Effects Of Diindolylmethane Research Articles

Indole-3 Carbinol and Diindolylmethane Mitigated β-Amyloid-Induced Neurotoxicity and Acetylcholinesterase Enzyme Activity: In Silico, In Vitro, and Network Pharmacology Study.

Background: Alzheimer's disease (AD) is a neurodegenerative disease characterized by beta-amyloid (Aβ) deposition and increased acetylcholinesterase (AchE) enzyme activities. Indole 3 carbinol (I3C) and diindolylmethane (DIM) are reported to have neuroprotective activities against various neurological diseases, including ischemic stroke, Parkinson's disease, neonatal asphyxia, depression, stress, neuroinflammation, and excitotoxicity, except for AD. In the present study, we have investigated the anti-AD effects of I3C and DIM. Methods: Docking and molecular dynamic studies against AchE enzyme and network pharmacological studies were conducted for I3C and DIM. I3C and DIM's neuroprotective effects against self and AchE-induced Aβ aggregation were investigated. The neuroprotective effects of I3C and DIM against Aβ-induced neurotoxicity were assessed in SH-S5Y5 cells by observing cell viability and ROS. Results: Docking studies against AchE enzyme with I3C and DIM show binding efficiency of -7.0 and -10.3, respectively, and molecular dynamics studies revealed a better interaction and stability between I3C and AchE and DIM and AchE. Network pharmacological studies indicated that I3C and DIM interacted with several proteins involved in the pathophysiology of AD. Further, I3C and DIM significantly inhibited the AchE (IC50: I3C (18.98 µM) and DIM (11.84 µM)) and self-induced Aβ aggregation. Both compounds enhanced the viability of SH-S5Y5 cells that are exposed to Aβ and reduced ROS. Further, I3C and DIM show equipotential neuroprotection when compared to donepezil. Conclusions: Our findings indicate that both I3C and DIM show anti-AD effects by inhibiting the Aβ induced neurotoxicity and AchE activities.

3, 3’-diindolylmethane enhances macrophage efferocytosis and subsequently relieves visceral pain via the AhR/Nrf2/Arg-1-mediated arginine metabolism pathway

Background3, 3’-diindolylmethane (DIM), a classical aryl hydrocarbon receptor (AhR) agonist, has been shown to relieve neuropathic pain, but few studies have reported the efficacy of DIM in visceral pain under colitis condition. PurposeThis study aimed to investigate the effect and mechanism of DIM on visceral pain under colitis condition. MethodsCytotoxicity was performed using the MTT assay. RT-qPCR and ELISA assays were applied to determine the expression and release of algogenic substance P (SP), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). Flow cytometry was used to examine the apoptosis and efferocytosis. The expression of Arg-1-arginine metabolism-related enzymes was detected using western blotting assays. ChIP assays were used to examine the binding of Nrf2 to Arg-1. Mouse models of dextran sulfate sodium (DSS) were established to illustrate the effect of DIM and validate the mechanism in vivo. ResultsDIM did not directly affect expressions and release of algogenic SP, NGF and BDNF in enteric glial cells (EGCs). However, when co-cultured with DIM-pre-treated RAW264.7 cells, the release of SP and NGF was decreased in lipopolysaccharides-stimulated EGCs. Furthermore, DIM increased the number of PKH67+ F4/80+ cells in the co-culture system of EGCs and RAW264.7 cells in vitro and alleviated visceral pain under colitis condition by regulating levels of SP and NGF as well as values of electromyogram (EMG), abdominal withdrawal reflex (AWR) and tail-flick latency (TFL) in vivo, which was significantly inhibited by efferocytosis inhibitor. Subsequently, DIM was found to down-regulate levels of intracellular arginine, up-regulate levels of ornithine, putrescine and Arg-1 but not extracellular arginine or other metabolic enzymes, and polyamine scavengers reversed the effect of DIM on efferocytosis and release of SP and NGF. Moving forward, Nrf2 transcription and the binding of Nrf2 to Arg-1-0.7 kb was enhanced by DIM, AhR antagonist CH223191 abolished the promotion of DIM on Arg-1 and efferocytosis. Finally, nor-NOHA validated the importance of Arg-1-dependent arginine metabolism in DIM-alleviated visceral pain. ConclusionDIM enhances macrophage efferocytosis in an arginine metabolism-dependent manner via “AhR-Nrf2/Arg-1” signals and inhibits the release of SP and NGF to relieve visceral pain under colitis condition. These findings provide a potential therapeutic strategy for the treatment of visceral pain in patients with colitis.

Indole-3-Carbinol: Occurrence, Health-Beneficial Properties, and Cellular/Molecular Mechanisms.

Indole-3-carbinol (I3C) is a bioactive phytochemical abundant in cruciferous vegetables. One of its main in vivo metabolites is 3,3'-diindolylmethane (DIM), formed by the condensation of two molecules of I3C. Both I3C and DIM alter multiple signaling pathways and related molecules controlling diverse cellular events, including oxidation, inflammation, proliferation, differentiation, apoptosis, angiogenesis, and immunity. There is a growing body of evidence from both in vitro and in vivo models that these compounds possess strong potential to prevent several forms of chronic disease such as inflammation, obesity, diabetes, cardiovascular disease, cancer, hypertension, neurodegenerative diseases, and osteoporosis. This article reviews current knowledge of the occurrence of I3C in nature and foods, along with the beneficial effects of I3C and DIM concerning prevention and treatment of human chronic diseases, focusing on preclinical studies and their mechanisms of action at cellular and molecular levels.

3,3'-Diindolylmethane Augments 5-Fluorouracil-InducedGrowth Suppression in Gastric Cancer Cells through Suppression of the Akt/GSK-3β and WNT/Beta-Catenin.

Gastric cancer (GC) is one of the most lethal cancers in South Korea, and it is a cancer of concern worldwide. 5-fluorouracil (5-Fu) is commonly used as the first-line therapy for advanced GC; however, its side effects often limit the dosage range and impair patients' quality of life. Due to the limitations of current chemotherapy, new anticancer therapies are urgently needed. 3,3'-diindolylmethane (DIM) has been reported to have the ability to protect against various types of cancer. Our study aimed to elucidate the anticancer effect of DIM in GC when treated with the chemotherapeutic agent 5-Fu. In our results, combined treatment with DIM and 5-Fu resulted in higher apoptosis and lower cell proliferation than treatment with 5-Fu in SNU484 and SNU638 cell lines. Furthermore, when DIM and 5-Fu were administered together, cell invasion was diminished by mediated E-cadherin, MMP-9, and uPA; p-Akt and p-GSK-3β levels were reduced more significantly than when 5-Fu was administered alone. Moreover, in the Wnt signaling pathway, combined treatment of DIM and 5-Fu diminished β-catenin levels in the nucleus and inhibited cyclin D1and c-Myc protein levels. The Akt inhibitor, wortmannin, further inhibited the levels of β-catenin and c-Myc that were inhibited by DIM and 5-Fu. Furthermore, an animal xenograft model demonstrated that DIM combined with 5-Fu considerably reduced tumor growth without any toxic effects by regulating the Akt/GSK-3β and β-catenin levels. Our findings suggest that DIM significantly potentiates the anticancer effects of 5-Fu by targeting the Akt/GSK-3β and WNT/β-catenin because the combination therapy is more effective than 5-Fu alone, thereby offering an innovative potential therapy for patients with GC.

Intestinal epithelium aryl hydrocarbon receptor is involved in stress sensitivity and maintaining depressive symptoms

The aryl hydrocarbon receptor (AhR) is a key regulator in the microbiome-gut-brain axis, and AhR-active microbial metabolites modulate multiple neuronal responses. We recently demonstrated that 3,3’-diindolylmethane (DIM) and 1,4-dihydroxy-2-naphthoic acid (DHNA), two selective AhR modulators (SAhRMs), act as antidepressants in female mice. Thus, to examine the role of intestinal AhR in depression, anxiety, and spatial learning, this study employed transgenic mice in which the AhR was knockout only in the intestinal epithelium (AhRΔIEC). Additionally, this study examined whether the antidepressant effects of dietary DIM and DHNA is mediated by intestinal AhR. AhRΔIEC and WT female mice were fed daily with vehicle, 20 mg/kg DIM or DHNA for three weeks prior to four weeks of unpredictable chronic mild stress (UCMS). Mice were examined for weight gain, anhedonia-like behavior (sucrose preference test), anxiety levels (open field, light/dark, elevated plus maze, novelty-induced hypophagia, and marble burying tests), and spatial learning (Morris water maze). UCMS reduced weight gain in AhRΔIECs, but not WTs. Moreover, UCMS initially reduced sucrose preference in both AhRΔIECs and WTs, but over 4 weeks of UCMS, AhRΔIECs develop resilience to UCMS-induced anhedonia. Additionally, AhRΔIECs exhibit slightly reduced anxiety in certain tests and faster spatial learning. DIM and DHNA acted as antidepressants in both AhRΔIECs and WTs. Thus, this study suggests that intestinal AhR plays differential roles, mitigating stress effects on weight gain, and increasing stress effects on mood. However, the site of antidepressant action of SAhRMs, such as DIM and DHNA, is not dependent on the expression of intestinal AhR.

3,3′-Diindolylmethane improves antitumor immune responses of PD-1 blockade via inhibiting myeloid-derived suppressor cells

BackgroundImmune checkpoint inhibitors that target programmed cell death protein 1 (PD-1) have obtained encouraging results, but a fraction of tumor patients failed to respond to anti-PD-1 treatment due to the existence of multiple immune suppressive elements such as myeloid-derived suppressor cells (MDSCs). Traditional Chinese medicine or natural products from medicinal plants could enhance immunity and may be helpful for cancer immunotherapy. As a digestive metabolite from cruciferous plants, 3,3′-diindolylmethane (DIM) has been widely used in chemotherapy, but its influence on cancer immunotherapy remains unclear. Here we investigate the function of DIM on MDSCs and examine the therapeutic effects of DIM in conjunction with PD-1 antibody against mouse tumors.MethodsFlow cytometry analysis, Western blot analysis and qRT-PCR assay were used to examine the inhibitory effects and mechanisms of DIM on MDSCs in vitro and in vivo. The therapeutic effects of DIM on cancer immunotherapy by PD-1 antibody were evaluated in mouse models of breast cancer and melanoma tumor.ResultsDIM exerted the inhibitory effect on MDSCs via downregulating miR-21 level and subsequently activating PTEN/PIAS3-STAT3 pathways. Adoptive transfer of MDSCs impaired the therapeutic effects of DIM, indicating that the antitumor activity of DIM might be due to the suppression of MDSCs. Furthermore, in mouse models of breast cancer and melanoma tumor, the addition of DIM can enhance the therapeutic effect of PD-1 antibody through promoting T cells responses, and thereby inhibiting tumor growth.ConclusionsOverall, the strategy based on the combination treatment of anti-PD-1 antibody and DIM may provide a new approach for cancer immunotherapy. Cruciferae plants-rich diet which contains high amount of DIM precursor may be beneficial for cancer patients that undergo the anti-PD-1 treatment.

3,3′-Diindolylmethane and 1,4-dihydroxy-2-naphthoic acid prevent chronic mild stress induced depressive-like behaviors in female mice

BackgroundCurrent pharmaceutical treatments for depression are sometimes ineffective and may have unwanted side effects that interfere with patient compliance. This study examined the potential antidepressant-like effects of dietary- and microbial-derived aryl hydrocarbon receptor (AhR) ligands, 3,3′-diindolylmethane (DIM) and 1,4-dihydroxy-2-naphthoic acid (1,4-DHNA). MethodsFemale C57BL/6 mice were subjected to unpredictable chronic mild stress (UCMS) or were unstressed. For three weeks prior to UCMS mice were fed daily with vehicle or 20 mg/kg DIM, 1,4-DHNA or AhR-inactive isomer 3,7-DHNA; another group was subjected to two weeks UCMS before ligand administration began. Mice were examined for anhedonia-like behavior as measured by the sucrose preference test. Additionally, anxiety levels of the mice were examined before UCMS and ligand administration began and at the end in the open field, light/dark, elevated plus maze, novelty-induced hypophagia, and marble burying tests. At the end of the experiment they were also examined in the Morris water maze (MWM) task. ResultsBoth DIM and 1,4-DHNA, but not 3,7-DHNA, successfully prevented and reversed UCMS-induced anhedonia-like behavior. Furthermore, both DIM and DHNA had little to no effect on anxiety levels and did not induce spatial learning deficits. LimitationsAdditional studies are required to determine to what degree the antidepressant-like effects of DIM and 1,4-DHNA can be attributed to their activities as AhR ligands. ConclusionsOur findings indicate that dietary and microbial-derived AhR ligands may have clinical applications as potential antidepressants. Future studies are necessary to elucidate the role of AhR in depression-like states and the underlying mechanisms of action.

3,3′-Diindolylmethane attenuates inflammation and fibrosis in radiation-induced lung injury by regulating NF-κB/TGF-β/Smad signaling pathways

Objective: This study aims to investigate the protective effect of 3,3′-diindolylmethane (DIM) on the radiation-induced lung injury (RILI) model and to explore its possible mechanism. Methods: A mouse model of RILI was established by thoracic irradiation, and dexamethasone was used as a positive drug to investigate the effect of DIM on RILI mice. Lung histopathology was analyzed by HE staining and Masson staining. Then the levels of inflammatory cytokines (TGF-β, TNF-α, IL-1β, and IL-6), inflammatory cell counts, and activity of MPO were detected. The expression of TGFβ1/Smad signaling pathway-related proteins was determined by immunohistochemistry. qPCR was used to analyze the mRNA expression levels of inflammatory factors, α‑SMA and COL1A1. The expression of COX-2, NF-κB, IκBα, PI3K, and Akt proteins was assessed by Western blot. Results: Histopathological staining of lung tissues showed that DIM administration alleviated the pulmonary inflammation and fibrosis caused by RILI. Moreover, the content of inflammatory factors such as IL-1β and IL-6, the expression of NF-κB pathway-related proteins, and the counts of inflammatory cells were inhibited in lung tissue, indicating that DIM can inhibit the NF-κB pathway to reduce inflammation. In addition, DIM could down-regulate the mRNA levels of α-SMA, COL1A1, and downregulate TGFβ1, Smad3, and p-Smad2/3 in lung tissues. Conclusion: Our study confirms that DIM has the potential to treat RILI in vivo by inhibiting fibrotic and inflammatory responses in lung tissue through the TGFβ/Smad and NF-κB dual pathways, respectively.

3,3’-Diindolylmethane Enhances Paclitaxel Sensitivity by Suppressing DNMT1-Mediated KLF4 Methylation in Breast Cancer

Paclitaxel (PTX) is a first-line chemotherapeutic drug for the treatment of breast cancer, but drug resistance seriously limits its clinical use. The aim of the present work was to explore the effect of 3,3’-diindolylmethane (DIM) on PTX sensitivity and its possible mechanism in breast cancer. The expression of Krüppel-like factor 4 (KLF4) and DNA-methyltransferase 1 (DNMT1) in breast cancer tissues were assessed by immunohistochemistry and Western blotting. The methylation of KLF4 was evaluated by the MassARRAY platform. The lentivirus carrying KLF4 and DNMT1 gene or shRNA targeting DNMT1 were used to overexpress KLF4 or knockdown DNMT1 in MCF-7 and T47D breast cancer cells and the role of KLF4 and DNMT1 in regulation of PTX sensitivity was investigated. The effect of PTX on inhibiting the proliferation of MCF-7 and T47D cells was measured by CCK-8 assay. Flow cytometry was used to examine cell apoptosis. The expression of mRNA and protein was evaluated by qRT-PCR and Western blotting analysis, respectively. Our data showed that the expression of DNMT1 was increased, and the methylation level of CpG sites (−148 bp) in the KLF4 promoter was increased while the KLF4 expression was significantly decreased in breast cancer tissues. Overexpression of KLF4 increased the sensitivity of MCF-7 and T47D cells to PTX. DNMT1 increased the methylation of the KLF4 promoter and decrease the expression of KLF4. Knockdown of DNMT1 increased the sensitivity of MCF-7 and T47D cells to PTX. DIM enhanced the PTX sensitivity of MCF-7 and T47D cells, decreased the expression of DNMT1 and the methylation level of KLF4 promoter, thus increasing the level of KLF4. Furthermore, overexpression of DNMT1 attenuated the effect of DIM on the regulation of PTX sensitivity. Collectively, our data indicated that DNMT1-mediated hypermethylation of KLF4 promoter leads to downregulation of KLF4 in breast cancer. The level of KLF4 is correlated with the sensitivity of MCF-7 and T47D cells to PTX. DIM could enhance the antitumor efficacy of PTX on MCF-7 and T47D cells by regulating DNMT1 and KLF4.

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.

Dietary glucosinolates inhibit splenic inflammation in high fat/cholesterol diet-fed C57BL/6 mice.

BACKGROUND/OBJECTIVESObesity is associated with chronic inflammation. The spleen is the largest organ of the lymphatic system and has an important role in immunity. Obesity-induced inflammatory responses are triggered by Toll-like receptor (TLR)-myeloid differentiation primary response 88 (MyD88) pathway signaling. Phenethyl isothiocyanate (PEITC) and 3,3′-diindolylmethane (DIM), major dietary glucosinolates present in cruciferous vegetables, have been reported to produce anti-inflammatory effects on various diseases. However, the effects of PEITC and DIM on the obesity-induced inflammatory response in the spleen are unclear. The purpose of this study was to examine the anti-inflammatory effects of PEITC and DIM on the spleen and their mechanism in high fat/cholesterol diet (HFCD)-fed C57BL/6 mice.MATERIALS/METHODSWe established an animal model of HFCD-induced obesity using C57BL/6 mice. The mice were divided into six groups: normal diet with AIN-93G diet (CON), high fat diet (60% calories from fat) with 1% cholesterol (HFCD), HFCD with PEITC 30 mg/kg/day or 75 mg/kg/day (HFCD+P30, HFCD+P75), and HFCD with DIM 1.5 mg/kg/day or 7.5 mg/kg/day (HFCD+D1.5, HFCD+D7.5). Enzyme-linked immunosorbent assay was used to evaluate pro-inflammatory cytokine secretion. Western blot and quantitative polymerase chain reaction were used to analyze protein and mRNA levels of nuclear factor kappa B (NF-κB) p65, interleukin 6 (IL-6), cyclooxygenase 2 (COX-2), TLR2, TLR4, and MyD88 in spleen tissue.RESULTSSerum IL-6 levels were significantly higher in the HFCD group than in groups fed a HFCD with PEITC or DIM. Levels of NF-κB p65 protein and TLR2/4, MyD88, NF-κB p65, IL-6, and COX-2 mRNA were significantly higher in the HFCD group than in the CON group and were reduced by the PEITC and DIM supplements.CONCLUSIONSPEITC- and DIM-supplemented diets improved splenic inflammation by modulating the TLR2/4-MyD88 pathway in HFCD-fed mice. We suggest that dietary glucosinolates may at least partially improve obesity-induced inflammation of the spleen.

3,3'-diindolylmethane exerts antiproliferation and apoptosis induction by TRAF2-p38 axis in gastric cancer.

3,3'-diindolylmethane (DIM), an active phytochemical derivative extracted from cruciferous vegetables, possesses anticancer effects. However, the underlying anticancer mechanism of DIM in gastric cancer remains unknown. Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2), one of the signal transduction proteins, plays critical role in proliferation and apoptosis of human gastric cancer cells, but there are still lack of practical pharmacological modulators for potential clinical application. Here, we further explored the role of TRAF2 in inhibiting cell proliferation and inducing apoptosis by DIM in human gastric cancer BGC-823 and SGC-7901 cells. After treating BGC-823 and SGC-7901 cells with DIM for 24 h, cell proliferation, apoptosis and TRAF2-related protein were measured. Our findings showed that DIM inhibited the expressions of TRAF2, activated p-p38 and its downstream protein p-p53, which were paralleled with DIM-triggered cells proliferation, inhibition and apoptosis induction. These effects of DIM were reversed by TRAF2 overexpression or p38 mitogen-activated protein kinase (MAPK)-specific inhibitor (SB203580). Taken together, our data suggest that regulating TRAF2/p38 MAPK signaling pathway is essential for inhibiting gastric cancer proliferation and inducing apoptosis by DIM. These findings broaden the understanding of the pharmacological mechanism of DIM's action as a new modulator of TRAF2, and provide a new therapeutic target for human gastric cancer.

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.

Anti-inflammatory activity of diindolylmethane alleviates Riemerella anatipestiferinfection in ducks.

3,3'-Diindolylmethane (DIM) is found in cruciferous vegetables and is used to treat various inflammatory diseases because of its potential anti-inflammatory effects. To investigate effects of DIM in Riemerella anatipestifer-infected ducks which induce upregulation of inflammatory cytokines, ducks were treated orally with DIM at dose of 200 mg/kg/day and infected the following day with R. anatipestifer. Infected and DIM-treated ducks exhibited 14% increased survival rate and significantly decreased bacterial burden compared to infected untreated ducks. Next, the effect on the expression level of inflammatory cytokines (interleukin [IL]-17A, IL-17F, IL-6, IL-1β) of both in vitro and in vivo DIM-treated groups was monitored by quantitative reverse-transcription PCR (qRT-PCR). Generally, the expression levels of the cytokines were significantly reduced in DIM-treated splenic lymphocytes stimulated with killed R. anatipestifer compared to stimulated untreated splenic lymphocytes. Similarly, the expression levels of the cytokines were significantly reduced in the spleens and livers of DIM-treated R. anatipestifer-infected ducks compared to infected untreated ducks. This study demonstrated the ameliorative effects of DIM in ducks infected with R. anatipestifer. Thus, DIM can potentially be used to prevent and/or treat R. anatipestifer infection via inhibition of inflammatory cytokine expression.

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.

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.

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.

Nanoformulated Bioactive Compounds Derived from Different Natural Products Combat Pancreatic Cancer Cell Proliferation.

PurposeThis study was designed to determine the potential effect of nanoencapsulated bioactive compounds from different natural sources on human pancreatic cancer.BackgroundPancreatic cancer carries the highest fatality rate among all human cancers because of its high metastatic potential and late presentation at the time of diagnosis. Hence there is a need for improved methods to prevent and treat it. Natural products, such as 3, 3′-diindolylmethane (DIM) and ellagic acid (EA) demonstrated anticancer efficacy against various cancer types. However, DIM is insoluble. Hence, using nanotechnology to encapsulate these compounds in combination with EA might improve their physical and chemical properties and their delivery to the cancer cells.MethodsHuman pancreatic cancer cells, namely SUIT2-luciferase transfected, were used to examine the effects of DIM or EA and their nanoformulation in poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) [PLGA-PEG] nanoparticles (NPs) on SUIT2-luciferase cell viability/proliferation over 24 hrs. Additionally, effects on tumor weight and angiogenesis were determined using the chick chorioallantoic membrane (CAM) tumor implant model.ResultsBoth DIM and EA PLGA-PEG NPs resulted in rapid suppression of pancreatic cancer cell viability/proliferation within 24 hrs (P < 0.01), while the non-encapsulated DIM and EA did not show any significant effect on SUIT2 cancer cell viability or cell proliferation (MTT assay). In the CAM pancreatic cancer cell (SUIT2) implant model, results showed a greater suppression of tumor weight (P < 0.01), tumor cell viability, and tumor angiogenesis (P < 0.01) for DIM NPs and EA NPs and their combinations versus DIM or EA alone.ConclusionNanoformulation of DIM and EA resulted in a more effective suppression of pancreatic cancer cell viability, pancreatic tumor weight, implanted cancer cell viability, and tumor angiogenesis as compared with these bioactive compounds alone.

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.