Tamene Melkamu

Alteration of microRNA expression in vinyl carbamate-induced mouse lung tumors and modulation by the chemopreventive agent indole-3-carbinol.

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.

Regulation of TLR2 expression and function in human airway epithelial cells

Toll-like receptor (TLR1–6) mRNAs are expressed in normal human bronchial epithelial cells with higher basal levels of TLR3. TLR2 mRNA and plasma membrane protein expression was enhanced by pretreatment with Poly IC, a synthetic double-stranded RNA (dsRNA) known to activate TLR3. Poly IC also enhanced mRNA expression of adaptor molecules (MyD88 and TIRAP) and coreceptors (Dectin-1 and CD14) involved in TLR2 signaling. Additionally, mRNA expression of TLR3 and dsRNA-sensing proteins MDA5 and RIG-I increased following Poly IC treatment. In contrast, basal mRNA expression of TLR5 and TLR2 coreceptor CD36 was reduced by 77% and 62%, respectively. ELISA of apical and basolateral solutions from Poly IC-stimulated monolayers revealed significantly higher levels of IL-6 and GM-CSF compared with the TLR2 ligand PAM3CSK4. Pretreatment with anti-TLR2 blocking antibody inhibited the PAM3CSK4-induced increase in IL-6 secretion after Poly IC exposure. An increase in IL-6 secretion was also observed in cells stimulated with Alternaria extract after pretreatment with Poly IC. However, IL-6 secretion was not stimulated by zymosan or lipothechoic acid (LTA). These data demonstrated that upregulation of TLR2 following exposure to dsRNA enhances functional responses of the airway epithelium to certain (PAM3CSK4), but not all (zymosan, LTA) TLR2 ligands and that this is likely due to differences in coreceptor expression.

Inhibition of vinyl carbamate-induced pulmonary adenocarcinoma by indole-3-carbinol and myo-inositol in A/J mice

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.

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.

ATP release and Ca2+ signalling by human bronchial epithelial cells following Alternaria aeroallergen exposure.

•  Exposure of human bronchial epithelial (HBE) cells to fungal aeroallergens derived from Alternaria alternata stimulates Ca2+‐dependent and Ca2+‐independent ATP release across the apical membrane. •  The Ca2+‐dependent component was blocked by inhibitors of both ATP uptake and transport of exocytotic vesicles to the plasma membrane. •  Treatment with inhibitors that target cysteine proteases significantly blocked Ca2+‐dependent ATP release evoked by Alternaria in normal HBE cells, but not in cells derived from asthmatic patients. •  The magnitude of ATP release and associated intracellular Ca2+ mobilization was significantly greater in bronchial epithelial cells obtained from patients with asthma. •  These findings establish a novel role for ATP release as a mechanism underlying Alternaria aeroallergen activation of airway mucosal immunity and that cells derived from patients with asthma exhibit greater responsiveness to these allergens.

TLR3 activation evokes IL-6 secretion, autocrine regulation of Stat3 signaling and TLR2 expression in human bronchial epithelial cells

Human bronchial epithelial cells exposed to synthetic double-stranded RNA (poly I:C) exhibited increased IL-6 and RANTES secretion and TLR2 expression that was inhibited following TLR3 silencing. Increased NF-κB and Stat3 phosphorylation were detected after poly I:C exposure and pretreatment with neutralizing antibody targeting IL-6 receptor α (IL-6Rα -nAb) or blocking Jak2 and Stat3 activity inhibited Stat3 phosphorylation. TLR2 up-regulation by poly I:C was also reduced by IL-6Rα-nAb and inhibitors of Jak2, Stat3 and NF-κB phosphorylation, whereas RANTES secretion was unaffected, but abolished following NF-κB inhibition. Treatment with exogenous IL-6 failed to increase TLR2. These findings demonstrate that TLR3 activation differentially regulates TLR expression through autocrine signaling involving IL-6 secretion, IL-6Rα activation and subsequent phosphorylation of Stat3. The results also indicate that NF-κB and Stat3 are required for TLR3-dependent up-regulation of TLR2 and that its delayed expression was due to a requirement for IL-6-dependent Stat3 activation.

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.

Abstract B41: A mouse model for inflammation-driven lung tumorigenesis

The link between chronic inflammation and cancer development has lately gained extensive epidemiological and experimental support, particularly in organs like liver, stomach, colon, and lung. Although experimental models are available to study inflammation-driven tumorigenesis in the liver, stomach, and colon, no such models are available for lung cancer. Therefore, the objective of the present study is to develop a model for inflammation-driven lung tumorigenesis. A/J mice received four intraperitoneal injections (two doses/week) of the tobacco smoke carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) at a dose level of 50 mg/kg body weight. Beginning one week after the last dose of NNK, the mice were given weekly intranasal instillations of lipopolysaccharide (LPS, 10ug/mouse), a potent inflammation-inducing agent, until the end of the study at week 27. Upon termination of the study, tumors on the surface of the lung were counted, their size determined and histopathologically analyzed. Moreover, we analyzed alterations in the expression of inflammation- and tumorigenesis-related proteins and microRNAs (miRs) by Western immunoblotting and qRT-PCR, respectively. Chronic exposure NNK-treated mice to LPS increased the number and size of lung tumors (47.3 ± 11 tumors/mouse for the NNK plus LPS group versus 28 ± 8 tumors/mouse in the NNK only group). Furthermore, lung tumors in the NNK plus LPS treated mice were significantly larger than the lung tumors of the mice challenged with NNK alone. Multiplicities of lung tumors in LPS-treated mice were similar to those mice given the vehicle. Multiplicities of the histopathological lesions pulmonary hyperplastic foci, adenoma, adenoma with dysplasia and adenocarcinoma in the NNK only group were 3.0 ± 1.2, 7.1 ± 2.9, 1.3 ± 1.1, and 0.3 ± 0.5/mouse, respectively. The corresponding frequencies of the lesions in the NNK plus LPS group were 3.1 ± 1.8, 11.8 ± 5.2, 3.9 ± 1.8, and 0.6 ± 0.7/mouse. Western immunoblotting studies showed that LPS exposure of NNK-treated mice enhanced activation of NF-kappaB, STAT3, and Akt but caused down-regulation of the tumor suppressor protein PTEN. Further, analyses of levels of five carcinogenesis-related microRNAs (miR-21, miR-146a, miR-150, miR-155 and miR-21, and miR-181b-1) showed increases in the expressions of miR-155 and miR-21. Levels of miR-155 and miR-21 were increased by 2.3-fold and 1.9-fold, respectively, in mice treated with NNK plus LPS group as compared to mice treated with NNK. Moreover, levels of miR-155 and miR-21 were increased by 10.5-fold and 4.7-fold, respectively, in LPS treated mice compared to vehicle-treated mice. In summary, our results demonstrate that LPS-induced chronic lung inflammation enhances lung tumorigenesis. This model could be a vey useful tool to understand the role inflammation in lung tumorigenesis and for identification of anti-inflammatory and lung cancer chemopreventive agents. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B41.