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Attenuation of reactive oxygen species by antioxidants suppresses hypoxia-induced epithelial-mesenchymal transition and metastasis of pancreatic cancer cells

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Abstract

Hypoxia has been shown to promote metastasis of cancer cells through induction of epithelial-mesenchymal transition (EMT). It is also known to cause generation of reactive oxygen species (ROS). We investigated here the role of ROS in hypoxia-induced EMT and whether attenuation of ROS by antioxidants suppresses hypoxia-induced EMT and metastasis of human pancreatic cancer cells in a xenograft nude mouse model. PANC-1 and MiaPaCa-2 cells exposed to hypoxia (1 % O2) showed increased ROS generation and characteristic changes of EMT such as morphological changes, enhanced invasiveness, and upregulation of EMT regulators, SLUG, SNAI1 and TWIST. The antioxidants N-acetylcysteine (NAC) and ebselen significantly suppressed EMT and the expression of EMT regulators during hypoxia. NAC abrogated activation of HIF-1α and NF-κB, both of which were found to play an active role in hypoxia-induced EMT. Administration of NAC to nude mice with orthotopic tumors suppressed the expression of EMT regulators in hypoxic areas and significantly inhibited hepatic metastasis. Together, the present findings demonstrate that attenuation of ROS by antioxidants suppresses hypoxia-induced EMT and metastatic phenotype, suggesting that antioxidants may be of therapeutic value in treating pancreatic cancers.

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Abbreviations

EMT:

Epithelial-mesenchymal transition

ROS:

Reactive oxygen species

NAC:

N-acetylcysteine

HIF:

Hypoxia-inducible factor

PDH:

Prolyl hydroxylases

Glut-1:

Glucose transporter-1

VEGF:

Vascular endothelial growth factor

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

NF-κB:

Nuclear factor-κB

ZO-1:

Zonula occludens-1

TGF-β:

Transforming growth factor-β

References

  1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, Thun MJ (2008) Cancer statistics, 2008. CA Cancer J Clin 58:71–96. doi:10.3322/CA.2007.0010

    Article  PubMed  Google Scholar 

  2. Baxter NN, Whitson BA, Tuttle TM (2007) Trends in the treatment and outcome of pancreatic cancer in the United States. Ann Surg Oncol 14:1320–1326. doi:10.1245/s10434-006-9249-8

    Article  PubMed  Google Scholar 

  3. Koong AC, Mehta VK, Le QT, Fisher GA, Terris DJ, Brown JM, Bastidas AJ, Vierra M (2000) Pancreatic tumors show high levels of hypoxia. Int J Rad Oncol Biol Phys 48:919–922. doi:10.1016/S0360-3016(00)00803-8

    Article  CAS  Google Scholar 

  4. Semenza GL (2008) Hypoxia-inducible factor 1 and cancer pathogenesis. IUBMB Life 60:591–597. doi:10.1002/iub.93

    Article  PubMed  CAS  Google Scholar 

  5. Sun HC, Qiu ZJ, Liu J, Sun J, Jiang T, Huang KJ, Yao M, Huang C (2007) Expression of hypoxia-inducible factor-1 alpha and associated proteins in pancreatic ductal adenocarcinoma and their impact on prognosis. Int J Oncol 30:1359–1367

    PubMed  CAS  Google Scholar 

  6. Semenza GL (2010) Defining the role of hypoxia-inducible factor 1 in cancer biology and therapeutics. Oncogene 29:625–634. doi:10.1038/onc.2009.441

    Article  PubMed  CAS  Google Scholar 

  7. Klimova T, Chandel NS (2008) Mitochondrial complex III regulates hypoxic activation of HIF. Cell Death Differ 15:660–666. doi:10.1038/sj.cdd.4402307

    Article  PubMed  CAS  Google Scholar 

  8. Wu WS (2006) The signaling mechanism of ROS in tumor progression. Cancer Metastasis Rev 25:695–705. doi:10.1007/s10555-006-9037-8

    Article  PubMed  CAS  Google Scholar 

  9. Wu Y, Deng J, Rychahou PG, Qiu S, Evers BM, Zhou BP (2009) Stabilization of Snail by NF-κB is required for inflammation-induced cell migration and invasion. Cancer Cell 15:416–428. doi:10.1016/j.ccr.2009.03.016

    Article  PubMed  CAS  Google Scholar 

  10. Cannito S, Novo E, Compagnone A, Valfrè di Bonzo L, Busletta C, Zamara E, Paternostro C, Povero D, Bandino A, Bozzo F, Cravanzola C, Bravoco V, Colombatto S, Parola M (2008) Redox mechanisms switch on hypoxia-dependent epithelial-mesenchymal transition in cancer cells. Carcinogenesis 29:2267–2278. doi:10.1093/carcin/bgn216

    Article  PubMed  CAS  Google Scholar 

  11. Peinado H, Olmeda D, Cano A (2007) Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? Nat Rev Cancer 7:415–428. doi:10.1038/nrc2131

    Article  PubMed  CAS  Google Scholar 

  12. Thiery JP, Sleeman JP (2006) Complex networks orchestrate epithelial-mesenchymal transitions. Nat Rev Mol Cell Biol 7:131–142. doi:10.1038/nrm1835

    Article  PubMed  CAS  Google Scholar 

  13. Zavadil J, Böttinger EP (2005) TGF-beta and epithelial-to-mesenchymal transitions. Oncogene 24:5764–5774. doi:10.1038/sj.onc.1208927

    Article  PubMed  CAS  Google Scholar 

  14. Sullivan R, Graham CH (2007) Hypoxia-driven selection of the metastatic phenotype. Cancer Metastasis Rev 26:319–331. doi:10.1007/s10555-007-9062-2

    Article  PubMed  CAS  Google Scholar 

  15. Imai T, Horiuchi A, Wang C, Oka K, Ohira S, Nikaido T, Konishi I (2003) Hypoxia attenuates the ex18pression of E-cadherin via up-regulation of Snail in ovarian carcinoma cells. Am J Pathol 163:1437–1447. doi:10.1016/S0002-9440(10)63501-8

    Article  PubMed  CAS  Google Scholar 

  16. Kurrey NK, Amit K, Bapat SA (2005) Snail and SLUG are major determinants of ovarian cancer invasiveness at the transcription level. Gynecol Oncol 97:155–165. doi:10.1016/j.ygyno.2004.12.043

    Article  PubMed  CAS  Google Scholar 

  17. Yang MH, Wu MZ, Chiou SH, Chen PM, Chang SY, Liu CJ, Teng SC, Wu KJ (2008) Direct regulation of TWIST by HIF-1alpha promotes metastasis. Nat Cell Biol 10:295–305. doi:10.1038/ncb1691

    Article  PubMed  CAS  Google Scholar 

  18. Alves CC, Carneiro F, Hoefler H, Becker KF (2009) Role of the epithelial-mesenchymal transition regulator SLUG in primary human cancers. Front Biosci 14:3041–3050

    CAS  Google Scholar 

  19. Gort EH, van Haaften G, Verlaan I, Groot AJ, Plasterk RH, Shvarts A, Suijkerbuijk KP, van Laar T, van der Wall E, Raman V, van Diest PJ, Tijsterman M, Vooijs M (2008) The TWIST1 oncogene is a direct target of hypoxia-inducible factor-2alpha. Oncogene 27:1501–1510. doi:10.1038/sj.onc.1210795

    Article  PubMed  CAS  Google Scholar 

  20. Aitio ML (2006) N-acetylcysteine–passe–partout or much ado about nothing? Br J Clin Pharmacol 61:5–15. doi:10.1111/j.1365-2125.2005.02523.x

    Article  PubMed  CAS  Google Scholar 

  21. Block KI, Koch AC, Mead MN, Tothy PK, Newman RA, Gyllenhaal C (2007) Impact of antioxidant supplementation on chemotherapeutic efficacy: a systematic review of the evidence from randomized controlled trials. Cancer Treat Rev 33:407–418. doi:10.1016/j.ctrv.2007.01.005

    Article  PubMed  CAS  Google Scholar 

  22. Millea PJ (2009) N-acetylcysteine: multiple clinical applications. Am Fam Physician 80:265–269

    PubMed  Google Scholar 

  23. Seril DN, Liao J, Ho KL, Yang CS, Yang GY (2002) Inhibition of chronic ulcerative colitis-associated colorectal adenocarcinoma development in a murine model by N-acetylcysteine. Carcinogenesis 23:993–1001. doi:10.1093/carcin/23.6.993

    Article  PubMed  CAS  Google Scholar 

  24. Cai T, Fassina G, Morini M, Aluigi MG, Masiello L, Fontanini G, D’Agostini F, De Flora S, Noonan DM, Albini A (1999) N-acetylcysteine inhibits endothelial cell invasion and angiogenesis. Lab Invest 79:1151–1159

    PubMed  CAS  Google Scholar 

  25. Felton VM, Borok Z, Willis BC (2009) N-acetylcysteine inhibits alveolar epithelial- mesenchymal transition. Am J Physiol Lung Cell Mol Physiol 297:L805–L812. doi:10.1152/ajplung.00009.2009

    Article  PubMed  CAS  Google Scholar 

  26. Rhyu DY, Yang Y, Ha H, Lee GT, Song JS, Uh ST, Lee HB (2005) Role of reactive oxygen species in TGF-b1-induced mitogen-activated protein kinase activation and epithelial-mesenchymal transition in renal tubular epithelial cells. J Am Soc Nephrol 16:667–675. doi:10.1681/ASN.2004050425

    Article  PubMed  CAS  Google Scholar 

  27. Ishikawa K, Takenaga K, Akimoto M, Koshikawa N, Yamaguchi A, Imanishi H, Nakada K, Honma Y, Hayashi J (2008) ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis. Science 320:661–664. doi:10.1126/science.1156906

    Article  PubMed  CAS  Google Scholar 

  28. Ito S, Koshikawa N, Mochizuki S, Takenaga K (2007) 3-methyladenine suppresses cell migration and invasion of HT1080 fibrosarcoma cells through inhibiting phosphoinositide 3-kinases independently of autophagy inhibition. Int J Oncol 31:261–268

    PubMed  CAS  Google Scholar 

  29. Koshikawa N, Maejima C, Miyazaki K, Nakagawara A, Takenaga K (2006) Hypoxia selects for high-metastatic Lewis lung carcinoma cells overexpressing Mcl-1 and exhibiting reduced apoptotic potential in solid tumors. Oncogene 25:917–928. doi:10.1038/sj.onc.1209128

    Article  PubMed  CAS  Google Scholar 

  30. Yang H, Fang G, Huang X, Yu J, Hsieh CL, Grossniklaus HE (2008) In vivo xenograft murine human uveal melanoma model develops hepatic micrometastases. Melanoma Res 18:95–103. doi:10.1097/CMR.0b013e3282f628df

    Article  PubMed  Google Scholar 

  31. Chandel NS, Maltepe E, Goldwasser E, Mathieu CE, Simon MC, Schumacker PT (1998) Mitochondrial reactive oxygen species trigger hypoxia-induced transcription. Proc Natl Acad Sci USA 95:11715–11720. doi:10.1073/pnas.95.20.11715

    Article  PubMed  CAS  Google Scholar 

  32. Cannito S, Novo E, di Bonzo LV, Busletta C, Colombatto S, Parola M (2010) Epithelial-mesenchymal transition: from molecular mechanisms, redox regulation to implications in human health and disease. Antioxid Redox Signal 12:1383–1430. doi:10.1089/ars.2009.2737

    Article  PubMed  CAS  Google Scholar 

  33. Casas E, Kim J, Bendesky A, Ohno-Machado L, Wolfe CJ, Yang J (2011) Snail2 is an essential mediator of TWIST1-induced epithelial mesenchymal transition and metastasis. Cancer Res 71:245–254. doi:10.1158/0008-5472.CAN-10-2330

    Article  PubMed  CAS  Google Scholar 

  34. Koshikawa N, Hayashi J, Nakagawara A, Takenaga K (2009) Reactive oxygen species-generating mitochondrial DNA mutation up-regulates hypoxia-inducible factor-1alpha gene transcription via phosphatidylinositol 3-kinase-Akt/protein kinase C/histone deacetylase pathway. J Biol Chem 284:33185–33194. doi:10.1074/jbc.M109.054221

    Article  PubMed  CAS  Google Scholar 

  35. Galanis A, Pappa A, Giannakakis A, Lanitis E, Dangaj D, Sandaltzopoulos R (2008) Reactive oxygen species and HIF-1 signaling in cancer. Cancer Lett 266:12–20. doi:10.1016/j.canlet.2008.02.028

    Article  PubMed  CAS  Google Scholar 

  36. Chua YL, Dufour E, Dassa EP, Rustin P, Jacobs HT, Taylor CT, Hagen T (2010) Stabilization of hypoxia-inducible factor-1alpha protein in hypoxia occurs independently of mitochondrial reactive oxygen species production. J Biol Chem 285:31277–31284. doi:10.1074/jbc.M110.158485

    Article  PubMed  CAS  Google Scholar 

  37. Cummins EP, Berra E, Comerford KM, Ginouves A, Fitzgerald KT, Seeballuck F, Godson C, Nielsen JE, Moynagh P, Pouyssegur J, Taylor CT (2006) Prolyl hydroxylase-1 negatively regulates IkappaB kinase-beta, giving insight into hypoxia-induced NFkappaB activity. Proc Natl Acad Sci USA 103:18154–18159. doi:10.1073/pnas.0602235103

    Article  PubMed  CAS  Google Scholar 

  38. Culver C, Sundqvist A, Mudie S, Melvin A, Xirodimas D, Rocha S (2010) Mechanism of hypoxia-induced NF-kappaB. Mol Cell Biol 30:4901–4921. doi:10.1128/MCB.00409-10

    Article  PubMed  CAS  Google Scholar 

  39. Lluis JM, Buricchi F, Chiarugi P, Morales A, Fernandez-Checa JC (2007) Dual role of mitochondrial reactive oxygen species in hypoxia signaling: activation of nuclear factor-kappaB via c-SRC and oxidant-dependent cell death. Cancer Res 67:7368–7377. doi:10.1158/0008-5472.CAN-07-0515

    Article  PubMed  CAS  Google Scholar 

  40. Cheng ZX, Sun B, Wang SJ, Gao Y, Zhang YM, Zhou HX, Jia G, Wang YW, Kong R, Pan SH, Xue DB, Jiang HC, Bai XW (2011) Nuclear factor-κB-dependent epithelial to mesenchymal transition induced by HIF-1α activation in pancreatic cancer cells under hypoxic conditions. PLoS One 6:e23752

    Article  PubMed  CAS  Google Scholar 

  41. Ohuchida K, Mizumoto K, Ohhashi S, Yamaguchi H, Konomi H, Nagai E, Yamaguchi K, Tsuneyoshi M, Tanaka M (2007) TWIST, a novel oncogene, is upregulated in pancreatic juice. Int J Cancer 120:1634–1640. doi:10.1002/ijc.22295

    Article  PubMed  CAS  Google Scholar 

  42. Hotz B, Arndt M, Dullat S, Bhargava S, Buhr HJ, Hotz HG (2007) Epithelial to mesenchymal transition: expression of the regulators Snail, SLUG, and TWIST in pancreatic cancer. Clin Cancer Res 13:4769–4776. doi:10.1158/1078-0432.CCR-06-2926

    Article  PubMed  CAS  Google Scholar 

  43. Yin T, Wang C, Liu T, Zhao G, Zha Y, Yang M (2007) Expression of snail in pancreatic cancer promotes metastasis and chemoresistance. J Surg Res 141:196–203. doi:10.1016/j.jss.2006.09.027

    Article  PubMed  CAS  Google Scholar 

  44. Cates JM, Byrd RH, Fohn LE, Tatsas AD, Washington MK, Black CC (2009) Epithelial-mesenchymal transition markers in pancreatic ductal adenocarcinoma. Pancreas 38:e1–e6. doi:10.1097/MPA.0b013e3181878b7f

    Article  PubMed  CAS  Google Scholar 

  45. van Zandwijk N, Dalesio O, Pastorino U, de Vries N, van Tinteren H, For the European Organization for Research and Treatment of Cancer Head and Neck and Lung Cancer Cooperative Groups (2000) EUROSCAN, a randomized trial of vitamin A and N-acetylcysteine in patients with head and neck cancer or lung cancer. J Natl Cancer Inst 92:977–986. doi:10.1093/jnci/92.12.977

    Article  PubMed  Google Scholar 

  46. Sharifi N (2009) Commentary: antioxidants for cancer: new tricks for an old dog? Oncologist 14:213–215. doi:10.1634/theoncologist.2008-0219

    Article  PubMed  Google Scholar 

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Acknowledgments

The authors thank Ms. Mika Nada and Ms. Miki Asazu for their valuable technical assistance. This work was supported in part by Grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to Keizo Takenaga), and from Shimane University “S-TAKUMI Medical Nanotechnology” Project (to Keizo Takenaga).

Conflict of interest

No potential conflicts of interest were disclosed.

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Authors and Affiliations

  1. Department of Digestive and General Surgery, Faculty of Medicine, Shimane University, 89-1 Enya, Izumo, Shimane, 693-8501, Japan

    Yoshihide Shimojo, Tsunehiro Hisanaga, Tsuneo Tanaka & Yoshitsugu Tajima

  2. Department of Life Science, Faculty of Medicine, Shimane University, 89-1 Enya, Izumo, Shimane, 693-8501, Japan

    Yoshihide Shimojo, Miho Akimoto, Tsunehiro Hisanaga, Yoshio Honma & Keizo Takenaga

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  1. Yoshihide Shimojo
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  2. Miho Akimoto
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Correspondence to Keizo Takenaga.

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Shimojo, Y., Akimoto, M., Hisanaga, T. et al. Attenuation of reactive oxygen species by antioxidants suppresses hypoxia-induced epithelial-mesenchymal transition and metastasis of pancreatic cancer cells. Clin Exp Metastasis 30, 143–154 (2013). https://doi.org/10.1007/s10585-012-9519-8

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  • DOI: https://doi.org/10.1007/s10585-012-9519-8

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