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. 2011 Sep;23(9):1466-72.
doi: 10.1016/j.cellsig.2011.04.008. Epub 2011 Apr 28.

AMPK-mediated autophagy is a survival mechanism in androgen-dependent prostate cancer cells subjected to androgen deprivation and hypoxia

Rishi Raj Chhipa  1 , Yue Wu, Clement Ip
Affiliations

AMPK-mediated autophagy is a survival mechanism in androgen-dependent prostate cancer cells subjected to androgen deprivation and hypoxia

Rishi Raj Chhipa et al. Cell Signal. 2011 Sep.

Abstract

The present study was designed to investigate (i) the role of AMPK activation in inducing autophagy in androgen-dependent prostate cancer cells subjected to androgen deprivation and hypoxia, and (ii) whether autophagy offers a survival advantage under these harsh conditions. Low androgen and low oxygen are two co-existing conditions frequently found in prostate cancer tissue following surgical or medical castration. In LNCaP cells, androgen deprivation and hypoxia together boosted AMPK activation to a higher level than that seen with either condition alone. The augmented AMPK response was associated with improved viability and the induction of autophagy. These observations suggest that a threshold of AMPK activity has to be attained in order to trigger autophagy, since neither androgen deprivation nor hypoxia by itself was capable of pushing AMPK activity past that threshold. Beclin-1 was identified as a potential downstream _target of AMPK in turning on the autophagic cascade. If autophagy was blocked by chemical inhibition or RNA interference of key regulators, e.g., AMPK or beclin-1, more cells would die by apoptosis. The occurrence of autophagy is thus a survival mechanism for androgen-dependent prostate cancer cells to escape from an androgen-deprived and hypoxic subsistence.

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Figures

Fig. 1
Fig. 1
Effect of androgen deprivation and hypoxia on AMPK activation and cell viability. (A) Western blot of phosphorylated and total AMPKα in normoxia/hypoxia, and with or without 1 nM testosterone. The numbers indicate the relative protein level as determined by densitometry and normalized to total AMPKα. Hypoxia was confirmed by the expression of HIF1α. (B) Cell viability as determined by the MTT assay. NX, normoxia; HX, hypoxia; +T, with 1 nM testosterone; −T, without 1 nM testosterone. *P<0.05 compared to the HX+T value.
Fig. 2
Fig. 2
Effect of Compound C on AMPK activation and cell viability. (A) Compound C mediated inhibition of AMPK phosphorylation in hypoxic cells, with or without testosterone. (B) Compound C mediated inhibition of cell viability in androgen-deprived and hypoxic cells. *P<0.05.
Fig. 3
Fig. 3
Induction of cytoprotective autophagy by androgen deprivation and hypoxia. (A) Western blot of p62 (a specific substrate of autophagic degradation) in normoxic or hypoxic cells, and with or without testosterone. (B) Western blot of LC3-II, Atg5-Atg12 and beclin-1 (all autophagic markers) in hypoxic cells, with or without testosterone. The ratio of LC3-II to LC3-I is shown in a bar graph format directly under each of the Western blot lanes. The numbers below the Atg5–Atg12 and beclin-1 Western blots indicate the relative protein level as determined by densitometry and normalized to GADPH. (C) Effect of 3-methyladenine (3-MA) on cell viability in hypoxic cells, with or without testosterone. 3-MA is an inhibitor of autophagy. *P<0.05 compared to the corresponding value in the presence of 3-MA. (D) 3-MA mediated inhibition of autophagic marker expression in androgen-deprived and hypoxic cells.
Fig. 4
Fig. 4
Role of AMPK in autophagy induction in androgen-deprived and hypoxic cells. (A) Effect of Compound C on GFP-LC3 puncta formation (indicated by arrow) in normoxic or hypoxic cells, both in the absence of testosterone. For interpretation of the color in the figure, the reader is referred to the web version of the article. (B) Effect of AMPK siRNA transfection on phospho-AMPKα and LC3 –II expression. (C) Inhibition of LC3 puncta formation in androgen-deprived and hypoxic cells by AMPK siRNA transfection. GFP-LC3 puncta (indicated by arrow) can be visualized in control siRNA transfected cells. For each micrograph shown, between 5 to 8 fields were observed in each experiment. (D) Percentage of autophagic cells, as calculated based on LC-3 puncta positive cells, in various conditions. P<0.05, n=3 independent experiments.
Fig. 5
Fig. 5
Role of autophagy as a survival mechanism. (A) Increase of apoptotic cell death, as measured by DNA fragmentation, in androgen-deprived and hypoxic cells by AMPK siRNA transfection. The experiment was performed in triplicate. *P<0.05 compared to the corresponding value in the presence of testosterone. (B) Effect of AMPK siRNA transfection on beclin-1 expression in androgen-deprived and hypoxic cells. (C) Effect of BECN1 shRNA transfection on LC3-II expression and PARP cleavage in hypoxia, with or without testosterone. The ratio of LC3-II to LC3-I is shown in a bar graph format directly under each of the Western blot lanes. The numbers below the PARP Western blot indicate the relative level of cleaved PARP as determined by densitometry and normalized to GADPH.
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