Carbonic anhydrase-IX inhibition enhances the efficacy of hexokinase II inhibitor for hepatocellular carcinoma in a murine model
Hypoxic conditions, which large or infiltrative hypovascular tumors may encounter, also produce acidic environments. Carbonic anhydrase-IX (CA-IX), an enzyme involved in lowering pH, is overexpressed in hepatocellular carcinoma (HCC). In the present study, whether inhibition of CA-IX enhances the efficacy of a hexokinase II inhibitor in an in vivo murine model was examined and its prognostic implication in HCC patients was investigated. CA-IX expression was evaluated using quantitative real-time PCR and western blot analysis using human HCC cell lines. 3-bromopyruvate (3-BP), a hexokinase II inhibitor, and acetazolamide, a carbonic anhydrase inhibitor, were used to target hexokinase II and CA-IX in vitro and in vivo, respectively. A human HCC cell line (Huh-7) was tested as a subcutaneous tumor model in BALB/c nu/nu mice. The prognostic role of CA-IX was evaluated in the TCGA database. Quantitative real-time PCR and western blot analysis revealed that CA-IX expression was activated in the presence of 3-BP. Further analysis showed that introducing an additional stress by treating the orally active CA-IX inhibitor (acetazolamide) can synergistically increase the efficacy of 3-BP in vivo, which was confirmed using a mouse model. We also found that HCC patients with high CA-IX expression show poor overall survival in TCGA database. These results indicate CA-IX is a promising therapeutic target for enhancing the efficacy of 3-BP and can be a prognostic factor for HCC.
KeywordsHepatocellular carcinoma Acetazolamide 3-BP Apoptosis Hypoxia
This study was funded by grants from the SNUH Research Fund (No. 04-2011-0660) and the Liver Research Foundation of Korea.
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interest.
- Budak-Alpdogan T, Chen B, Warrier A, Medina DJ, Moore D, Bertino JR (2009) Retinoblastoma tumor suppressor gene expression determines the response to sequential flavopiridol and doxorubicin treatment in small-cell lung carcinoma. Clin Cancer Res 15(4):1232–1240. https://doi.org/10.1158/1078-0432.CCR-08-0810 CrossRefGoogle Scholar
- Ganapathy-Kanniappan S, Geschwind JF, Kunjithapatham R, Buijs M, Syed LH, Rao PP, Ota S, Kwak BK, Loffroy R, Vali M (2010) 3-Bromopyruvate induces endoplasmic reticulum stress, overcomes autophagy and causes apoptosis in human HCC cell lines. Anticancer Res 30(3):923–935Google Scholar
- Kivela AJ, Parkkila S, Saarnio J, Karttunen TJ, Kivela J, Parkkila AK et al (2000) Expression of transmembrane carbonic anhydrase isoenzymes IX and XII in normal human pancreas and pancreatic tumours. Histochem Cell Biol 114(3):197–204Google Scholar
- Swinson DE, Jones JL, Richardson D, Wykoff C, Turley H, Pastorek J et al (2003) Carbonic anhydrase IX expression, a novel surrogate marker of tumor hypoxia, is associated with a poor prognosis in non-small-cell lung cancer. J Clin Oncol 21(3):473–482. https://doi.org/10.1200/JCO.2003.11.132 CrossRefGoogle Scholar
- Tezuka M, Hayashi K, Kubota K, Sekine S, Okada Y, Ina H, Irie T (2007) Growth rate of locally recurrent hepatocellular carcinoma after transcatheter arterial chemoembolization: comparing the growth rate of locally recurrent tumor with that of primary hepatocellular carcinoma. Dig Dis Sci 52(3):783–788. https://doi.org/10.1007/s10620-006-9537-y CrossRefGoogle Scholar
- van den Beucken T, Magagnin MG, Savelkouls K, Lambin P, Koritzinsky M, Wouters BG (2007) Regulation of Cited2 expression provides a functional link between translational and transcriptional responses during hypoxia. Radiother Oncol 83(3):346–352. https://doi.org/10.1016/j.radonc.2007.04.026 CrossRefGoogle Scholar
- Weinstein-Oppenheimer CR, Henriquez-Roldan CF, Davis JM, Navolanic PM, Saleh OA, Steelman LS et al (2001) Role of the Raf signal transduction cascade in the in vitro resistance to the anticancer drug doxorubicin. Clin Cancer Res 7(9):2898–2907Google Scholar
- Yu SJ, Yoon JH, Lee JH, Myung SJ, Jang ES, Kwak MS, Cho EJ, Jang JJ, Kim YJ, Lee HS (2011) Inhibition of hypoxia-inducible carbonic anhydrase-IX enhances hexokinase II inhibitor-induced hepatocellular carcinoma cell apoptosis. Acta Pharmacol Sin 32(7):912–920. https://doi.org/10.1038/aps.2011.24 CrossRefGoogle Scholar