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Autophagy pathways activated in response to PDT contribute to cell resistance against ROS damage.

Dewaele M, Martinet W, Rubio N, Verfaillie T, de Witte PA, Piette J, Agostinis P - J. Cell. Mol. Med. (2010)

Bottom Line: Paradoxically, genetic loss of MA improved clearance of oxidized proteins and reduced photokilling.We found that up-regulation of chaperone-mediated autophagy (CMA) in unstressed Atg(-/-) cells compensated for MA loss and increased cellular resistance to PDT.These results disclose a stress-specific recruitment of autophagy pathways with cytoprotective function and unravel CMA as the dominant defence mechanism against PDT.

View Article: PubMed Central - PubMed

Affiliation: Cell Death Research and Therapy Laboratory, Department of Molecular Cell Biology, Faculty of Medicine, Katholieke Universiteit Leuven, Leuven, Belgium.

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Rapid inhibition of Akt-mTOR pathway after PDT. All controls represent incubation with hypericin without irradiation. (A) Analysis of the Akt-mTOR pathway activation-status in MEF as a function of time after PDT. Left: representative Western blot analysis indicating phosphorylated and total levels of Akt, p70S6Kinase and S6 ribosomal protein and LC3 conversion after PDT (n= 3). Right: Densitometric quantification of the corresponding Western blot analysis on the left. (B) Left: Representative Western blot analysis of the activation status of S6 ribosomal protein in HeLa Neo or HeLa GPx4 cells following PDT (n= 3). Right: Densitometric quantification of the corresponding Western blot analysis on the left. Arbitrary densitometric units represent phosphorylation relative to the expression level and normalized to the control condition (A upper graph and B) or expression level of Akt normalized to the expression level of control (A lower graph).
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fig02: Rapid inhibition of Akt-mTOR pathway after PDT. All controls represent incubation with hypericin without irradiation. (A) Analysis of the Akt-mTOR pathway activation-status in MEF as a function of time after PDT. Left: representative Western blot analysis indicating phosphorylated and total levels of Akt, p70S6Kinase and S6 ribosomal protein and LC3 conversion after PDT (n= 3). Right: Densitometric quantification of the corresponding Western blot analysis on the left. (B) Left: Representative Western blot analysis of the activation status of S6 ribosomal protein in HeLa Neo or HeLa GPx4 cells following PDT (n= 3). Right: Densitometric quantification of the corresponding Western blot analysis on the left. Arbitrary densitometric units represent phosphorylation relative to the expression level and normalized to the control condition (A upper graph and B) or expression level of Akt normalized to the expression level of control (A lower graph).

Mentions: Because induction of MA is negatively regulated by the Akt-mTOR (mammalian target of rapamycin) pathway [5], we investigated the impact of our ROS-based therapy on this core signalling pathway. Activation of p70S6K by phosphorylation on the mTOR-specific serine residue Thr389 and subsequent phosphorylation/activation of the p70S6K substrate S6 provides a valuable measure of mTOR activity [32]. As soon as 1 hr after PDT, p70S6K became progressively dephosphorylated on Thr389 and phospho-S6 levels declined subsequently (Fig. 2A). Intriguingly, S6 phosphorylation level nearly recovered to control levels 16 hrs after irradiation, suggesting the modulation of S6 phosphorylation by additional p70S6K-independent mechanisms.


Autophagy pathways activated in response to PDT contribute to cell resistance against ROS damage.

Dewaele M, Martinet W, Rubio N, Verfaillie T, de Witte PA, Piette J, Agostinis P - J. Cell. Mol. Med. (2010)

Rapid inhibition of Akt-mTOR pathway after PDT. All controls represent incubation with hypericin without irradiation. (A) Analysis of the Akt-mTOR pathway activation-status in MEF as a function of time after PDT. Left: representative Western blot analysis indicating phosphorylated and total levels of Akt, p70S6Kinase and S6 ribosomal protein and LC3 conversion after PDT (n= 3). Right: Densitometric quantification of the corresponding Western blot analysis on the left. (B) Left: Representative Western blot analysis of the activation status of S6 ribosomal protein in HeLa Neo or HeLa GPx4 cells following PDT (n= 3). Right: Densitometric quantification of the corresponding Western blot analysis on the left. Arbitrary densitometric units represent phosphorylation relative to the expression level and normalized to the control condition (A upper graph and B) or expression level of Akt normalized to the expression level of control (A lower graph).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4373339&req=5

fig02: Rapid inhibition of Akt-mTOR pathway after PDT. All controls represent incubation with hypericin without irradiation. (A) Analysis of the Akt-mTOR pathway activation-status in MEF as a function of time after PDT. Left: representative Western blot analysis indicating phosphorylated and total levels of Akt, p70S6Kinase and S6 ribosomal protein and LC3 conversion after PDT (n= 3). Right: Densitometric quantification of the corresponding Western blot analysis on the left. (B) Left: Representative Western blot analysis of the activation status of S6 ribosomal protein in HeLa Neo or HeLa GPx4 cells following PDT (n= 3). Right: Densitometric quantification of the corresponding Western blot analysis on the left. Arbitrary densitometric units represent phosphorylation relative to the expression level and normalized to the control condition (A upper graph and B) or expression level of Akt normalized to the expression level of control (A lower graph).
Mentions: Because induction of MA is negatively regulated by the Akt-mTOR (mammalian target of rapamycin) pathway [5], we investigated the impact of our ROS-based therapy on this core signalling pathway. Activation of p70S6K by phosphorylation on the mTOR-specific serine residue Thr389 and subsequent phosphorylation/activation of the p70S6K substrate S6 provides a valuable measure of mTOR activity [32]. As soon as 1 hr after PDT, p70S6K became progressively dephosphorylated on Thr389 and phospho-S6 levels declined subsequently (Fig. 2A). Intriguingly, S6 phosphorylation level nearly recovered to control levels 16 hrs after irradiation, suggesting the modulation of S6 phosphorylation by additional p70S6K-independent mechanisms.

Bottom Line: Paradoxically, genetic loss of MA improved clearance of oxidized proteins and reduced photokilling.We found that up-regulation of chaperone-mediated autophagy (CMA) in unstressed Atg(-/-) cells compensated for MA loss and increased cellular resistance to PDT.These results disclose a stress-specific recruitment of autophagy pathways with cytoprotective function and unravel CMA as the dominant defence mechanism against PDT.

View Article: PubMed Central - PubMed

Affiliation: Cell Death Research and Therapy Laboratory, Department of Molecular Cell Biology, Faculty of Medicine, Katholieke Universiteit Leuven, Leuven, Belgium.

Show MeSH
Related in: MedlinePlus