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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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Attenuation of MA enhances PDT-induced cytotoxicity. All controls represent incubation with hypericin without irradiation unless otherwise stated. (A) Quantification of GFP-puncta after PDT-treatment in HeLa cells transiently transfected with GFP-LC3. A subset of representative cells was analysed for their punctuated pattern in control cells and 6 hrs after treatment in the absence or presence of 10 mM 3MA. Cells were digitally processed with the ImageJ software and manually supervised. The bar values are indicated in the graph.*P < 0.05 under the Student’s t-test, ns: not significant. (B) HeLa cells were PDT-treated in the absence or presence of 10 mM 3MA and Western blot analysis of caspase 3 activation and PARP cleavage was carried out at the indicated time-points after irradiation. Representative Western blot is shown (n= 3). Actin was used to monitor equal loading. (C) Flow cytometric cell cycle analysis determining the apoptotic SubG1 fraction in PDT-treated HeLa cells in the presence or absence of 10 mM 3MA. The graph represents the mean ± S.D. of three independent experiments carried out in duplicate. *P < 0.05 under the Student’s t-test. 3MA was added to the cells immediately after irradiation to avoid toxicity caused by the light exposure of the inhibitor and remained in the culture medium throughout the incubation time. Controls with or without 3MA were harvested at the last evaluated time-point (A–C). (D) HeLa cells were transfected with Atg5 siRNA or scrambled (non-targeting) siRNA (Scr siRNA) before PDT-treatment. At the indicated time-points after PDT cell lysates were subjected to Western blot analysis with the indicated antibodies. Representative Western blot is shown (n= 3). Actin was used to monitor equal loading. (E) Apoptotic SubG1 fraction in PDT-treated HeLa cells transfected with control or Atg5 siRNA. The graph represents the mean ± S.D. of two independent experiments carried out in duplicate. *P < 0.05 under the Student’s t-test. (F) Left: Total cell lysates were analysed for the presence of carbonylated protein side chains with OxyBlot Protein Oxidation Detection Kit after PDT in scrambled-siRNA or Atg5-siRNA transfected HeLa cells. Actin was used as a loading control. Right: The graph represents the densitometric values of the OxyBlot normalized to actin and expressed as fold increase to controls. During OxyBlot procedure, hypericin was omitted from the controls to minimize effects of background irradiation during manipulation of the samples.
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fig03: Attenuation of MA enhances PDT-induced cytotoxicity. All controls represent incubation with hypericin without irradiation unless otherwise stated. (A) Quantification of GFP-puncta after PDT-treatment in HeLa cells transiently transfected with GFP-LC3. A subset of representative cells was analysed for their punctuated pattern in control cells and 6 hrs after treatment in the absence or presence of 10 mM 3MA. Cells were digitally processed with the ImageJ software and manually supervised. The bar values are indicated in the graph.*P < 0.05 under the Student’s t-test, ns: not significant. (B) HeLa cells were PDT-treated in the absence or presence of 10 mM 3MA and Western blot analysis of caspase 3 activation and PARP cleavage was carried out at the indicated time-points after irradiation. Representative Western blot is shown (n= 3). Actin was used to monitor equal loading. (C) Flow cytometric cell cycle analysis determining the apoptotic SubG1 fraction in PDT-treated HeLa cells in the presence or absence of 10 mM 3MA. The graph represents the mean ± S.D. of three independent experiments carried out in duplicate. *P < 0.05 under the Student’s t-test. 3MA was added to the cells immediately after irradiation to avoid toxicity caused by the light exposure of the inhibitor and remained in the culture medium throughout the incubation time. Controls with or without 3MA were harvested at the last evaluated time-point (A–C). (D) HeLa cells were transfected with Atg5 siRNA or scrambled (non-targeting) siRNA (Scr siRNA) before PDT-treatment. At the indicated time-points after PDT cell lysates were subjected to Western blot analysis with the indicated antibodies. Representative Western blot is shown (n= 3). Actin was used to monitor equal loading. (E) Apoptotic SubG1 fraction in PDT-treated HeLa cells transfected with control or Atg5 siRNA. The graph represents the mean ± S.D. of two independent experiments carried out in duplicate. *P < 0.05 under the Student’s t-test. (F) Left: Total cell lysates were analysed for the presence of carbonylated protein side chains with OxyBlot Protein Oxidation Detection Kit after PDT in scrambled-siRNA or Atg5-siRNA transfected HeLa cells. Actin was used as a loading control. Right: The graph represents the densitometric values of the OxyBlot normalized to actin and expressed as fold increase to controls. During OxyBlot procedure, hypericin was omitted from the controls to minimize effects of background irradiation during manipulation of the samples.

Mentions: Having shown that MA is stimulated following PDT in different cells, we decided to investigate the functional role of MA on PDT-mediated cell death in apoptosis-competent cells. Although the inhibitor of autophagosome formation 3MA [32] alone was not cytotoxic, its addition in cells exposed to PDT inhibited GFP-LC3 puncta formation (Fig. 3A), enhanced caspase 3 activation and PARP processing (Fig. 3B) and significantly increased apoptosis (Fig. 3C). An siRNA-mediated knockdown of Atg5, resulted in the reduction of the Atg5-Atg12 complex and the attenuation of MA stimulation after PDT as demonstrated by reduced LC3-conversion (Fig. 3D). Consistent with the results obtained with the inhibitor, Atg5 knockdown boosted caspase 3 cleavage and PARP processing, increased apoptotic SubG1 fraction (Fig. 3D and E) and enhanced overall photokilling (data not shown). Note that as compared to our previous studies in HeLa cells [35], the light dose used in this study was purposely milder (i.e. 2.7 J/cm2) in order to measure possible pro-death inducing effects of MA inhibition.


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)

Attenuation of MA enhances PDT-induced cytotoxicity. All controls represent incubation with hypericin without irradiation unless otherwise stated. (A) Quantification of GFP-puncta after PDT-treatment in HeLa cells transiently transfected with GFP-LC3. A subset of representative cells was analysed for their punctuated pattern in control cells and 6 hrs after treatment in the absence or presence of 10 mM 3MA. Cells were digitally processed with the ImageJ software and manually supervised. The bar values are indicated in the graph.*P < 0.05 under the Student’s t-test, ns: not significant. (B) HeLa cells were PDT-treated in the absence or presence of 10 mM 3MA and Western blot analysis of caspase 3 activation and PARP cleavage was carried out at the indicated time-points after irradiation. Representative Western blot is shown (n= 3). Actin was used to monitor equal loading. (C) Flow cytometric cell cycle analysis determining the apoptotic SubG1 fraction in PDT-treated HeLa cells in the presence or absence of 10 mM 3MA. The graph represents the mean ± S.D. of three independent experiments carried out in duplicate. *P < 0.05 under the Student’s t-test. 3MA was added to the cells immediately after irradiation to avoid toxicity caused by the light exposure of the inhibitor and remained in the culture medium throughout the incubation time. Controls with or without 3MA were harvested at the last evaluated time-point (A–C). (D) HeLa cells were transfected with Atg5 siRNA or scrambled (non-targeting) siRNA (Scr siRNA) before PDT-treatment. At the indicated time-points after PDT cell lysates were subjected to Western blot analysis with the indicated antibodies. Representative Western blot is shown (n= 3). Actin was used to monitor equal loading. (E) Apoptotic SubG1 fraction in PDT-treated HeLa cells transfected with control or Atg5 siRNA. The graph represents the mean ± S.D. of two independent experiments carried out in duplicate. *P < 0.05 under the Student’s t-test. (F) Left: Total cell lysates were analysed for the presence of carbonylated protein side chains with OxyBlot Protein Oxidation Detection Kit after PDT in scrambled-siRNA or Atg5-siRNA transfected HeLa cells. Actin was used as a loading control. Right: The graph represents the densitometric values of the OxyBlot normalized to actin and expressed as fold increase to controls. During OxyBlot procedure, hypericin was omitted from the controls to minimize effects of background irradiation during manipulation of the samples.
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fig03: Attenuation of MA enhances PDT-induced cytotoxicity. All controls represent incubation with hypericin without irradiation unless otherwise stated. (A) Quantification of GFP-puncta after PDT-treatment in HeLa cells transiently transfected with GFP-LC3. A subset of representative cells was analysed for their punctuated pattern in control cells and 6 hrs after treatment in the absence or presence of 10 mM 3MA. Cells were digitally processed with the ImageJ software and manually supervised. The bar values are indicated in the graph.*P < 0.05 under the Student’s t-test, ns: not significant. (B) HeLa cells were PDT-treated in the absence or presence of 10 mM 3MA and Western blot analysis of caspase 3 activation and PARP cleavage was carried out at the indicated time-points after irradiation. Representative Western blot is shown (n= 3). Actin was used to monitor equal loading. (C) Flow cytometric cell cycle analysis determining the apoptotic SubG1 fraction in PDT-treated HeLa cells in the presence or absence of 10 mM 3MA. The graph represents the mean ± S.D. of three independent experiments carried out in duplicate. *P < 0.05 under the Student’s t-test. 3MA was added to the cells immediately after irradiation to avoid toxicity caused by the light exposure of the inhibitor and remained in the culture medium throughout the incubation time. Controls with or without 3MA were harvested at the last evaluated time-point (A–C). (D) HeLa cells were transfected with Atg5 siRNA or scrambled (non-targeting) siRNA (Scr siRNA) before PDT-treatment. At the indicated time-points after PDT cell lysates were subjected to Western blot analysis with the indicated antibodies. Representative Western blot is shown (n= 3). Actin was used to monitor equal loading. (E) Apoptotic SubG1 fraction in PDT-treated HeLa cells transfected with control or Atg5 siRNA. The graph represents the mean ± S.D. of two independent experiments carried out in duplicate. *P < 0.05 under the Student’s t-test. (F) Left: Total cell lysates were analysed for the presence of carbonylated protein side chains with OxyBlot Protein Oxidation Detection Kit after PDT in scrambled-siRNA or Atg5-siRNA transfected HeLa cells. Actin was used as a loading control. Right: The graph represents the densitometric values of the OxyBlot normalized to actin and expressed as fold increase to controls. During OxyBlot procedure, hypericin was omitted from the controls to minimize effects of background irradiation during manipulation of the samples.
Mentions: Having shown that MA is stimulated following PDT in different cells, we decided to investigate the functional role of MA on PDT-mediated cell death in apoptosis-competent cells. Although the inhibitor of autophagosome formation 3MA [32] alone was not cytotoxic, its addition in cells exposed to PDT inhibited GFP-LC3 puncta formation (Fig. 3A), enhanced caspase 3 activation and PARP processing (Fig. 3B) and significantly increased apoptosis (Fig. 3C). An siRNA-mediated knockdown of Atg5, resulted in the reduction of the Atg5-Atg12 complex and the attenuation of MA stimulation after PDT as demonstrated by reduced LC3-conversion (Fig. 3D). Consistent with the results obtained with the inhibitor, Atg5 knockdown boosted caspase 3 cleavage and PARP processing, increased apoptotic SubG1 fraction (Fig. 3D and E) and enhanced overall photokilling (data not shown). Note that as compared to our previous studies in HeLa cells [35], the light dose used in this study was purposely milder (i.e. 2.7 J/cm2) in order to measure possible pro-death inducing effects of MA inhibition.

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