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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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Pulmonary Nrf2 expression and activation caused by urethane.(A) Western blot analysis of total Nrf2 protein in uninvolved (UN) and tumor tissues compared to saline control in lung at 22 wk (57 kDa determined). Nuclear translocation of Nrf2 in urethane-treated lungs at 22 wk was determined by Western blot analysis in whole lung nuclear extracts (10 µg). Lamin B1 level was determined as an internal control. (B) Binding activity of nuclear protein (5 µg) to [γ32P]ATP end-labeled oligonucleotide probe containing ARE consensus sequence was determined. Gel shift analysis demonstrated increased total ARE binding activity (arrows) of lung nuclear proteins after urethane treatment in Nrf2+/+ mice. Supershifted bands (arrow head) indicate specific binding activity of nuclear Nrf2 or small Maf on ARE determined by addition of anti-Nrf2 or –Maf (F/G/K) antibodies to the reaction. Representative digitized bands of Western blotting and gel shift analysis (n = 2/group) are presented. (C) Immunohistochemistry for Nrf2 in saline- (a) or urethane-treated (c,e) tissue sections. Greater localization of Nrf2 proteins (brown dots) in growing adenomas and in conducting airway and alveolar epithelial cells was found in Nrf2+/+ mice. Nrf2 was located mainly in airway epithelial cells of vehicle control lungs. Lung sections from Nrf2-/- mice (e) are shown as a negative control for Nrf2. H&E-stained lower magnification of lung sections treated with vehicle (b) or urethane (d,f) are depicted for general histology. Representative images showing intermediate degree of Nrf2 staining for each treatment group are presented (n = 3/group). AV = alveoli, BR = bronchi or bronchiole, TB = terminal bronchiole, BV = blood vessel, Bar = 100 µm.
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pone-0026590-g004: Pulmonary Nrf2 expression and activation caused by urethane.(A) Western blot analysis of total Nrf2 protein in uninvolved (UN) and tumor tissues compared to saline control in lung at 22 wk (57 kDa determined). Nuclear translocation of Nrf2 in urethane-treated lungs at 22 wk was determined by Western blot analysis in whole lung nuclear extracts (10 µg). Lamin B1 level was determined as an internal control. (B) Binding activity of nuclear protein (5 µg) to [γ32P]ATP end-labeled oligonucleotide probe containing ARE consensus sequence was determined. Gel shift analysis demonstrated increased total ARE binding activity (arrows) of lung nuclear proteins after urethane treatment in Nrf2+/+ mice. Supershifted bands (arrow head) indicate specific binding activity of nuclear Nrf2 or small Maf on ARE determined by addition of anti-Nrf2 or –Maf (F/G/K) antibodies to the reaction. Representative digitized bands of Western blotting and gel shift analysis (n = 2/group) are presented. (C) Immunohistochemistry for Nrf2 in saline- (a) or urethane-treated (c,e) tissue sections. Greater localization of Nrf2 proteins (brown dots) in growing adenomas and in conducting airway and alveolar epithelial cells was found in Nrf2+/+ mice. Nrf2 was located mainly in airway epithelial cells of vehicle control lungs. Lung sections from Nrf2-/- mice (e) are shown as a negative control for Nrf2. H&E-stained lower magnification of lung sections treated with vehicle (b) or urethane (d,f) are depicted for general histology. Representative images showing intermediate degree of Nrf2 staining for each treatment group are presented (n = 3/group). AV = alveoli, BR = bronchi or bronchiole, TB = terminal bronchiole, BV = blood vessel, Bar = 100 µm.

Mentions: Compared to saline controls at 22 wk, lung Nrf2 protein was increased in tumor tissues and in remaining uninvolved lung tissues (UN: adjacent, non-tumor bearing regions) of Nrf2+/+ mice (Figure 4A). Nuclear levels of Nrf2 (Figure 4A) and total ARE binding activity of nuclear proteins (Figure 4B) were also higher in tumor-bearing lungs. Specific binding activities of Nrf2 and its dimerization partner small Maf on ARE were also heightened by urethane (Figure 4B). Abundant Nrf2 was located throughout the epithelium lining bronchial/bronchiolar and alveolar airways in control mice (Figure 4Ca). At 22 wk after urethane, the Nrf2-bearing cell populations were apparent in hyperplastic foci and growing adenomas in addition to their increase in the airway epithelium (Figure 4Cc).


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)

Pulmonary Nrf2 expression and activation caused by urethane.(A) Western blot analysis of total Nrf2 protein in uninvolved (UN) and tumor tissues compared to saline control in lung at 22 wk (57 kDa determined). Nuclear translocation of Nrf2 in urethane-treated lungs at 22 wk was determined by Western blot analysis in whole lung nuclear extracts (10 µg). Lamin B1 level was determined as an internal control. (B) Binding activity of nuclear protein (5 µg) to [γ32P]ATP end-labeled oligonucleotide probe containing ARE consensus sequence was determined. Gel shift analysis demonstrated increased total ARE binding activity (arrows) of lung nuclear proteins after urethane treatment in Nrf2+/+ mice. Supershifted bands (arrow head) indicate specific binding activity of nuclear Nrf2 or small Maf on ARE determined by addition of anti-Nrf2 or –Maf (F/G/K) antibodies to the reaction. Representative digitized bands of Western blotting and gel shift analysis (n = 2/group) are presented. (C) Immunohistochemistry for Nrf2 in saline- (a) or urethane-treated (c,e) tissue sections. Greater localization of Nrf2 proteins (brown dots) in growing adenomas and in conducting airway and alveolar epithelial cells was found in Nrf2+/+ mice. Nrf2 was located mainly in airway epithelial cells of vehicle control lungs. Lung sections from Nrf2-/- mice (e) are shown as a negative control for Nrf2. H&E-stained lower magnification of lung sections treated with vehicle (b) or urethane (d,f) are depicted for general histology. Representative images showing intermediate degree of Nrf2 staining for each treatment group are presented (n = 3/group). AV = alveoli, BR = bronchi or bronchiole, TB = terminal bronchiole, BV = blood vessel, Bar = 100 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198791&req=5

pone-0026590-g004: Pulmonary Nrf2 expression and activation caused by urethane.(A) Western blot analysis of total Nrf2 protein in uninvolved (UN) and tumor tissues compared to saline control in lung at 22 wk (57 kDa determined). Nuclear translocation of Nrf2 in urethane-treated lungs at 22 wk was determined by Western blot analysis in whole lung nuclear extracts (10 µg). Lamin B1 level was determined as an internal control. (B) Binding activity of nuclear protein (5 µg) to [γ32P]ATP end-labeled oligonucleotide probe containing ARE consensus sequence was determined. Gel shift analysis demonstrated increased total ARE binding activity (arrows) of lung nuclear proteins after urethane treatment in Nrf2+/+ mice. Supershifted bands (arrow head) indicate specific binding activity of nuclear Nrf2 or small Maf on ARE determined by addition of anti-Nrf2 or –Maf (F/G/K) antibodies to the reaction. Representative digitized bands of Western blotting and gel shift analysis (n = 2/group) are presented. (C) Immunohistochemistry for Nrf2 in saline- (a) or urethane-treated (c,e) tissue sections. Greater localization of Nrf2 proteins (brown dots) in growing adenomas and in conducting airway and alveolar epithelial cells was found in Nrf2+/+ mice. Nrf2 was located mainly in airway epithelial cells of vehicle control lungs. Lung sections from Nrf2-/- mice (e) are shown as a negative control for Nrf2. H&E-stained lower magnification of lung sections treated with vehicle (b) or urethane (d,f) are depicted for general histology. Representative images showing intermediate degree of Nrf2 staining for each treatment group are presented (n = 3/group). AV = alveoli, BR = bronchi or bronchiole, TB = terminal bronchiole, BV = blood vessel, Bar = 100 µm.
Mentions: Compared to saline controls at 22 wk, lung Nrf2 protein was increased in tumor tissues and in remaining uninvolved lung tissues (UN: adjacent, non-tumor bearing regions) of Nrf2+/+ mice (Figure 4A). Nuclear levels of Nrf2 (Figure 4A) and total ARE binding activity of nuclear proteins (Figure 4B) were also higher in tumor-bearing lungs. Specific binding activities of Nrf2 and its dimerization partner small Maf on ARE were also heightened by urethane (Figure 4B). Abundant Nrf2 was located throughout the epithelium lining bronchial/bronchiolar and alveolar airways in control mice (Figure 4Ca). At 22 wk after urethane, the Nrf2-bearing cell populations were apparent in hyperplastic foci and growing adenomas in addition to their increase in the airway epithelium (Figure 4Cc).

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