23 Oxidized proteins can be detected by Western blotting because the protein-bound carbonyl groups react with 2,4-dinitrophenylhydrazine (DNPH) and can be recognized by anti-2,4-dinitrophenol antibodies (anti-DNP). The most widely studied oxidative stress-induced modification of proteins is the formation of carbonyl groups in some amino-acid side chains. 18, 19, 20, 21, 22 Therefore, we hypothesized that oxidative stress associated with PDT induces selective oxidation of specific target proteins, which could play a key role in the induction of apoptosis. Recent studies support the concept that oxidative damage to proteins during oxidative stress and ageing is selective, and that proteins with specific sensitivity to oxidation can regulate cellular signalling events, including apoptosis. 12 However, the critical events and specific targets triggering apoptosis after PDT remain to be investigated. 17 It has been reported that the apoptotic response depends on the nature of the photosensitizer, the subcellular localization of the sensitizer, the cell types used and the treatment conditions.
QUANTITATION OF PROTEIN CARBONYLATION BY DOT BLOT SERIES
10, 11, 12 In cell culture, the oxidative stress induced by PDT triggers a series of events, including activation of phospholipase A2 and C, 13 release of nitric oxide, 14 activation of stress kinases, 12 nuclear factor kB, 15 and increased expression of early response genes 16 and heat-shock proteins (HSP's). 8, 9 Apoptotic and necrotic pathways are both involved in cell death after PDT. The interaction of light with photosensitizer molecules in the presence of molecular oxygen catalyses the formation of reactive oxygen species (ROS) within cells to produce the cytotoxic effect. 7 It utilizes a bimodal protocol including a combination of photosensitizer and visible light. 6 One of them is photodynamic therapy (PDT), an approved treatment for several types of cancer as well as for age-related macular degeneration. 5 Many of the chemical and physical treatments capable of inducing apoptosis also evoke oxidative stress. Recent evidence suggests that oxidative stress is a potential common mediator of apoptosis. 1, 2 Apoptosis is triggered by precise signals that induce crucial biochemical changes in target cells, including activation of caspases, mitochondrial depolarization, nucleosomal DNA fragmentation and alterations in the pattern of protein expression and stability. Inappropriate apoptosis is associated with many diseases, including cancer. Apoptotic mechanisms participate in the removal of toxicologically or genetically damaged cells. 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.Īpoptosis is an active highly conserved process that plays an important role in the development, homeostasis and maintenance of multicellular organisms. 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. Interestingly, all carbonylated proteins except calreticulin and enolase- α showed a pI shift in the proteome maps. 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. We previously reported that photodynamic therapy (PDT) using Purpurin-18 (Pu-18) induces apoptosis in HL60 cells.
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