Pediatric Surgery International

, Volume 35, Issue 12, pp 1369–1378 | Cite as

Epidermal growth factor receptor/heme oxygenase-1 axis is involved in chemoresistance to cisplatin and pirarubicin in HepG2 cell lines and hepatoblastoma specimens

  • Takashi KobayashiEmail author
  • Masayuki Kubota
  • Yoshiaki Kinoshita
  • Yuki Arai
  • Toshiyuki Oyama
  • Naoki Yokota
  • Koichi Saito
  • Yasunobu Matsuda
  • Mami Osawa
Original Article



To investigate the possibility that the antioxidant stress protein Heme oxygenase-1 (HO-1) is involved in the acquisition of chemoresistance in cisplatin and pirarubicin (CITA) therapy.


Human hepatoblastoma-derived cell line (HepG2) was used to generate a knockdown cell line of HO-1 by small interfering RNA (siRNA). Expression of HO-1, epidermal growth factor receptor (EGFR), Akt, and extracellular signal-regulated kinase1/2 (ERK1/2) was examined by Western blot. The cytotoxic effect of cisplatin, pirarubicin, and EGFR inhibitor was examined by trypan blue staining. In human hepatoblastoma specimens (n = 5), changes of HO-1 expression were examined immunohistochemically before and after CITA therapy.


HO-1 expression in HepG2 cells was increased by the treatment of cisplatin (CDDP) and pirarubicin (THP) dose-dependently. In HO-1 knockdown HepG2 cells, the HO-1 was not expressed and the percentage of trypan blue-positive cells (dead cells) was significantly increased after treatment of CDDP and THP. The EGFR inhibitor decreased the levels of HO-1, phospho-Akt and phospho-ERK1/2 in HepG2 cells. Combination treatment of EGFR inhibitor with CDDP and THP increased the cytotoxic effect in HepG2 cells. In human hepatoblastoma specimens, 4 of the 5 patients (80%) showed HO-1 expression changed much stronger in the viable tumor cells after CITA therapy.


The cytotoxic effects of CDDP and THP were both enhanced under HO-1 knockdown conditions as well as under conditions that inhibit the activation pathway of HO-1 by EGFR inhibitors. EGFR/HO-1 axis may be involved in acquiring chemoresistance in HepG2 cell lines as well as in human hepatoblastoma.


Hepatoblastoma Cisplatin Pirarubicin Heme oxygenase-1 Epidermal growth factor receptor 



This study was supported in part by a Grant-in-Aid for Scientific Research (17H04270F).


  1. 1.
    Latini G, Gallo F, De Felice C (2004) Birth characteristics and hepatoblastoma risk in young children. Cancer 101(1):210CrossRefGoogle Scholar
  2. 2.
    Meyers RL (2007) Tumors of the liver in children. Surg Oncol 16(3):195–203CrossRefGoogle Scholar
  3. 3.
    Kremer N, Walther AE, Tiao GM (2014) Management of hepatoblastoma: an update. Curr Opin Pediatr 26(3):362–369CrossRefGoogle Scholar
  4. 4.
    Hishiki T, Matsunaga T, Sasaki F, Yano M, Ida K, Horie H, Kondo S, Watanabe K, Oue T, Tajiri T, Kamimatsuse A, Ohnuma N, Hiyama E (2011) Outcome of hepatoblastomas treated using the Japanese Study Group for Pediatric Liver Tumor (JPLT) protocol-2: report from the JPLT. Pediatr Surg Int 27(1):1–8CrossRefGoogle Scholar
  5. 5.
    Malogolowkin MH, Katzenstein H, Krailo MD, Chen Z, Bowman L, Reynolds M, Finegold M, Greffe B, Rowland J, Newman K, Womer RB, London WB, Castleberry RP (2006) Intensified platinum therapy is an ineffective strategy for improving outcome in pediatric patients with advanced hepatoblastoma. J Clin Oncol 24(18):2879–2884CrossRefGoogle Scholar
  6. 6.
    Zsiros J, Brugieres L, Brock P, Roebuck D, Maibach R, Zimmermann A, Childs M, Pariente D, Laithier V, Otte JB, Branchereau S, Aronson D, Rangaswami A, Ronghe M, Casanova M, Sullivan M, Morland B, Czauderna P, Perilongo G, International Childhood Liver Tumours Strategy G (2013) Dose-dense cisplatin-based chemotherapy and surgery for children with high-risk hepatoblastoma (SIOPEL-4): a prospective, single-arm, feasibility study. Lancet Oncol 14(9):834–842CrossRefGoogle Scholar
  7. 7.
    Hiyama E, Ueda Y, Onitake Y, Kurihara S, Watanabe K, Hishiki T, Tajiri T, Ida K, Yano M, Kondo S, Oue T, Japanese Study Group for Pediatric Liver T (2013) A cisplatin plus pirarubicin-based JPLT2 chemotherapy for hepatoblastoma: experience and future of the Japanese Study Group for Pediatric Liver Tumor (JPLT). Pediatr Surg Int 29(10):1071–1075CrossRefGoogle Scholar
  8. 8.
    Trobaugh-Lotrario AD, Katzenstein HM (2012) Chemotherapeutic approaches for newly diagnosed hepatoblastoma: past, present, and future strategies. Pediatr Blood Cancer 59(5):809–812CrossRefGoogle Scholar
  9. 9.
    Furfaro AL, Traverso N, Domenicotti C, Piras S, Moretta L, Marinari UM, Pronzato MA, Nitti M (2016) The Nrf2/HO-1 axis in cancer cell growth and chemoresistance. Oxid Med Cell Longev 2016:1958174CrossRefGoogle Scholar
  10. 10.
    Lopez-Terrada D, Cheung SW, Finegold MJ, Knowles BB (2009) Hep G2 is a hepatoblastoma-derived cell line. Hum Pathol 40(10):1512–1515CrossRefGoogle Scholar
  11. 11.
    Matsuo T, Miyata Y, Mitsunari K, Yasuda T, Ohba K, Sakai H (2017) Pathological significance and prognostic implications of heme oxygenase 1 expression in non-muscle-invasive bladder cancer: Correlation with cell proliferation, angiogenesis, lymphangiogenesis and expression of VEGFs and COX-2. Oncol Lett 13(1):275–280CrossRefGoogle Scholar
  12. 12.
    Temma K, Akera T, Chugun A, Kondo H, Hagane K, Hirano S (1993) Comparison of cardiac actions of doxorubicin, pirarubicin and aclarubicin in isolated guinea-pig heart. Eur J Pharmacol 234(2–3):173–181CrossRefGoogle Scholar
  13. 13.
    Hirano S, Wakazono K, Agata N, Iguchi H, Tone H (1994) Comparison of cardiotoxicity of pirarubicin, epirubicin and doxorubicin in the rat. Drugs Exp Clin Res 20(4):153–160PubMedGoogle Scholar
  14. 14.
    Na HK, Surh YJ (2014) Oncogenic potential of Nrf2 and its principal target protein heme oxygenase-1. Free Radic Biol Med 67:353–365CrossRefGoogle Scholar
  15. 15.
    Paine A, Eiz-Vesper B, Blasczyk R, Immenschuh S (2010) Signaling to heme oxygenase-1 and its anti-inflammatory therapeutic potential. Biochem Pharmacol 80(12):1895–1903CrossRefGoogle Scholar
  16. 16.
    Owuor ED, Kong AN (2002) Antioxidants and oxidants regulated signal transduction pathways. Biochem Pharmacol 64(5–6):765–770CrossRefGoogle Scholar
  17. 17.
    Levitzki A, Gazit A (1995) Tyrosine kinase inhibition: an approach to drug development. Science 267(5205):1782–1788CrossRefGoogle Scholar
  18. 18.
    Kuroda H, Takeno M, Murakami S, Miyazawa N, Kaneko T, Ishigatsubo Y (2010) Inhibition of heme oxygenase-1 with an epidermal growth factor receptor inhibitor and cisplatin decreases proliferation of lung cancer A549 cells. Lung Cancer 67(1):31–36CrossRefGoogle Scholar
  19. 19.
    Yoshida T, Okamoto I, Iwasa T, Fukuoka M, Nakagawa K (2008) The anti-EGFR monoclonal antibody blocks cisplatin-induced activation of EGFR signaling mediated by HB-EGF. FEBS Lett 582(30):4125–4130CrossRefGoogle Scholar
  20. 20.
    Bang YJ, Van Cutsem E, Feyereislova A, Chung HC, Shen L, Sawaki A, Lordick F, Ohtsu A, Omuro Y, Satoh T, Aprile G, Kulikov E, Hill J, Lehle M, Ruschoff J, Kang YK, To GATI (2010) Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial. Lancet 376(9742):687–697CrossRefGoogle Scholar
  21. 21.
    Ikeda M, Shimizu S, Sato T, Morimoto M, Kojima Y, Inaba Y, Hagihara A, Kudo M, Nakamori S, Kaneko S, Sugimoto R, Tahara T, Ohmura T, Yasui K, Sato K, Ishii H, Furuse J, Okusaka T (2016) Sorafenib plus hepatic arterial infusion chemotherapy with cisplatin versus sorafenib for advanced hepatocellular carcinoma: randomized phase II trial. Ann Oncol 27(11):2090–2096CrossRefGoogle Scholar
  22. 22.
    du Bois A, Kristensen G, Ray-Coquard I, Reuss A, Pignata S, Colombo N, Denison U, Vergote I, Del Campo JM, Ottevanger P, Heubner M, Minarik T, Sevin E, de Gregorio N, Bidzinski M, Pfisterer J, Malander S, Hilpert F, Mirza MR, Scambia G, Meier W, Nicoletto MO, Bjorge L, Lortholary A, Sailer MO, Merger M, Harter P, Consortium AGOSGlGCIENoGOTGI (2016) Standard first-line chemotherapy with or without nintedanib for advanced ovarian cancer (AGO-OVAR 12): a randomised, double-blind, placebo-controlled phase 3 trial. Lancet Oncol 17(1):78–89CrossRefGoogle Scholar
  23. 23.
    Hwang IG, Jang JS, Oh SY, Lee S, Kwon HC, Lee GW, Go S, Kang MH, Cha YJ, Kang JH (2012) A phase II trial of Erlotinib in combination with gemcitabine and cisplatin in advanced pancreatic cancer. Invest New Drugs 30(6):2371–2376CrossRefGoogle Scholar
  24. 24.
    Nogueira-Rodrigues A, Moralez G, Grazziotin R, Carmo CC, Small IA, Alves FV, Mamede M, Erlich F, Viegas C, Triginelli SA, Ferreira CG (2014) Phase 2 trial of erlotinib combined with cisplatin and radiotherapy in patients with locally advanced cervical cancer. Cancer 120(8):1187–1193CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Takashi Kobayashi
    • 1
    Email author
  • Masayuki Kubota
    • 1
    • 4
  • Yoshiaki Kinoshita
    • 1
  • Yuki Arai
    • 1
  • Toshiyuki Oyama
    • 1
  • Naoki Yokota
    • 1
  • Koichi Saito
    • 1
  • Yasunobu Matsuda
    • 2
  • Mami Osawa
    • 3
  1. 1.Department of Pediatric SurgeryNiigata University Graduate School of Medical and Dental SciencesNiigataJapan
  2. 2.Department of Medical TechnologyNiigata University Graduate School of Health SciencesNiigataJapan
  3. 3.Division of Digestive and General SurgeryNiigata University Graduate School of Medical and Dental SciencesNiigataJapan
  4. 4.Kokuraminami Medical Care HospitalKitakyu-syuJapan

Personalised recommendations