Xuemin Qian

Indole-3-carbinol inhibited tobacco smoke carcinogen-induced lung adenocarcinoma in A/J mice when administered during the post-initiation or progression phase of lung tumorigenesis

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.

Enhanced inhibition of lung adenocarcinoma by combinatorial treatment with indole-3-carbinol and silibinin in A/J mice

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.

Lipopolysaccharide Enhances Mouse Lung Tumorigenesis: A Model for Inflammation-Driven Lung Cancer

The association between pulmonary inflammation and lung cancer is well established. However, currently there are no appropriate models that recapitulate inflammation-related lung cancer in humans. In the present study, we examined, in 2 tumor bioassays, enhancement by bacterial lipopolysaccharide (LPS) of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)–induced lung tumorigenesis in A/J mice. Mice that were treated with NNK alone developed 29.6 ± 9.8 and 36.2 ± 4.1 lung tumors per mouse in experiments 1 and 2, respectively. Chronic intranasal instillation of LPS to NNK-treated mice increased the multiplicity of lung tumors to 47.3 ± 16.1 and 51.2 ± 4.8 lung tumors per mouse in experiments 1 and 2, corresponding to a significant increase by 60% and 41%, respectively. Moreover, administration of LPS to NNK-pretreated mice significantly increased the multiplicity of larger tumors and histopathologically more advanced lesions (adenoma with dysplasia and adenocarcinoma), macrophage recruitment to the peritumoral area, and expression of inflammation-, cell proliferation-, and survival-related proteins. Overall, our findings demonstrated the promise of the NNK-LPS-A/J mice model to better understand inflammation-driven lung cancer, dissect the molecular pathways involved, and identify more effective preventive and therapeutic agents against lung cancer.

Dimethylaminoparthenolide, a water soluble parthenolide, suppresses lung tumorigenesis through down-regulating the STAT3 signaling pathway.

Lung cancer is the most fatal cancer and development of agents that suppress lung tumorigenesis is a crucial strategy to reduce mortality related to this disease. In the present study, we showed, using an in vitro model of lung tumorigenesis, that dimethylamino-parthenolide (DMAPT), a water soluble parthenolide analog, selectively inhibited the growth and survival of premalignant and malignant cells with minimal effects on parental immortalized cells. These effects were paralleled by suppression of pSTAT3, Mcl-1 and cyclin D1 and PARP cleavage, suggesting that the antiproliferative and apoptotic effects of DMAPT could be mediated, at least in part, via suppression of the STAT3 signaling pathway. Moreover, in tobacco smoke carcinogen-induced lung tumor bioassay in mice, intranasal instillation of low doses of DMAPT significantly reduced the overall lung tumor multiplicity by 39%. Interestingly, the drug was specifically effective (62% reduction) against bigger lung tumors (> 2 mm), which have a higher potential to develop into lung adenocarcinoma. Western immunoblotting analyses of mouse lung tissues indicated significantly lower level of pSTAT3 and Mcl-1 in the carcinogen plus DMAPT group relative to the group treated with the carcinogen only. Given the evidence that STAT3 is activated in more than half of lung cancers and it regulates genes involved in cell proliferation, survival and angiogenesis, DMAPT is a promising agent for lung cancer chemoprevention in subjects who are at high risk of developing this devastating disease.

Combinations of indole-3-carbinol and silibinin suppress inflammation-driven mouse lung tumorigenesis by modulating critical cell cycle regulators

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.

DNA Adduct Formation of 2-Amino-9H-pyrido[2,3-b]indole and 2-Amino-3,4-dimethylimidazo[4,5-f]quinoline in Mouse Liver and Extrahepatic Tissues During a Subchronic Feeding Study

Tobacco smoking is a risk factor for cancers of the liver and gastrointestinal (GI) tract, but the causal agents responsible for these cancers are uncertain. 2-Amino-9H-pyrido[2,3-b]indole (AαC) is an abundant heterocyclic aromatic amine present in tobacco smoke. AαC is a liver carcinogen and both a transgene mutagen and inducer of aberrant crypt foci in the colon of mice. We hypothesize that AαC may contribute to DNA damage and tumorigenesis in these organs of smokers. The potential of AαC to induce DNA adduct formation in liver, organs of the GI tract, lung, and urinary bladder, which are target organs of cancer in smokers, was examined using the C57BL/6 mouse as an animal model. AαC (400 or 800 ppm) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) (300 ppm), a liver and colon carcinogen in C57BL/6 mice, were given in the diet for up to 12 weeks. Liquid chromatography/mass spectrometry was employed to measure DNA adducts. The major DNA adducts of both carcinogens were identified as deoxyguanosine-C8 adducts. The levels of formation of AαC- and MeIQ-DNA adducts were similar in liver and extrahepatic tissues when adjusted for dose. The highest levels of adducts occurred in liver, followed by urinary bladder, and then in cecum and colon; lower DNA adduct levels were formed in the lung and pancreas following 12 weeks of feeding. The high levels of AαC adduct formed in liver, GI tract, and bladder of C57BL/6 mice reinforce the notion that AαC may contribute to DNA damage and cancer of these organs in smokers.

DNA Adducts in Aldehyde Dehydrogenase-Positive Lung Stem Cells of A/J Mice Treated with the Tobacco Specific Lung Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

Lung cancer is the leading cause of cancer death in the world. Evidence suggests that lung cancer could originate from mutations accumulating in a subpopulation of self-renewing cells, lung stem cells. Aldehyde dehydrogenase (ALDH) is a marker of stem cells. To investigate the presence of DNA modifications in these cells, we isolated ALDH-positive lung cells from A/J mice exposed to the lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Using LC-NSI-HRMS/MS-PRM, O(6)-methyl-G, 7-POB-G, and O(2)-POB-dT were positively identified in ALDH-positive cell DNA. This is the first example of detection of carcinogen-DNA adducts in lung stem cells, supporting the hypothesis of their role in lung carcinogenesis.

Dietary Diindolylmethane Suppresses Inflammation-Driven Lung Squamous Cell Carcinoma in Mice

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. Cancer Prev Res; 8(1); 77–85. ©2014 AACR.

Intranasal delivery of liposomal indole-3-carbinol improves its pulmonary bioavailability

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.

Chemoprevention of lung tumorigenesis by intranasally administered diindolylmethane in A/J mice

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.