Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2005 Jul;96(7):394-402.
doi: 10.1111/j.1349-7006.2005.00065.x.

Mechanism of HIF-1alpha-dependent suppression of hypoxia-induced apoptosis in squamous cell carcinoma cells

Eri Sasabe  1 , Yukihiro Tatemoto, Dechao Li, Tetsuya Yamamoto, Tokio Osaki
Affiliations

Mechanism of HIF-1alpha-dependent suppression of hypoxia-induced apoptosis in squamous cell carcinoma cells

Eri Sasabe et al. Cancer Sci. 2005 Jul.

Abstract

The transcriptional factor hypoxia-inducible factor-1 (HIF-1) plays an important role in solid tumor cell growth and survival. Overexpression of HIF-1alpha has been demonstrated in many human tumors and predicts a poor response to chemoradiotherapy. We examined the HIF-1alpha-induced survival pathways in human oral squamous cell carcinoma cell (OSCC) lines. The results showed that forced expression of HIF-1alpha suppressed hypoxia-induced apoptosis of OSCC lines by inhibiting cytochrome c release from mitochondria. Overexpression of HIF-1alpha inhibited the generation of reactive oxygen species (ROS), elevation of intracellular Ca(2+) concentration, reduction of mitochondrial membrane potential, and cytosolic accumulation of cytochrome c, which resulted in the inactivation of caspase-9 and caspase-3. In addition, antiapoptotic Bcl-2 and Bcl-X(L) levels were increased and pro-apoptotic Bax and Bak levels were decreased in the HIF-1alpha-overexpressing OSCC line. Overexpression of HIF-1alpha also increased the levels of phosphorylation of Akt and extracellular signal-regulated kinases (ERK). These findings indicate that HIF-1alpha prevents apoptotic cell death through two mechanisms, including inhibition of cytochrome c release and activation of Akt and ERK.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Expression of HIF‐1α and VEGF proteins in the cell lines examined. (a) Cells were cultured under normoxic (20% O2; N) and hypoxic (1% O2; H) conditions for 24 h, and whole cell extracts obtained from cells were subjected to western blot analysis for HIF‐1α protein expression. (b) VEGF protein levels in the whole cell lysates were determined using ELISA. The experiment was repeated three times and this is a representative example.
Figure 2
Figure 2
Effects of forced expression of HIF‐1α on apoptosis of OSC‐4 cells. OSC‐4 cells transfected with vector alone (mock) or HIF‐1α cDNA (HIF‐1α) were cultured under normoxic and hypoxic conditions for 24 h. (a) Whole cell extracts from the cells were subjected to western blot analysis for HIF‐1α protein expression. (b) ELISA for VEGF protein levels. (c) Apoptotic cell death was determined by flow cytometric analysis after cell incubation under normoxic and hypoxic conditions for 24 h. (d) DNA fragmentation. After incubation under normoxic and hypoxic conditions for 24 h, cells were stained with Hoechst 33258.
Figure 3
Figure 3
The [Ca2+]i, generation of ROS and Δψm in control cells and HIF‐1α‐overexpressing cells. Both cell types were cultured under normoxic and hypoxic conditions for 24 h. (a) [Ca2+]i was determined by the fluorescence obtained from Fluo‐3 AM using a FACScan cytometer. (b) ROS levels were measured using flow cytometry after loading cells with DCFH‐DA. (c) Δψm was measured by the relative fluorescence intensities of rhodamine‐123 using flow cytometry. The experiment was repeated three times and this is a representative example. Values are the mean ± SEM of determinations made in three independent cultures. *P < 0.05 compared with values for control cells (mock).
Figure 4
Figure 4
Effects of forced expression of HIF‐1α on the expression of Bcl‐2 family proteins and cytochrome c release. (a) Bcl‐2, Bcl‐XL, Bax and Bak protein levels in OSC‐4 cells after each indicated treatment were determined by western blots of total cell lysates. (b) Both cell types were cultured under normoxic and hypoxic conditions for 24 h and cytosolic and mitochondrial fractions were prepared for immunoblotting of cytochrome c. The experiment was repeated three times and this is a representative example.
Figure 5
Figure 5
Effects of forced expression of HIF‐1α on the activation of caspase‐9 and ‐3. (a) Both cell types were cultured under normoxic and hypoxic conditions for 24 h, and cell lysates were prepared for immunoblotting of the processed and non‐processed caspase‐9 and ‐3. (b) Hydrolyzing activities of caspase‐9 and ‐3. Cell extracts obtained from cells after the indicated cultivation conditions were assessed for their ability to hydrolyze the peptides Ac‐LEHD‐pNA and Ac‐DEVD‐pNA. Values are the mean ± SEM of determinations made in three independent cultures. *P < 0.05 compared with values for control cells (mock). (c) Both cell types were pretreated with 50 µM caspase‐9 inhibitor Z‐LEHD‐FNK or caspase‐3 inhibitor Z‐DEVD‐FMK for 1 h, and cultured under normoxic and hypoxic conditions for 24 h. Apoptotic cell death was determined by flow cytometric analysis. The experiment was repeated three times and this is a representative example. Values are the mean ± SEM of determinations made in three independent cultures. *P < 0.05 compared with values for untreated cultures (control).
Figure 6
Figure 6
Effects of forced expression of HIF‐1α on the expression levels of PI3K, Akt and ERK and their phosphorylation levels. (a) Both cell types were cultured under normoxic and hypoxic conditions for 24 h. Cell lysates were then prepared and western blot analysis was performed to determine the levels of PI3K, Akt, p‐Akt (Ser473), total ERK1/2 and p‐ERK1/2. (b) Phosphorylation of Bad was determined by western blotting using antiphosphorylated Bad (Ser112 and Ser136) antibodies. (c) Hetero‐oligomerization between Bad, 14‐3‐3, Bcl‐2 and Bcl‐XL. Co‐immunoprecipitates were obtained using antibodies against each Bcl‐2 family protein and the obtained immunoprecipitates were blotted using each indicated antibody. The experiment was repeated three times and this is a representative example.
Figure 7
Figure 7
Effects of LY294002 and PD98059 on the expression of HIF‐1α, p‐Akt, Akt, p‐ERK, ERK and apoptosis. (a) The transfectants were pretreated with LY294002 (40 µM) and PD98059 (20 µM) for 30 min and cultured under normoxic and hypoxic conditions for 30 min to 6 h. Cell lysates were then prepared, and western blot analysis was performed. (b) Apoptotic cell death was analyzed by a FACScan cytometer after each treatment under normoxic and hypoxic conditions for 24 h. The experiment was repeated three times and this is a representative example. Values are the mean ± SEM of determinations made in three independent cultures. *P < 0.05 compared with values for untreated cultures (control).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia‐inducible factor 1 is a basic‐helix‐loop‐helix‐PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci USA 1995; 92: 5510–4. - PMC - PubMed
    1. Salceda S, Caro J. Hypoxia‐inducible factor‐1α (HIF‐1α) protein is rapidly degraded by the ubiquitin‐proteasome system under normoxic conditions. J Biol Chem 1997; 272: 22642–7. - PubMed
    1. Huang LE, Gu J, Schau M, Bunn HF. Regulation of hypoxia‐inducible factor 1α is mediated by an O2‐dependent degradation domain via the ubiquitin‐proteasome pathway. Proc Natl Acad Sci USA 1998; 95: 7987–92. - PMC - PubMed
    1. Sutter CH, Laughner E, Semenza GL. Hypoxia‐inducible factor 1α protein expression is controlled by oxygen‐regulated ubiquitination that is disrupted by deletions and missense mutations. Proc Natl Acad Sci USA 2000; 97: 4748–53. - PMC - PubMed
    1. Guillemin K, Krasnow MA. The hypoxic response: huffing HIFing. Cell 1997; 89: 9–12. - PubMed

MeSH terms

Substances