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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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Bronchoalveolar lavage analysis (BAL) and body weight changes at a pre-neoplastic stage.(A) BAL analysis found significantly higher concentration of total protein and numbers of monocytes, lymphocytes, phagocytic macrophages, and epithelial cells in Nrf2-/- mice at 9 and/or 11 wk after urethane treatment. Neutrophilic myeloperoxidase (MPO) activity in BAL fluids was also significantly higher in Nrf2-/- mice than in Nrf2+/+ mice. Mean±SD are presented (n = 3/group for vehicle, n = 5−9/group for urethane). *, p<0.05 vs. genotype-matched controls. +, p<0.05 vs. treatment-matched Nrf2+/+ mice. (B) Necrotic lung cell lysis and death was assessed by lactate dehydrogenase (LDH) activity in aliquots of BAL fluids using a colorimetric assay. Mean±SD are presented (n = 3/group for vehicle, n = 5−9/group for urethane). *, p<0.05 vs. genotype-matched controls. +, p<0.05 vs. treatment-matched Nrf2+/+ mice. (C) Percent whole body weight changes monitored during and after saline or urethane treatment. Mean±SD are presented (n = 9−24 in vehicle groups, n = 64−82 in urethane groups). *, p<0.05 Nrf2+/+ saline-treated vs. urethane-treated mice (p<0.05). #, p<0.05 Nrf2-/- saline-treated vs. urethane-treated mice. +, p<0.05 urethane-treated Nrf2+/+ mice vs. urethane-treated Nrf2-/- mice (p<0.05).
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pone-0026590-g001: Bronchoalveolar lavage analysis (BAL) and body weight changes at a pre-neoplastic stage.(A) BAL analysis found significantly higher concentration of total protein and numbers of monocytes, lymphocytes, phagocytic macrophages, and epithelial cells in Nrf2-/- mice at 9 and/or 11 wk after urethane treatment. Neutrophilic myeloperoxidase (MPO) activity in BAL fluids was also significantly higher in Nrf2-/- mice than in Nrf2+/+ mice. Mean±SD are presented (n = 3/group for vehicle, n = 5−9/group for urethane). *, p<0.05 vs. genotype-matched controls. +, p<0.05 vs. treatment-matched Nrf2+/+ mice. (B) Necrotic lung cell lysis and death was assessed by lactate dehydrogenase (LDH) activity in aliquots of BAL fluids using a colorimetric assay. Mean±SD are presented (n = 3/group for vehicle, n = 5−9/group for urethane). *, p<0.05 vs. genotype-matched controls. +, p<0.05 vs. treatment-matched Nrf2+/+ mice. (C) Percent whole body weight changes monitored during and after saline or urethane treatment. Mean±SD are presented (n = 9−24 in vehicle groups, n = 64−82 in urethane groups). *, p<0.05 Nrf2+/+ saline-treated vs. urethane-treated mice (p<0.05). #, p<0.05 Nrf2-/- saline-treated vs. urethane-treated mice. +, p<0.05 urethane-treated Nrf2+/+ mice vs. urethane-treated Nrf2-/- mice (p<0.05).

Mentions: Microscopic analysis of bronchoalveolar lavage (BAL) cells indicated pulmonary cytotoxicity including lysis at 9–11 wk after initial urethane injection in Nrf2-/- and Nrf2+/+ mice. Increases in mean numbers of BAL epithelial cells and leukocytes, as well as total protein concentration, were significantly greater in Nrf2-/- than Nrf2+/+ mice after 9–11 wk (Figure 1-A). By 11 wk, BAL cell aggregation was mostly resolved in both strains, but cell lysis remained in Nrf2-/- mice with concurrent manifestation of significantly higher large atypical macrophages showing phagocytosis or necrosis-like features than in Nrf2+/+ mice (Figure 1A; Figure S1). Significantly greater membrane rupture was also found in BAL cells from Nrf2-/- mice than Nrf2+/+ mice (Figure S1). At 9 wk, BAL cell clustering accompanied cytolysis, and this feature was markedly greater in Nrf2-/- mice compared to Nrf2+/+ mice (Figure S1). This observation is assumed to be associated with excess viscous secretion such as mucus as suggested by aggregation of cells with secreted mucus in Alcian Blue (pH 2.5)/periodic acid-Schiff (AB/PAS)-stained lungs (Figure S1), or by procoagulant activity due to fibrin deposition in mononuclear phagocytes which occurs during lung inflammation [16], [17]. Infiltrated neutrophils were obvious but not quantifiable due to extensive clustering and lysis of BAL cells in Nrf2-/- mice; consistent with this observation, neutrophil myeloperoxidase (MPO) activity was significantly higher in Nrf2-/- mice than in Nrf2+/+ mice treated with urethane (Figure 1A). Urethane-induced increase in lactate dehydrogenase (LDH) activity, a quantitative indicator of necrotic cell lysis, was also significantly greater in BAL returns from Nrf2-/- compared to Nrf2+/+ mice (Figure 1B).


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)

Bronchoalveolar lavage analysis (BAL) and body weight changes at a pre-neoplastic stage.(A) BAL analysis found significantly higher concentration of total protein and numbers of monocytes, lymphocytes, phagocytic macrophages, and epithelial cells in Nrf2-/- mice at 9 and/or 11 wk after urethane treatment. Neutrophilic myeloperoxidase (MPO) activity in BAL fluids was also significantly higher in Nrf2-/- mice than in Nrf2+/+ mice. Mean±SD are presented (n = 3/group for vehicle, n = 5−9/group for urethane). *, p<0.05 vs. genotype-matched controls. +, p<0.05 vs. treatment-matched Nrf2+/+ mice. (B) Necrotic lung cell lysis and death was assessed by lactate dehydrogenase (LDH) activity in aliquots of BAL fluids using a colorimetric assay. Mean±SD are presented (n = 3/group for vehicle, n = 5−9/group for urethane). *, p<0.05 vs. genotype-matched controls. +, p<0.05 vs. treatment-matched Nrf2+/+ mice. (C) Percent whole body weight changes monitored during and after saline or urethane treatment. Mean±SD are presented (n = 9−24 in vehicle groups, n = 64−82 in urethane groups). *, p<0.05 Nrf2+/+ saline-treated vs. urethane-treated mice (p<0.05). #, p<0.05 Nrf2-/- saline-treated vs. urethane-treated mice. +, p<0.05 urethane-treated Nrf2+/+ mice vs. urethane-treated Nrf2-/- mice (p<0.05).
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Related In: Results  -  Collection

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pone-0026590-g001: Bronchoalveolar lavage analysis (BAL) and body weight changes at a pre-neoplastic stage.(A) BAL analysis found significantly higher concentration of total protein and numbers of monocytes, lymphocytes, phagocytic macrophages, and epithelial cells in Nrf2-/- mice at 9 and/or 11 wk after urethane treatment. Neutrophilic myeloperoxidase (MPO) activity in BAL fluids was also significantly higher in Nrf2-/- mice than in Nrf2+/+ mice. Mean±SD are presented (n = 3/group for vehicle, n = 5−9/group for urethane). *, p<0.05 vs. genotype-matched controls. +, p<0.05 vs. treatment-matched Nrf2+/+ mice. (B) Necrotic lung cell lysis and death was assessed by lactate dehydrogenase (LDH) activity in aliquots of BAL fluids using a colorimetric assay. Mean±SD are presented (n = 3/group for vehicle, n = 5−9/group for urethane). *, p<0.05 vs. genotype-matched controls. +, p<0.05 vs. treatment-matched Nrf2+/+ mice. (C) Percent whole body weight changes monitored during and after saline or urethane treatment. Mean±SD are presented (n = 9−24 in vehicle groups, n = 64−82 in urethane groups). *, p<0.05 Nrf2+/+ saline-treated vs. urethane-treated mice (p<0.05). #, p<0.05 Nrf2-/- saline-treated vs. urethane-treated mice. +, p<0.05 urethane-treated Nrf2+/+ mice vs. urethane-treated Nrf2-/- mice (p<0.05).
Mentions: Microscopic analysis of bronchoalveolar lavage (BAL) cells indicated pulmonary cytotoxicity including lysis at 9–11 wk after initial urethane injection in Nrf2-/- and Nrf2+/+ mice. Increases in mean numbers of BAL epithelial cells and leukocytes, as well as total protein concentration, were significantly greater in Nrf2-/- than Nrf2+/+ mice after 9–11 wk (Figure 1-A). By 11 wk, BAL cell aggregation was mostly resolved in both strains, but cell lysis remained in Nrf2-/- mice with concurrent manifestation of significantly higher large atypical macrophages showing phagocytosis or necrosis-like features than in Nrf2+/+ mice (Figure 1A; Figure S1). Significantly greater membrane rupture was also found in BAL cells from Nrf2-/- mice than Nrf2+/+ mice (Figure S1). At 9 wk, BAL cell clustering accompanied cytolysis, and this feature was markedly greater in Nrf2-/- mice compared to Nrf2+/+ mice (Figure S1). This observation is assumed to be associated with excess viscous secretion such as mucus as suggested by aggregation of cells with secreted mucus in Alcian Blue (pH 2.5)/periodic acid-Schiff (AB/PAS)-stained lungs (Figure S1), or by procoagulant activity due to fibrin deposition in mononuclear phagocytes which occurs during lung inflammation [16], [17]. Infiltrated neutrophils were obvious but not quantifiable due to extensive clustering and lysis of BAL cells in Nrf2-/- mice; consistent with this observation, neutrophil myeloperoxidase (MPO) activity was significantly higher in Nrf2-/- mice than in Nrf2+/+ mice treated with urethane (Figure 1A). Urethane-induced increase in lactate dehydrogenase (LDH) activity, a quantitative indicator of necrotic cell lysis, was also significantly greater in BAL returns from Nrf2-/- compared to Nrf2+/+ mice (Figure 1B).

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