Anion exchanger inhibitor DIDS induces human poorly-differentiated malignant hepatocellular carcinoma HA22T cell apoptosis

  • Chung-Jung Liu
  • Jin-Ming Hwang
  • Trang-Tiau Wu
  • Yi-Hsien Hsieh
  • Cheng-Chung Wu
  • Yih-Shou Hsieh
  • Chang-Hai Tsai
  • Hsi-Chin Wu
  • Chih-Yang Huang
  • Jer-Yuh Liu


Anion exchangers (AEs) of the Cl-/HCO3- exchanger family contribute to the regulation of intracellular acid-base balance. Recently, we found that anion exchanger 2 (AE2) was significantly expressed in human hepatocellular carcinoma (HCC) and in poorly-differentiated human HCC HA22T/VGH cells. In the present study, we further explored the pharmacological function of AE in four human HCC cell lines (SK-Hep-1, HA22T/VGH, HepG2, and Hep3B) following the treatment of 4,4’-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), an AEs specific inhibitor. After administrations with 400–1000 μM of DIDS, cell proliferation was greatly inhibited in a dose-dependent manner from 47.5 to 65.0% in higher malignant HA22T/VGH cells, but not in other cell lines. The results of 4,6-diamidino-2-phenylindole (DAPI) staining, DNA fragmentation and flow cytometric analysis further revealed that cell apoptosis induced by DIDS was also observed in HA22T/VGH cells. Therefore, these findings suggested that AE may be involved, in part, in the proliferation and survival of HA22T cells and could be a new potential therapeutic target against specific human HCC.


HA22T hepatocellular carcinoma cells Anion exchanger 2 DIDS Apoptosis Proliferation Anion transport activity 



This work was supported in part by grants from the National Science Council, Republic of China (NSC 95–2320-B-040-043) and from Chung Shan Medical University, Republic of China (CSMU 95-OM-A-110, CSMU 95-OM-B-009 and CSMU 95-OM-B-027).


  1. 1.
    Alper SL (2006) Molecular physiology of SLC4 anion exchangers. Exp Physiol 91:153–161PubMedCrossRefGoogle Scholar
  2. 2.
    Romero MF (2005) Molecular pathophysiology of SLC4 bicarbonate transporters. Curr Opin Nephrol Hypertens 14:495–501PubMedCrossRefGoogle Scholar
  3. 3.
    Tanner MJ (1997) The structure and function of band 3 (AE1): recent developments (review) Mol Membr Biol 14:155–165Google Scholar
  4. 4.
    Stewart AK, Kurschat CE, Burns D et al (2007) Transmembrane domain histidines contribute to regulation of AE2-mediated anion exchange by pH. Am J Physiol Cell Physiol 292:C909–C918PubMedCrossRefGoogle Scholar
  5. 5.
    Stuart-Tilley A, Sardet C, Pouyssegur J et al (1994) Immunolocalization of anion exchanger AE2 and cation exchanger NHE-1 in distinct adjacent cells of gastric mucosa. Am J Physiol 266:C559–C568PubMedGoogle Scholar
  6. 6.
    Alper SL, Stuart-Tilley A, Simmons CF et al (1994) The fodrin–ankyrin cytoskeleton of choroid plexus preferentially colocalizes with apical Na+K(+)-ATPase rather than with basolateral anion exchanger AE2. J Clin Invest 93:1430–1438PubMedCrossRefGoogle Scholar
  7. 7.
    Alper SL, Rossmann H, Wilhelm S et al (1992) Expression of AE2 anion exchanger in mouse intestine. Am J Physiol 277:G321–G332Google Scholar
  8. 8.
    Stuart-Tiley AK, Shmukler BE, Brown D et al (1998) Immunolocalization and tissue-specific splicing of AE2 anion exchanger in mouse kidney. J Am Soc Nephrol 9:946–959Google Scholar
  9. 9.
    Dudeja PK, Hafez N, Tyagi S et al (1999) Expression of the Na+/H+ and Cl-/HCO-3 exchanger isoforms in proximal and distal human airways. Am J Physiol 276:L971–L978PubMedGoogle Scholar
  10. 10.
    Kopito RR, Lee BS, Simmons DM et al (1989) Regulation of intracellular pH by a neuronal homolog of the erythrocyte anion exchanger. Cell 59:927–937PubMedCrossRefGoogle Scholar
  11. 11.
    Kobayashi S, Morgans CW, Casey JR et al (1994) AE3 anion exchanger isoforms in the vertebrate retina: developmental regulation and differential expression in neurons and glia. J Neurosci 14:6266–6279PubMedGoogle Scholar
  12. 12.
    Yannoukakos D, Stuart-Tilley A, Fernandez HA et al (1994) Molecular cloning, expression, and chromosomal localization of two isoforms of the AE3 anion exchanger from human heart. Circ Res 75:603–614PubMedGoogle Scholar
  13. 13.
    Humphreys BD, Jiang L, Chernova MN et al (1995) Hypertonic activation of AE2 anion exchanger in Xenopus oocytes via NHE-mediated intracellular alkalinization. Am J Physiol 268:C201–C209PubMedGoogle Scholar
  14. 14.
    Fejes-Toth G, Rusvai E, Cleaveland ES et al (1998) Regulation of AE2 mRNA expression in the cortical collecting duct by acid/base balance. Am J Physiol 274:F596–F601PubMedGoogle Scholar
  15. 15.
    Garcia C, Montuenga LM, Medina JF et al (1998) In situ detection of AE2 anion-exchanger mRNA in the human liver. Cell Tissue Res 291:481–488PubMedCrossRefGoogle Scholar
  16. 16.
    Martinez-Anso E, Castillo JE, Diez J et al (1994) Immunohistochemical detection of chloride/bicarbonate anion exchangers in human liver. Hepatology 19:1400–1406PubMedCrossRefGoogle Scholar
  17. 17.
    Wu TT, Hsieh YH, Wu CC et al (2006) Overexpression of anion exchanger 2 in human hepatocellular carcinoma. Chin J Physiol 49:192–198PubMedGoogle Scholar
  18. 18.
    Tian J, Tang Z, Xue Q (1999) Expressions of the metastasis-associated factors of a new human hepatocellular carcinoma cell line with highly metastatic potential]. Zhonghua Yi Xue Za Zhi 79:470–472PubMedGoogle Scholar
  19. 19.
    Chuma M, Sakamoto M, Yasuda J et al (2004) Overexpression of cortactin is involved in motility and metastasis of hepatocellular carcinoma. J Hepatol 41:629–636PubMedCrossRefGoogle Scholar
  20. 20.
    Farazi PA, DePinho RA (2006) Hepatocellular carcinoma pathogenesis: from genes to environment. Nat Rev Cancer 6:674–687PubMedCrossRefGoogle Scholar
  21. 21.
    Wu TT, Hsieh YH, Hsieh YS et al (2007) Reduction of PKCalpha decreases cell proliferation, migration, and invasion of human malignant hepatocellular carcinoma. J Cell Biochem 2007 (in press)Google Scholar
  22. 22.
    Hsieh YH, Wu TT, Huang CY et al (2007) p38 mitogen-activated protein kinase pathway is involved in protein kinase Calpha-regulated invasion in human hepatocellular carcinoma cells. Cancer Res 67:4320–4327PubMedCrossRefGoogle Scholar
  23. 23.
    Aden DP, Fogel A, Plotkin S et al (1979) Controlled synthesis of HBsAg in a differentiated human liver carcinoma-derived cell line. Nature 282:615–616PubMedCrossRefGoogle Scholar
  24. 24.
    Chang C, Lin Y, O-Lee TW et al (1983) Induction of plasma protein secretion in a newly established human hepatoma cell line. Mol Cell Biol 3:1133–1137PubMedGoogle Scholar
  25. 25.
    Montcourrier P, Mangeat PH, Valembois C et al (1994) Characterization of very acidic phagosomes in breast cancer cells and their association with invasion. J Cell Sci 107:2381–2391PubMedGoogle Scholar
  26. 26.
    Mooren FC, Domschke W, Kinne RK et al (2001) Non-invasive single cell pH measurements in the isolated perfused pancreas. Clin Exp Pharmacol Physiol 28:463–465PubMedCrossRefGoogle Scholar
  27. 27.
    Bosch FX, Ribes J, Diaz M et al (2004) Primary liver cancer: worldwide incidence and trends. Gastroenterology 127:S5–S16PubMedCrossRefGoogle Scholar
  28. 28.
    Llovet JM, Burroughs A, Bruix J (2003) Hepatocellular carcinoma. Lancet 362:1907–1917PubMedCrossRefGoogle Scholar
  29. 29.
    Rotin D, Steele-Norwood D, Grinstein S et al (1989) Requirement of the Na+/H+ exchanger for tumor growth. Cancer Res 49:205–211PubMedGoogle Scholar
  30. 30.
    Reshkin SJ, Bellizzi A, Caldeira S et al (2000) Na+/H+ exchanger-dependent intracellular alkalinization is an early event in malignant transformation and plays an essential role in the development of subsequent transformation-associated phenotypes. FASEB J 14:2185–2197PubMedCrossRefGoogle Scholar
  31. 31.
    Denker SP, Barber DL (2002) Ion transport proteins anchor and regulate the cytoskeleton. Curr Opin Cell Biol 14:214–220PubMedCrossRefGoogle Scholar
  32. 32.
    Rich IN, Worthington-White D, Garden OA et al (2000) Apoptosis of leukemic cells accompanies reduction in intracellular pH after targeted inhibition of the Na(+)/H(+) exchanger. Blood 95:1427–1434PubMedGoogle Scholar
  33. 33.
    Di Sario A, Bendia E, Omenetti A et al (2007) Selective inhibition of ion transport mechanisms regulating intracellular pH reduces proliferation and induces apoptosis in cholangiocarcinoma cells. Dig Liver Dis 39:60–69PubMedCrossRefGoogle Scholar
  34. 34.
    Wong P, Kleemann HW, Tannock IF (2002) Cytostatic potential of novel agents that inhibit the regulation of intracellular pH. Br J Cancer 87:238–245PubMedCrossRefGoogle Scholar
  35. 35.
    Park HJ, Lyons JC, Ohtsubo T et al (1999) Acidic environment causes apoptosis by increasing caspase activity. Br J Cancer 80:1892–1897PubMedCrossRefGoogle Scholar
  36. 36.
    Matsuyama S, Llopis J, Deveraux QL et al (2000) Changes in intramitochondrial and cytosolic pH: early events that modulate caspase activation during apoptosis. Nat Cell Biol 2:318–325PubMedCrossRefGoogle Scholar
  37. 37.
    Waibel M, Kramer S, Lauber K et al (2007) Mitochondria are not required for death receptor-mediated cytosolic acidification during apoptosis. Apoptosis 12:623–630PubMedCrossRefGoogle Scholar
  38. 38.
    Martinez-Zaguilan R, Seftor EA, Seftor RE et al (1996) Acidic pH enhances the invasive behavior of human melanoma cells. Clin Exp Metastasis 14:176–186PubMedCrossRefGoogle Scholar
  39. 39.
    Cuvier C, Jang A, Hill RP (1997) Exposure to hypoxia, glucose starvation and acidosis: effect on invasive capacity of murine tumor cells and correlation with cathepsin (L + B) secretion. Clin Exp Metastasis 15:19–25PubMedCrossRefGoogle Scholar
  40. 40.
    Doppler W, Jaggi R, Groner B (1987) Induction of v-mos and activated Ha-ras oncogene expression in quiescent NIH 3T3 cells causes intracellular alkalinisation and cell-cycle progression. Gene 54:147–153PubMedCrossRefGoogle Scholar
  41. 41.
    Webb SD, Sherratt JA, Fish RG (1999) Mathematical modelling of tumour acidity: regulation of intracellular pH. J Theor Biol 196:237–250PubMedCrossRefGoogle Scholar
  42. 42.
    Kraus M, Wolf B (1996) Implications of acidic tumor microenvironment for neoplastic growth and cancer treatment: a computer analysis. Tumour Biol 17:133–154PubMedGoogle Scholar
  43. 43.
    Lagana A, Vadnais J, Le PU et al (2000) Regulation of the formation of tumor cell pseudopodia by the Na(+)/H(+) exchanger NHE1. J Cell Sci 113:3649–3662PubMedGoogle Scholar
  44. 44.
    Gillies RJ, Raghunand N, Karczmar GS et al (2002) MRI of the tumor microenvironment. J Magn Reson Imaging 16:430–450PubMedCrossRefGoogle Scholar
  45. 45.
    Radinsky R (1995) Modulation of tumor cell gene expression and phenotype by the organ-specific metastatic environment. Cancer Metastasis Rev 14:323–338PubMedCrossRefGoogle Scholar
  46. 46.
    Izumi H, Torigoe T, Ishiguchi H et al (2003) Cellular pH regulators: potentially promising molecular targets for cancer chemotherapy. Cancer Treat Rev 29:541–549PubMedCrossRefGoogle Scholar
  47. 47.
    Melillo G, Semenza GL (2006) Meeting report: exploiting the tumor microenvironment for therapeutics. Cancer Res 66:4558–4560PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2007

Authors and Affiliations

  • Chung-Jung Liu
    • 1
  • Jin-Ming Hwang
    • 2
  • Trang-Tiau Wu
    • 3
  • Yi-Hsien Hsieh
    • 1
  • Cheng-Chung Wu
    • 4
  • Yih-Shou Hsieh
    • 1
  • Chang-Hai Tsai
    • 5
  • Hsi-Chin Wu
    • 6
  • Chih-Yang Huang
    • 7
    • 8
    • 9
  • Jer-Yuh Liu
    • 1
  1. 1.Institute of Biochemistry and Biotechnology, Medical CollegeChung Shan Medical UniversityTaichungTaiwan
  2. 2.School of Applied Chemistry, Health Care and Management College Chung Shan Medical UniversityTaichungTaiwan
  3. 3.Department of Surgery, School of Medicine, Medical College Chung Shan Medical UniversityTaichungTaiwan
  4. 4.Department of General SurgeryTaichung Veterans General Hospital TaichungTaiwan
  5. 5.Department of Healthcare Administration Asia UniversityTaichungTaiwan
  6. 6.School of medicineChina Medical University and HospitalTaichungTaiwan
  7. 7.Graduate Institute of Chinese Medical ScienceChina Medical UniversityTaichungTaiwan
  8. 8.Institute of Medical Science China Medical UniversityTaichungTaiwan
  9. 9.Department of Health and Nutrition BiotechnologyAsia UniversityTaichungTaiwan

Personalised recommendations