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ABSTRACT: Silibinin inhibits mouse lung tumorigenesis in part by targeting tumor microenvironment. Tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) can be pro- or anti-tumorigenic, but in lung cancer cell lines they induce pro-inflammatory enzymes cyclooxygenase 2 (COX2) and inducible nitric oxide synthase (iNOS). Accordingly, here we examined mechanism of silibinin action on TNF-α + IFN-γ (hereafter referred as cytokine mixture) elicited signaling in tumor-derived mouse lung epithelial LM2 cells. Both signal transducers and activators of the transcription (STAT)3 (tyr705 and ser727) and STAT1 (tyr701) were activated within 15 min of cytokine mixture exposure, while STAT1 (ser727) activated after 3 h. Cytokine mixture also activated Erk1/2 and caused an increase in both COX2 and iNOS levels. Pretreatment of cells with a MEK, NF-κB, and/or epidermal growth factor receptor (EGFR) inhibitor inhibited cytokine mixture-induced activation of Erk1/2, NF-κB, or EGFR, respectively, and strongly decreased phosphorylation of STAT3 and STAT1 and expression of COX2 and iNOS. Also, janus family kinases (JAK)1 and JAK2 inhibitors specifically decreased cytokine-induced iNOS expression, suggesting possible roles of JAK1, JAK2, Erk1/2, NF-κB, and EGFR in cytokine mixture-caused induction of COX2 and iNOS expression via STAT3/STAT1 activation in LM2 cells. Importantly, silibinin pretreatment inhibited cytokine mixture-induced phosphorylation of STAT3, STAT1, and Erk1/2, NF-κB-DNA binding, and expression of COX2, iNOS, matrix metalloproteinases (MMP)2, and MMP9, which was mediated through impairment of STAT3 and STAT1 nuclear localization. Silibinin also inhibited cytokine mixture-induced migration of LM2 cells. Together, we showed that STAT3 and STAT1 could be valuable chemopreventive and therapeutic targets within the lung tumor microenvironment in addition to being targets within tumor itself, and that silibinin inhibits their activation as a plausible mechanism of its efficacy against lung cancer. © 2011 Wiley Periodicals, Inc.
Molecular Carcinogenesis 08/2011; 51(10):832-42. · 3.16 Impact Factor
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ABSTRACT: Worldwide, lung cancer kills more people than breast, colon and prostate cancer combined. Alterations in macrophage number and function during lung tumorigenesis suggest that these immune effector cells stimulate lung cancer growth. Evidence from cancer models in other tissues suggests that cancer cells actively recruit growth factor-producing macrophages through a reciprocal signaling pathway. While the levels of lung macrophages increase during tumor progression in mouse models of lung cancer, and high pulmonary macrophage content correlates with a poor prognosis in human non-small cell lung cancer, the specific role of alveolar macrophages in lung tumorigenesis is not clear.
After culturing either an immortalized lung macrophage cell line or primary murine alveolar macrophages from naïve and lung-tumor bearing mice with primary tumor isolates and immortalized cell lines, the effects on epithelial proliferation and cellular kinase activation were determined. Insulin-like growth factor-1 (IGF-1) was quantified by ELISA, and macrophage conditioned media IGF-1 levels manipulated by IL-4 treatment, immuno-depletion and siRNA transfection.
Primary macrophages from both naïve and lung-tumor bearing mice stimulated epithelial cell proliferation. The lungs of tumor-bearing mice contained 3.5-times more IGF-1 than naïve littermates, and media conditioned by freshly isolated tumor-educated macrophages contained more IGF-1 than media conditioned by naïve macrophages; IL-4 stimulated IGF-1 production by both macrophage subsets. The ability of macrophage conditioned media to stimulate neoplastic proliferation correlated with media IGF-1 levels, and recombinant IGF-1 alone was sufficient to induce epithelial proliferation in all cell lines evaluated. Macrophage-conditioned media and IGF-1 stimulated lung tumor cell growth in an additive manner, while EGF had no effect. Macrophage-derived factors increased p-Erk1/2, p-Akt and cyclin D1 levels in neoplastic cells, and the combined inhibition of both MEK and PI3K ablated macrophage-mediated increases in epithelial growth.
Macrophages produce IGF-1 which directly stimulates neoplastic proliferation through Erk and Akt activation. This observation suggests that combining macrophage ablation therapy with IGF-1R, MEK and/or PI3K inhibition could improve therapeutic response in human lung cancer. Exploring macrophage-based intervention could be a fruitful avenue for future research.
Molecular Cancer 06/2011; 10:76. · 3.99 Impact Factor
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ABSTRACT: Sustained nitric oxide (NO) generation positively correlates with lung cancer development and progression. Herein, we genetically confirmed this role of iNOS and evaluated the chemopreventive efficacy of silibinin in carcinogen-treated B6/129 wild-type (WT) and iNOS(-/-) mice.
Male B6/129-Nos2(tm1Lau) (iNOS(-/-)) and B6/129PF2 WT mice were injected i.p. with 1 mg/g body weight urethane once weekly for 7 consecutive weeks, followed by silibinin gavage (742 mg/kg body weight) for 5 d/wk for 18 weeks.
Quantification of micro-CT data in real-time showed that silibinin significantly decreases urethane-induced tumor number and size in WT mice, consistent with measurements made ex vivo at study termination. Genetic ablation of iNOS decreased urethane-induced tumor multiplicity by 87% (P < 0.001) compared to WT mice. Silibinin decreased tumor multiplicity by 71% (P < 0.01) in WT mice, but did not show any such considerable effect in iNOS(-/-) mice. Tumors from WT mice expressed more iNOS (P < 0.01) but almost similar eNOS and nNOS than those in silibinin-treated mice. In these tumors, silibinin moderately (P < 0.01) inhibited cell proliferation but strongly (P < 0.01) reduced the number of newly formed nestin-positive microvessels. Silibinin decreased VEGFR2 level, and STAT3 and NF-κB activation in tumors.
The lack of effect of silibinin in iNOS(-/-) mice suggests that silibinin exerts most of its chemopreventive and angiopreventive effects through its inhibition of iNOS expression in lung tumors. Our results support iNOS as a potential target for controlling lung cancer, and demonstrate the value of real-time noninvasive micro-CT imaging modality for evaluating the efficacy of lung cancer chemopreventive agents.
Clinical Cancer Research 02/2011; 17(4):753-61. · 7.74 Impact Factor
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ABSTRACT: The inflammatory cytokines tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ) stimulate production of the inflammatory mediators prostaglandin E₂ (PGEγ), prostacyclin (PGIγ), and nitric oxide (NO) in cultured lung epithelial cells. Pretreatment of these cells with the selective MEK1/2 (mitogen-activated protein kinase/extracellular signal-regulated kinase [ERK] kinase 1/2) inhibitor U0126 blocked ERK1/2 activation and inhibited cytokine-induced production of these inflammatory mediators. Primary bronchiolar epithelial Clara cells treated with TNFα and IFNγ also produced increased PGE₂, PGI₂, and NO, and PG and NO production was decreased by MEK inhibition. U0126 differentially affected cyclooxygenase (COX)-1, COX-2, and inducible NO synthase (iNOS) expression in cell lines, however, suggesting that MEK1/2 regulates prostanoid and NO production by means other than inducing their biosynthetic enzymes. Functionally, inhibition of MEK1/2 caused G1 cell cycle arrest and decreased cyclin D1 expression, but these effects were not related to decreased prostanoid production. These results indicate separate proinflammatory and proliferative roles for ERK1/2 in lung epithelial cells. During lung tumor formation in vivo, ERK1/2 phosphorylation increased as lung tumors progressed. Since tumor-derived cells were more sensitive than nontumorigenic cells to the antiproliferative effects of U0126, MEK1/2 inhibition may serve as an attractive chemotherapeutic target.
Experimental Lung Research 11/2010; 36(9):558-71. · 1.22 Impact Factor
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ABSTRACT: Tumor-associated macrophages (TAMs) encourage and coordinate neoplastic growth. In late stage human lung adenocarcinoma, TAMs exhibited mixed M1 (classical; argI(low)iNOS(high)) and M2 (alternative; argI(high)iNOS(low)) polarization based on arginine metabolism. In several murine cancer models including chemically and genetically-induced primary lung tumors, prostate tumors, colon xenografts, and lung metastases, TAMs expressed argI(high)iNOS(low) early during tumor formation; argI(low)iNOS(high) polarization also occurred during malignancy in some models. In a chemically-induced lung tumor model, macrophages expressed argI(high)iNOS(low) within one week after carcinogen treatment, followed by similar polarization of bone marrow-derived monocytes (BDMCs) a few days later. TAMs surrounding murine prostate tumors also expressed argI(high)iNOS(low) early during tumorigenesis, indicating that this polarization is not unique to neoplastic lungs. In a human colon cancer xenograft model, the primary tumor was surrounded by argI(high)iNOS(low)-expressing TAMs, and BDMCs also expressed argI(high)iNOS(low), but pulmonary macrophages adopted argI(high)iNOS(low) polarization only after tumors metastasized to the lungs. Persistence of tumors is required to maintain TAM polarization. Indeed, in both conditional mutant Kras- and FGF10-driven models of lung cancer, mice expressing the transgene develop lung tumors that regress rapidly when the transgene is silenced. Furthermore, pulmonary macrophages expressed argI(high)iNOS(low) on tumor induction, but then returned to argI(low) iNOS(low) (no polarization) after tumors regressed. Manipulating TAM function or depleting TAMs may provide novel therapeutic strategies for preventing and treating many types of cancer.
American Journal Of Pathology 06/2010; 176(6):2972-85. · 4.89 Impact Factor
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ABSTRACT: Alveolar macrophages and BDMCs undergo sequential biochemical changes during the chronic inflammatory response to chemically induced lung carcinogenesis in mice. Herein, we examine two chronic lung inflammation models-repeated exposure to BHT and infection with Mycobacterium tuberculosis-to establish whether similar macrophage phenotype changes occur in non-neoplastic pulmonary disease. Exposure to BHT or M. tuberculosis results in pulmonary inflammation characterized by an influx of macrophages, followed by systemic effects on the BM and other organs. In both models, pulmonary IFN-gamma and IL-4 production coincided with altered polarization of alveolar macrophages. Soon after BHT administration or M. tuberculosis infection, IFN-gamma content in BALF increased, and BAL macrophages became classically (M1) polarized, as characterized by increased expression of iNOS. As inflammation progressed in both models, the amount of BALF IFN-gamma content and BAL macrophage iNOS expression decreased, and BALF IL-4 content and macrophage arginase I expression rose, indicating alternative/M2 polarization. Macrophages present in M. tuberculosis-induced granulomas remained M1-polarized, implying that these two pulmonary macrophage populations, alveolar and granuloma-associated, are exposed to different activating cytokines. BDMCs from BHT-treated mice displayed polarization profiles similar to alveolar macrophages, but BDMCs in M. tuberculosis-infected mice did not become polarized. Thus, only alveolar macrophages in these two models of chronic lung disease exhibit a similar progression of polarization changes; polarization of BDMCs was specific to BHT-induced pulmonary inflammation, and polarization of granuloma macrophages was specific to the M. tuberculosis infection.
Journal of leukocyte biology 04/2010; 88(1):159-68. · 4.99 Impact Factor
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ABSTRACT: Human lung cancer patients exhibit different KRAS mutations depending on smoking status. In a mouse model of human cancer, A/J and BALB/cBy mice treated with the tobacco carcinogen, 3-methylcholanthrene (MCA), followed by butylated hydroxytoluene (BHT)-elicited chronic inflammation develop a high multiplicity of lung tumors.
DNA was isolated from MCA-induced lung tumors in A/J and BALB/cByJ mice. Kras codon 12 sequences from these tumors were compared to those in human lung tumors from smokers and never-smokers.
The distribution of Kras codon 12 mutations in MCA-induced A/J lung tumors is strikingly similar to those found in adenocarcinomas from human smokers. In contrast, codon 12 mutations in BALB/cBy mice contain predominantly G --> D mutations, which is the most common mutation in never smokers.
A single lung carcinogen induces different tumor initiating mutations in different strains of mice. This may be useful for investigating the role of specific KRAS mutations in adenocarcinoma pathogenesis in smokers versus never smokers, identifying mechanisms that select for certain KRAS mutations and developing new drugs that specifically target cells with different KRAS mutations.
Journal of thoracic oncology: official publication of the International Association for the Study of Lung Cancer 02/2010; 5(2):254-7. · 4.55 Impact Factor
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ABSTRACT: Alternative (M2) macrophage activation is associated with tumor development in many tumor types, including those in the lung. Herein the biological consequences of forcing classical (M1) or alternative (M2) macrophage activation on lung tumor development are examined.
Urethane-induced lung tumor multiplicity and size were compared in IFN-gamma(-/-) mice which lack M1 macrophage activation, IL-4Ralpha(-/-) mice which lack M2 macrophage activation, and wild-type BALB/cJ (background strain of the IFN-gamma(-/-) and IL-4Ralpha(-/-) mice) mice. Tumor-associated macrophage (TAM) and bone marrow-derived monocyte (BDMC) activation were each examined.
The TAMs and BDMCs in the IFN-gamma(-/-) mice exhibited M2 activation, and their lung tumors were significantly larger than those in the wild-type mice. In contrast, urethane-treated IL-4Ralpha(-/-) mice, whose TAMs and BDMCs were M1 activated, developed smaller tumors than the wild-type mice.
Altered innate immunity can diminish or accelerate lung tumor progression in response to defective cytokine signaling.
Anticancer research 12/2009; 29(12):5095-101. · 1.73 Impact Factor
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ABSTRACT: Cancer susceptibility results from interactions between sensitivity and resistance alleles. We employed murine chromosome substitution strains to study how resistance alleles affected sensitive alleles during chemically-induced lung carcinogenesis. The C57BL/6J-Chr#(A/J) strains, constructed by selectively breeding sensitive A/J and resistant C57BL/6J (B6) mice, each contain one pair of A/J chromosomes within an otherwise B6 genome. Pas1, the major locus responsible for this differential strain response to urethane carcinogenesis, resides on Chr 6, but C57BL/6J-Chr6(A/J) mice (hereafter CSS-6) developed few tumors following a single urethane injection, which demonstrates epistatic interactions with other B6 alleles. CSS6 mice developed dozens of lung tumors after chronic urethane exposure, however, indicating that these epistatic interactions could be overcome by repeated carcinogen administration. Unlike A/J, but similar to B6 mice, CSS6 mice were resistant to lung carcinogenesis induced by 3-methylcholanthrene (MCA). Tumor multiplicity increased if BHT administration followed urethane exposure, showing that a Chr 6 gene(s) regulates sensitivity to chemically-induced tumor promotion. Unlike A/J tumors (predominantly codon 61 A-->T transversions), Kras mutations in tumors induced by urethane in CSS-6 mice were similar to B6 tumors (codon 61 A-->G transitions). DNA repair genes not located on Chr 6 may determine the nature of Kras mutations. CSS-6 mice are a valuable resource for testing the ability of candidate genes to modulate lung carcinogenesis.
International Journal of Cancer 07/2009; 126(1):125-32. · 5.44 Impact Factor
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ABSTRACT: The latency period for lung tumor progression offers a window of opportunity for therapeutic intervention. Herein, we studied the effect of oral silibinin (742 mg/kg body weight, 5 d/wk for 10 weeks) on the growth and progression of established lung adenocarcinomas in A/J mice. Silibinin strongly decreased both tumor number and tumor size, an antitumor effect that correlates with reduced antiangiogenic activity. Silibinin reduced microvessel size (50%, P < 0.01) with no change in the number of tumor microvessels and reduced (by 30%, P < 0.05) the formation of nestin-positive microvessels in tumors. Analysis of several proteins involved in new blood vessel formation showed that silibinin decreased the tumor expression of interleukin-13 (47%) and tumor necrosis factor-alpha (47%), and increased tissue inhibitor of metalloproteinase-1 (2-fold) and tissue inhibitor of metalloproteinase-2 (7-fold) expression, without significant changes in vascular endothelial growth factor levels. Hypoxia- inducible factor-1 alpha expression and nuclear localization were also decreased by silibinin treatment. Cytokines secreted by tumor cells and tumor-associated macrophages regulate angiogenesis by activating nuclear factor-kappaB (NF-kappaB) and signal transducers and activators of transcription (STAT). Silibinin decreased the phosphorylation of p65NF-kappaB (ser276, 38%; P < 0.01) and STAT-3 (ser727, 16%; P < 0.01) in tumor cells and decreased the lung macrophage population. Angiopoietin-2 (Ang-2) and Ang-receptor tyrosine kinase (Tie-2) expression were increased by silibinin. Therapeutic efficacy of silibinin in lung tumor growth inhibition and regression by antiangiogenic mechanisms seem to be mediated by decreased tumor-associated macrophages and cytokines, inhibition of hypoxia-inducible factor-1 alpha, NF-kappaB, and STAT-3 activation, and up-regulation of the angiogenic inhibitors, Ang-2 and Tie-2.
Cancer Prevention Research 01/2009; 2(1):74-83. · 4.91 Impact Factor
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Laura K Zerbe,
Lori D Dwyer-Nield,
Jason M Fritz,
Elizabeth F Redente,
Robert J Shroyer,
Elizabeth Conklin,
Shawn Kane,
Chris Tucker,
S Gail Eckhardt,
Daniel L Gustafson,
Kenneth K Iwata, Alvin M Malkinson
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ABSTRACT: Erlotinib, a small molecule inhibitor of the tyrosine kinase (TK) domain of epidermal growth factor receptor (EGFR), increases survival of advanced non-small cell lung cancer patients who failed standard chemotherapy (Phase III study). We evaluated whether erlotinib is also effective at an early stage of primary lung tumorigenesis in a carcinogen-induced lung tumor model in mice.
Sixteen weeks after carcinogen (urethane) injection, when small self-contained adenomas are evident, male and female A/J mice were treated IP with 10 mg/kg erlotinib or Captisol vehicle daily over 3.5 weeks (15 mice per group). The efficacy, metabolism and mechanism of action of erlotinib were evaluated.
Erlotinib reduced tumor burden in males by twofold compared to vehicle (12.7 +/- 1.2 vs 26.2 +/- 2.5 mg, respectively; p < 0.0001), while tumor burden in erlotinib-treated females slightly increased compared to vehicle by 21% (15.1 +/- 1.2 vs 11.9 +/- 0.9 mg, respectively; p < 0.05). Tumor multiplicity, in contrast, was unaffected by erlotinib. The levels of erlotinib that accumulated in plasma, lung tumor tissue and adjacent uninvolved (UI) lung were comparable in males and females. Males, however, accumulated more OSI-420, an active and pharmacologically equipotent metabolite of erlotinib, than females in plasma, lung tumors, and UI lung. In both genders, 80% of tumors contained Kras mutations at codon 61, but no EGFR mutations were detected. The cellular distribution and concentration of EGFR were also similar between genders. In control mice, however, phosphorylated EGFR (pEGFR) levels were nearly 2.5-fold higher in males compared to females in UI lungs and sevenfold higher in lung tumors. Further, erlotinib decreased the contents of pEGFR in UI lungs and lung tumors, particularly in males.
Adenomas from male mice in this early lung cancer model are responsive to erlotinib treatment, possibly because of a greater dependence of male tumor growth on the EGFR pathway compared to females. Importantly, these results indicate that small lung adenomas from male mice that utilize EGFR signaling but also harbor Kras mutations shrink in response to erlotinib, suggesting that erlotinib may be beneficial for some patients very early during lung cancer progression.
Cancer Chemotherapy and Pharmacology 09/2008; 62(4):605-20. · 2.83 Impact Factor
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ABSTRACT: Microarray experiments designed to identify genes differentially expressed in the E11.5 lung and trachea showed that melanoma inhibitory activity (Mia1) was expressed only in the lung. Mia1 was abundantly expressed during early lung development, but was virtually absent by the end of gestation. Distal embryonic lung epithelium showed high levels of Mia1 expression, which was suppressed by treatment with either retinoic acid or the FGF signaling antagonist SU5402. Late-gestation fetuses in which lung epithelial hyperplasia was induced by misexpression of FGF7 or FGF10 showed continued expression of Mia1 in areas of aberrant morphogenesis. Mia1 expression was also significantly increased in urethane-induced lung adenomas. Treatment of E18.5 lung explants with exogenous MIA caused significant reductions in the expression of the lung differentiation markers Sftpa, Sftpb, Sftpc, and Abca3. Bitransgenic mice expressing MIA under the control of the SFTPC promoter after E16.5, the age when Mia1 is normally silenced, died from respiratory failure at birth with morphologically immature lungs associated with reduced levels of saturated phosphatidylcholine and mature SP-B. Microarray analysis showed significant reductions in the expression of Sftpa, Sftpb, Abca3, Aqp5, Lzp-s, Scd2, and Aytl2 in lungs misexpressing MIA. These results suggest that the silencing of Mia1 that occurs in late gestation may be required for maturation of the surfactant system.
Developmental Biology 05/2008; 316(2):441-55. · 4.07 Impact Factor
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ABSTRACT: One area of intensive investigation is to understand complex cellular and signaling interactions in the tumor microenvironment. Using a novel, although straightforward, microarray approach, we defined a gene expression signature from the lung tumor microenvironment in the murine A/J-urethane model of human lung adenocarcinoma. The tumor microenvironment is reflected by the composition of the cell types present and alterations in mRNA levels, resulting in a "Field Effect" around the tumor. The genes composing the Field Effect expression signature include proteases and their inhibitors, inflammation markers, and immune signaling molecules. By several criteria, the Field Effect expression signature can be attributed to the macrophage lineage, suggesting a qualitative change in the expression pattern of tumor-associated macrophages (TAM) observed in lung tumors. The protein expression levels for a number of Field Effect genes were verified by Western blot analysis of lung homogenates, and for their expression in macrophages and parenchymal cells outside of the tumors by immunohistochemistry. In addition, the Field Effect expression signature was used to classify bronchoalveolar lavage (BAL) cells from tumor-bearing or age-matched control mice. Using a variety of statistical measures, the Field Effect expression signature correctly classified the BAL cells >94% of the time. Finally, the protein levels for several Field Effect genes were higher in cell-free BAL fluid, indicating they may be secreted by the TAMs. This work suggests that TAMs generate a unique gene expression signature within the tumor microenvironment, and this signature could potentially be used for identifying lung cancer from BAL cells and/or fluid.
Cancer Research 01/2008; 68(1):34-43. · 7.86 Impact Factor
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ABSTRACT: Our previous proteomic investigation of lung neoplasia in vitro demonstrated a high concentration of the lung cancer biomarker and splicing factor, hnRNP A2/B1, in the transformed mouse lung epithelial cell line, E9. Since changes in pre-mRNA splicing profoundly affect neoplastic progression, we examined hnRNP A2/B1 expression in chemically induced primary mouse lung tumors, an in vivo model of pulmonary adencocarcinoma. Tumor hnRNP A2/B1 content and spatial distribution assessed by immunohistochemistry varied with stage of progression, genetic background, and whether tumors were induced by a single agent (urethane) or by 2-stage initiation/promotion (3-methylcholanthrene/butylated hydroxytoluene) carcinogenesis. To address mechanisms governing hnRNP A2/B1 expression changes, we utilized in vitro models. hnRNP A2/B1 protein was overexpressed in E9, the spontaneous tranformant of immortalized but non-neoplastic E10 cells, but expression was not strictly a function of enhanced proliferative rate in neoplastic cells. Elevated mRNA content was positively associated with cell division in both E10 and E9, but hnRNP A2/B1 protein levels decreased in proliferating E10 cells. The increased mRNA reflected enhanced mRNA stability, as shown by measuring time-dependent mRNA decay after inhibiting transcription. Dysregulation of hnRNP A2/B1 expression during lung neoplasia in vivo thus depends on complex gene-environmental interactions that affect cell type-specific changes in mRNA processing and, most probably, the rates of translation and/or protein degradation.
Molecular Carcinogenesis 12/2007; 46(11):887-900. · 3.16 Impact Factor
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ABSTRACT: Little is known about how the composition of stromal cells within the lung cancer microenvironment varies during tumor progression. We examined by immunohistochemistry each of six different stromal cell populations during the development of chemically induced primary lung cancer in mice. Blood vessels were seen even in microscopic lesions, and their numbers increased with tumor size. Neutrophils infiltrated the alveoli of tumor-bearing lungs and within the periphery of macroscopic adenomas and adenocarcinomas. The numbers of peritumoral lymphocytes and macrophages increased during oncogeny, but quantitative changes in mast cells and fibroblasts were not evident. Because macrophage depletion reduces tumor growth and these cells are thus important to tumorigenesis, we also investigated their phenotype. Pulmonary macrophages expressed arginase I (subtype M2) but not inducible nitric-oxide synthase in lungs with premalignant lesions, whereas macrophages in carcinoma-bearing lungs expressed inducible nitric-oxide synthase (subtype M1) but not arginase I. Local pulmonary stimuli did not seem responsible for this shift in macrophage activation state because monocytes still residing within the bone marrow adopted these expression patterns before entering the circulation, presumably in response to tumor-derived signals. These biochemical markers of macrophage activation states would have diagnostic and/or therapeutic value if analogous systemic shifts occur in humans.
American Journal Of Pathology 03/2007; 170(2):693-708. · 4.89 Impact Factor
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ABSTRACT: Inbred mice vary in their susceptibility to develop macroscopic, chemically-induced, pulmonary neoplasias. It is not known, however, whether microscopic lesions appear in resistant strains but do not grow or if no early lesions arise at all. We show herein that resistant C57BL/6J (B6) and intermediately resistant BALB/cByJ (BALB) mice form very few urethane-induced early microadenomas (i.e. adenomas larger than hyperplasic foci, but detectable only by light microscopy). Additionally, while all urethane-induced microadenomas in sensitive A/J mice gave rise to adenomas, most microscopic tumors induced in BALB mice by 2-stage, 3-methylcholanthrene/butylated hydroxytoluene carcinogenesis spontaneously regressed. The formation of microscopic lesions is thus genetically dependent, but whether they continue to grow or regress depends on how they were induced.
Cancer Letters 10/2006; 241(2):197-202. · 4.24 Impact Factor
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Amy M Meyer,
Lori D Dwyer-Nield,
Gregory Hurteau,
Robert L Keith,
Yanli Ouyang,
Brian M Freed,
Lori R Kisley,
Mark W Geraci,
Joseph V Bonventre,
Raphael A Nemenoff, Alvin M Malkinson
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ABSTRACT: Administration of butylated hydroxytoluene (BHT) to mice causes lung damage characterized by the death of alveolar type I pneumocytes and the proliferation and subsequent differentiation of type II cells to replace them. Herein, we demonstrate this injury elicits an inflammatory response marked by chemokine secretion, alveolar macrophage recruitment, and elevated expression of enzymes in the eicosanoid pathway. Cytosolic phospholipase A(2) (cPLA(2)) catalyzes release of arachidonic acid from membrane phospholipids to initiate the synthesis of prostaglandins and other inflammatory mediators. A role for cPLA(2) in this response was examined by determining cPLA(2) expression and enzymatic activity in distal respiratory epithelia and macrophages and by assessing the consequences of cPLA(2) genetic ablation. BHT-induced lung inflammation, particularly monocyte infiltration, was depressed in cPLA(2) null mice. Monocyte chemotactic protein-1 (MCP-1) content in bronchoalveolar lavage fluid increases after BHT treatment but before monocyte influx, suggesting a causative role. Bronchiolar Clara cells isolated from cPLA(2) null mice secrete less MCP-1 than Clara cells from wild-type mice, consistent with the hypothesis that cPLA(2) is required to secrete sufficient MCP-1 to induce an inflammatory monocytic response.
AJP Lung Cellular and Molecular Physiology 07/2006; 290(6):L1260-6. · 3.66 Impact Factor
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ABSTRACT: Silibinin, a flavanone from milk thistle, inhibits the growth of tumors in several rodent models. We examined the effects of dietary silibinin on the growth, progression, and angiogenesis of urethane-induced lung tumors in mice.
A/J mice (15 per group) were injected with urethane (1 mg/g body weight) or saline alone and fed normal diets for 2 weeks, after which they were fed diets containing different doses of silibinin (0%-1% [wt/wt] silibinin) for 18 or 27 weeks. Immunohistochemistry and Western blot analysis were used to examine angiogenesis and enzymatic markers of inflammation, proliferation, and apoptosis. All statistical tests were two-sided.
Urethane-injected mice exposed to silibinin had statistically significantly lower lung tumor multiplicities than urethane-injected mice fed the control diet lacking silibinin (i.e., control mice). Mice that received urethane and 1% (wt/wt) dietary silibinin for 18 weeks had 93% fewer large (i.e., 1.5-2.5-mm-diameter) lung tumors than control mice (mean number of tumors/mouse: 27 in the urethane group versus 2 in the urethane + 1% silibinin group, difference = 25 tumors/mouse, 95% confidence interval [CI] = 13 to 37 tumors/mouse, P = .005). Lung tumors of silibinin-fed mice had 41%-74% fewer cells positive for the cell proliferation markers proliferating cell nuclear antigen and cyclin D1 than lung tumors of control mice. Tumor microvessel density was reduced by up to 89% with silibinin treatment (e.g., 56 microvessels/400x field in tumors from control mice versus 6 microvessels/400x field in tumors from urethane + 1% silibinin-treated mice [difference = 50 microvessels/400x field, 95% CI = 46 to 54 microvessels/400x field; P<.001]). Silibinin decreased lung tumor expression of vascular endothelial growth factor (VEGF) and of inducible nitric oxide synthase and cyclooxygenase-2, two enzymes that promote lung tumor growth and progression by inducing VEGF expression.
Silibinin inhibits lung tumor angiogenesis in an animal model and merits investigation as a chemopreventive agent for suppressing lung cancer progression.
CancerSpectrum Knowledge Environment 07/2006; 98(12):846-55. · 14.07 Impact Factor
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ABSTRACT: Because chronic pulmonary diseases predispose to lung neoplasia, the identification of the molecular mechanisms involved could provide novel preventive, diagnostic, and therapeutic strategies. Toll-like receptors (TLRs) transduce exogenous and endogenous signals into the production of inflammatory cytokines to coordinate adaptive immune responses. To determine the role of Tlr4 in chronic lung inflammation, we compared lung permeability, leukocyte infiltration, and nuclear factor kappa B (NFkappaB) and activator protein 1 (AP-1) DNA binding in butylated hydroxytoluene (BHT)-treated (four weekly injections of 125-200 mg/kg each) inbred mouse strains with functional Tlr4 (OuJ and BALB) and mutated Tlr4 (HeJ and BALB(Lps-d)). We also measured primary tumor formation in these mice after single-carcinogen injection (3-methylcholanthrene; 10 microg/kg), followed by BHT treatment (six weekly injections of 125-200 mg/kg each). Mice with functional Tlr4 had reduced lung permeability, leukocyte inflammation, and primary tumor formation (BALB(Lps-d), mean = 22.3 tumors/mouse, versus BALB, mean = 13.9 tumors/mouse, difference = 8.4 tumors/mouse, 95% confidence interval = 4.6 to 12.1 tumors/mouse; P = .025) compared with mice with mutated Tlr4. NFkappaB DNA binding activity was higher in OuJ than in HeJ mice; however, AP-1 activity was elevated in HeJ mice. To our knowledge, this is the first model to demonstrate a modulatory role for Tlr4 in chronic lung inflammation and tumorigenesis.
CancerSpectrum Knowledge Environment 01/2006; 97(23):1778-81. · 14.07 Impact Factor
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Robert S Stearman,
Lori Dwyer-Nield,
Laura Zerbe,
Stacy A Blaine,
Zeng Chan,
Paul A Bunn,
Gary L Johnson,
Fred R Hirsch,
Daniel T Merrick,
Wilbur A Franklin,
Anna E Baron,
Robert L Keith,
Raphael A Nemenoff, Alvin M Malkinson,
Mark W Geraci
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ABSTRACT: Human adenocarcinoma (AC) is the most frequently diagnosed human lung cancer, and its absolute incidence is increasing dramatically. Compared to human lung AC, the A/J mouse-urethane model exhibits similar histological appearance and molecular changes. We examined the gene expression profiles of human and murine lung tissues (normal or AC) and compared the two species' datasets after aligning approximately 7500 orthologous genes. A list of 409 gene classifiers (P value <0.0001), common to both species (joint classifiers), showed significant, positive correlation in expression levels between the two species. A number of previously reported expression changes were recapitulated in both species, such as changes in glycolytic enzymes and cell-cycle proteins. Unexpectedly, joint classifiers in angiogenesis were uniformly down-regulated in tumor tissues. The eicosanoid pathway enzymes prostacyclin synthase (PGIS) and inducible prostaglandin E(2) synthase (PGES) were joint classifiers that showed opposite effects in lung AC (PGIS down-regulated; PGES up-regulated). Finally, tissue microarrays identified the same protein expression pattern for PGIS and PGES in 108 different non-small cell lung cancer biopsies, and the detection of PGIS had statistically significant prognostic value in patient survival. Thus, the A/J mouse-urethane model reflects significant molecular details of human lung AC, and comparison of changes in orthologous gene expression may provide novel insights into lung carcinogenesis.
American Journal Of Pathology 12/2005; 167(6):1763-75. · 4.89 Impact Factor
