title: Inhibition of hypoxia-inducible carbonic anhydrase-IX enhances hexokinase II inhibitor-induced hepatocellular carcinoma cell apoptosis.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul 110-744, Korea.
authors.forename: Su-jong
authors.initials: SJ
authors.lastname: Yu
authors.forename: Jung-hwan
authors.initials: JH
authors.lastname: Yoon
authors.forename: Jeong-hoon
authors.initials: JH
authors.lastname: Lee
authors.forename: Sun-jung
authors.initials: SJ
authors.lastname: Myung
authors.forename: Eun-sun
authors.initials: ES
authors.lastname: Jang
authors.forename: Min-sun
authors.initials: MS
authors.lastname: Kwak
authors.forename: Eun-ju
authors.initials: EJ
authors.lastname: Cho
authors.forename: Ja-june
authors.initials: JJ
authors.lastname: Jang
authors.forename: Yoon-jun
authors.initials: YJ
authors.lastname: Kim
authors.forename: Hyo-suk
authors.initials: HS
authors.lastname: Lee
mesh_terms: D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D000071231:Carbonic Anhydrase IX; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology*; D002256:Carbonic Anhydrases / Q000235:genetics / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic; D056945:Hep G2 Cells; D006593:Hexokinase / Q000037:antagonists & inhibitors*; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics; D011773:Pyruvates / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000235:genetics; D013449:Sulfonamides / Q000494:pharmacology*; D000096926:Benzenesulfonamides
chemical_list: D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents; D000071231:Carbonic Anhydrase IX; D002257:Carbonic Anhydrase Inhibitors; D002256:Carbonic Anhydrases; D006593:Hexokinase; D011773:Pyruvates; D034741:RNA, Small Interfering; D013449:Sulfonamides; C097944:4-(2-aminoethyl)benzenesulfonamide; D000096926:Benzenesulfonamides; C017092:bromopyruvate; C089226:CA9 protein, human