Skip to main content

Advertisement

SpringerLink
Log in

Nrf2 inhibition sensitizes cholangiocarcinoma cells to cytotoxic and antiproliferative activities of chemotherapeutic agents

  • Original Article
  • Published:
Tumor Biology

Abstract

Nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcription factor regulating antioxidant, cytoprotective, and metabolic enzymes, plays important roles in drug resistance and proliferation in cancer cells. The present study was aimed to examine the expression of Nrf2 in connection with chemotherapeutic drug sensitivity on cholangiocarcinoma (CCA) cells. The basal levels of Nrf2 protein in cytosol and nuclear fractions of CCA cells were determined using Western blot analysis. Nrf2 mRNA expression of KKU-M156 and KKU-100 cells, representatives of low and high-Nrf2-expressing CCA cells, were silenced using siRNA. After knockdown of Nrf2, the sensitivity of those cells to the cytotoxicity of cisplatin (Cis) was enhanced in association with the increased release of AIF and downregulation of Bcl-xl in both cells. Also, knockdown of Nrf2 suppressed the replicative capability of those cells in colony-forming assay and enhanced their sensitivity to antiproliferative activity of Cis and 5-fluorouracil. The chemosensitizing effect was associated with the suppressed expression of Nrf2-regulated and Cis-induced antioxidant and metabolic genes including NQO1, HO-1, GCLC, TXN, MRP2, TKT, and G6PD. In cell cycle analysis, Nrf2 knockdown cells were arrested at G0/G1 phase and combination with Cis increased the accumulation of cells at S phase. The suppression of KKU-M156 cell proliferation was associated with the downregulation of cyclin D1 and increased level of p21. Inhibition of Nrf2 could be a novel strategy in enhancing antitumor activity of chemotherapeutic agent in control of resistant cancer.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

5-FU:

5-Fluorouracil

AO/EB:

Acridine orange and ethidium bromide

CCA:

Cholangiocarcinoma

Cis:

Cisplatin

NT:

Non-target siRNA

References

  1. Khan SA, Toledano MB, Taylor-Robinson SD. Epidemiology, risk factors, and pathogenesis of cholangiocarcinoma. HPB (Oxford). 2008;10:77–82.

    Article  CAS  Google Scholar 

  2. Khan SA, Davidson BR, Goldin RD, Heaton N, Karani J, Pereira SP, et al. Guidelines for the diagnosis and treatment of cholangiocarcinoma: an update. Gut. 2012;61:1657–69.

    Article  CAS  PubMed  Google Scholar 

  3. Butthongkomvong K, Sirachainan E, Jhankumpha S, Kumdang S, Sukhontharot OU. Treatment outcome of palliative chemotherapy in inoperable cholangiocarcinoma in Thailand. Asian Pac J Cancer Prev. 2013;14:3565–8.

    Article  PubMed  Google Scholar 

  4. Valle J, Wasan H, Palmer DH, Cunningham D, Anthoney A, Maraveyas A, et al. Cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer. N Engl J Med. 2010;362:1273–81.

    Article  CAS  PubMed  Google Scholar 

  5. Kansanen E, Kuosmanen SM, Leinonen H, Levonen AL. The keap1-nrf2 pathway: mechanisms of activation and dysregulation in cancer. Redox Biol. 2013;1:45–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Wang XJ, Sun Z, Villeneuve NF, Zhang S, Zhao F, Li Y, et al. Nrf2 enhances resistance of cancer cells to chemotherapeutic drugs, the dark side of nrf2. Carcinogenesis. 2008;29:1235–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Surh YJ, Kundu JK, Na HK. Nrf2 as a master redox switch in turning on the cellular signaling involved in the induction of cytoprotective genes by some chemopreventive phytochemicals. Planta Med. 2008;74:1526–39.

    Article  CAS  PubMed  Google Scholar 

  8. Kensler TW, Wakabayashi N, Biswal S. Cell survival responses to environmental stresses via the keap1-nrf2-are pathway. Annu Rev Pharmacol Toxicol. 2007;47:89–116.

    Article  CAS  PubMed  Google Scholar 

  9. Hayes JD, Dinkova-Kostova AT. The nrf2 regulatory network provides an interface between redox and intermediary metabolism. Trends Biochem Sci. 2014;39:199–218.

    Article  CAS  PubMed  Google Scholar 

  10. Hu R, Xu C, Shen G, Jain MR, Khor TO, Gopalkrishnan A, et al. Gene expression profiles induced by cancer chemopreventive isothiocyanate sulforaphane in the liver of c57bl/6j mice and c57bl/6j/nrf2 (−/−) mice. Cancer Lett. 2006;243:170–92.

    Article  CAS  PubMed  Google Scholar 

  11. Mitsuishi Y, Taguchi K, Kawatani Y, Shibata T, Nukiwa T, Aburatani H, et al. Nrf2 redirects glucose and glutamine into anabolic pathways in metabolic reprogramming. Cancer Cell. 2012;22:66–79.

    Article  CAS  PubMed  Google Scholar 

  12. Hayes JD, McMahon M. Nrf2 and keap1 mutations: permanent activation of an adaptive response in cancer. Trends Biochem Sci. 2009;34:176–88.

    Article  CAS  PubMed  Google Scholar 

  13. Ma X, Zhang J, Liu S, Huang Y, Chen B, Wang D. Nrf2 knockdown by shrna inhibits tumor growth and increases efficacy of chemotherapy in cervical cancer. Cancer Chemother Pharmacol. 2012;69:485–94.

    Article  CAS  PubMed  Google Scholar 

  14. Lister A, Nedjadi T, Kitteringham NR, Campbell F, Costello E, Lloyd B, et al. Nrf2 is overexpressed in pancreatic cancer: Implications for cell proliferation and therapy. Mol Cancer. 2011;10:37.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Samatiwat P, Prawan A, Senggunprai L, Kukongviriyapan V: Repression of nrf2 enhances antitumor effect of 5-fluorouracil and gemcitabine on cholangiocarcinoma cells. Naunyn Schmiedebergs Arch Pharmacol 2015;388:601–12.

  16. Hu XF, Yao J, Gao SG, Wang XS, Peng XQ, Yang YT, et al. Nrf2 overexpression predicts prognosis and 5-fu resistance in gastric cancer. Asian Pac J Cancer Prev. 2013;14:5231–5.

    Article  PubMed  Google Scholar 

  17. Onodera Y, Motohashi H, Takagi K, Miki Y, Shibahara Y, Watanabe M, et al. Nrf2 immunolocalization in human breast cancer patients as a prognostic factor. Endocrinol Relat Cancer. 2014;21:241–52.

    Article  CAS  Google Scholar 

  18. Yang H, Wang W, Zhang Y, Zhao J, Lin E, Gao J, et al. The role of nf-e2-related factor 2 in predicting chemoresistance and prognosis in advanced non-small-cell lung cancer. Clin Lung Cancer. 2011;12:166–71.

    Article  PubMed  Google Scholar 

  19. Zhang P, Singh A, Yegnasubramanian S, Esopi D, Kombairaju P, Bodas M, et al. Loss of kelch-like ech-associated protein 1 function in prostate cancer cells causes chemoresistance and radioresistance and promotes tumor growth. Mol Cancer Ther. 2010;9:336–46.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Homma S, Ishii Y, Morishima Y, Yamadori T, Matsuno Y, Haraguchi N, et al. Nrf2 enhances cell proliferation and resistance to anticancer drugs in human lung cancer. Clin Cancer Res. 2009;15:3423–32.

    Article  CAS  PubMed  Google Scholar 

  21. Jiang T, Chen N, Zhao F, Wang XJ, Kong B, Zheng W, et al. High levels of nrf2 determine chemoresistance in type ii endometrial cancer. Cancer Res. 2010;70:5486–96.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Heiss EH, Schachner D, Zimmermann K, Dirsch VM. Glucose availability is a decisive factor for nrf2-mediated gene expression. Redox Biol. 2013;1:359–65.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Yonglitthipagon P, Pairojkul C, Chamgramol Y, Loukas A, Mulvenna J, Bethony J, et al. Prognostic significance of peroxiredoxin 1 and ezrin-radixin-moesin-binding phosphoprotein 50 in cholangiocarcinoma. Hum Pathol. 2012;43:1719–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Sripa B, Leungwattanawanit S, Nitta T, Wongkham C, Bhudhisawasdi V, Puapairoj A, et al. Establishment and characterization of an opisthorchiasis-associated cholangiocarcinoma cell line (kku-100). World J Gastroenterol. 2005;11:3392–7.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Bunthot S, Obchoei S, Kraiklang R, Pirojkul C, Wongkham S, Wongkham C. Overexpression of claudin-4 in cholangiocarcinoma tissues and its possible role in tumor metastasis. Asian Pac J Cancer Prev. 2012;13(Suppl):71–6.

    PubMed  Google Scholar 

  26. Buranrat B, Prawan A, Kukongviriyapan U, Kongpetch S, Kukongviriyapan V. Dicoumarol enhances gemcitabine-induced cytotoxicity in high nqo1-expressing cholangiocarcinoma cells. World J Gastroenterol. 2010;16:2362–70.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Suphim B, Prawan A, Kukongviriyapan U, Kongpetch S, Buranrat B, Kukongviriyapan V. Redox modulation and human bile duct cancer inhibition by curcumin. Food Chem Toxicol. 2010;48:2265–72.

    Article  CAS  PubMed  Google Scholar 

  28. Kongpetch S, Kukongviriyapan V, Prawan A, Senggunprai L, Kukongviriyapan U, Buranrat B. Crucial role of heme oxygenase-1 on the sensitivity of cholangiocarcinoma cells to chemotherapeutic agents. PLoS One. 2012;7, e34994.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Aneknan P, Kukongviriyapan V, Prawan A, Kongpetch S, Sripa B, Senggunprai L. Luteolin arrests cell cycling, induces apoptosis and inhibits the jak/stat3 pathway in human cholangiocarcinoma cells. Asian Pac J Cancer Prev. 2014;15:5071–6.

    Article  PubMed  Google Scholar 

  30. Norberg E, Gogvadze V, Vakifahmetoglu H, Orrenius S, Zhivotovsky B. Oxidative modification sensitizes mitochondrial apoptosis-inducing factor to calpain-mediated processing. Free Radic Biol Med. 2010;48:791–7.

    Article  CAS  PubMed  Google Scholar 

  31. Franken NA, Rodermond HM, Stap J, Haveman J, van Bree C. Clonogenic assay of cells in vitro. Nat Protoc. 2006;1:2315–9.

    Article  CAS  PubMed  Google Scholar 

  32. Liu PP, Liao J, Tang ZJ, Wu WJ, Yang J, Zeng ZL, et al. Metabolic regulation of cancer cell side population by glucose through activation of the akt pathway. Cell Death Differ. 2014;21:124–35.

    Article  PubMed  Google Scholar 

  33. DeBerardinis RJ, Lum JJ, Hatzivassiliou G, Thompson CB. The biology of cancer: metabolic reprogramming fuels cell growth and proliferation. Cell Metab. 2008;7:11–20.

    Article  CAS  PubMed  Google Scholar 

  34. Crescenzi E, Chiaviello A, Canti G, Reddi E, Veneziani BM, Palumbo G. Low doses of cisplatin or gemcitabine plus photofrin/photodynamic therapy: disjointed cell cycle phase-related activity accounts for synergistic outcome in metastatic non-small cell lung cancer cells (h1299). Mol Cancer Ther. 2006;5:776–85.

    Article  CAS  PubMed  Google Scholar 

  35. Florea AM, Busselberg D. Cisplatin as an anti-tumor drug: cellular mechanisms of activity, drug resistance and induced side effects. Cancers (Basel). 2011;3:1351–71.

    Article  CAS  Google Scholar 

  36. Fu M, Wang C, Li Z, Sakamaki T, Pestell RG. Minireview: Cyclin d1: normal and abnormal functions. Endocrinology. 2004;145:5439–47.

    Article  CAS  PubMed  Google Scholar 

  37. Gartel AL. P21(waf1/cip1) and cancer: a shifting paradigm? Biofactors. 2009;35:161–4.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the Office of the Higher Education Commission through SHeP-GMS of Khon Kaen University, grants in-aid from Khon Kaen University and Faculty of Medicine (IN58130), and a scholarship from the Royal Golden Jubilee Ph.D. Program (PS). We thank Miss S. Srichanwang of Research Affair, Faculty of Medicine, for technical assistance of flow cytometry work, Professor Y. Nawa of Publication Clinic, Khon Kaen University for English language assistance.

Author information

Authors and Affiliations

  1. Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002, Thailand

    Papavee Samatiwat, Auemduan Prawan, Laddawan Senggunprai & Veerapol Kukongviriyapan

  2. Liver Fluke and Cholangiocarcinoma Research Center, Khon Kaen University, Khon Kaen, 40002, Thailand

    Auemduan Prawan, Laddawan Senggunprai & Veerapol Kukongviriyapan

  3. Department of Physiology, Faculty of Medicine, Khon Kaen University, Khon Kaen, 40002, Thailand

    Upa Kukongviriyapan

Authors
  1. Papavee Samatiwat
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Auemduan Prawan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Laddawan Senggunprai
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Upa Kukongviriyapan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Veerapol Kukongviriyapan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Veerapol Kukongviriyapan.

Ethics declarations

Conflicts of interest

None

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Fig. S1

Nrf2 knockdown enhanced the sensitivity of KKU-100 cells to Cis in association with altered apoptotic protein expression. The cells were transfected with siNrf2 or NT for 24 h followed by treatment with 5 μM Cis for another 18 h. Whole cell extracts were prepared for Western blotting to determine AIF, cytochrome c and Bcl-xl levels using β-actin as a loading control. (GIF 70 kb)

High Resolution (TIF 325 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Samatiwat, P., Prawan, A., Senggunprai, L. et al. Nrf2 inhibition sensitizes cholangiocarcinoma cells to cytotoxic and antiproliferative activities of chemotherapeutic agents. Tumor Biol. 37, 11495–11507 (2016). https://doi.org/10.1007/s13277-016-5015-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13277-016-5015-0

Keywords

Search

Navigation