Immediate Protein Targets of Photodynamic Treatment in Carcinoma Cells

Department of Membrane Enzymology, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands.
Journal of Proteome Research (Impact Factor: 4.25). 09/2008; 7(9):3868-78. DOI: 10.1021/pr800189q
Source: PubMed


Oxidative stress induced in tumor cells undergoing photodynamic treatment (PDT) leads to extensive modification of many proteins in these cells. Protein oxidation mainly gives rise to formation of carbonyls and oxidized thiols. The immediate targets of PDT-induced protein oxidation in A431 tumor cells have been identified using a proteomic approach involving selective biotinylation, affinity purification and mass spectrometric identification of modified proteins. In all, 314 proteins were shown to undergo PDT-mediated oxidative modifications. While abundant structural proteins and chaperones represented a significant fraction of the carbonylated proteins, labeling of proteins containing oxidized thiols allowed identification of many proteins at low abundance and those involved in signaling and redox homeostasis. On the basis of the identification of these proteins, several likely mechanisms of PDT-induced triggering of apoptosis were put forward. This may not only lead to a further understanding of the complex network of cellular responses to oxidative stress, but it may also help in detailed targeting of photodynamic treatment applied to cancer.

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Available from: Maarten R Egmond, May 27, 2014
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    • "s upon oxidative stress . In a similar study , tumor cells were subject to photodynamic treatment ( PDT ) , which leads to oxidative modifications in cells . Samples were enriched by biotin – avidin affinity chromatography and separated and visualized by 2D - gel . MS identified 314 proteins , including carbonylation and reversible sulfenylation ( Tsaytler et al . , 2008 ) ."
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    ABSTRACT: Proteomics techniques are continuously being developed to further understanding of biology and disease. Many of the pathways that are relevant to disease mechanisms rely on the identification of post-translational modifications (PTMs) such as phosphorylation, acetylation, and glycosylation. Much attention has also been focused on oxidative PTMs which include protein carbonyls, protein nitration, and the incorporation of fatty acids and advanced glycation products to amino acid side chains, amongst others. The introduction of these PTMs in the cell can occur due to the attack of reactive oxygen and nitrogen species (ROS and RNS, respectively) on proteins. ROS and RNS can be present as a result of normal metabolic processes as well as external factors such as UV radiation, disease, and environmental toxins. The imbalance of ROS and RNS with antioxidant cellular defenses leads to a state of oxidative stress, which has been implicated in many diseases. Redox proteomics techniques have been used to characterize oxidative PTMs that result as a part of normal cell signaling processes as well as oxidative stress conditions. This review highlights many of the redox proteomics techniques which are currently available for several oxidative PTMs and brings to the reader's attention the application of redox proteomics for understanding disease pathogenesis in neurodegenerative disorders and others such as cancer, kidney, and heart diseases.
    Full-text · Article · Jul 2014 · Mass Spectrometry Reviews
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    • "For instance, carbonylated peptides can be captured by a solid-phase hydrazide reagent (Roe et al., 2007) or by immobilized oxalyldihydrazide on a microchip (Hollins, Soper, & Feng, 2012). In any case, the enriched proteins can be further digested and the peptides obtained identified by LC-MS/ MS (Soreghan et al., 2003; Grimsrud et al., 2007; Meany et al., 2007; Mirzaei & Regnier, 2007; Tsaytler et al., 2008). Using a biotin-hydrazide based approach, Soreghan and co-workers identified 100 carbonylated proteins, including low abundance receptors, in brain homogenates of mice of different ages (Soreghan et al., 2003). "
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    ABSTRACT: Detection and quantification of protein carbonyls present in biological samples has become a popular, albeit indirect, method to determine the existence of oxidative stress. Moreover, the rise of proteomics has allowed the identification of the specific proteins targeted by protein carbonylation. This review discusses these methodologies and proteomic strategies and then focuses on the relationship between protein carbonylation and aging and the parameters that may explain the increased sensitivity of certain proteins to protein carbonylation. © 2013 Wiley Periodicals, Inc. Mass Spec Rev.
    Full-text · Article · Jan 2014 · Mass Spectrometry Reviews
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    • "Antibody microarrays based on immunofluorescent labeling of the most important signaling proteins have recently been created [13] and used, for example, for cancer diagnosis [14] [15]. The proteomic approach has also been used in some studies of photodynamic cell injury [10] [11]. However, PDT doses used in these works were generally about LD50 or higher. "
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    ABSTRACT: Photodynamic therapy (PDT) that induces oxidative stress and cell death is used for tumor destruction in oncology. To characterize early molecular events in photosensitized glioblastoma cells, we studied expression of 224 proteins after sublethal PDT that doesn't kill but wounds cells. Cultured glioblastoma D54Mg cells were photosensitized with 5-aminolevulinic acid so that cell survival was 95-100%. At following 0.5-5.5h protein expression and phosphorylation was assayed using proteomic antibody microarrays. Within the first post-treatment hour we observed phosphorylation of protein kinase Raf, adhesion-related kinases FAK and Pyk2, and microtubule-associated protein tau. Protein kinase Cγ and microtubule-associated protein MAP-1B were overexpressed. Dystrophin, calponin, and vinculin, components of the actin cytoskeleton scaffold, microtubule-associated proteins MAP2 and CNP, cytokeratins 4 and 7 were down-regulated that indicated changes in adhesion and cell shape. Down-regulation of cyclins A, D1 and D3, c-Myc, checkpoint proteins chk1/2 and up-regulation of Smad4 could arrest the cell cycle. Overexpression of Bcl-xL and down-regulation of caspase 9 demonstrated anti-apoptotic response. At 2h post-treatment protein expression changed lesser but at 5.5h levels of PKCγ and β-synuclein and phosphorylation of Raf, FAK, Pyk2, and tau increased again. Sub-lethal PDT induces complex response of glioblastoma cells including changes in activity and expression of proteins involved in adhesion-mediated signaling, signal transduction, cytoskeleton remodeling, cell cycle regulation and anti-apoptotic processes. Multiple reactions of various cellular subsystems including adhesion, cytoskeleton, signal transduction, cell cycle, and apoptosis are integrated into the general cell response to a sublethal impact.
    Full-text · Article · Mar 2012 · Biochimica et Biophysica Acta
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