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Tumor Biology

, Volume 37, Issue 8, pp 11495–11507 | Cite as

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

  • Papavee Samatiwat
  • Auemduan Prawan
  • Laddawan Senggunprai
  • Upa Kukongviriyapan
  • Veerapol Kukongviriyapan
Original Article

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.

Keywords

Nrf2 Chemosensitizing Cholangiocarcinoma Cisplatin Antioxidant Antiproliferation 

Abbreviations

5-FU

5-Fluorouracil

AO/EB

Acridine orange and ethidium bromide

CCA

Cholangiocarcinoma

Cis

Cisplatin

NT

Non-target siRNA

Notes

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.

Compliance with ethical standard

Conflicts of interest

None

Supplementary material

13277_2016_5015_Fig7_ESM.gif (71 kb)
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)

13277_2016_5015_MOESM1_ESM.tif (326 kb)
High Resolution (TIF 325 kb)

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Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2016

Authors and Affiliations

  • Papavee Samatiwat
    • 1
  • Auemduan Prawan
    • 1
    • 2
  • Laddawan Senggunprai
    • 1
    • 2
  • Upa Kukongviriyapan
    • 3
  • Veerapol Kukongviriyapan
    • 1
    • 2
  1. 1.Department of Pharmacology, Faculty of MedicineKhon Kaen UniversityKhon KaenThailand
  2. 2.Liver Fluke and Cholangiocarcinoma Research CenterKhon Kaen UniversityKhon KaenThailand
  3. 3.Department of Physiology, Faculty of MedicineKhon Kaen UniversityKhon KaenThailand

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