Hypoxia-Induced Suppression of Antiapoptotic Bcl-2 Expression in Human Bladder Tumor Cells Is Regulated by
Tae Jin Cho1,2, Da-Hyun Lee1, Bo-Hwa Choi1
1Department of Pharmacology, Hypoxia-Related Disease Research Center, Inha Research Institute for Medical Sciences, Incheon, Korea.
Purpose:
Adenosine monophosphate-activated protein kinase (AMPK) is thought to inhibit cell proliferation or promote cell death, but the details remain unclear. In this study, we propose that AMPK inhibits the expression of anti-apoptotic B-cell lymphoma 2 (Bcl-2) by relying on the hypoxia-inducible factor 1 alpha (HIF-1α)-induced caveolin-1 (Cav-1) expression pathway in noninvasive human bladder tumor (RT4) cells.
Methods:
In cells exposed to a hypoxic environment (0.5% oxygen), the levels of expression and phospho-activity of the relevant signaling enzymes were examined via Western blots and reverse transcription-polymerase chain reaction. Cell proliferation was assessed using a Cell Counting Kit-8 assay.
Results:
The level of expression of Cav-1 was very low or undetectable in RT4 cells. Hypoxia was associated with significantly decreased cell growth, along with marked induction of HIF-1α and Cav-1 expression; additionally, it suppressed the expression of the antiapoptotic marker Bcl-2 while leaving AMPK activity unchanged. Under hypoxic conditions, HIF-1α acts as a transcription factor for Cav-1 mRNA gene expression. The cell growth and Bcl-2 expression suppressed under hypoxia were reversed along with decreases in the induced HIF-1α and Cav-1 levels by AMPK activation with metformin (1mM) or phenformin (0.1mM). In addition, pretreatment with AMPK small interfering RNA not only increased the hypoxia-induced expression of HIF-1α and Cav-1, but also reversed the suppression of Bcl-2 expression. These results suggest that HIF-1α and Cav-1 expression in hypoxic environments is regulated by basal AMPK activity; therefore, the inhibition of Bcl-2 expression cannot be expected when AMPK activity is suppressed, even if Cav-1 expression is elevated.
Conclusion:
For the first time, we find that AMPK activation can regulate HIF-1α induction as well as HIF-1α-induced Cav1 expression, and the hypoxia-induced inhibitory effect on the antiapoptotic pathway in RT4 cells is due to Cav-1-dependent AMPK activity.
Insights
Adenosine monophosphate-activated protein kinase (AMPK) activation inhibits hypoxia-induced caveolin-1 (Cav-1) and hypoxia-inducible factor 1 alpha (HIF-1α) expression, thereby suppressing anti-apoptotic B-cell lymphoma 2 (Bcl-2) in bladder cancer cells.
Area of Science:
- Cellular signaling pathways
- Cancer biology
- Molecular mechanisms of apoptosis
Background:
- Adenosine monophosphate-activated protein kinase (AMPK) plays a role in cell metabolism and survival.
- The precise mechanisms by which AMPK influences cell proliferation and apoptosis, particularly in cancer, are not fully elucidated.
- Caveolin-1 (Cav-1) and hypoxia-inducible factor 1 alpha (HIF-1α) are implicated in tumor growth and survival.
Purpose of the Study:
- To investigate the role of AMPK in regulating cell proliferation and apoptosis in noninvasive human bladder tumor (RT4) cells.
- To determine if AMPK affects the expression of anti-apoptotic B-cell lymphoma 2 (Bcl-2) via the HIF-1α-induced Cav-1 pathway.
- To elucidate the molecular mechanisms underlying hypoxia-induced changes in bladder cancer cells.
Main Methods:
- Western blot analysis to assess protein expression and phospho-activity.
- Reverse transcription-polymerase chain reaction (RT-PCR) for gene expression analysis.
- Cell Counting Kit-8 assay to evaluate cell proliferation.
- Manipulation of AMPK activity using pharmacological activators (metformin, phenformin) and small interfering RNA (siRNA).
Main Results:
- Hypoxia significantly decreased cell growth and suppressed B-cell lymphoma 2 (Bcl-2) expression in RT4 cells.
- Hypoxia induced the expression of hypoxia-inducible factor 1 alpha (HIF-1α) and caveolin-1 (Cav-1), with HIF-1α acting as a transcription factor for Cav-1.
- Adenosine monophosphate-activated protein kinase (AMPK) activation reversed hypoxia-induced suppression of cell growth and Bcl-2 expression by reducing HIF-1α and Cav-1 levels.
- AMPK activation was found to regulate HIF-1α induction and HIF-1α-induced Cav-1 expression.
Conclusions:
- AMPK activation plays a critical role in regulating hypoxia-induced HIF-1α and Cav-1 expression in bladder cancer cells.
- The hypoxia-induced inhibition of the anti-apoptotic pathway in RT4 cells is mediated by Cav-1-dependent AMPK activity.
- These findings provide novel insights into the molecular mechanisms governing bladder tumor cell behavior under hypoxic conditions.
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