Selectivity of protein carbonylation in the apoptotic response to oxidative stress associated with photodynamic therapy: a cell biochemical and proteomic investigation.
We previously reported that photodynamic therapy (PDT) using Purpurin-18 (Pu-18) induces apoptosis in HL60 cells. Using flow cytometry, two-dimensional electrophoresis coupled with immunodetection of carbonylated proteins and mass spectrometry, we now show that PDT-induced apoptosis is associated with increased reactive oxygen species generation, glutathione depletion, changes in mitochondrial transmembrane potential, simultaneous downregulation of mitofilin and carbonylation of specific proteins: glucose-regulated protein-78, heat-shock protein 60, heat-shock protein cognate 71, phosphate disulphide isomerase, calreticulin, beta-actin, tubulin-alpha-1-chain and enolase-alpha. Interestingly, all carbonylated proteins except calreticulin and enolase-alpha showed a pI shift in the proteome maps. Our results suggest that PDT with Pu-18 perturbs the normal redox balance and shifts HL60 cells into a state of oxidative stress, which systematically induces the carbonylation of specific chaperones. As these proteins normally produce a prosurvival signal during oxidative stress, we hypothesize that their carbonylation represents a signalling mechanism for apoptosis induced by PDT.
Magi
B
B
Department of Molecular Biology, University of Siena, via Fiorentina 1, Siena 53100, Italy.
Ettorre
A
A
Liberatori
S
S
Bini
L
L
Andreassi
M
M
Frosali
S
S
Neri
P
P
Pallini
V
V
Di Stefano
A
A
D000199:Actins / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D014408:Biomarkers, Tumor; D037282:Calreticulin / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005434:Flow Cytometry; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D010778:Photochemotherapy; D011166:Porphyrins / Q000494:pharmacology*; D040901:Proteomics; D017382:Reactive Oxygen Species / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism
D000199:Actins; D014408:Biomarkers, Tumor; D037282:Calreticulin; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D011166:Porphyrins; D017382:Reactive Oxygen Species; D025521:Tumor Suppressor Proteins; C059051:purpurin 18; D020169:Caspases; C095157:ENO1 protein, human; D010751:Phosphopyruvate Hydratase