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Low dose radiation upregulates Ras/p38 and NADPH oxidase in mouse colon two months after exposure

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Abstract

Background

Exposure to ionizing is known to cause persistent cellular oxidative stress and NADPH oxidase (Nox) is a major source of cellular oxidant production. Chronic oxidative stress is associated with a myriad of human diseases including gastrointestinal cancer. However, the roles of NADPH oxidase in relation of long-term oxidative stress in colonic epithelial cells after radiation exposure are yet to be clearly established.

Methods and results

Mice were exposed either to sham or to 0.5 Gy γ radiation, and NADPH oxidase, oxidative stress, and related signaling pathways were assessed in colon samples 60 days after exposure. Radiation exposure led to increased expression of colon-specific NADPH oxidase isoform, Nox1, as well as upregulation of its modifiers such as Noxa1 and Noxo1 at the mRNA and protein level. Co-immunoprecipitation experiments showed enhanced binding of Rac1, an activator of NADPH oxidase, to Nox1. Increased 4-hydroxynonenal, 8-oxo-dG, and γH2AX along with higher protein carbonylation levels suggest increased oxidative stress after radiation exposure. Immunoblot analysis demonstrates upregulation of Ras/p38 pathway, and Gata6 and Hif1α after irradiation. Increased staining of β-catenin, cyclinD1, and Ki67 after radiation was also observed.

Conclusions

In summary, data show that exposure to a low dose of radiation was associated with upregulation of NADPH oxidase and its modifiers along with increased Ras/p38/Gata6 signaling in colon. When considered along with oxidative damage and proliferative markers, our observations suggest that the NADPH oxidase pathway could be playing a critical role in propagating long-term oxidative stress after radiation with implications for colon carcinogenesis.

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Abbreviations

NADPH:

Nicotinamide adenine dinucleotide phosphate-reduced

Nox:

NADPH oxidase

Noxo1:

NADPH oxidase organizer 1

Noxa1:

NADPH oxidase activator 1

Hif1α:

Hypoxia inducible factor 1

Pak1-PBD:

P21 activated kinase-protein binding domain

Rac1:

Ras-related C3 botulinum toxin substrate 1

Cdc42:

Cell division cycle 42

ROS:

Reactive Oxygen Species

4HNE:

4-Hydroxynonenal

DSB:

Double Strand Break

Mapk:

Mitogen-activated protein kinases

IACUC:

Institutional Animal Care and Use Committee

DAB:

3,3'Diaminobenzidine

SDS-PAGE:

Sodium dodecyl-sulfate polyacrylamide gel electrophoresis

DNPH:

2,4-Dinitrophenylhydrazine

SEM:

Standard Errors of the Mean

RT-PCR:

Reverse transcription-polymerase chain reaction

References

  1. Bedard K, Krause KH (2007) The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiol Rev 87:245–313

    CAS  PubMed  Google Scholar 

  2. Ambasta RK, Kumar P, Griendling KK, Schmidt HH, Busse R, Brandes RP (2004) Direct interaction of the novel Nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase. J Biol Chem 279:45935–45941

    CAS  PubMed  Google Scholar 

  3. Kawahara T, Lambeth JD (2007) Molecular evolution of Phox-related regulatory subunits for NADPH oxidase enzymes. BMC Evol Biol 7:178

    PubMed  PubMed Central  Google Scholar 

  4. Datta K, Suman S, Fornace AJJ (2014) Radiation persistently promoted oxidative stress, activated mTOR via PI3K/Akt, and downregulated autophagy pathway in mouse intestine. Int J Biochem Cell Biol 57:167–176

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Robbins ME, Zhao W (2004) Chronic oxidative stress and radiation-induced late normal tissue injury: a review. Int J Radiat Biol 80:251–259

    CAS  PubMed  Google Scholar 

  6. Pazhanisamy SK, Li H, Wang Y, Batinic-Haberle I, Zhou D (2011) NADPH oxidase inhibition attenuates total body irradiation-induced haematopoietic genomic instability. Mutagenesis 26:431–435

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Wang Y, Liu L, Pazhanisamy SK, Li H, Meng A, Zhou D (2010) Total body irradiation causes residual bone marrow injury by induction of persistent oxidative stress in murine hematopoietic stem cells. Free Radic Biol Med 48:348–356

    CAS  PubMed  Google Scholar 

  8. Datta K, Suman S, Kallakury BV, Fornace AJJ (2012) Exposure to heavy ion radiation induces persistent oxidative stress in mouse intestine. PLoS ONE 7:e42224

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Hauck AK, Bernlohr DA (2016) Oxidative stress and lipotoxicity. J Lipid Res 57:1976–1986

    CAS  PubMed  PubMed Central  Google Scholar 

  10. England K, Cotter TG (2005) Direct oxidative modifications of signalling proteins in mammalian cells and their effects on apoptosis. Redox Rep 10:237–245

    CAS  PubMed  Google Scholar 

  11. Cox AD, Fesik SW, Kimmelman AC, Luo J, Der CJ (2014) Drugging the undruggable RAS: mission possible. Nat Rev Drug Discov 13:828–851

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Ferro E, Goitre L, Retta SF, Trabalzini L (2012) The interplay between ROS and Ras GTPases: physiological and pathological implications. J Signal Transduct 2012:365769

    PubMed  Google Scholar 

  13. Skonieczna M, Hejmo T, Poterala-Hejmo A, Cieslar-Pobuda A, Buldak RJ (2017) NADPH oxidases: insights into selected functions and mechanisms of action in cancer and stem cells. Oxid Med Cell Longev 2017:9420539

    PubMed  PubMed Central  Google Scholar 

  14. Zhang J, Wang X, Vikash V, Ye Q, Wu D, Liu Y, Dong W (2016) ROS and ROS-mediated cellular signaling. Oxid Med Cell Longev 2016:4350965

    PubMed  PubMed Central  Google Scholar 

  15. Bosco EE, Mulloy JC, Zheng Y (2009) Rac1 GTPase: a “Rac” of all trades. Cell Mol Life Sci 66:370–374

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Suman S, Rodriguez OC, Winters TA, Fornace AJJ, Albanese C, Datta K (2013) Therapeutic and space radiation exposure of mouse brain causes impaired DNA repair response and premature senescence by chronic oxidant production. Aging (Albany NY) 5:607–622

    PubMed  PubMed Central  Google Scholar 

  17. Suman S, Kallakury BV, Fornace AJJ, Datta K (2015) Protracted upregulation of Leptin and IGF1 is associated with activation of PI3K/Akt and JAK2 pathway in mouse intestine after ionizing radiation exposure. Int J Biol Sci 11:274–283

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Suman S, Kumar S, N’Gouemo P, Datta K (2016) Increased DNA double-strand break was associated with downregulation of repair and upregulation of apoptotic factors in rat hippocampus after alcohol exposure. Alcohol 54:45–50

    CAS  PubMed  Google Scholar 

  19. Suman S, Kumar S, Fornace AJ, Datta K (2016) Space radiation exposure persistently increased leptin and IGF1 in serum and activated leptin-IGF1 signaling axis in mouse intestine. Sci Rep 6:31853

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Datta K, Hyduke DR, Suman S, Moon BH, Johnson MD, Fornace AJJ (2012) Exposure to ionizing radiation induced persistent gene expression changes in mouse mammary gland. Radiat Oncol 7:205

    CAS  PubMed  PubMed Central  Google Scholar 

  21. van Houdt WJ, de Bruijn MT, Emmink BL, Raats D, Hoogwater FJ, Borel Rinkes IH, Kranenburg O (2010) Oncogenic K-ras activates p38 to maintain colorectal cancer cell proliferation during MEK inhibition. Cell Oncol 32:245–257

    PubMed  PubMed Central  Google Scholar 

  22. Li HS, Zhou YN, Li L, Li SF, Long D, Chen XL, Zhang JB, Feng L, Li YP (2019) HIF-1α protects against oxidative stress by directly targeting mitochondria. Redox Biol 25:101109

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Crawford DR, Davies KJ (1994) Adaptive response and oxidative stress. Environ Health Perspect 102(Suppl 10):25–28

    PubMed  PubMed Central  Google Scholar 

  24. Azzam EI, Jay-Gerin JP, Pain D (2012) Ionizing radiation-induced metabolic oxidative stress and prolonged cell injury. Cancer Lett 327:48–60

    CAS  PubMed  Google Scholar 

  25. Henderson TO, Oeffinger KC, Whitton J, Leisenring W, Neglia J, Meadows A, Crotty C, Rubin DT, Diller L, Inskip P, Smith SA, Stovall M, Constine LS, Hammond S, Armstrong GT, Robison LL, Nathan PC (2012) Secondary gastrointestinal cancer in childhood cancer survivors: a cohort study. Ann Intern Med 156:757–766

    PubMed  PubMed Central  Google Scholar 

  26. Rapiti E, Fioretta G, Verkooijen HM, Zanetti R, Schmidlin F, Shubert H, Merglen A, Miralbell R, Bouchardy C (2008) Increased risk of colon cancer after external radiation therapy for prostate cancer. Int J Cancer 123:1141–1145

    CAS  PubMed  Google Scholar 

  27. Coant N, Ben Mkaddem S, Pedruzzi E, Guichard C, Tréton X, Ducroc R, Freund JN, Cazals-Hatem D, Bouhnik Y, Woerther PL, Skurnik D, Grodet A, Fay M, Biard D, Lesuffleur T, Deffert C, Moreau R, Groyer A, Krause KH, Daniel F, Ogier-Denis E (2010) NADPH oxidase 1 modulates WNT and NOTCH1 signaling to control the fate of proliferative progenitor cells in the colon. Mol Cell Biol 30:2636–2650

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Juhasz A, Markel S, Gaur S, Liu H, Lu J, Jiang G, Wu X, Antony S, Wu Y, Melillo G, Meitzler JL, Haines DC, Butcher D, Roy K, Doroshow JH (2017) NADPH oxidase 1 supports proliferation of colon cancer cells by modulating reactive oxygen species-dependent signal transduction. J Biol Chem 292:7866–7887

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Ramonaite R, Skieceviciene J, Kiudelis G, Jonaitis L, Tamelis A, Cizas P, Borutaite V, Kupcinskas L (2013) Influence of NADPH oxidase on inflammatory response in primary intestinal epithelial cells in patients with ulcerative colitis. BMC Gastroenterol 13:159

    PubMed  PubMed Central  Google Scholar 

  30. Lomax ME, Cunniffe S, O’Neill P (2004) 8-OxoG retards the activity of the ligase III/XRCC1 complex during the repair of a single-strand break, when present within a clustered DNA damage site. DNA Repair (Amst) 3:289–299

    CAS  PubMed  Google Scholar 

  31. Ohno M, Sakumi K, Fukumura R, Furuichi M, Iwasaki Y, Hokama M, Ikemura T, Tsuzuki T, Gondo Y, Nakabeppu Y (2014) 8-oxoguanine causes spontaneous de novo germline mutations in mice. Sci Rep 4:4689

    PubMed  PubMed Central  Google Scholar 

  32. Nakabeppu Y (2014) Cellular levels of 8-oxoguanine in either DNA or the nucleotide pool play pivotal roles in carcinogenesis and survival of cancer cells. Int J Mol Sci 15:12543–12557

    PubMed  PubMed Central  Google Scholar 

  33. Zhong H, Yin H (2015) Role of lipid peroxidation derived 4-hydroxynonenal (4-HNE) in cancer: focusing on mitochondria. Redox Biol 4:193–199

    CAS  PubMed  Google Scholar 

  34. Suzuki YJ, Carini M, Butterfield DA (2010) Protein carbonylation. Antioxid Redox Signal 12:323–325

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Adachi Y, Shibai Y, Mitsushita J, Shang WH, Hirose K, Kamata T (2008) Oncogenic Ras upregulates NADPH oxidase 1 gene expression through MEK-ERK-dependent phosphorylation of GATA-6. Oncogene 27:4921–4932

    CAS  PubMed  Google Scholar 

  36. Brewer AC, Sparks EC, Shah AM (2006) Transcriptional regulation of the NADPH oxidase isoform, Nox1, in colon epithelial cells: role of GATA-binding factor(s). Free Radic Biol Med 40:260–274

    CAS  PubMed  Google Scholar 

  37. Lee YH, Bae HC, Noh KH, Song KH, Ye SK, Mao CP, Lee KM, Wu TC, Kim TW (2015) Gain of HIF-1α under normoxia in cancer mediates immune adaptation through the AKT/ERK and VEGFA axes. Clin Cancer Res 21:1438–1446

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Kwon SJ, Song JJ, Lee YJ (2005) Signal pathway of hypoxia-inducible factor-1alpha phosphorylation and its interaction with von Hippel-Lindau tumor suppressor protein during ischemia in MiaPaCa-2 pancreatic cancer cells. Clin Cancer Res 11:7607–7613

    CAS  PubMed  Google Scholar 

  39. Khandrika L, Lieberman R, Koul S, Kumar B, Maroni P, Chandhoke R, Meacham RB, Koul HK (2009) Hypoxia-associated p38 mitogen-activated protein kinase-mediated androgen receptor activation and increased HIF-1alpha levels contribute to emergence of an aggressive phenotype in prostate cancer. Oncogene 28:1248–1260

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Tchakarska G, Sola B (2020) The double dealing of cyclin D1. Cell Cycle 19:163–178

    CAS  PubMed  Google Scholar 

  41. Zhang B, Li YL, Zhao JL, Zhen O, Yu C, Yang BH, Yu XR (2018) Hypoxia-inducible factor-1 promotes cancer progression through activating AKT/Cyclin D1 signaling pathway in osteosarcoma. Biomed Pharmacother 105:1–9

    CAS  PubMed  Google Scholar 

  42. Luo Y, Li M, Zuo X, Basourakos SP, Zhang J, Zhao J, Han Y, Lin Y, Wang Y, Jiang Y, Lan L (2018) β-catenin nuclear translocation induced by HIF-1α overexpression leads to the radioresistance of prostate cancer. Int J Oncol 52:1827–1840

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Zeller E, Hammer K, Kirschnick M, Braeuning A (2013) Mechanisms of RAS/β-catenin interactions. Arch Toxicol 87:611–632

    CAS  PubMed  Google Scholar 

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Acknowledgements

We are thankful to Ms. Pelagie Ake for animal facility supports.

Funding

This study was supported in part by National Aeronautics and Space Administration (NASA) Grant Numbers NNX13AD58G, NNX09AU95G, and 80NSSC22K1279. The authors acknowledge the Lombardi Comprehensive Cancer Shared Resources (MSR), which are in part supported by award number P30CA051008 (P.I. Louis Weiner) from the National Cancer Institute.

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Authors and Affiliations

  1. Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, 20057, USA

    Santosh Kumar, Shubhankar Suman, Bo-Hyun Moon, Albert J Fornace Jr. & Kamal Datta

  2. Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Research Building, Room E518, 3970 Reservoir Rd., NW, Washington, DC, 20057, USA

    Albert J Fornace Jr. & Kamal Datta

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  1. Santosh Kumar
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Correspondence to Kamal Datta.

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All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. A protocol approved by the Georgetown University Animal Care and Use Committee (GUACUC) was followed for the care and use of animals. This article does not contain any studies with human participants performed by any of the authors.

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Kumar, S., Suman, S., Moon, BH. et al. Low dose radiation upregulates Ras/p38 and NADPH oxidase in mouse colon two months after exposure. Mol Biol Rep 50, 2067–2076 (2023). https://doi.org/10.1007/s11033-022-08186-3

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