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Hypoxia and cytokines regulate carbonic anhydrase 9 expression in hepatocellular carcinoma cells in vitro
Feray Kockar, Hatice Yildrim, Rahsan Ilikci Sagkan, Carsten Hagemann, Yasemin Soysal, Jelena Anacker, Ahmed Ayad Hamza, Dirk Vordermark, Michael Flentje, Harun M Said
Feray Kockar, Hatice Yildrim, Department of Biology, Faculty of Art and Science, Balikesir University, 10145 Balikesir, Turkey
Rahsan Ilikci Sagkan, Department of Immunology and Allergy Diseases, Gulhane Military Medical Academy, Etlik, 06018 Ankara, Turkey
Carsten Hagemann, Tumorbiology Laboratory, Department of Neurosurgery, University of Würzburg, D-97080 Würzburg, Germany
Yasemin Soysal, Faculty of Medicine, Department of Medical Genetics, Afyon Kocatepe University, 03100 Afyonkarahisar, Turkey
Jelena Anacker, Department of Obstetrics and Gynaecology, University of Würzburg, D-97080 Würzburg, Germany
Ahmed Ayad Hamza, School of Medicine, Southern Illinois University, Springfield, IL 62794-9620, United States
Dirk Vordermark, Department of Radiation Oncology, University-Halle-Wittenberg, 06110 Halle, Germany
Michael Flentje, Harun M Said, Department of Radiation Oncology, University of Wuerzburg, D-97080 Würzburg, Germany
Author contributions: Kockar F and Said HM were the primary authors and performed the in vitro hypoxia experiments, supplied the in vitro mRNA, protein lysates and nuclear extracts, performed the western blots, densitometric analysis of the results and participated in the study design; Hagemenn C, Soysal Y, Hamza AA were co-authors and participated in the study design; Kockar F and Said HM coordinated the group and contributed to the development of the experimental strategy; Anacker J designed the primers used for reverse transcription polymerase chain reaction and participated in the study design and evaluation; Hagemenn C, Vordermark D and Flentje M also participated in the study design; and all authors read and approved the manuscript.
Supported by Deutsche Forschungsgemeinschaft DFG, VO 871/2-3, to Vordermark D; and the IZKF Würzburg, B25, to Hagemenn C; Turkish Research Council (TUBITAK) Project, TBAG 105T326, to Kockar F and Yildrim H; Balikesir University Research Project, 2008/15, to Sagkan RI
Correspondence to: Dr. Harun M Said, PhD, Department of Radiation Oncology, University of Wurzburg, Josef-Schneider-Str. 11, D-97080 Wurzburg, Germany. said@scientist.com
Telephone: +49-16-37531174 Fax: +49-16-37531174
Received: August 30, 2011
Revised: May 30, 2012
Accepted: June 5, 2012
Published online: June 10, 2012
AIM: To study the expression of carbonic anhydrase (CA) 9 in human hepatocellular carcinoma (HCC) cells.
METHODS: We studied CA9 protein, CA9 mRNA and hypoxia-inducible factor-1 alpha (HIF-1α) protein levels in Hep3B cells exposed in different parallel approaches. In one of these approaches, HCC cells were exposed to extreme in vitro hypoxia (24 h 0.1% O2) without or with interleukin (IL)-1, IL-6, tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-β) stimulation for the same hypoxic exposure time or exposed to normoxic oxygenation conditions without or with cytokine stimulation.
RESULTS: The tumour cell line analysed showed a strong hypoxic CA9 mRNA expression pattern in response to prolonged severe hypoxia with cell-line specific patterns and a marked induction of CA9 protein in response to severe hypoxia. These results were paralleled by the results for HIF-1α protein under identical oxygenation conditions with a similar expression tendency to that displayed during the CA9 protein expression experimental series. Continuous stimulation with the cytokines, IL-1, IL-6, TNF-α and TGF-β, under normoxic conditions significantly increased the carbonic anhydrase 9 expression level at both the protein and mRNA level, almost doubling the CA9 mRNA and CA9 and HIF-1α protein expression levels found under hypoxia. The findings from these experiments indicated that hypoxia is a positive regulator of CA9 expression in HCC, and the four signal transduction pathways, IL-1, IL-6, TNF-α and TGF-β, positively influence CA9 expression under both normoxic and hypoxic conditions.
CONCLUSION: These findings may potentially be considered in the design of anti- cancer therapeutic approaches involving hypoxia-induced or cytokine stimulatory effects on expression. In addition, they provide evidence of the stimulatory role of the examined cytokine families resulting in an increase in CA9 expression under different oxygenation conditions in human cancer, especially HCC, and on the role of the CA9 gene as a positive disease regulator in human cancer.
