Effect of normoxic acidosis on HIF-1α protein expression and activity in human liver cancer cell line HepG2 / 第二军医大学学报

ABSTRACT

Objective: To investigate the effect of normoxic acidosis on HIF-1α protein expression and activity in human liver cancer cell line HepG2, so as to assess the role of acidosis in tumor growth. Methods: HepG2 cells were cultured in vitro; the cell growth was assessed by MTT assay after exposure to normoxic acidosis culture media (AP, pH 6.5) for 20 h, and the results were compared with those of the control cells (SD, pH 7.2). Nuclear extractions were performed in the cultured cells, and HIF-1α protein level and HIF-1 DNA binding activities were examined by immunocytochemistry, Western blotting, and HIF-1α Transcription Factor Assay Kit (TransAM™). The protein level of VEGF (vascular endothelial growth factor) was detected in whole cell extractions by Western blotting assay. Results: MTT assay demonstrated that the cell survival rate was (98.71±1.79)% after treatment with normoxic acidosis culture media (AP, pH 6.5). In comparison with SD group, we noted a slight decrease of cell viability in AP group (P>0.05). Western blotting analysis showed that the levels of HIF-1α and VEGF protein increased by (9.34±1.67) folds and (3.42±0.83) folds, respectively (P<0.01). Moreover, normoxic acidosis greatly enhanced HIF-1α DNA-binding activity compared to SD groups (P<0.01). Conclusion: Normoxic acidosis can evidently increase HIF-1α protein level and DNA binding activity in HepG2 cells. Furthermore, the protein level of VEGF, an important target gene of HIF-1, is also increased. Therefore it is suggested that, in addition to hypoxia, acidosis may also play an important role in induction of HIF-1 in cancer.