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

Abstract

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.

Figure 1

Inhibition HIF-1α mRNA expression in A549 by siRNA. A549 were transfected with the siRNA-expressing EGFP-pSUPER vector or a HIF-1 expression plasmid. Cells were allowed to recover in regular culture medium for 20 h after transfection, then exposed to a normoxic or hypoxic environment for an additional 24 h. Experiments were performed five times with the similar results (n = 5 in each group). $, P < 0.05 compared to control and HIF-1α siRNA; #, P < 0.05 compared to normoxic or hypoxic HIF-1α siRNA; *, P < 0.05 compared to hypoxic mock-transfected and scrambled HIF-1α control siRNA transfection. siRNA: HIF-1α plasmid transfected, Mock: mock transfected, HIF: HIF-1α expression plasmid transfected.

Figure 2

Suppression and over-expression of HIF-1α: western analysis. A549 cells were transfected with HIF-1α siRNA vector or a HIF-1 expression plasmid. Cells were allowed to recover in regular culture medium for 20 h after transfection, then exposed to normoxia or hypoxia for 24 h, at which point cellular protein extracts were prepared (n = 5 in each group). Proteins were separated by SDS-PAGE and transferred to membrane for western blot analysis. siRNA: HIF-1α plasmid transfected, Mock: mock transfected, HIF: HIF-1α expression plasmid transfected.

Figure 3

Suppression and over-expression of HIF-1α: graphical format. Densitometric analysis was performed on the western blot shown in figure 3 and displayed here in graphical format. $, P < 0.05 compared to control and HIF-1α siRNA; #, P < 0.05 compared to normoxic and hypoxic HIF-1α siRNA; *, P < 0.05 compared to hypoxic mock transfected and scrambled HIF-1α control siRNA transfection. siRNA: HIF-1α plasmid transfected, Mock: mock transfected, HIF: HIF-1α expression plasmid transfected.

Figure 4

siRNA-mediated knock-down of HIF-1α expression leads to the suppression of three key glycolysis genes. Expression of three key genes in the glycosis pathway, GLUT1, HK1, and PGK1 was monitored by real-time RT-PCR. Cells were transfected with HIF-1α siRNA EGFP-pSUPER vector, scrambled HIF-1α control siRNA, or an expression plasmid encoding HIF-1α. After 20 h, transfected cells were exposed to hypoxia for 24 h (n = 5 in each group). $, P < 0.05 compared to normoxic control; #, P < 0.05 compared to normoxic and hypoxic HIF-1α siRNA; *, P < 0.05 compared to hypoxic mock-transfected and scrambled HIF-1α control siRNA transfection. siRNA: HIF-1α siRNA vector transfected, Mock: mock transfected, HIF: HIF-1α expression plasmid transfected.

Figure 5

Quantitation of the cell culture medium pH. The media from cell cultures were collected after transfection of the cells with HIF-1α siRNA vector or a HIF-1α expression plasmid and exposure to hypoxic conditions for 24 h. The pH of the cell culture medium measured immediately after samples collected (n = 5 in each group). $, P < 0.05 compared to normoxic mock and HIF-1α siRNA transfected; *, P < 0.05 compared to hypoxic HIF-1α siRNA and HIF-1α expression plasmid with HEPES; #, P < 0.05 compared to hypoxic HIF-1αexpression plasmid with or without 2-DG. siRNA: HIF-1α siRNA EGFP-pSUPER vector transfected, Mock: mock-transfected, HIF: HIF-1α expression plasmid transfected, HIF+2-DG: Cells were transfected with HIF-1α expression plasmid and cultured with 2-DG, HIF+HEPES: Cells were transfected with HIF-1α expression plasmid and cultured with medium containing HEPES.

Figure 6

Measurement of lactate levels in the cell culture medium. The media from cell cultures were collected after transfection of the cells with HIF-1α siRNA vector or a HIF-1α expression plasmid and exposure to hypoxic conditions for 24 h. Lactate concentration was measured using a commercial kit (n = 5 in each group). $, P < 0.05 compared to normoxic mock transfected and HIF-1α siRNA; *, P < 0.05 compared to hypoxic HIF-1α siRNA and HIF-1α expression vector with HEPES; #, P < 0.05 compared to hypoxic HIF-1α expression vector with or without 2-DG. siRNA: A549 cells transfected HIF-1α siRNA vector, Mock: mock-transfected, HIF: A549 cells transfected with HIF-1α expression plasmid, HIF+2-DG: Cells transfected with HIF-1α expression plasmid and cultured with 2-DG, HIF+HEPES: Cells transfected with HIF-1α expression plasmid and cultured with medium containing HEPES.

Figure 7

Detection of apoptosis by flow cytometry: histogram analysis. A549 cells were transfected with HIF-1α siRNA vector or HIF-1 expression plasmid. Cells were allowed to recover in regular culture medium for 20 h after transfection, then exposed to normoxic or hypoxic environment for 24 h with or without additional HEPES (25 mM) or the glycolytic inhibitor, 2-DG (5 mM) (n = 5 in each group). Cells were collected and stained with Apo2.7 monoclonal antibody (7A6). Shown are representative results from one of five independent experiments. siRNA: A549 cells transfected HIF-1α siRNA vector, Mock: mock-transfected, HIF: A549 cells transfected with HIF-1α expression plasmid, HIF+2-DG: Cells transfected with HIF-1α expression plasmid and cultured with 2-DG, HIF+HEPES: Cells transfected with HIF-1α expression plasmid and cultured with medium containing HEPES.

Figure 8

Detection of apoptosis by flow cytometry: graphical format. Cells were treated and stained as described for Fig. 7. Shown is a graphical representation of this data with statistical analysis from a representative of 5 independent experiments. $, P < 0.05 compared to normoxic mock transfected and HIF-1α siRNA; *, P < 0.05 compared to hypoxic HIF-1α siRNA and HIF-1α expression vector with HEPES; #, P < 0.05 compared to hypoxic HIF-1α expression vector with or without 2-DG. siRNA: A549 cells transfected HIF-1α siRNA vector, Mock: mock-transfected, HIF: A549 cells transfected with HIF-1α expression plasmid, HIF+2-DG: Cells transfected with HIF-1α expression plasmid and cultured with 2-DG, HIF+HEPES: Cells transfected with HIF-1α expression plasmid and cultured with medium containing HEPES.

Figure 9

Detection of apoptosis by TUNEL: Staining. A549 cells were cultured on cover slides and transfected with different plasmids. Cells were allowed to recover in regular culture medium for 20 h after transfection, then exposed to normoxic or hypoxic atmosphere for 24 h with or without HEPES (25 mM) or the glycolytic inhibitor, 2-DG (5 mM) (n = 5 in each group). Only occasional normoxic cells were positive for TUNEL staining, (A). Hypoxia increased the amount of apoptosis of A549 cells, (D). Over-expression of HIF-1α further increased this pro-apoptotic effect of hypoxia, (F). However, siRNA suppression of HIF-1α expression rescued A549 cells from hypoxia-induced apoptosis, (C); but scrambled siRNA did not have this effect, (E). The pro-apoptotic effect of HIF-1α was also inhibited by the glycolytic inhibitor, 2-DG, (G). Increasing the buffering capacity of the culture medium with HEPES also inhibited the pro-apoptotic effect of HIF-1α, (H). Shown are representative photomicrographs from five independent experiments. Figure 9 was statistics of the five independent experiments. siRNA: A549 cells transfected HIF-1α siRNA vector, Mock: mock-transfected, HIF: A549 cells transfected with HIF-1α expression plasmid, HIF+2-DG: Cells transfected with HIF-1α expression plasmid and cultured with 2-DG, HIF+HEPES: Cells transfected with HIF-1α expression plasmid and cultured with medium containing HEPES. Scale bar: 100 μm.

Figure 10

Detection of apoptosis by TUNEL: graphical format. TUNEL staining was performed on cells as described above. Shown is a graphical representation of the staining shown in Fig. 9. $, P < 0.05 compared to normoxic mock transfected and HIF-1α siRNA; *, P < 0.05 compared to hypoxic HIF-1α siRNA and HIF-1α expression vector with HEPES; #, P < 0.05 compared to hypoxic HIF-1α expression vector with or without 2-DG. siRNA: A549 cells transfected HIF-1α siRNA vector, Mock: mock-transfected, HIF: A549 cells transfected with HIF-1α expression plasmid, HIF+2-DG: Cells transfected with HIF-1α expression plasmid and cultured with 2-DG, HIF+HEPES: Cells transfected with HIF-1α expression plasmid and cultured with medium containing HEPES.