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Lee W. Wattenberg, who spent his entire career at the University of Minnesota, was a true pioneer in the field of chemoprevention. This paper is a tribute to his groundbreaking research which uncovered the cancer prevention properties of many dietary compounds, including those discussed here in some detail-indole-3-carbinol and diindolylmethane. These compounds occur as glucosinolate conjugates in cruciferous vegetables and are released when one chews or otherwise macerates the vegetable. They have numerous beneficial effects including the ability to prevent cancer in laboratory animals treated with carcinogens. We review some of the early work on indole-3-carbinol and diindolylmethane which spurred subsequent studies on their efficacy and molecular mechanisms of prevention. We also present unique data on field conditions that affect levels of their glucosinolate precursors in vegetables and on the release of diindolylmethane in people who consume cruciferous vegetables., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chronic inflammation is an important risk factor for lung cancer. Therefore, identification of chemopreventive agents that suppress inflammation-driven lung cancer is indispensable. We studied the efficacy of combinations of indole-3-carbinol (I3C) and silibinin (Sil), 20 µmol/g diet each, against mouse lung tumors induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and driven by lipopolysaccharide (LPS), a potent inflammatory agent and constituent of tobacco smoke. Mice treated with NNK + LPS developed 14.7±4.1 lung tumors/mouse, whereas mice treated with NNK + LPS and given combinations of I3C and Sil had 7.1±4.5 lung tumors/mouse, corresponding to a significant reduction of 52%. Moreover, the number of largest tumors (>1.0mm) was significantly reduced from 6.3±2.9 lung tumors/mouse in the control group to 1.0±1.3 and 1.6±1.8 lung tumors/mouse in mice given I3C + Sil and I3C alone, respectively. These results were paralleled by significant reductions in the level of proinflammatory and procarcinogenic proteins (pSTAT3, pIκBα and COX-2) and proteins that regulate cell proliferation (pAkt, cyclin D1, CDKs 2, 4, 6 and pRB). Further studies in premalignant bronchial cells showed that the antiproliferative effects of I3C + Sil were higher than the individual compounds and these effects were mediated by targeting cyclin D1, CDKs 2, 4 and 6 and pRB. I3C + Sil suppressed cyclin D1 by reducing its messenger RNA level and by enhancing its proteasomal degradation. Our results showed the potential lung cancer chemopreventive effects of I3C + Sil in smokers/former smokers with chronic pulmonary inflammatory conditions., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Inflammatory conditions of the lung such as chronic obstructive pulmonary disease (COPD) are known to increase lung cancer risk, particularly lung squamous cell carcinoma (LSCC). In the present study, we developed a mouse model of inflammation-driven LSCC that was induced by N-nitroso-trischloroethylurea (NTCU) and enhanced by lipopolysaccharide (LPS), a potent proinflammatory agent contained in tobacco and tobacco smoke, and determined the chemopreventive effects of BioResponse diindolylmethane (DIM) in the same model. Compared with mice treated with NTCU alone, mice treated with the combination of NTCU and LPS had a 9-fold increase in the number of bronchioles with LSCC. Also, compared with mice treated with LPS alone, mice treated with NTCU plus LPS showed significantly increased expression of the inflammatory cytokines IL1α, IL6, and TNFα (all three increased about 7-fold). Parallel to the increased cytokine gene expression, the NTCU plus LPS-treated group exhibited significantly enhanced activation of NF-κB, STAT3, ERK, p-38, and Akt, expression of p53, COX-2, and Mcl-1, and NF-κB- and STAT3-DNA binding in the lung. Dietary administration of DIM (10 μmol/g diet or 2,460 ppm) to mice treated with NTCU plus LPS reduced the incidence of LSCC by 2-fold, suppressed activation/expression of proinflammatory and procarcinogenic proteins and NF-κB- and STAT3-DNA binding, but not the expression of cytokines and p53. This study highlights the potential significance of our mouse model to identify promising drugs or dietary agents for the chemoprevention of human LSCC and that DIM is a very good candidate for clinical lung cancer chemoprevention trials., (©2014 American Association for Cancer Research.)

Indole-3-carbinol (I3C), a constituent of commonly consumed Brassica vegetables, has been shown to have anticancer effects in a variety of preclinical models of lung cancer. However, it has shown only limited efficacy in clinical trials, likely due to its poor oral bioavailability. Intranasal administration of I3C has the potential to enhance the pulmonary accumulation of the drug, thereby improving its availability at the target site of action. In this study, we developed a liposomal formulation of I3C and evaluated its lung delivery and chemopreventive potential in tobacco smoke carcinogen [4-(methylnitro-samino)-1-(3-pyridyl)-1-butanone (NNK)]-treated mice. Intranasal administration of I3C liposomes led to a ∼100-fold higher lung exposure of I3C than the oral route of administration. Further, intranasal delivery of liposomal I3C led to a significant reduction (37%; p<0.05) in the levels of the DNA adduct formation induced by NNK treatment. Liposomal I3C also significantly increased (by 10-fold) the expression of CYP1A1, a cytochrome P450 enzyme known to increase the detoxification of chemical carcinogens by enhancing their metabolism. Overall, our findings demonstrate that intranasal administration of liposomal I3C has the potential to significantly improve the efficacy of I3C for lung cancer chemoprevention., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The main reasons for the failure of most chemopreventive agents during clinical trials are poor in vivo bioavailability and dose-limiting side effects. One potential approach to surmount these problems in lung cancer chemoprevention trials could be direct delivery of agents into the pulmonary tissue. In this study, we assessed the efficacy of intranasally delivered bio-response diindolylmethane (BRD) against 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in mice. Mice treated with NNK (two doses of 50mg/kg at an interval of a week, intraperitoneal) developed 16.3±2.9 lung tumors per mouse. Post-carcinogen administration of BRD, via intranasal instillation, for 24 weeks, twice a week, at a dose of 2mg per mouse (0.6mg pure diindolylmethane per mouse) reduced the lung tumor multiplicity to 4.6±2.2 tumors per mouse (72% reduction). Likewise, large tumors (>1mm) were almost completely abolished and multiplicities of tumors with a size of 0.5-1mm were reduced by 74%. Tumor volume was also reduced by 82%. Further studies using an in vitro model of lung tumorigenesis showed that BRD exhibited pronounced antiproliferative and apoptotic effects in premalignant and malignant bronchial cells but only minimal effects in parental immortalized cells through, at least in part, suppression of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. These results showed the potent lung tumor inhibitory activities of low doses of BRD given via intranasal instillation and, therefore, intranasal delivery of BRD holds a great promise for lung cancer chemoprevention in subjects at high risk to develop lung cancer.

We studied the chemopreventive efficacy of indole-3-carbinol (I3C), a phytochemical found in cruciferous vegetables, to inhibit tobacco carcinogen-induced lung adenocarcinoma in A/J mice when given following post-initiation or progression protocol. Moreover, we assessed the potential mechanisms responsible for the anticancer effects of I3C. Post-initiation administration of I3C decreased the multiplicity of surface tumors as well as all forms of histopathological lesions, including adenocarcinoma, whereas administration of the compound during tumor progression failed to decrease the multiplicity of surface tumors and early forms of microscopic lesions but reduced the frequency of adenocarcinoma. Mechanistic studies in A549 lung adenocarcinoma cells indicated that the lung cancer preventive effects of I3C are mediated, at least in part, via modulation of the receptor tyrosine kinase/PI3K/Akt signaling pathway., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

In earlier studies, we demonstrated the efficacy of indole-3-carbinol (I3C) against lung adenocarcinoma in A/J mice. However, these effects were accompanied by reductions in body weight gain. We therefore assessed if combinations of low doses of I3C with silibinin could inhibit lung tumorigenesis without causing undesirable side effects. In in vitro assays with A549 and H460 lung cancer cells, exposure of the cells to a mixture of low concentrations of I3C (50 μM) plus silibinin (50 μM) for 72 h caused inhibition of cell growth and extracellular signal-regulated kinase (ERK) and Akt activation and induction of apoptosis, whereas the individual agents did not have any effect. In mice pretreated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and given I3C (10 μmol/g diet) plus silibinin (7 μmol/g diet), multiplicities of tumors on the surface of the lung and adenocarcinoma were reduced by 60 and 95%, respectively. The individual effects of I3C and silibinin were relatively weaker: 43 and 36% reductions, respectively, in the multiplicity of tumors on the surface of the lung and 83 and 50% reductions, respectively, in the number of adenocarcinoma. Also, the expression of phospho-Akt, phospho-ERK and cyclin D1 and poly (ADP-ribose) polymerase cleavage were strongly modulated by I3C plus silibinin than by I3C or silibinin alone, suggesting that the chemopreventive activities of the mixture could be mediated, at least partly, via modulation of the level of these proteins. Taken together, our findings showed that mixtures of I3C and silibinin are more potent than the individual compounds for the chemoprevention of lung cancer in A/J mice.

In an extension of our earlier studies, we examined the inhibitory effects of N-acetyl-S-(N-2-phenethylthiocarbamoyl)-l-cysteine (PEITC-NAC), myo-inositol (MI) and indole-3-carbinol (I3C) or 3,3'-diindolylmethane (DIM), alone and in combination, on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) plus benzo[a]pyrene (BaP)-induced A/J mouse lung tumorigenesis and proliferation of A549 cells and human bronchial epithelial cells (HBECs) and relevant potential mechanisms. Mice treated with NNK plus BaP and fed non-supplemented diet had 13.0 + or - 4.1 lung tumors per mouse. Dietary feeding of mice with PEITC-NAC (5 mumol/g diet), I3C (5 mumol/g diet) or MI (56 mumol/g diet), beginning at 50% in the carcinogen treatment phase, significantly reduced tumor multiplicity to 8.2 + or - 2.0, 8.4 + or - 1.5 and 6.8 + or - 1.7 tumors per mouse, respectively. In mice given combinations of the chemopreventive agents, lung tumor multiplicity was significantly reduced to 6.3 + or - 2.2, 4.9 + or - 1.8, 4.8 + or - 1.9 and 3.6 + or - 1.4 by PEITC-NAC plus I3C, PEITC-NAC plus MI, I3C plus MI or PEITC-NAC plus I3C plus MI, respectively. Post-carcinogen administration of combinations of the agents also caused significant but weaker effects. Assessment of the anti-proliferative effects of the individual agents or their combinations showed significant reductions in the proliferation of cigarette smoke condensate (CSC)-pretreated HBEC (reduction by 30-41% at 48 h and 41-58% at 72 h) and A549 cells (30-43% at 48 h and 40-59% at 72 h), but not in dimethyl sulfoxide-pretreated HBEC. Combinatorial treatment with the agents also caused marked reductions in the activation of Akt, extracellular signal-regulated kinase and nuclear factor-kappaB in lung tumor tissues, CSC-pretreated HBEC and A549 cells. In conclusion, our studies demonstrated the promise of combinations of PEITC-NAC, I3C/DIM and MI for the chemoprevention of lung carcinogenesis in current and former smokers.

In previous studies, we reported that indole-3-carbinol (I3C) and myo-inositol (MI) inhibit lung adenoma induced by tobacco smoke carcinogens in A/J mice. In this paper, we extended our work and examined the effects of I3C (70 or 30 micromol/g diet) and MI (56 micromol/g diet) against vinyl carbamate (VC)-induced lung adenocarcinoma by administering the agents from 1 week after the second of two injections of VC until termination of the study at week 18. The higher dose of I3C decreased multiplicities of tumors on the surface of the lung (26%, P = 0.0005), carcinoma incidence (38%), multiplicity (67%, P < 0.0001) and size (complete abolition of carcinoma with an area of >1.0 cm(2)) as well as adenoma with cellular pleomorphism (46%, P < 0.0001). The lower dose of I3C was less effective. MI decreased multiplicities of pulmonary surface tumors (20%, P = 0.0005), adenoma with cellular pleomorphism (40%, P < 0.0001) and lung adenoma (52%, P < 0.0001) and the proportion of the biggest carcinoma (carcinoma with an area of >1.0 cm(2), P < 0.05). Immunoblot analyses of lung tissues for potential target identification showed that I3C (70 micromol/g diet) inhibits IkappaBalpha degradation, nuclear factor-kappaB activation, expression of cyclooxygenase-2, phospho-Akt and fatty acid synthase (FAS) and activates caspase-3 and poly ADP ribose polymerase cleavage. The effect of MI was limited to inhibition of phospho-Akt and FAS expression. Our data show that I3C and MI inhibit lung carcinoma and provide a basis for future evaluation of these compounds in clinical trials as chemopreventive agents for current and former smokers.

MicroRNAs (miRNAs) are small, non-protein-coding RNAs that can function as tumor suppressors or oncogenes. Deregulation of miRNA expression has been reported in lung cancer. However, modulation of miRNA expression by chemopreventive agents remains to be defined. In the present study, we examined if the chemopreventive agent indole-3-carbinol (I3C) reversed vinyl carbamate (VC)-induced deregulation of miRNA levels in lung tissues of female A/J mice. Lung tissues were obtained from a previous chemoprevention study, in which mice were treated with VC and given I3C in the diet for 15 weeks. Microarray studies revealed alterations in the expression of a number of miRNAs in lung tumors relative to that of normal lungs. miR-21, mir-31, miR-130a, miR-146b and miR-377 were consistently upregulated, whereas miR-1 and miR-143 were downregulated in lung tumors relative to normal lungs. In mice treated with VC and given I3C in the diet, levels of miR-21, mir-31, miR-130a, miR-146b and miR-377 were reduced relative to the level in mice treated with the carcinogen only. The results of the microarray study were confirmed by quantitative reverse transcription-polymerase chain reaction and gel analysis of polymerase chain reaction products. Further studies with miR-21 indicated that phosphatase and tensin homolog, programmed cell death 4 and rich protein with Kazal motifs are potential targets for the oncogenic effect of miR-21 and the chemopreventive activity of I3C. Taken together, we showed here that miRNAs are deregulated during VC-induced mouse lung tumorigenesis and their levels are modulated by I3C. Therefore, miRNAs and their target genes are promising biomarkers for the diagnosis of lung cancer and efficacy of chemopreventive/chemotherapeutic agents.

Recently, we reported inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) plus benzo(a)pyrene (BaP)-induced lung tumorigenesis in A/J mice by indole-3-carbinol (I3C; 112 micromol/g diet) administered beginning at 50% in the carcinogen treatment phase. In this study, we examined the dose-dependent and postcarcinogen tumor-inhibitory activities of I3C. A mixture of NNK plus BaP (2 micromol each) administered by gavage as eight biweekly doses caused 21.1 +/- 5.2 lung tumors per mouse. Carcinogen-treated mice given diets containing I3C at 1, 10, 30, 71, and 112 micromol/g, beginning at 50% in the carcinogen treatment phase, had 17.9 +/- 6.1, 10.4 +/- 3.7, 9.8 +/- 5.1, 5.2 +/- 4.0, and 2.5 +/- 2.4 lung tumors per mouse, corresponding to reductions by 15%, 51%, 53%, 75%, and 88%, respectively. All reductions, except at the lowest dose level (1 micromol I3C/g diet), were significant (P < 0.001). Similarly, administration of I3C (112 micromol/g diet) beginning 1 week after the last dose of the carcinogen significantly reduced NNK plus BaP-induced lung tumor multiplicity to 5.6 +/- 3.5, corresponding to a reduction by 74%. Analyses of cell proliferation and apoptosis markers revealed that I3C reduced the number of Ki-67-positive cells and expression of proliferating cell nuclear antigen, phospho-Akt, and phospho-BAD and increased cleavage of poly(ADP-ribose) polymerase, suggesting that the lung tumor inhibitory effects of I3C were mediated, at least partly, through inhibition of cell proliferation and induction of apoptosis. These results clearly show the efficacy of I3C in the prevention of tobacco carcinogen-induced lung tumorigenesis in A/J mice and provide a basis for future evaluation of this compound in clinical trials as a chemopreventive agent for current and former smokers.

We tested the chemopreventive efficacy of indole-3-carbinol (I3C), a constituent of Brassica vegetables, and its major condensation product, 3,3'-diindolylmethane (DIM), against lung tumorigenesis induced by a mixture of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (BaP) in A/J mice. The mixture of NNK plus BaP (2 micromol each) was administered by gavage as eight weekly doses, whereas I3C (112 micromol/g diet) and DIM (2 and 30 micromol/g diet in experiments 1 and 2, respectively) were given in the diet for 23 weeks beginning at 50% of carcinogen treatment. I3C reduced NNK plus BaP-induced tumor multiplicity by 78% in experiment 1 and 86% in experiment 2; the respective reductions in tumor multiplicity by DIM were 5% and 66%. Using a quantitative proteomics method, isobaric tags for relative and absolute quantitation (iTRAQ) coupled with mass spectrometry, we identified and quantified at least 250 proteins in lung tissues. Of these proteins, nine showed differences in relative abundance in lung tissues of carcinogen-treated versus untreated mice: fatty acid synthase, transketolase, pulmonary surfactant-associated protein C (SP-C), L-plastin, annexin A1, and haptoglobin increased, whereas transferrin, alpha-1-antitrypsin, and apolipoprotein A-1 decreased. Supplementation of the diet of carcinogen-treated mice with I3C reduced the level of SP-C, L-plastin, annexin A1, and haptoglobin to that of untreated controls. These results were verified using immunoblotting. We show here that tumor-associated signature proteins are increased during NNK plus BaP-induced lung carcinogenesis, and I3C inhibits this effect, suggesting that the lung tumor chemopreventive activity of I3C might be related to modulation of carcinogen-induced alterations in protein levels.