Diindolylmethane Treatment Research Articles

3,3'-Diindolylmethane inhibits Th17 cell differentiation via impairing IRF-7-mediated plasmacytoid dendritic cell activation in imiquimod-induced psoriasis mice.

Psoriasis is a paradigmatic condition characterised by a heightened autoimmune response and chronic inflammation. However, the exact nature and the pathological causes behind it are still unknown. Growing evidence suggest dysregulated cytokine network as a result of over-activated T cells and plasmacytoid dendritic cells (pDCs) as the critical drivers in the development of psoriasis. In the present study, we aimed to investigate the therapeutic efficacy of 3,3'-diindolylmethane (DIM) on pDC activation and Th17 cell development in imiquimod (IMQ)-induced psoriasis mice. Our in vitro research investigated the IRF-7 signalling in pDCs that explained the reduced expression of the transcription factor IRF-7 responsible for pDC activation as a result of DIM treatment. Concurrently, DIM treatment decreased the release of Th17 cell polarising cytokines (IFN-α, IL-23, and IL-6) by pDCs which validated a reduction in differentiated pathogenic Th17 cell population and associated cytokine IL-17A in IMQ-induced psoriatic mice. Thus, our recent findings provide therapeutic evidence in targeting the early potential contributors for psoriasis treatment by preventing IRF-7-mediated pDC activation and Th17 cell development in IMQ-induced psoriasis mice.

Therapeutic Potential of Diindolylmethane and Empagliflozin in DMBA-induced Breast Cancer: on Body Weight and Tumor Volume

Breast cancer remains a major global health concern, necessitating the exploration of novel therapeutic approaches. This study aimed to investigate the effects of Diindolylmethane (DIM) and Empagliflozin on body weight and tumor volume in a 7,12-Dimethylbenz[a]anthracene (DMBA)-induced breast cancer model. Female Sprague-Dawley rats were randomly divided into Five groups: Negative Control, Positive control, Standard treated, DIM + Empagliflozin lower doe(Treatment 1) and DIM + Empagliflozin higher dose (Treatment 2). The animals were monitored for changes in body weight, and tumor volume was measured using callipers. Our findings revealed that both DIM and Empagliflozin treatment significantly attenuated body weight gain compared to the Positive control group. Moreover, DIM and Empagliflozin exhibited substantial inhibitory effects on tumor volume. These results suggest that DIM and Empagliflozin may have potential therapeutic benefits in the management of breast cancer. DIM's anti-proliferative and pro-apoptotic properties, along with Empagliflozin's ability to modulate glucose metabolism, likely contribute to their observed effects on tumor growth. The synergistic effect of the combination treatment indicates a promising strategy for enhancing therapeutic efficacy. This study provides valuable insights into the potential use of DIM and Empagliflozin as adjunctive treatments for breast cancer. Further investigations are warranted to elucidate the underlying mechanisms and evaluate the long-term safety and efficacy of these agents. These findings contribute to the development of targeted therapeutic approaches and support the exploration of DIM and Empagliflozin in future clinical trials for breast cancer management.

Diindolylmethane Ameliorates Ischemic Stroke-Induced Brain Injury by Peripheral and Central Mechanisms.

Diindolylmethane (DIM), a major acid condensation product of Indole-3- carbinol, is known to inhibit platelet aggregation and thrombosis. The drugs with antiplatelet and antithrombotic activities are used to treat ischemic stroke. The present study investigated the role of DIM on platelet aggregation inhibitory properties in middle cerebral artery occluded (MCAO) rats. DIM (12.5, 25, and 50 mg/kg) was orally administered to MCAO rats for 3 days. Platelet aggregation, platelet cyclic adenosine monophosphate (cAMP), reactive oxygen species (ROS), hydrogen peroxide (H2O2), and serum cyclooxygenase (COX-1), thromboxane B2 (TXB2), and prostaglandin E2 (PGE2), and inflammatory markers were estimated. Further brain structural and functional recovery was evaluated by measuring cerebral blood flow, neurological deficits, brain infarction, blood-brain barrier (BBB) leakage, brain water content, and histological abnormalities. DIM significantly ameliorated adenosine diphosphate (ADP), collagen, thrombin, and arachidonic acid-induced platelet aggregation by inhibiting COX-1, TXB2, and PGE2 and elevating cAMP. Further, DIM also alleviated platelet-mediated oxidative stress (ROS and H2O2) and reduced the serum inflammatory markers, tumor necrosis factor-α (TNF-α) and interleukin -6 (IL-6), and increased anti-inflammatory cytokine, IL-10, in MCAO rats. DIM treatment confers neuroprotection in MCAO rats by inhibition of platelet aggregation, platelet-mediated oxidative stress, and inflammation. Correspondingly, DIM improved cerebral blood flow and reduced neurological deficits, brain infarction, BBB leakage, brain water content, and histopathological abnormalities indicating the preservation of brain structural integrity. Thus, the present study provided preclinical evidence of DIM neuroprotection against ischemic stroke.

Hepatoprotective Effects of a Natural Flavanol 3,3'-Diindolylmethane against CCl4-Induced Chronic Liver Injury in Mice and TGFβ1-Induced EMT in Mouse Hepatocytes via Activation of Nrf2 Cascade.

Hepatic fibrosis is a form of irregular wound-healing response with acute and chronic injury triggered by the deposition of excessive extracellular matrix. Epithelial–mesenchymal transition (EMT) is a dynamic process that plays a crucial role in the fibrogenic response and pathogenesis of liver fibrosis. In the present study, we postulated a protective role of 3,3′-diindolylmethane (DIM) against TGF-β1 mediated epithelial–mesenchymal transition (EMT) in vitro and carbon tetrachloride (CCl4)-induced liver fibrosis in mice. TGF-β1-induced AML-12 hepatocyte injury was evaluated by monitoring cell morphology, measuring reactive oxygen species (ROS) and mitochondrial membrane potential, and quantifying apoptosis, inflammatory, and EMT-related proteins. Furthermore, CCl4-induced liver fibrosis in mice was evaluated by performing liver function tests, including serum ALT and AST, total bilirubin, and albumin to assess liver injury and by performing H&E and Sirius red staining to determine the degree of liver fibrosis. Immunoblotting was performed to determine the expression levels of inflammation, apoptosis, and Nrf2/HO-1 signaling-related proteins. DIM treatment significantly restored TGF-β1-induced morphological changes, inhibited the expression of mesenchymal markers by activating E-cadherin, decreased mitochondrial membrane potential, reduced ROS intensity, and upregulated levels of Nrf2-responsive antioxidant genes. In the mouse model of CCl4-induced liver fibrosis, DIM remarkably attenuated liver injury and liver fibrosis, as reflected by the reduced ALT and AST parameters with increased serum Alb activity and fewer lesions in H&E staining. It also mitigated the fibrosis area in Sirius red and Masson staining. Taken together, our results suggest a possible molecular mechanism of DIM by suppressing TGF-β1-induced EMT in mouse hepatocytes and CCl4-induced liver fibrosis in mice.

Anti-Virulence Activity of 3,3'-Diindolylmethane (DIM): A Bioactive Cruciferous Phytochemical with Accelerated Wound Healing Benefits.

Antimicrobial resistance is among the top global health problems with antibacterial resistance currently representing the major threat both in terms of occurrence and complexity. One reason current treatments of bacterial diseases are ineffective is the occurrence of protective and resistant biofilm structures. Phytochemicals are currently being reviewed for newer anti-virulence agents. In the present study, we aimed to investigate the anti-virulence activity of 3,3′-diindolylmethane (DIM), a bioactive cruciferous phytochemical. Using a series of in vitro assays on major Gram-negative pathogens, including transcriptomic analysis, and in vivo porcine wound studies as well as in silico experiments, we show that DIM has anti-biofilm activity. Following DIM treatment, our findings show that biofilm formation of two of the most prioritized bacterial pathogens Acinetobacter baumannii and Pseudomonas aeruginosa was inhibited respectively by 65% and 70%. Combining the antibiotic tobramycin with DIM enabled a high inhibition (94%) of P. aeruginosa biofilm. A DIM-based formulation, evaluated for its wound-healing efficacy on P. aeruginosa-infected wounds, showed a reduction in its bacterial bioburden, and wound size. RNA-seq was used to evaluate the molecular mechanism underlying the bacterial response to DIM. The gene expression profile encompassed shifts in virulence and biofilm-associated genes. A network regulation analysis showed the downregulation of 14 virulence-associated super-regulators. Quantitative real-time PCR verified and supported the transcriptomic results. Molecular docking and interaction profiling indicate that DIM can be accommodated in the autoinducer- or DNA-binding pockets of the virulence regulators making multiple non-covalent interactions with the key residues that are involved in ligand binding. DIM treatment prevented biofilm formation and destroyed existing biofilm without affecting microbial death rates. This study provides evidence for bacterial virulence attenuation by DIM.

3,3'-Diindolylmethane Enhances Fluorouracil Sensitivity via Inhibition of Pyrimidine Metabolism in Colorectal Cancer.

Chemoresistance limits treatment outcomes in colorectal cancer (CRC) patients. A dimeric metabolite of indole-3-carbinol, 3,3′-diindolylmethane (DIM) is abundant in cruciferous vegetables and has shown anticancer efficacy. The role of DIM in regulating chemosensitivity in CRC remains unknown. In this study, we demonstrated that DIM treatment inhibits the malignant progression of CRC. RNA sequencing indicated that pyrimidine synthesis genes are attenuated by DIM treatment. Stable 13C-labeled glucose tracing revealed that DIM inhibits de novo pyrimidine biosynthesis in CRC. DIM increases 5-FU cytotoxicity in CRC via regulation of the expression of pyrimidine metabolism-related genes. DIM synergizes with 5-FU to enhance its inhibitory effects on CRC both in vivo and in vitro. Our results suggest that DIM improves the therapeutic outcomes of FU-based chemotherapy in CRCs by inhibiting pyrimidine metabolism, identifying a new strategy for clinical therapy.

3,3'-Diindolylmethane induces ferroptosis by BAP1-IP3R axis in BGC-823 gastric cancer cells.

To investigate the effect and potential mechanism of 3,3'-diindolylmethane (DIM) on ferroptosis against gastric cancer, cells proliferation, lipid reactive oxygen species (ROS) and GSH level were measured in the BGC-823 gastric cancer cells after DIM treatment. Western blotting was used to detect the expression of SLC7A11, GPX4, IP3R and BAP1. Results showed that DIM could induce ferroptosis in the BGC-823 gastric cancer cells via upregulating lipid-ROS level and decreasing GSH generation. Besides, DIM also significantly reduced the protein level of SLC7A11 and GPX4, which was an important regulator of ferroptosis. In addition, DIM promoted the protein level of BAP1 and IP3R in a concentration-dependent manner in the BGC-823 gastric cancer cells. The knockdown of BAP1 could reduce IP3R level and DIM-induced ferroptosis of gastric cancer cells. Taken together, these results indicated that DIM could induce ferroptosis to exert anti-cancer effects via BAP1-IP3R axis, suggesting its effective therapeutic potential in gastric cancer.

AKT inhibitor AZD5363 suppresses stemness and promotes anti-cancer activity of 3,3′-diindolylmethane in human breast cancer cells

3,3′-diindolylmethane (DIM) is a dimer compound converted from Indoly-3-carbinol that had been studied as promising chemo-preventive agent against breast cancer. In this study, we observed that proportion of CD133+Nanog+ subpopulation in MCF-7 cells was significantly increased after DIM administration with up-regulated AKT activity by using CyTOF assay. SPADE analysis revealed this stem-like subpopulation exhibited apoptosis-resistance property against DIM treatment. By combining with AKT inhibitor AZD5363, DIM induced CD133 expression could be suppressed. In addition, a combination treatment of MCF-7 and MDA-MB-231 breast cancer cells with DIM and AZD5363 showed synergistic decreases in cell proliferation and induced apoptosis. Furthermore, results from imaging flow cytometry suggested that FoxO3a nuclear localization and PUMA expression could be improved by combination of AZD5363 with DIM. Taken together, the above observations suggested that the combination of AZD5363 with DIM could be developed as potential therapy for breast cancer.

3,3′-Diindolylmethane Suppresses the Growth of Hepatocellular Carcinoma by Regulating Its Invasion, Migration, and ER Stress-Mediated Mitochondrial Apoptosis

Hepatocellular carcinoma (HCC) is the leading cause of cancer-related death worldwide with limited treatment options. Biomarker-based active phenolic flavonoids isolated from medicinal plants might shed some light on potential therapeutics for treating HCC. 3,3′-diindolylmethane (DIM) is a unique biologically active dimer of indole-3-carbinol (I3C), a phytochemical compound derived from Brassica species of cruciferous vegetables—such as broccoli, kale, cabbage, and cauliflower. It has anti-cancer effects on various cancers such as breast cancer, prostate cancer, endometrial cancer, and colon cancer. However, the molecular mechanism of DIM involved in reducing cancer risk and/or enhancing therapy remains unknown. The aim of the present study was to evaluate anti-cancer and therapeutic effects of DIM in human hepatoma cell lines Hep3B and HuhCell proliferation was measured with MTT and trypan blue colony formation assays. Migration, invasion, and apoptosis were measured with Transwell assays and flow cytometry analyses. Reactive oxygen species (ROS) intensity and the loss in mitochondrial membrane potential of Hep3B and Huh7 cells were determined using dihydroethidium (DHE) staining and tetramethylrhodamine ethyl ester dye. Results showed that DIM significantly suppressed HCC cell growth, proliferation, migration, and invasion in a concentration-dependent manner. Furthermore, DIM treatment activated caspase-dependent apoptotic pathway and suppressed epithelial–mesenchymal transition (EMT) via ER stress and unfolded protein response (UPR). Taken together, our results suggest that DIM is a potential anticancer drug for HCC therapy by targeting ER-stress/UPR.

Exogenous 3,3'-Diindolylmethane Improves Vanadium Stress Tolerance in Brassica napus Seedling Shoots by Modulating Antioxidant Enzyme Activities.

3,3′-diindolylmethane (DIM) belongs to a family of indole glucosinolate compounds that have been shown to improve Brassica napus growth through the modulation of reactive oxygen species when applied exogenously. The B. napus cultivar AV Garnet was previously identified as a vanadium-sensitive cultivar. Therefore, in this study we investigated whether exogenous DIM could improve the vanadium tolerance of AV Garnet. We performed the following experiments: seed germination assessment, dry weight assessment, cell viability assay, chlorophyll content assay, malondialdehyde (MDA) assay, conjugated diene (CD) content assay, hydrogen peroxide (H2O2) content assay, superoxide (O2−) content determination, methylglyoxal (MG) content determination, hydroxyl radical (·OH) concentration determination, ascorbate peroxidase (APX) activity assay, superoxide dismutase (SOD) activity assay, glyoxalase I (Gly I) activity assay, glutathione S-transferase (GST) activity assay and inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis for vanadium content determination. Under vanadium stress, exogenous DIM increased the seed germination percentage, shoot dry weight, cell viability and chlorophyll content. Exogenous DIM also led to a decrease in MDA, CD, H2O2, O2−, MG and ·OH, under vanadium stress in the shoots. Furthermore, DIM application led to an increase in the enzymatic activities of APX, SOD, Gly I and GST under vanadium stress. Interestingly, under vanadium stress, DIM treatment did not alter vanadium content in B. napus shoots. Our results indicate that exogenous application of DIM can improve B. napus seedling shoot growth and biomass under vanadium stress by priming the antioxidant enzymes via reactive oxygen species (ROS) signaling.

3,3'-Diindolylmethane induces gastric cancer cells death via STIM1 mediated store-operated calcium entry.

3,3'-Diindolylmethane (DIM), a natural phytochemicals isolated from cruciferous vegetables, has been reported to inhibit human gastric cancer cells proliferation and induce cells apoptosis as well as autophagy, but its mechanisms are still unclear. Store-operated calcium entry (SOCE) is a main Ca2+ influx pathway in various of cancers, which is activated by the depletion of endoplasmic reticulum (ER) Ca2+ store. Stromal interaction molecular 1 (STIM1) is the necessary component of SOCE. In this study, we focus on to examine the regulatory mechanism of SOCE on DIM-induced death in gastric cancer. After treating the human BGC-823 and SGC-7901 gastric cancer cells with DIM, cellular proliferation was determined by MTT, apoptosis and autophagy were detected by flow cytometry or Hoechst 33342 staining. The expression levels of related proteins were evaluated by Western blotting. Free cytosolilc Ca2+ level was assessed by fluorescence monitoring under a laser scanning confocal microscope. The data have shown that DIM could significantly inhibit proliferation and induce apoptosis as well as autophagy in two gastric cancer cell lines. After DIM treatment, the STIM1-mediated SOCE was activated by upregulating STIM1 and decreasing ER Ca2+ level. Knockdown STIM1 with siRNA or pharmacological inhibition of SOCE attenuated DIM induced apoptosis and autophagy by inhibiting p-AMPK mediated ER stress pathway. Our data highlighted that the potential of SOCE as a promising target for treating cancers. Developing effective and selective activators targeting STIM1-mediated SOCE pathway will facilitate better therapeutic sensitivity of phytochemicals acting on SOCE in gastric cancer. Moreover, more research should be performed to validate the efficacy of combination chemotherapy of anti-cancer drugs targeting SOCE for clinical application.

PTEN/Akt Signaling-Mediated Activation of the Mitochondrial Pathway Contributes to the 3,3'-Diindolylmethane-Mediated Antitumor Effect in Malignant Melanoma Cells.

3,3'-diindolylmethane (DIM) has an anticancer activity, but the role DIM plays on malignant melanoma cells and its specific mechanism is unclear. We studied the biological effects of DIM on malignant melanoma cells and the related mechanism and the results showed that DIM significantly suppressed cell proliferation and induced apoptosis in malignant melanoma cells. In addition, the expression levels of phosphatase and tensin homolog deleted on chromosome ten (PTEN), Bax, Bid, cleaved caspase-3, and cleaved caspase-9 were increased after DIM treatment. In A2058 PTENmut cells, DIM-mediated inhibition of proliferation and DIM-induced apoptosis were attenuated. Additionally, the overexpression and knockdown of PTEN could regulate such effects of DIM in malignant melanoma cells. Furthermore, DIM exerted growth-inhibiting and apoptosis-inducing effects in vivo. This study demonstrated that DIM has antitumor effect in human malignant melanoma cells through the mitochondrial apoptotic pathway activated by PTEN/Akt signaling.

3,3'-Diindolylmethane Enhances Tumor Regression After Radiation Through Protecting Normal Cells to Modulate Antitumor Immunity

PurposePreclinical and clinical data indicate that radiation therapy acts as an immune modifier, having both immune-stimulatory and immunosuppressive effects on the tumor-immune microenvironment (TIME). 3.3’-diindolylmethane (DIM) sensitizes tumor cells to radiation and protects mice from lethal doses of total body irradiation. We hypothesize that protecting nontumoral cells from the adverse effects of radiation treatment (RT) may help to correct immunosuppression resulting from radiation. Methods and MaterialsWe generated tumor graft models using immune-competent and immune-deficient mouse strains. Narrow-beamed radiation was targeted to tumor sites using shielding. Tumor regression was monitored after DIM and RT versus RT alone. The effects of DIM on the efficacy of RT were assessed using immunohistochemistry staining and gene expression profiling. Complete blood counts, clonogenic cell survival assays, and global gene expression profiling of cultured cells were performed to study DIM’s radioprotective effects on normal cells. ResultsDIM enhanced tumor regression after RT in immune-competent but not immune-deficient mice. Data indicated that DIM increased intratumoral immune cells after RT, contributing to enhanced immunologic responses such as adhesion and antigen processing. DIM protected normal cells from radiation-induced immediate injuries in vitro and in vivo. Transcriptomic profiling of cultured cells showed that DIM treatment mildly increased expression of some genes that are normally induced after radiation, such as genes involved in cell cycle arrest and apoptosis. ConclusionsIn this study, using cultured cells and preclinical breast cancer models, we show that DIM protects normal cells from radiation-induced immediate cellular injury and combination treatment of DIM and radiation potentiates antitumor immune responses and enhances the efficacy of RT.

DIM mitigates the development of experimental autoimmune encephalomyelitis by maintaining the stability and suppressive function of regulatory T cells

Recent studies have revealed that indoles, dietary ligands of the aryl hydrocarbon receptor (AhR), have immunomodulatory characteristics of balancing the differentiation of regulatory T cells (Tregs) and Th17 cells in multiple autoimmune diseases.In this study, we aimed to investigate the potency of the indole, 3,3′-diindolylmethane (DIM), on the stability and suppressive function of Tregs in experimental autoimmune encephalomyelitis (EAE). Furthermore, we used the AhR antagonist CH223191 to verify that DIM exerts its effects on Tregs through the activation of AhR.We found that DIM treatment significantly alleviated the severity of EAE by maintaining the stability and suppressive function of Tregs instead of facilitating the differentiation of Tregs. Thus, these DIM-treated Tregs might indirectly inhibit the generation of Th17 cells and the production of proinflammatory cytokines. And we confirmed the critical role of AhR in the EAE model.Our study further investigated the mechanisms by which dietary indoles promote Treg activity in the EAE model. DIM may act as a novel therapeutic to restrain autoimmune inflammation in multiple sclerosis.

Abstract 6513: 3,3'-diindolylmethane enhances tumor regression after radiation through protecting normal cells to modulate anti-tumor immunity

Abstract Preclinical and clinical data indicate that radiotherapy acts as an immune modifier, having both immunostimulatory and immunosuppressive effects on the tumor immune microenvironment (TIME). 3.3'-Diindolylmethane (DIM), the acid-catalyzed dimer of the bioactive indole found in cruciferous vegetables, has long been known for its anticancer properties and has been proposed for use as a cancer prevention agent. The current study characterizes how DIM influences TIME and tumor regression after fractionated radiation treatment using preclinical breast cancer models. We first compared tumor graft models using immune-competent and immune-deficient mouse strains to find out whether immune cells are required for DIM's effects. Tumor targeted radiation was done using narrow-beam radiation with shielding. Tumor regression was monitored after DIM and radiation combined treatment and radiation alone. Our results indicated that DIM enhanced tumor regression after radiation treatment in immune-competent but not immuno-deficient mice. Data from nCounter® PanCancer immune profiling panel showed DIM increased intratumoral immune cells including cytotoxic T cells, neutrophils and macrophages after radiation and enhanced immunological processes such as adhesion and antigen processing. Expression of several MHC Class II molecules and CD74, a gene encoding chaperone and transport cofactor that assists MHC II molecules folding and presenting, significantly increased in the combined treatment of radiation and DIM. The results showing that DIM treatment alone does not affect tumor growth suggested that DIM's radioprotective effect on non-tumoral cells, including stromal cells and immune cells, plays a critical role in promoting a microenvironment that favors anti-tumor immunity. Additional experiments showed that DIM protected normal cells from radiation-caused immediate injuries in vitro and in vivo. In summary, DIM's radioprotective effects on immune cells and other normal cells are important in promoting a TIME that favors anti-tumor immunity. As a result, DIM increases the efficacy of radiation treatment on tumor regression. This study supports the development of DIM as an adjunct agent for radiation treatment. Citation Format: Renxiang Chen, Lijun Li, Yun-Tien Lin, Arslon Humayun, Albert J. Fornace Jr., Heng-Hong Li. 3,3'-diindolylmethane enhances tumor regression after radiation through protecting normal cells to modulate anti-tumor immunity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6513.

Modulatory Effect of Indoles on the Expression of miRNAs Regulating G1/S Cell Cycle Phase in Breast Cancer Cells.

Indole-3-carbinol (I3C) is a naturally occurring glucosinolate found in Brassica vegetables that is usually converted in gastric acidic environment to the efficient metabolite 3,3'-diindolylmethane (DIM). Both indoles (I3C and DIM) are known chemopreventive agents for various cancers including breast cancer. This study aimed to investigate the influence of both indoles on the tumor suppressor miRNAs (let-7a-e, miR-15a, miR-16, miR-17-5p, miR-19a, and miR-20a) and oncomiRs (miR-181a, miR-181b, miR-210, miR-221, and miR-106a), which are controlling the cell cycle key regulators: cyclin-dependent kinases (CDKs), CDK inhibitor p27Kip1, and cyclin D1. Our results indicated that both indoles generally elevated the expression of the tumor suppressor miRNAs let-7a-e, miR-19a, miR-17-5p, and miR-20a and decreased the expression of the oncomiR list. Both indoles were able to significantly suppress the expression of CDK4 and CDK6 as well as the apoptotic markers Bcl-2 and survivin. Both indoles decreased cyclin-D1 protein, where I3C decreased cytoplasmic and nuclear cyclin-D1 significantly. Cytoplasmic and nuclear P27Kip1 showed overexpression following treatment with I3C higher than that detected following DIM treatment. This study provides a mechanistic elucidation of the previously reported cell cycle arrest by I3C and DIM in breast cancer cells suggesting that this effect could be through modulation of miRNAs expression that, in turn, regulates the genetic network controlling the G1/S phase in cell cycle progression.

Effect of 3,3'-Diindolylmethane on Pulmonary Injury Following Thoracic Irradiation in CBA Mice.

The molecule 3,3'-diindolylmethane (DIM) is small, a major bioactive metabolite of indole-3 carbinol (13C), and a phytochemical compound from cruciferous vegetables released upon exposure to the gut acid environment. DIM is a proposed anti-cancer agent and was previously demonstrated to prevent radiation damage in the bone marrow and the gastrointestinal tract. Here we investigated the effect of DIM on radiation-induced injury to the lung in a murine model through untargeted metabolomics and gene expression studies of select genes. CBA mice were exposed to thoracic irradiation (17.5 Gy). Mice were treated with vehicle or DIM (250 mg kg, subcutaneous injection) on days -1 pre-irradiation through +14 post-irradiation. DIM induced a significant improvement in survival by day 150 post-irradiation. Fibrosis-related gene expression and metabolomics were examined using lung tissue from days 15, 45, 60, 90, and 120 post-irradiation. Our qRT-PCR experiments showed that DIM treatment reduced radiation-induced late expression of collagen Iα and the cell cycle checkpoint proteins p21/waf1 (CDKN1A) and p16ink (CDKN2A). Metabolomic studies of lung tissue demonstrated a significant dampening of radiation-induced changes following DIM treatment. Metabolites associated with pro-inflammatory responses and increased oxidative stress, such as fatty acids, were suppressed by DIM treatment compared to irradiated samples. Together these data suggest that DIM reduces radiation-induced sequelae in the lung.

Indole-3-Carbinol Derivative DIM Mitigates Carbon Tetrachloride-Induced Acute Liver Injury in Mice by Inhibiting Inflammatory Response, Apoptosis and Regulating Oxidative Stress.

3,3′-Diindolylmethane (DIM), a metabolic product of indole-3-carbinol extracted from cruciferous vegetables exhibits anti-inflammatory and anti-cancer properties. Earlier, the product has been demonstrated to possess anti-fibrotic properties; however, its protective effects on liver injury have not been clearly elucidated. In this study, we postulated the effects and molecular mechanisms of action of DIM on carbon tetrachloride (CCl4)-induced liver injury in mice. Acute liver injury was induced by a single intraperitoneal administration of CCl4 (1 ml/kg) into mice. DIM was injected via subcutaneous route for three days at various doses (2.5, 5 and 10 mg/kg) before CCl4 injection. Mice were sacrificed and serum was collected for quantification of serum transaminases. The liver was collected and weighed. Treatment with DIM significantly reduced serum transaminases levels (AST and ALT), tumor necrosis factor-α (TNF-α) and reactive oxygen species (ROS). CCl4- induced apoptosis was inhibited by DIM treatment by the reduction in the levels of cleaved caspase-3 and Bcl2 associated X protein (Bax). DIM treated mice significantly restored Cytochrome P450 2E1, nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression in CCl4 treated mice. In addition, DIM downregulated overexpression of hepatic nuclear factor kappa B (NF-κB) and inhibited CCl4 mediated apoptosis. Our results suggest that the protective effects of DIM against CCl4- induced liver injury are due to the inhibition of ROS, reduction of pro-inflammatory mediators and apoptosis.

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.

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.