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Targeted deletion of Nrf2 reduces urethane-induced lung tumor development in mice.

Bauer AK, Cho HY, Miller-Degraff L, Walker C, Helms K, Fostel J, Yamamoto M, Kleeberger SR - PLoS ONE (2011)

Bottom Line: However, permanent Nrf2 activation in human lung carcinomas promotes pulmonary malignancy and chemoresistance.Nrf2 modulated expression of genes involved cell-cell signaling, glutathione metabolism and oxidative stress response, and immune responses during early stage neoplasia.Our results were consistent with the concept that Nrf2 over-activation is an adaptive response of cancer conferring resistance to anti-cancer drugs and promoting malignancy.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan, United States of America. alison.bauer@ucdenver.edu

ABSTRACT
Nrf2 is a key transcription factor that regulates cellular redox and defense responses. However, permanent Nrf2 activation in human lung carcinomas promotes pulmonary malignancy and chemoresistance. We tested the hypothesis that Nrf2 has cell survival properties and lack of Nrf2 suppresses chemically-induced pulmonary neoplasia by treating Nrf2(+/+) and Nrf2(-/-) mice with urethane. Airway inflammation and injury were assessed by bronchoalveolar lavage analyses and histopathology, and lung tumors were analyzed by gross and histologic analysis. We used transcriptomics to assess Nrf2-dependent changes in pulmonary gene transcripts at multiple stages of neoplasia. Lung hyperpermeability, cell death and apoptosis, and inflammatory cell infiltration were significantly higher in Nrf2(-/-) mice compared to Nrf2(+/+) mice 9 and 11 wk after urethane. Significantly fewer lung adenomas were found in Nrf2(-/-) mice than in Nrf2(+/+) mice at 12 and 22 wk. Nrf2 modulated expression of genes involved cell-cell signaling, glutathione metabolism and oxidative stress response, and immune responses during early stage neoplasia. In lung tumors, Nrf2-altered genes had roles in transcriptional regulation of cell cycle and proliferation, carcinogenesis, organismal injury and abnormalities, xenobiotic metabolism, and cell-cell signaling genes. Collectively, Nrf2 deficiency decreased susceptibility to urethane-induced lung tumorigenesis in mice. Cell survival properties of Nrf2 were supported, at least in part, by reduced early death of initiated cells and heightened advantage for tumor cell expansion in Nrf2(+/+) mice relative to Nrf2(-/-) mice. Our results were consistent with the concept that Nrf2 over-activation is an adaptive response of cancer conferring resistance to anti-cancer drugs and promoting malignancy.

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Early stage tumorigenesis at 12 wk.(A) H&E-staining demonstrate pulmonary hyperplastic regions and early tumor development (arrow heads) 12 wk after the first urethane injection. Greater tumor cell proliferation in sporadically small adenomatous regions was found in Nrf2+/+ mice than Nrf2-/- mice as indicated by denser proliferating cell nuclear antigen (PCNA) localization in relatively more advanced tumors (inset). Representative light photomicrographs showing intermediate magnitude of pathology for each treatment group are presented (n = 3−8/group for H&E, n = 3/group for PCNA). AV, alveoli; TB, terminal bronchiole; BV, blood vessel; UN, uninvolved region. Arrow heads, tumor; arrows, PCNA-positive nuclei. Bars indicate 100 µm. (B) Numerous apoptotic airway cells in Nrf2-/- mice were identified by TUNEL assay on paraffin-embedded lung tissue sections. TUNEL-positive nuclei of epithelial, endothelial, and smooth muscle cells on proximal lung sections were counted and normalized to airway surface (mm2) using digital image processing software. Few TUNEL-stained cells were found in vehicle control mice. Mean±SD are presented (n = 3/group). +, p<0.05 vs. urethane-treated Nrf2+/+ mice. (C) Differential early tumor formation between Nrf2+/+ and Nrf2-/- mice. Average number of tumors (≥200 µm) per whole lung from each mouse was assessed in serial sections of paraffin-embedded lungs fixed with 10% NBF. Mean±SD are presented (n = 13−14/group). +, p<0.05 vs. urethane-treated Nrf2+/+ mice.
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pone-0026590-g002: Early stage tumorigenesis at 12 wk.(A) H&E-staining demonstrate pulmonary hyperplastic regions and early tumor development (arrow heads) 12 wk after the first urethane injection. Greater tumor cell proliferation in sporadically small adenomatous regions was found in Nrf2+/+ mice than Nrf2-/- mice as indicated by denser proliferating cell nuclear antigen (PCNA) localization in relatively more advanced tumors (inset). Representative light photomicrographs showing intermediate magnitude of pathology for each treatment group are presented (n = 3−8/group for H&E, n = 3/group for PCNA). AV, alveoli; TB, terminal bronchiole; BV, blood vessel; UN, uninvolved region. Arrow heads, tumor; arrows, PCNA-positive nuclei. Bars indicate 100 µm. (B) Numerous apoptotic airway cells in Nrf2-/- mice were identified by TUNEL assay on paraffin-embedded lung tissue sections. TUNEL-positive nuclei of epithelial, endothelial, and smooth muscle cells on proximal lung sections were counted and normalized to airway surface (mm2) using digital image processing software. Few TUNEL-stained cells were found in vehicle control mice. Mean±SD are presented (n = 3/group). +, p<0.05 vs. urethane-treated Nrf2+/+ mice. (C) Differential early tumor formation between Nrf2+/+ and Nrf2-/- mice. Average number of tumors (≥200 µm) per whole lung from each mouse was assessed in serial sections of paraffin-embedded lungs fixed with 10% NBF. Mean±SD are presented (n = 13−14/group). +, p<0.05 vs. urethane-treated Nrf2+/+ mice.

Mentions: Immunohistochemical detection of proliferating cell nuclear antigen (PCNA, a cell proliferation marker [18]) at 12 wk demonstrated that PCNA-positive cells in G1/S phase were localized extensively in small adenomas, focal alveolar hyperplastic lesions, injured perivascular and peribronchial regions, and nodular lymphoid aggregates in adventitia of blood vessels. Compared to Nrf2-/-, more abundant and intense localization of PCNA were found in small adenomatous regions of Nrf2+/+ mice (Figure 2A). PCNA was sporadic in conducting airway epithelium of saline controls (Figure 2A). Consistent with differential BAL cell necrosis at 9–11 wk, the number of apoptotic lung cells determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) in proximal pulmonary sections was more highly elevated in Nrf2-/- mice compared to Nrf2+/+ mice at 12 wk (Figure 2B). TUNEL-positive cells included epithelial, endothelial, and smooth muscle cells. Few apoptotic cells were found in lungs of vehicle control mice.


Targeted deletion of Nrf2 reduces urethane-induced lung tumor development in mice.

Bauer AK, Cho HY, Miller-Degraff L, Walker C, Helms K, Fostel J, Yamamoto M, Kleeberger SR - PLoS ONE (2011)

Early stage tumorigenesis at 12 wk.(A) H&E-staining demonstrate pulmonary hyperplastic regions and early tumor development (arrow heads) 12 wk after the first urethane injection. Greater tumor cell proliferation in sporadically small adenomatous regions was found in Nrf2+/+ mice than Nrf2-/- mice as indicated by denser proliferating cell nuclear antigen (PCNA) localization in relatively more advanced tumors (inset). Representative light photomicrographs showing intermediate magnitude of pathology for each treatment group are presented (n = 3−8/group for H&E, n = 3/group for PCNA). AV, alveoli; TB, terminal bronchiole; BV, blood vessel; UN, uninvolved region. Arrow heads, tumor; arrows, PCNA-positive nuclei. Bars indicate 100 µm. (B) Numerous apoptotic airway cells in Nrf2-/- mice were identified by TUNEL assay on paraffin-embedded lung tissue sections. TUNEL-positive nuclei of epithelial, endothelial, and smooth muscle cells on proximal lung sections were counted and normalized to airway surface (mm2) using digital image processing software. Few TUNEL-stained cells were found in vehicle control mice. Mean±SD are presented (n = 3/group). +, p<0.05 vs. urethane-treated Nrf2+/+ mice. (C) Differential early tumor formation between Nrf2+/+ and Nrf2-/- mice. Average number of tumors (≥200 µm) per whole lung from each mouse was assessed in serial sections of paraffin-embedded lungs fixed with 10% NBF. Mean±SD are presented (n = 13−14/group). +, p<0.05 vs. urethane-treated Nrf2+/+ mice.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198791&req=5

pone-0026590-g002: Early stage tumorigenesis at 12 wk.(A) H&E-staining demonstrate pulmonary hyperplastic regions and early tumor development (arrow heads) 12 wk after the first urethane injection. Greater tumor cell proliferation in sporadically small adenomatous regions was found in Nrf2+/+ mice than Nrf2-/- mice as indicated by denser proliferating cell nuclear antigen (PCNA) localization in relatively more advanced tumors (inset). Representative light photomicrographs showing intermediate magnitude of pathology for each treatment group are presented (n = 3−8/group for H&E, n = 3/group for PCNA). AV, alveoli; TB, terminal bronchiole; BV, blood vessel; UN, uninvolved region. Arrow heads, tumor; arrows, PCNA-positive nuclei. Bars indicate 100 µm. (B) Numerous apoptotic airway cells in Nrf2-/- mice were identified by TUNEL assay on paraffin-embedded lung tissue sections. TUNEL-positive nuclei of epithelial, endothelial, and smooth muscle cells on proximal lung sections were counted and normalized to airway surface (mm2) using digital image processing software. Few TUNEL-stained cells were found in vehicle control mice. Mean±SD are presented (n = 3/group). +, p<0.05 vs. urethane-treated Nrf2+/+ mice. (C) Differential early tumor formation between Nrf2+/+ and Nrf2-/- mice. Average number of tumors (≥200 µm) per whole lung from each mouse was assessed in serial sections of paraffin-embedded lungs fixed with 10% NBF. Mean±SD are presented (n = 13−14/group). +, p<0.05 vs. urethane-treated Nrf2+/+ mice.
Mentions: Immunohistochemical detection of proliferating cell nuclear antigen (PCNA, a cell proliferation marker [18]) at 12 wk demonstrated that PCNA-positive cells in G1/S phase were localized extensively in small adenomas, focal alveolar hyperplastic lesions, injured perivascular and peribronchial regions, and nodular lymphoid aggregates in adventitia of blood vessels. Compared to Nrf2-/-, more abundant and intense localization of PCNA were found in small adenomatous regions of Nrf2+/+ mice (Figure 2A). PCNA was sporadic in conducting airway epithelium of saline controls (Figure 2A). Consistent with differential BAL cell necrosis at 9–11 wk, the number of apoptotic lung cells determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) in proximal pulmonary sections was more highly elevated in Nrf2-/- mice compared to Nrf2+/+ mice at 12 wk (Figure 2B). TUNEL-positive cells included epithelial, endothelial, and smooth muscle cells. Few apoptotic cells were found in lungs of vehicle control mice.

Bottom Line: However, permanent Nrf2 activation in human lung carcinomas promotes pulmonary malignancy and chemoresistance.Nrf2 modulated expression of genes involved cell-cell signaling, glutathione metabolism and oxidative stress response, and immune responses during early stage neoplasia.Our results were consistent with the concept that Nrf2 over-activation is an adaptive response of cancer conferring resistance to anti-cancer drugs and promoting malignancy.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan, United States of America. alison.bauer@ucdenver.edu

ABSTRACT
Nrf2 is a key transcription factor that regulates cellular redox and defense responses. However, permanent Nrf2 activation in human lung carcinomas promotes pulmonary malignancy and chemoresistance. We tested the hypothesis that Nrf2 has cell survival properties and lack of Nrf2 suppresses chemically-induced pulmonary neoplasia by treating Nrf2(+/+) and Nrf2(-/-) mice with urethane. Airway inflammation and injury were assessed by bronchoalveolar lavage analyses and histopathology, and lung tumors were analyzed by gross and histologic analysis. We used transcriptomics to assess Nrf2-dependent changes in pulmonary gene transcripts at multiple stages of neoplasia. Lung hyperpermeability, cell death and apoptosis, and inflammatory cell infiltration were significantly higher in Nrf2(-/-) mice compared to Nrf2(+/+) mice 9 and 11 wk after urethane. Significantly fewer lung adenomas were found in Nrf2(-/-) mice than in Nrf2(+/+) mice at 12 and 22 wk. Nrf2 modulated expression of genes involved cell-cell signaling, glutathione metabolism and oxidative stress response, and immune responses during early stage neoplasia. In lung tumors, Nrf2-altered genes had roles in transcriptional regulation of cell cycle and proliferation, carcinogenesis, organismal injury and abnormalities, xenobiotic metabolism, and cell-cell signaling genes. Collectively, Nrf2 deficiency decreased susceptibility to urethane-induced lung tumorigenesis in mice. Cell survival properties of Nrf2 were supported, at least in part, by reduced early death of initiated cells and heightened advantage for tumor cell expansion in Nrf2(+/+) mice relative to Nrf2(-/-) mice. Our results were consistent with the concept that Nrf2 over-activation is an adaptive response of cancer conferring resistance to anti-cancer drugs and promoting malignancy.

Show MeSH
Related in: MedlinePlus