Abstract
BackgroundHypoxia-inducible transcription factor-1α (HIF-1α), 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-1α on apoptosis by modulating HIF-1α gene expression in A549 cells through both siRNA knock-down and over-expression.MethodsA549 cells were transfected with a HIF-1α siRNA plasmid or a HIF-1α 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.ResultsKnocking down expression of HIF-1α 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-1α accelerated glycolysis in A549 cells, decreased the pH of the culture medium, and enhanced hypoxia-induced apoptosis. These effects of HIF-1α on glycolysis, pH of the medium, and apoptosis were reversed by treatment with the glycolytic inhibitor, 2-DG. Apoptosis induced by HIF-1α over-expression was partially inhibited by increasing the buffering capacity of the culture medium by adding HEPES.ConclusionDuring hypoxia in A549 cells, HIF-1α promotes activity of the glycolysis pathway and decreases the pH of the culture medium, resulting in increased cellular apoptosis.
Highlights
Hypoxia-inducible transcription factor-1α (HIF-1α), which plays an important role in controlling the hypoxia-induced glycolysis pathway, is a "master" gene in the tissue hypoxia response during tumor development
We investigated whether controlling HIF-1α expression regulated apoptosis of the lung adenocarcinoma cell line, A549, during hypoxia, and whether this effect of HIF-1α was dependent upon glycolysis
Down-regulation of HIF-1α gene expression in A549 cells with small interfering RNA (siRNA) As shown in Figure 1, in A549 cells transfected with the siRNA EGFP-pSUPER vector system, the hypoxia-induced expression of HIF-1α mRNA was significantly suppressed
Summary
Hypoxia-inducible transcription factor-1α (HIF-1α), which plays an important role in controlling the hypoxia-induced glycolysis pathway, is a "master" gene in the tissue hypoxia response during tumor development. Giatromanolaki et al [4] reported that HIF-1α overexpression is common in NSCLC, and that this is related to the up-regulation of various angiogenic factors and associated with poor prognosis. They suggested that HIF pathway might be an important therapeutic target for NSCLC. These results are at odds with those of Volm et al [5], who demonstrated that NSCLC patients with HIF-1α-positive carcinomas had significantly longer median survival times than patients with HIF-1α-negative carcinomas. As apoptosis is one of the most important mechanisms mediating cancer remission, investigation into the role of HIF-1α in apoptosis of lung cancer may provide useful information for the resolution of these controversies
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