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.


CONCLUSIONS
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.
Luo
FengMing
F
West China Hospital, Sichuan University, Chengdu, China. luofengming@hotmail.com
Liu
XiaoJing
X
Yan
NaiHong
N
Li
ShuangQing
S
Cao
GuiQun
G
Cheng
QingYing
Q
Xia
QingJie
Q
Wang
HongJing
H
D017209:Apoptosis; D015153:Blotting, Western; D002289:Carcinoma, Non-Small-Cell Lung / Q000378:metabolism; D045744:Cell Line, Tumor; D003470:Culture Media / Q000378:metabolism; D003847:Deoxyglucose / Q000737:chemistry; D015536:Down-Regulation; D005434:Flow Cytometry; D005822:Genetic Vectors; D051272:Glucose Transporter Type 1 / Q000378:metabolism; D006019:Glycolysis; D006531:HEPES / Q000737:chemistry; D006593:Hexokinase / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D000860:Hypoxia; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D020287:In Situ Nick-End Labeling; D007773:Lactates / Q000737:chemistry; D008175:Lung Neoplasms / Q000378:metabolism; D015233:Models, Statistical; D010100:Oxygen / Q000378:metabolism; D010735:Phosphoglycerate Kinase / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D012313:RNA / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection; D015854:Up-Regulation
D003470:Culture Media; D051272:Glucose Transporter Type 1; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007773:Lactates; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012313:RNA; D003847:Deoxyglucose; D006593:Hexokinase; D010735:Phosphoglycerate Kinase; D006531:HEPES; D010100:Oxygen