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Role of ER stress response in photodynamic therapy: ROS generated in different subcellular compartments trigger diverse cell death pathways.

Moserova I, Kralova J - PLoS ONE (2012)

Bottom Line: ER stress developed into unfolded protein response.Immediately after irradiation the PERK pathway was activated through phosphorylation of PERK, eIF2α and induction of transcription factors ATF4 and CHOP, which regulate stress response genes.PERK knockdown and PERK deficiency protected cells against mTPP(EG)4-mediated apoptosis, confirming the causative role of the PERK pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Virology, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic.

ABSTRACT
We have analyzed the molecular mechanisms of photoinduced cell death using porphyrins with similar structure differing only in the position of the ethylene glycol (EG) chain on the phenyl ring. Meta- and para-positioned EG chains targeted porphyrins to different subcellular compartments. After photoactivation, both types of derivatives induced death of tumor cells via reactive oxygen species (ROS). Para derivatives pTPP(EG)4 and pTPPF(EG)4 primarily accumulated in lysosomes activated the p38 MAP kinase cascade, which in turn induced the mitochondrial apoptotic pathway. In contrast, meta porphyrin derivative mTPP(EG)4 localized in the endoplasmic reticulum (ER) induced dramatic changes in Ca(2+) homeostasis manifested by Ca(2+) rise in the cytoplasm, activation of calpains and stress caspase-12 or caspase-4. ER stress developed into unfolded protein response. Immediately after irradiation the PERK pathway was activated through phosphorylation of PERK, eIF2α and induction of transcription factors ATF4 and CHOP, which regulate stress response genes. PERK knockdown and PERK deficiency protected cells against mTPP(EG)4-mediated apoptosis, confirming the causative role of the PERK pathway.

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Effect of Capn4 siRNA on the demise of PDT-treated cells.(A) Western blot analysis of capn4/capns1 and fodrin in 4T1 cells analyzed on the 3rd day after Capn4 siRNA transfection. Transfected cells were subjected to EG-porphyrin-mediated PDT, harvested and resolved by Western blot analysis. Equal protein loading is demonstrated by actin reprobing. (B) Viability of cells transfected with Capn4 siRNA. Transfected and control cells were subjected to EG-porphyrin-mediated PDT and cell viability was monitored 24 h later. The percentage of dead cells was expressed as the mean ± SD (n = 3). *P<0.05 represents the statistical difference between transfected PDT-treated cells vs. non-transfected or control siRNA-transfected PDT-treated cells.
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pone-0032972-g007: Effect of Capn4 siRNA on the demise of PDT-treated cells.(A) Western blot analysis of capn4/capns1 and fodrin in 4T1 cells analyzed on the 3rd day after Capn4 siRNA transfection. Transfected cells were subjected to EG-porphyrin-mediated PDT, harvested and resolved by Western blot analysis. Equal protein loading is demonstrated by actin reprobing. (B) Viability of cells transfected with Capn4 siRNA. Transfected and control cells were subjected to EG-porphyrin-mediated PDT and cell viability was monitored 24 h later. The percentage of dead cells was expressed as the mean ± SD (n = 3). *P<0.05 represents the statistical difference between transfected PDT-treated cells vs. non-transfected or control siRNA-transfected PDT-treated cells.

Mentions: To confirm the importance of the calpain activation in EG porphyrin-mediated phototoxicity, we knocked down the expression of Capn4 using specific siRNA. Transfection of Capn4 siRNA, but not control siRNA (NC), decreased the level of endogenous Capn4/Capns1 protein and reduced fodrin cleavage (Fig. 7A). Moreover, Capn4 siRNA significantly reduced the amount of dead cells in mTPP(EG)4-treated cells (Fig. 7B). Despite that knockdown of Capn4 decreased fodrin cleavage also in para derivatives, no effect on cell viability was observed (Fig. 7B). In summary, all these experiments confirm an important role of calpain activation in mTPP(EG)4 but not pTPP(EG)4 and pTPPF(EG)4-induced cell death.


Role of ER stress response in photodynamic therapy: ROS generated in different subcellular compartments trigger diverse cell death pathways.

Moserova I, Kralova J - PLoS ONE (2012)

Effect of Capn4 siRNA on the demise of PDT-treated cells.(A) Western blot analysis of capn4/capns1 and fodrin in 4T1 cells analyzed on the 3rd day after Capn4 siRNA transfection. Transfected cells were subjected to EG-porphyrin-mediated PDT, harvested and resolved by Western blot analysis. Equal protein loading is demonstrated by actin reprobing. (B) Viability of cells transfected with Capn4 siRNA. Transfected and control cells were subjected to EG-porphyrin-mediated PDT and cell viability was monitored 24 h later. The percentage of dead cells was expressed as the mean ± SD (n = 3). *P<0.05 represents the statistical difference between transfected PDT-treated cells vs. non-transfected or control siRNA-transfected PDT-treated cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3293927&req=5

pone-0032972-g007: Effect of Capn4 siRNA on the demise of PDT-treated cells.(A) Western blot analysis of capn4/capns1 and fodrin in 4T1 cells analyzed on the 3rd day after Capn4 siRNA transfection. Transfected cells were subjected to EG-porphyrin-mediated PDT, harvested and resolved by Western blot analysis. Equal protein loading is demonstrated by actin reprobing. (B) Viability of cells transfected with Capn4 siRNA. Transfected and control cells were subjected to EG-porphyrin-mediated PDT and cell viability was monitored 24 h later. The percentage of dead cells was expressed as the mean ± SD (n = 3). *P<0.05 represents the statistical difference between transfected PDT-treated cells vs. non-transfected or control siRNA-transfected PDT-treated cells.
Mentions: To confirm the importance of the calpain activation in EG porphyrin-mediated phototoxicity, we knocked down the expression of Capn4 using specific siRNA. Transfection of Capn4 siRNA, but not control siRNA (NC), decreased the level of endogenous Capn4/Capns1 protein and reduced fodrin cleavage (Fig. 7A). Moreover, Capn4 siRNA significantly reduced the amount of dead cells in mTPP(EG)4-treated cells (Fig. 7B). Despite that knockdown of Capn4 decreased fodrin cleavage also in para derivatives, no effect on cell viability was observed (Fig. 7B). In summary, all these experiments confirm an important role of calpain activation in mTPP(EG)4 but not pTPP(EG)4 and pTPPF(EG)4-induced cell death.

Bottom Line: ER stress developed into unfolded protein response.Immediately after irradiation the PERK pathway was activated through phosphorylation of PERK, eIF2α and induction of transcription factors ATF4 and CHOP, which regulate stress response genes.PERK knockdown and PERK deficiency protected cells against mTPP(EG)4-mediated apoptosis, confirming the causative role of the PERK pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Virology, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic.

ABSTRACT
We have analyzed the molecular mechanisms of photoinduced cell death using porphyrins with similar structure differing only in the position of the ethylene glycol (EG) chain on the phenyl ring. Meta- and para-positioned EG chains targeted porphyrins to different subcellular compartments. After photoactivation, both types of derivatives induced death of tumor cells via reactive oxygen species (ROS). Para derivatives pTPP(EG)4 and pTPPF(EG)4 primarily accumulated in lysosomes activated the p38 MAP kinase cascade, which in turn induced the mitochondrial apoptotic pathway. In contrast, meta porphyrin derivative mTPP(EG)4 localized in the endoplasmic reticulum (ER) induced dramatic changes in Ca(2+) homeostasis manifested by Ca(2+) rise in the cytoplasm, activation of calpains and stress caspase-12 or caspase-4. ER stress developed into unfolded protein response. Immediately after irradiation the PERK pathway was activated through phosphorylation of PERK, eIF2α and induction of transcription factors ATF4 and CHOP, which regulate stress response genes. PERK knockdown and PERK deficiency protected cells against mTPP(EG)4-mediated apoptosis, confirming the causative role of the PERK pathway.

Show MeSH
Related in: MedlinePlus