Original Article

Inhibition of hypoxia-inducible carbonic anhydrase-IX enhances hexokinase II inhibitor-induced hepatocellular carcinoma cell apoptosis

Authors: Su-jong Yu, Jung-hwan Yoon, Jeong-hoon Lee, Sun-jung Myung, Eun-sun Jang, Min-sun Kwak, Eun-ju Cho, Ja-june Jang, Yoon-jun Kim, Hyo-suk Lee
DOI: 10.1038/aps.2011.24

Abstract

Aim: The hypoxic condition within large or infiltrative hypovascular tumors produces intracellular acidification, which could activate many signaling pathways and augment cancer cell growth and invasion. Carbonic anhydrase-IX (CA-IX) is an enzyme lowering pH. This study is to examine whether hypoxia induces CA-IX in hepatocellular carcinoma (HCC) cells, and to evaluate its clinical implication in HCC patients.
Methods: Human HCC cell lines (Huh-7 and HepG2 cells) were used, and cell growth was assessed using MTS assay. CA-IX expression and apoptotic/kinase signaling were evaluated using immunoblotting. The cells were transfected with CA-IX-specific siRNA, or treated with its inhibitor 4-(2-aminoethyl) benzenesulfonamide (CAI#1), and/or the hexokinase II inhibitor, 3-bromopyruvate (3-BP). A clinic pathological analysis of 69 patients who underwent an HCC resection was performed using a tissue array.
Results: Incubation of HCC cells under hypoxia (1% O2, 5% CO2, 94% N2) for 36 h significantly increased CA-IX expression level. CAI#1 (400 μmol/L) or CA-IX siRNA (100 μmol/L) did not influence HCC cell growth and induce apoptosis. However, CAI#1 or CA-IX siRNA at these concentrations enhanced the apoptosis induced by 3-BP (100 μmol/L). This enhancement was attributed to increased ER stress and JNK activation, as compared with 3-BP alone. Furthermore, a clinic pathological analysis of 69 HCC patients revealed that tumor CA-IX intensity was inversely related to E-cadherin intensity.
Conclusion: Inhibition of hypoxia-induced CA-IX enhances hexokinase II inhibitor-induced HCC apoptosis. Furthermore, CA-IX expression profiles may have prognostic implications in HCC patients. Thus, the inhibition of CA-IX, in combination with a hexokinase II inhibitor, may be therapeutically useful in patients with HCCs that are aggressively growing in a hypoxic environment.
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