445 INHIBITION OF HYPOXIA-INDUCIBLE CARBONIC ANHYDRASE IX ENHANCES HEXOKINASE II INHIBITOR-INDUCED HEPATOCELLULAR CARCINOMA CELL APOPTOSIS

Abstract

Background and Aims: Cancer cells may adapt to pH changes and thereby, grow at low pHs. In acidic environments, many signals are activated to augment cancer cell growth and invasion. Carbonic anhydrase-9 (CA-IX), an enzyme involved in lowering pH, is known to be overexpressed in many cancer cells. Hypoxic condition, which large or infiltrative hypovascular tumor may encounter, also produces acidic environments. This study was to examine if CA-IX is hypoxia-inducibly expressed in hepatocellular carcinoma (HCC) cells, and to evaluate its clinical implication in HCC patients. Methods: Human HCC cell line (Huh-7) was used in this study. CAIX expression was evaluated using immunoblot analysis in cells grown either in a normoxic or hypoxic condition. Cells were treated with CA-IX inhibitor, 4-(2-aminoethyl)benzenesulfonamide, in the presence or absence of hexokinase II inhibitor, 3-bromopyruvate (3-BP), which has previously been demonstrated to induce HCC cell apoptosis more efficiently in hypoxic state than in normoxic state. Cell growth was assessed using the MTS assay, and apoptotic and kinase signaling were explored by immunoblot analysis. A clinico-pathological analysis in patients who had undergone surgical resection of HCC was evaluated by tissue array method and immunohistochemistry of CA-IX. Results: HCC cells expressed CA-IX, and its expression was increased in cells cultured under hypoxic condition as compared to normoxic cells. The CA-IX inhibitor alone did not induce HCC cell apoptosis, while the simultaneous treatment of this inhibitor with 3-BP enhanced 3-BPinduced apoptosis, in particular by activating caspase 9 more efficiently. This enhanced apoptosis was due to more augmented JNK activation in cells treated with both inhibitors as compared to 3-BP-treated cells. A clinico-pathological analysis revealed that tumoral CA-IX extent was positively related to Edmondson grade and its intensity was negatively correlated with recurrence free survival in HCC patients. Conclusions: This study demonstrates that the inhibition of hypoxiainducible CA-IX enhances hexokinase II inhibitor-induced HCC cell apoptosis, and that CA-IX expression profiles may have a prognostic implication in HCC patients. Thus, the blockage of this enzyme in combination with hexokinase II inhibitor may therapeutically be useful in patients with large or infiltrative hypovascular HCCs, which are aggressively growing under hypoxic environment.