Hypoxia and cytokines regulate carbonic anhydrase 9 expression in hepatocellular carcinoma cells in vitro

Abstract

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-1 alpha)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% O-2) without or with interleukin (IL)-1, IL-6, tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-ss) 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-1 alpha 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-alpha and TGF-ss, 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-1 alpha 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-alpha and TGF-ss, 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