Hypoxia-inducible transcription factor-1alpha promotes hypoxia-induced A549 apoptosis via a mechanism that involves the glycolysis pathway.

BACKGROUND: Hypoxia-inducible transcription factor-1alpha (HIF-1alpha), 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-1alpha on apoptosis by modulating HIF-1alpha gene expression in A549 cells through both siRNA knock-down and over-expression.

METHODS: A549 cells were transfected with a HIF-1alpha siRNA plasmid or a HIF-1alpha 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-1alpha inhibited the glycolysis pathway, increased the pH of the culture medium, and protected the cells from hypoxia-induced apoptosis. In contrast, over-expression of HIF-1alpha accelerated glycolysis in A549 cells, decreased the pH of the culture medium, and enhanced hypoxia-induced apoptosis. These effects of HIF-1alpha on glycolysis, pH of the medium, and apoptosis were reversed by treatment with the glycolytic inhibitor, 2-DG. Apoptosis induced by HIF-1alpha over-expression was partially inhibited by increasing the buffering capacity of the culture medium by adding HEPES.

CONCLUSION: During hypoxia in A549 cells, HIF-1alpha promotes activity of the glycolysis pathway and decreases the pH of the culture medium, resulting in increased cellular apoptosis.