Abstract
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, β-actin, tubulin-α-1-chain and enolase-α. Interestingly, all carbonylated proteins except calreticulin and enolase-α 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.
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Abbreviations
- anti-DNP:
-
anti-2,4dinitrophenol antibody
- CHAPS:
-
3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane-sulphonate
- 2-D:
-
two-dimensional gel electrophoresis
- DCFH-DA:
-
2′,7′-dichlorofluorescein diacetate
- DMSO:
-
dimethylsulphoxide
- DNPH:
-
2,4-dinitrophenylhydrazine
- Δψm:
-
Mitochondrial Transmembrane Potential
- DTE:
-
dithioerythritol
- DTT:
-
dithiothreitol
- FACS:
-
fluorescence-activated cell sorter
- FCS:
-
foetal calf serum
- GSH:
-
reduced glutathione
- HEPES buffer:
-
N-[2-hydroxyethyl]piperazine-N’-[2-ethanesulphonic acid]
- OPT:
-
o-phthaldialdehyde
- PBS:
-
phosphate-buffered saline
- PDT:
-
photodynamic therapy
- PI:
-
propidium iodide
- Pu-18:
-
Purpurin-18
- Rhod123:
-
Rhodamine123
- ROS:
-
reactive oxygen species
- SDS:
-
sodium dodecyl sulphate
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Acknowledgements
We thank Mr Leonardo Taurisano for excellent technical assistance, Dr Simona Tavarini and Dr Sandra Nuti for helpful discussion with regard to the cytofluorimetric analysis and Dr Peter Christie for the careful English revision. This research was supported in part by grants from the European Commission (QLK3-CT-2001-01495) and the Piano di Ateneo per la Ricerca of University of Siena.
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Magi, B., Ettorre, A., Liberatori, S. et al. Selectivity of protein carbonylation in the apoptotic response to oxidative stress associated with photodynamic therapy: a cell biochemical and proteomic investigation. Cell Death Differ 11, 842–852 (2004). https://doi.org/10.1038/sj.cdd.4401427
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DOI: https://doi.org/10.1038/sj.cdd.4401427
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