical hypernociception induced by CFA, mitigated nociceptive behavior of formalin-induced neurogenic and inflammatory pain and increased paw withdrawal latency assessed by the hot-plate test. Importantly, the DIM nanoencapsulation promoted a rapid initiation and prolonged the bioactive antinociceptive action (up to 8 h) as well as reduced the effective dose in comparison to its free form. In summary, this study reported that the NCs had adequate nanometric size, increased DIM stability and its antinociceptive action in different animal models, suggesting that the formulation may be a possible therapeutic alternative to the management of pain and inflammatory-related pathologies.

Strain-specific altered nicotine metabolism in 3,3'-diindolylmethane (DIM) exposed mice.

Two indole compounds, indole-3-carbinol (I3C) and its acid condensation product, 3,3'-diindolymethane (DIM), have been shown to suppress the expression of flavin-containing monooxygenases (FMO) and to induce some hepatic cytochrome P450s (CYPs) in rats. In liver microsomes prepared from rats fed I3C or DIM, FMO-mediated nicotine N-oxygenation was decreased, whereas CYP-mediated nicotine metabolism to nicotine iminium and subsequently to cotinine was unchanged. Therefore, it was hypothesized that in mice DIM would also suppress nicotine N-oxygenation without affecting CYP-mediated nicotine metabolism. Liver microsomes were produced from male and female C57BL/6J and CD1 mice fed 2500 parts per million (ppm) DIM for 14days. In liver microsomes from DIM-fed mice, FMO-mediated nicotine N-oxygenation did not differ from the controls, but CYP-mediated nicotine metabolism was significantly increased, with results varying by sex and strain. To confirm the effects of DIM in vivo, control and DIM-fed CD1 male mice were injected subcutaneously with nicotine, and the plasma concentrations of nicotine, cotinine and nicotine-N-oxide were measured over 30minutes. The DIM-fed mice showed greater cotinine concentrations compared with the controls 10minutes following injection. It is concluded that the effects of DIM on nicotine metabolism in vitro and in vivo differ between mice and rats and between mouse strains, and that DIM is an effective inducer of CYP-mediated nicotine metabolism in commonly studied mouse strains.

3,3′-Diindolylmethane Inhibits TNF-α- and TGF-β-Induced Epithelial–Mesenchymal Transition in Breast Cancer Cells

Epithelial–mesenchymal transition (EMT) is the initial event required by cancer cells. Thus, inhibition of the EMT process could have potential benefits for preventing the spread of cancers. The phytochemicals have been reported to have inhibitory activity against the EMT process in breast cancers, but the mechanism behind this effect has not been fully elucidated.3,3′-Diindolylmethane (DIM) is a major indole derived from bioactive compounds in cruciferous vegetables. In this study, we examined the effects of DIM cotreatment together with TNF-α/TGF-β on the EMT process as well as the mechanisms underlying its effects on human breast cancer cells. DIM significantly enhanced the mRNA and protein expression of E-cadherin and occludin in MCF-7 cells. The protein expression levels of E-cadherin and occludin in MCF-7 cells were significantly decreased after TNF-α/TGF-β treatment alone, but these effects were reversed by the DIM co-treatment. Furthermore, DIM with TNF-α/TGF-β co-treatment attenuated the phosphorylation of Smad2/3 and ERK1/2 proteins. DIM significantly inhibited the TNF-α/TGF-β-induced migration of breast cancer cells. Taken together, the results indicated that DIM effectively suppressed EMT processes through the inhibition of TNF-α/TGF-β-associated signaling pathways in breast cancer cells. Thus, DIM may be a novel preventive and/or therapeutic approach for the treatment of breast cancers.

3,3'‑Diindolylmethane mitigates lipopolysaccharide‑induced acute kidney injury in mice by inhibiting NOX‑mediated oxidative stress and the apoptosis of renal tubular epithelial cells.

3,3′-Diindolylmethane (DIM) is a naturally derived indole compound found in the Brassica family of vegetables. DIM has several beneficial effects, including anti-cancer, anti-inflammatory and anti-angiogenic functions. However, the effects of DIM on acute kidney injury (AKI) stimulated by lipopolysaccharide (LPS) are poorly studied. In this present study, male BALB/c mouse models of AKI were established using intraperitoneal injections of 10 mg/kg LPS. DIM (40 mg/kg) was administered intraperitoneally 24 and 2 h before LPS exposure. The results indicated that DIM significantly mitigated histopathological changes in the kidneys and improved the levels of blood urea nitrogen and serum creatinine. DIM also suppressed the LPS-induced production of reactive oxygen species and cell apoptosis. Furthermore, DIM treatment significantly decreased the expression of NADPH oxidase 2 (NOX2) and NOX4 in LPS-treated mice. Therefore, DIM may exert its renoprotective actions by inhibiting NOX-mediated oxidative stress and the apoptosis of renal tubular epithelial cells.

Indole Treatment Alleviates Intestinal Tissue Damage Induced by Chicken Coccidiosis Through Activation of the Aryl Hydrocarbon Receptor.

Indoles, as the ligands of aryl hydrocarbon receptor (AhR), have been shown to possess immune-modulating property in terms of the balancing between regulatory T cells (Treg) and T helper 17 cells (Th17) activities. In the present study, we examined the effects of dietary indoles, 3,3′-diindolylmethane (DIM) and indole-3-carbinol (I3C), on CD4+T cell population and functions in chickens. Furthermore, the effects of dietary DIM treatment on chicken coccidiosis caused by an apicomplexan parasite were investigated. Dietary treatment of healthy chickens with DIM and I3C induced increased CD4+CD25+ (Treg) cells and the mRNA expression of IL-10, while decreasing number of CD4+IL-17A+ (Th17) cells and Th17-related cytokines transcripts expression in the intestine. In addition, we explored the role of AhR in indole-treated splenic lymphocytes by using AhR antagonist and our results suggested that DIM is a ligand for chicken AhR. In chicken coccidiosis, treatment of DIM increased the ratio of Treg/Th17 cells and significantly reduced intestinal lesion although no significant changes in body weight and fecal oocyst production were noted compared to non-treated control group. These results indicate that DIM is likely to affect the ratios of Treg/Th17 reducing the level of local inflammatory response induced by Eimeria or facilitate repairing process of inflamed gut following Eimeria infection. The results described herein are thus consistent with the concept that AhR ligand modulates the T cell immunity through the alteration of Treg/Th17 cells with Treg dominance. To our knowledge, present study is the first scientific report showing the effects of dietary indole on T cell immunity in poultry species.

Physicochemical and Microstructural Properties of Polymerized Whey Protein Encapsulated 3,3'-Diindolylmethane Nanoparticles.

The fat-soluble antioxidant 3,3′-diindolylmethane (DIM), is a natural phytochemical found in Brassica vegetables, such as cabbage, broccoli, and Brussels sprouts. The stability of this compound is a major challenge for its applications. Polymerized whey protein (PWP)-based DIM nanoparticles were prepared at different mass ratios of protein and DIM by mixing PWP and DIM followed by ultrasound treatment for 4 min. All the nanoparticles were studied for particle size, zeta potential, rheological and microstructural properties, and storage stability. The mean particle size of the PWP-based nanoparticles was significantly increased (p < 0.05) by the addition of DIM at different mass ratios, ranging from 241.33 ± 14.82 to 270.57 ± 15.28 nm. Zeta potential values of all nanoparticles were highly negative (greater than ±30 mV), suggesting a stable solution due its electrostatic repulsive forces. All samples exhibited shear thinning behavior (n < 1), fitted with Sisko model (R2 > 0.997). Fourier Transform Infrared (FTIR)spectra revealed that the secondary structure was changed and the absorption intensity for hydrogen bonding got stronger by further incorporating DIM into PWP. Transmission electronic microscopy (TEM) images showed spherical and smooth surface shape of the PWP-based nanoparticles. DIM encapsulated by PWP showed enhanced stability at 4, 37 and 55 °C for 15 days evidenced by changes in mean particle size and color (a*-value and b*-value) compared with control (DIM only). In conclusion, the polymerized whey protein based 3,3′-diindolylmethane nanoparticles are stable and the encapsulation may protect the core material from oxidation.

Pharmacokinetic evaluation of medicinally important synthetic N,N′ diindolylmethane glucoside: Improved synthesis and metabolic stability

An improved route for the synthesis of N,N′-diindolyl methane (DIM) glycosides has been developed by using Fe/Al pillared clay catalyst. In-silico pharmacokinetics followed by in-vitro studies like aqueous solubility, lipophilicity, P-glycoprotein (P-gp) dependent ATPase activity, permeability, plasma protein binding, RBC partitioning, metabolic stability in different liver microsomes and its in-vitro-in-vivo extrapolation were conducted for the most potent derivative namely NGD16. The compound was found to have low solubility, optimum lipophilicity, no P-gp inhibitory activity, intermediate permeability, high plasma protein binding, low RBC partitioning, acceptable metabolic stability in rat liver microsomes (RLM) as well as human liver microsomes (HLM) with intermediate hepatic extraction ratio.

3, 3'-Diindolylmethane-encapsulated chitosan nanoparticles accelerate molecular events during chemical carcinogen-induced mammary cancer in Sprague Dawley rats.

3, 3'-Diindolylmethane (DIM) is a dietary indole compound; its medical application was limited because of poor bioavailability, unsatisfying dispersity, and rapid metabolism. To conquer this problem, nanoformulation of DIM was synthesized and investigated its mechanism-based chemotherapeutic potential. 7,12-Dimethylbenz(a)anthracene (DMBA) 25mg/kg b.wt initiated mammary carcinogenesis in rats, the investigational tumor model that closely resembles human mammary cancer. Rats had accessed after 8 weeks of tumor formation, DIM 10mg/kg b.wt. and DIM@CS-NP 0.5mg/kg b.wt. were administrated orally for 8 weeks. The treatment with DIM@CS-NP 0.5mg/kg b.wt. on DMBA-induced tumor-bearing rats was down-regulated Cyclin D1, Bcl-2 expression, and up-regulated proapoptotic proteins such as Bax, p53, Cytochrome-C, Caspase-9, and Caspase-3 as compared to DIM 10mg/kg b.wt. In addition, the mRNA expressions of Cyclin D1, Bcl-2 decreased and increased Bax, p53 expression, in immunohistochemical analysis decreased expressions of Cyclin D1 and PCNA in the treatment of DIM@CS-NP 0.5mg/kg b.wt. compared to DIM 10mg/kg b.wt. Histological analysis of tumor tissues shows abnormal in collagen deposition in with Masson's trichrome (MT) and Picrosirius red (PR) staining, the treatment of DIM@CS-NP 0.5mg/kg b.wt. reduced the collagen deposition as compared to DIM 10mg/kg b.wt. Our results clearly provide evidence that DIM@CS-NP exerts chemotherapeutic effect than DIM in DMBA model of mammary cancer by hold back anomalous tumor cell proliferation and inducing apoptosis to intervene through alterations of up-regulated and down-regulated molecules. Taken together, the data provide new evidence for mechanism action of DIM@CS-NP on mammary cancer.