Hypoxia-inducible transcription factor-Iα promotes hypoxia-induced A549 apoptosis via a mechanism that involves the glycolysis pathway

Background: Hypoxia-inducible transcription factor-1 alpha (HIF-1 alpha), which plays an important role in controlling the hypoxia-induced glycolysis pathway, is a "master" gene in the tissue hypoxia response during tumor development. However, its role in the apoptosis of non-small cell lung cancer remains unknown. Here, we have studied the effects of HIF-1 alpha on apoptosis by modulating HIF-1 alpha gene expression in A549 cells through both siRNA knock-down and over-expression. Methods: A549 cells were transfected with a HIF-1 alpha siRNA plasmid or a HIF-1 alpha expression vector. Transfected cells were exposed to a normoxic or hypoxic environment in the presence or absence of 25 mM HEPES and 2-deoxyglucose (2-DG) ( 5 mM). The expression of three key genes of the glycolysis pathway, glucose transporter type 1(GLUT1), phosphoglycerate kinase 1(PGK1), and hexokinase 1(HK1), were measured using real-time RT-PCR. Glycolysis was monitored by measuring changes of pH and lactate concentration in the culture medium. Apoptosis was detected by TUNEL assay and flow cytometry. Results: Knocking down expression of HIF-1 alpha inhibited the glycolysis pathway, increased the pH of the culture medium, and protected the cells from hypoxia-induced apoptosis. In contrast, overexpression of HIF-1 alpha accelerated glycolysis in A549 cells, decreased the pH of the culture medium, and enhanced hypoxia-induced apoptosis. These effects of HIF-1 alpha on glycolysis, pH of the medium, and apoptosis were reversed by treatment with the glycolytic inhibitor, 2-DG. Apoptosis induced by HIF-1 alpha over-expression was partially inhibited by increasing the buffering capacity of the culture medium by adding HEPES. Conclusion: During hypoxia in A549 cells, HIF-1 alpha promotes activity of the glycolysis pathway and decreases the pH of the culture medium, resulting in increased cellular apoptosis.