3,3'-Diindolylmethane (DIM) inhibits the growth and invasion of drug-resistant human cancer cells expressing EGFR mutants.
Rahimi M, Huang KL, Tang CK.
Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown University Medical Center, Washington, DC 20057, United States.
Cancer Lett. 2010 Sep 1;295(1):59-68.
Epidermal Growth Factor Receptor (EGFR) mutants are associated with resistance to chemotherapy, radiation, and targeted therapies. Here we found that the phytochemical 3,3'-Diindolylmethane (DIM) can inhibit the growth and also the invasion of breast cancer, glioma, and non-small cell lung cancer cells regardless of which EGFR mutant is expressed and the drug-resistant phenotype. DIM reduced an array of growth factor signaling pathways and altered cell cycle regulators and apoptotic proteins favoring cell cycle arrest and apoptosis. Therefore, DIM may be used in treatment regimens to inhibit cancer cell growth and invasion, and potentially overcome EGFR mutant-associated drug resistance. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.
PMID: 20299148 [PubMed - indexed for MEDLINE]
Anticancer properties of indole compounds: mechanism of apoptosis induction and role in chemotherapy.
Ahmad A, Sakr WA, Rahman KM.
Department of Pathology, Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201, USA.
Curr Drug Targets. 2010 Jun 1;11(6):652-66.
Indole compounds, obtained from cruciferous vegetables, have been investigated for their putative anti-cancer properties. Studies with indole-3-carbinol (I3C) and its dimeric product, 3, 3' diindolylmethane (DIM), have indicated efficacy of these compounds against a number of human cancers. Available as well as emerging data suggests that these compounds act on a number of cellular signaling pathways leading to their observed biological effects. Such pleiotropic effects of these compounds are also considered crucial for their chemosensitization activity wherein they help reduce the toxicity and resistance against conventional chemotherapeutic drugs. These observations have major clinical implications especially in chemotherapy. Through this review, we have attempted to update current understanding on the state of anti-cancer research involving indole compounds. We have also summarized the available literature on modulatory effects of indoles on molecular targets such as survivin, uPA/uPAR and signaling pathways such as the NF-kappaB pathway, which are important for the apoptosis-inducing and chemosensitizing properties of these compounds.
PMID: 20298156 [PubMed - in process]
Activation of Chk2 by 3,3' Diinolylmethane is required for causing G2/M cell cycle arrest in human ovarian cancer cells.
Kandala PK, Srivastava SK.
Texas Tech University Health Sciences Center.
Mol Pharmacol. 2010 May 5.
We evaluated the effect of 3,3'-diindolylmethane (DIM) in ovarian cancer cells. DIM treatment inhibited the growth of SKOV-3, TOV-21G and OVCAR-3 ovarian cancer cells in both dose and time-dependent manner with effective concentrations ranging from 40muM to 100muM. Growth inhibitory effects of DIM were mediated by cell cycle arrest in G2/M phase in all the three cell lines. G2/M arrest was associated with DNA damage as indicated by phosphorylation of H(2)A.X at Ser 139 and activation of Chk2 in all the three cell lines. Other G2/M regulatory molecules such as Cdc25C, Cdk1, Cyclin B1 were downregulated by DIM. Cycloheximide or Chk2 inhibitor pretreatment abrogated not only activation of Chk2 but also G2/M arrest and apoptosis mediated by DIM. To further establish the involvement of Chk2 in DIM-mediated G2/M arrest, cells were transfected with dominant negative Chk2 (DN-Chk2). Blocking Chk2 activation by DN-Chk2 completely protected cells from DIM- mediated G2/M arrest. These results were further confirmed in Chk2 knock out DT40 lymphoma cells where DIM failed to cause cell cycle arrest. These results clearly indicate the requirement of Chk2 activation to cause G2/M arrest by DIM in ovarian cancer cells. Moreover, blocking Chk2 activation also abrogates the apoptosis-inducing effects of DIM. Further our results show that DIM treatment cause ROS generation. Blocking ROS generation by NAC protect the cells from DIM- mediated G2/M arrest and apoptosis. Our results establish Chk2 as a potent molecular target of DIM in ovarian cancer cells and provide the rationale for further clinical investigation of DIM.
PMID: 20444961 [PubMed - as supplied by publisher]
Concurrent inhibition of NF-kappaB, cyclooxygenase-2, and epidermal growth factor receptor leads to greater anti-tumor activity in pancreatic cancer.
Ali S, Banerjee S, Schaffert JM, El-Rayes BF, Philip PA, Sarkar FH.
Division of Hematology/Oncology, Karmanos Cancer Institute, Wayne State University, Detroit, Michigan 48201, USA.
J Cell Biochem. 2010 May;110(1):171-81.
Inactivation of survival pathways such as NF-kappaB, cyclooxygenase (COX-2), or epidermal growth factor receptor (EGFR) signaling individually may not be sufficient for the treatment of advanced pancreatic cancer (PC) as suggested by recent clinical trials. 3,3'-Diindolylmethane (B-DIM) is an inhibitor of NF-kappaB and COX-2 and is a well-known chemopreventive agent. We hypothesized that the inhibition of NF-kappaB and COX-2 by B-DIM concurrently with the inhibition of EGFR by erlotinib will potentiate the anti-tumor effects of cytotoxic drug gemcitabine, which has been tested both in vitro and in vivo. Inhibition of viable cells in seven PC cell lines treated with B-DIM, erlotinib, or gemcitabine alone or their combinations was evaluated using 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Significant inhibition in cell viability was observed in PC cells expressing high levels of COX-2, EGFR, and NF-kappaB proteins. The observed inhibition was associated with an increase in apoptosis as assessed by ELISA. A significant down-regulation in the expression of COX-2, NF-kappaB, and EGFR in BxPC-3, COLO-357, and HPAC cells was observed, suggesting that simultaneous targeting of EGFR, NF-kappaB, and COX-2 is more effective than targeting either signaling pathway separately. Our in vitro results were further supported by in vivo studies showing that B-DIM in combination with erlotinib and gemcitabine was significantly more effective than individual agents. Based on our preclinical in vitro and in vivo results, we conclude that this multi-targeted combination could be developed for the treatment of PC patients whose tumors express high levels of COX-2, EGFR, and NF-kappaB. (c) 2010 Wiley-Liss, Inc.
PMID: 20213764 [PubMed - in process]
miR-146a suppresses invasion of pancreatic cancer cells.
Li Y, Vandenboom TG 2nd, Wang Z, Kong D, Ali S, Philip PA, Sarkar FH.
Departments of Pathology and Internal Medicine, Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Cancer Res. 2010 Feb 15;70(4):1486-95. Epub 2010 Feb 2.
The aggressive course of pancreatic cancer is believed to reflect its unusually invasive and metastatic nature, which is associated with epidermal growth factor receptor (EGFR) overexpression and NF-kappaB activation. MicroRNAs (miRNA) have been implicated in the regulation of various pathobiological processes in cancer, including metastasis in pancreatic cancer and in other human malignancies. In this study, we report lower expression of miR-146a in pancreatic cancer cells compared with normal human pancreatic duct epithelial cells. Reexpression of miR-146a inhibited the invasive capacity of pancreatic cancer cells with concomitant downregulation of EGFR and the NF-kappaB regulatory kinase interleukin 1 receptor-associated kinase 1 (IRAK-1). Cellular mechanism studies revealed crosstalk between EGFR, IRAK-1, IkappaBalpha, NF-kappaB, and MTA-2, a transcription factor that regulates metastasis. Treatment of pancreatic cancer cells with the natural products 3,3'-diinodolylmethane (DIM) or isoflavone, which increased miR-146a expression, caused a downregulation of EGFR, MTA-2, IRAK-1, and NF-kappaB, resulting in an inhibition of pancreatic cancer cell invasion. Our findings reveal DIM and isoflavone as nontoxic activators of a miRNA that can block pancreatic cancer cell invasion and metastasis, offering starting points to design novel anticancer agents.
PMID: 20124483 [PubMed - indexed for MEDLINE]
Chemopreventive agent 3,3'-diindolylmethane selectively induces proteasomal degradation of class I histone deacetylases.
Li Y, Li X, Guo B.
Department of Pharmaceutical Sciences, College of Pharmacy, North Dakota State University, Fargo, North Dakota 58108, USA.
Cancer Res. 2010 Jan 15;70(2):646-54.
3,3'-Diindolylmethane (DIM) is an anticancer agent that induces cell cycle arrest and apoptosis through unknown mechanisms. Here, we report that DIM can selectively induce proteasome-mediated degradation of class I histone deacetylases (HDAC1, HDAC2, HDAC3, and HDAC8) without affecting the class II HDAC proteins. DIM induced downregulation of class I HDACs in human colon cancer cells in vitro and in vivo in tumor xenografts. HDAC depletion relieved HDAC-mediated transcriptional inhibition of the cyclin-dependent kinase inhibitors p21WAF1 and p27KIP2, significantly increasing their expression and triggering cell cycle arrest in the G(2) phase of the cell cycle. Additionally, HDAC depletion was associated with an induction of DNA damage that triggered apoptosis. Our findings indicate that DIM acts to selectively target the degradation of class I HDACs.
PMID: 20068155 [PubMed - indexed for MEDLINE]
Oral administration of 3,3'-diindolylmethane inhibits lung metastasis of 4T1 murine mammary carcinoma cells in BALB/c mice.
Kim EJ, Shin M, Park H, Hong JE, Shin HK, Kim J, Kwon DY, Park JH.
Center for Efficacy Assessment and Development of Functional Foods and Drugs, Hallym University, Chuncheon, Korea.
J Nutr. 2009 Dec;139(12):2373-9. Epub 2009 Oct 28.
3,3'-diindolylmethane (DIM) is the major in vivo product of the acid-catalyzed oligomerization of indole-3-carbinol present in cruciferous vegetables, and it has been shown to exhibit anticancer properties. In this study, we assessed the effects of DIM on the metastasis of 4T1 mouse mammary carcinoma cells. In vitro culture studies showed that DIM dose-dependently inhibited the migration, invasion, and adhesion of 4T1 cells at concentrations of 0-10 micromol/L without attendant changes in cell viability. In an in vivo lung metastasis model, 4T1 cells (2 x 10(5) cells/mouse) were injected into the tail veins of syngeneic female BALB/c mice. Beginning on the second day, the mice were subjected to gavage with 0-10 mg DIM/(kg body weight x d) for 13 d. Oral DIM administration resulted in a marked reduction in the number of pulmonary tumor nodules. DIM treatment significantly reduced the levels of matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, and vascular cell adhesion molecule (VCAM)-1 and increased TIMP-2 levels in the sera and lungs of mice injected with 4T1 cells. Additionally, DIM treatment reduced the serum concentrations of interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)alpha. We have demonstrated that DIM profoundly inhibits the lung metastasis of 4T1 cells, which was accompanied by reduced levels of MMP, adhesion molecules, and proinflammatory cytokines. These results indicate that DIM has potential as an antimetastatic agent for the treatment of breast cancer.
PMID: 19864400 [PubMed - indexed for MEDLINE]
Up-regulation of miR-200 and let-7 by natural agents leads to the reversal of epithelial-to-mesenchymal transition in gemcitabine-resistant pancreatic cancer cells.
Li Y, VandenBoom TG 2nd, Kong D, Wang Z, Ali S, Philip PA, Sarkar FH.
Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Cancer Res. 2009 Aug 15;69(16):6704-12.
Pancreatic cancer is the fourth most common cause of cancer death in the United States, and the aggressiveness of pancreatic cancer is in part due to its intrinsic and extrinsic drug resistance characteristics, which are also associated with the acquisition of epithelial-to-mesenchymal transition (EMT). Emerging evidence also suggests that the processes of EMT are regulated by the expression status of many microRNAs (miRNA), which are believed to function as key regulators of various biological and pathologic processes during tumor development and progression. In the present study, we compared the expression of miRNAs between gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer cells and investigated whether the treatment of cells with "natural agents" [3,3'-diindolylmethane (DIM) or isoflavone] could affect the expression of miRNAs. We found that the expression of miR-200b, miR-200c, let-7b, let-7c, let-7d, and let-7e was significantly down-regulated in gemcitabine-resistant cells, which showed EMT characteristics such as elongated fibroblastoid morphology, lower expression of epithelial marker E-cadherin, and higher expression of mesenchymal markers such as vimentin and ZEB1. Moreover, we found that reexpression of miR-200 by transfection studies or treatment of gemcitabine-resistant cells with either DIM or isoflavone resulted in the down-regulation of ZEB1, slug, and vimentin, which was consistent with morphologic reversal of EMT phenotype leading to epithelial morphology. These results provide experimental evidence, for the first time, that DIM and isoflavone could function as miRNA regulators leading to the reversal of EMT phenotype, which is likely to be important for designing novel therapies for pancreatic cancer.
PMID: 19654291 [PubMed - indexed for MEDLINE]
3,3'-Diindolylmethane enhances chemosensitivity of multiple chemotherapeutic agents in pancreatic cancer.
Banerjee S, Wang Z, Kong D, Sarkar FH.
Department of Pathology, Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Cancer Res. 2009 Jul 1;69(13):5592-600.
Clinical management of pancreatic cancer is a major problem, which is in part due to both de novo and acquired resistance to conventional therapeutics. Here, we present in vitro and in vivo preclinical evidence in support of chemosensitization of pancreatic cancer cells by 3,3-diindolylmethane (DIM), a natural compound that can be easily obtained by consuming cruciferous vegetables. DIM pretreatment of pancreatic cancer cells led to a significantly increased apoptosis (P < 0.01) with suboptimal concentrations of chemotherapeutic agents (cisplatin, gemcitabine, and oxaliplatin) compared with monotherapy. It is known that resistance to chemotherapy in pancreatic cancer is associated with constitutively activated nuclear factor-kappaB (NF-kappaB), which becomes further activated by chemotherapeutic drugs. Our data provide mechanistic evidence for the first time showing that DIM potentiates the killing of pancreatic cancer cells by down-regulation of constitutive as well as drug-induced activation of NF-kappaB and its downstream genes (Bcl-xL, XIAP, cIAP, and survivin). Most importantly, using an orthotopic animal model, we found reduction in tumor size (P < 0.001) when DIM was given in combination with oxaliplatin compared with monotherapy. This was accompanied by loss of phospho-p65 and down-regulation of NF-kappaB activity and its downstream genes (Bcl-xL, survivin, and XIAP), which correlated with reduced cell proliferation (as assessed by Ki-67 immunostaining of tumor specimens) and evidence of apoptosis [as assessed by poly(ADP-ribose) polymerase cleavage and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining]. These results provide strong in vivo evidence in support of our hypothesis that DIM could abrogate chemotherapeutic drug (cisplatin, gemcitabine, and/or oxaliplatin)-induced activation of NF-kappaB, resulting in the chemosensitization of pancreatic tumors to conventional therapeutics.
PMID: 19531648 [PubMed - indexed for MEDLINE]
Bcl-2 family-mediated apoptotic effects of 3,3'-diindolylmethane (DIM) in human breast cancer cells.
Hong C, Firestone GL, Bjeldanes LF.
3,3'-Diindolylmethane (DIM) is a major in vivo derivative of the putative anticancer agent indole-3-carbinol (I3C), which is present in vegetables of the Brassica genus.
At concentrations above 10 mg, DIM inhibited DNA synthesis and cell proliferation in both estrogen receptor replete (MCF-7) and deficient (MDA-MB-231) human breast cancer cells in a concentration- and time-dependent manner. These antiproliferative effects were accompanied by characteristic indications of programmed cell death in both cell lines, including externalization of phosphatidylserine, chromatin condensation, and DNA fragmentation.
Furthermore, Western and Northern blot analyses, as well as coimmunoprecipitation assays, revealed that in both MCF-7 and MDA-MB-231 cells, DIM treatment decreased total transcript and protein levels of the apoptosis inhibitory protein Bcl-2, and the amount of Bcl-2 bound to the pro-apoptotic protein Bax. DIM treatment also caused an increase in Bax protein levels, but did not affect the level of Bax that was bound to Bcl-2.
As a functional test of the role of Bcl-2 down-regulation in the DIM-induced apoptotic response, ectopic expression of Bcl-2 in MCF-7 cells was shown to attenuate the apoptotic effect of DIM. These results demonstrate that DIM can induce apoptosis in breast cancer cells independent of estrogen receptor status by a process that is mediated by the modulated expression of the Bax/Bcl-2 family of apoptotic regulatory factors.
PMID: 11931841 [PubMed - as supplied by publisher]
Fate of indole-3-carbinol in cultured human breast tumor cells.
Staub RE, Feng C, Onisko B, Bailey GS, Firestone GL, Bjeldanes
Indole-3-carbinol (I3C), a natural component of Brassica vegetables, is a promising cancer preventive agent that can reduce the incidence of tumors in reproductive organs when administered in the diet.
Here we report on the metabolic fate of radiolabeled I3C in MCF-7 cells. I3C was surprisingly inert to metabolism by these cells with a half-life in medium of approximately 40 h. [(3)H]I3C levels in media declined at a similar rate whether incubation was with cultured cells or in cell-free medium. Neither [(3)H]I3C nor its modified products accumulated in MCF-7 cells and only low levels of intact I3C were detected in cellular fractions.
In contrast, I3C represented over 30% of the radioactivity in media even after 72 h. In cytosolic fractions, the 3-(cystein-S-ylmethyl) and 3-(glutathion-S-ylmethyl) conjugates of [(3)H]I3C were the primary conversion products identified after 16 h, representing approximately 50% and approximately 15% of the radioactivity in these fractions, respectively.
The reaction of I3C with thiols appears to be nonenzymatic since the cysteine conjugate is produced when I3C is incubated in cell-free medium containing additional cysteine. Both cellular and extracellular proteins were nonspecifically modified with [(3)H]I3C. In medium, proteins are radiolabeled even in the absence of cells, indicating again that enzymatic activation was not required. I3C was also oxidized to indole-3-carboxaldehyde and indole-3-carboxylic acid in culture medium independent of cells.
Unexpectedly, 3,3'-diindolylmethane (DIM), an I3C product with in vitro and in vivo biological activity, was detected in cellular fractions and appeared to accumulate in the nucleus, representing approximately 40% of this fraction after 72 h treatment. These findings suggest that MCF-7 cells do not vigorously metabolize I3C and that the major route of reaction is with cellular thiols such as glutathione and proteins. The accumulation of DIM in the nucleus suggests that this product may have a role in the cellular biological activities of I3C.3
PMID: 11849035 [PubMed - in process] Department of Nutritional Sciences and Toxicology, University of California, Berkeley, California 94720, USA.
Aryl hydrocarbon receptor-mediated antiestrogenic and antitumorigenic activity of diindolylmethane (DIM).
Chen I, McDougal A, Wang F, Safe S.
Phytochemicals such as indole-3-carbinol (I3C) and sulforaphane are components of cruciferous vegetables which exhibit antitumorigenic activity associated with altered carcinogen metabolism and detoxification. Diindolylmethane (DIM) is a major acid-catalyzed metabolite of I3C formed in the gut that binds to the aryl hydrocarbon receptor (AhR) and treatment of MCF-7 human breast cancer cells with 10-50 microM DIM resulted in rapid formation of the nuclear AhR complex and induction of CYP1A1 gene expression was observed at concentrations >50 microM. Previous studies have demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a high affinity AhR ligand, inhibits 17beta-estradiol (E2)-induced responses in MCF-7 cells and growth of E2-dependent 7,12-dimethylbenzanthracene (DMBA)-induced mammary tumors in female Sprague-Dawley rats.
Results of this study show that like TCDD, DIM inhibits E2-induced proliferation of MCF-7 cells, reporter gene activity in cells transiently transfected with an E2-responsive plasmid (containing a frog vitellogenin A2 gene promoter insert) and down-regulates the nuclear estrogen receptor. Moreover, DIM (5 mg/kg every other day) also inhibits DMBA-induced mammary tumor growth in Sprague-Dawley rats and this was not accompanied by induction of hepaticCYP1A1-dependent activity.
Thus, DIM represents a new class of relatively non-toxic AhR-based antiestrogens that inhibit E2-dependent tumor growth in rodents and current studies are focused on development of analogs for clinical treatment of breast cancer.
Selective cytostatic and cytotoxic effects of glucosinolates hydrolysis products on human colon cancer cells in vitro.
Gamet-Payrastre L, Lumeau S, Gasc N, Cassar G, Rollin P,
Glucosinolates hydrolysis products are attracting increasing attention since many studies have suggested that they may be involved in the anticarcinogenic property of cruciferous vegetables.
In this study, we show that diindolylmethane (DIM) and sulforaphane, produced during the hydrolysis of glucobrassicin and glucoraphanin, respectively, exert a dose-dependent cytotoxicity on human colon adenocarcinoma HT29 cells. Moreover, these products are able to inhibit quiescent cells to re-enter the cell cycle. Interestingly, our results clearly show that low doses of DIM and sulforaphane, although very effective on undifferentiated intestinal HT29 cells, do not affect the viability of the differentiated CaCo2 cells. The reversibility of their effects has also been tested and is discussed.
3,3'-Diindolylmethane induces apoptosis in human cancer cells.
Ge X, Yannai S, Rennert G, Gruener N, Fares FA.
3,3'-Diindolylmethane is a dimer of indole-3-carbinol formed both in vivo and in vitro. In this study, human cancer cells MCF-7 (with wild-type p53), T47-D (mutant p53), and Saos-2 (deficient in p53 gene), were used to examine the anticancer activities of 3,3'-diindolylmethane. The dose-dependent growth inhibitory effect was found in all these cell lines.
Exposure of the cells to 50 microM solution of 3,3'-diindolylmethane for 48 h, apoptosis (programmed cell death) was evidenced by the characteristic morphology of cell nuclei under fluorescence microscope and the DNA "ladder" in agarose gel electrophoresis. The percentage of apoptotic cells in each cell line was found to be 12% for MCF-7, 14% for T47D and 13% for Saos2 cells. Exposure of MCF-7 cells to 100 microM 3,3'-diindolylmethane for 24 h, 19% of apoptotic cells were detected by flow cytometry analysis. The lowest dose required for induction of apoptosis in MCF-7 cells was found to be 10 microM after 72 h incubation. Western blot showed that wild-type p53 protein was unchanged after MCF-7 cells had been exposed to 50 microM 3,3'-diindolylmethane for 8 h.
This study provides evidence that 3,3'-diindolylmethane induces apoptosis in human cancer cells and that the induction of apoptosis is independent of p53 pathway.
Inhibition of polycyclic aromatic hydrocarbon-induced neoplasia by naturally occurring indoles.
Wattenberg LW, Loub WD.
Indole-3-carbinol, 3,3'-diindolylmethane, and indole-3-acetonitrile, three indoles occurring in edible cruciferous vegetables, have been studied for their effects on 7,12-dimethylbenz(a)anthracene-induced mammary tumor formation in female Sprague-Dawley rats and on benzo(a)pyrene-induced neoplasia of the forestomach in female ICR/Ha mice.
When given by p.o. intubation 20 hr prior to 7,12-dimethylbenz(a)anthracene administration, indole-3-carbinol and 3,3'-diindolylmethane had an inhibitory effect on mammary tumor formation, but indole-3-acetonitrile was inactive. Indole-3-carbinol when added to the diet for 8 days prior to challenge with 7,12-dimethylbenz(a)anthracene inhibited mammary tumor formation, whereas indole-3-acetonitrile did not. Dietary administration of all three indoles inhibited benzo(a)pyrene-induced neoplasia of the forestomach in ICR/Ha mice.
The identification of dietary constituents that can inhibit chemical carcinogens ultimately may be of value in understanding the balance of factors that determines the neoplastic response to these cancer-producing agents in the environment.