?-Phenylethyl isothiocyanate mediated apoptosis: A proteomic investigation of early apoptotic protein changes

Autor: Choon Nam Ong, Peter Rose, Maxey C. M. Chung, Jason C. H. Neo
Rok vydání: 2005
Předmět:
Proteomics
Time Factors
Phenethyl isothiocyanate
Difference gel electrophoresis
Blotting
Western
HSP27 Heat-Shock Proteins
Apoptosis
Biochemistry
Mass Spectrometry
Cell Line
Heterogeneous-Nuclear Ribonucleoprotein K
Proto-Oncogene Proteins c-myc
Inhibitory Concentration 50
chemistry.chemical_compound
Hsp27
Isothiocyanates
Cell Line
Tumor
Heat shock protein
Image Processing
Computer-Assisted
Humans
Electrophoresis
Gel
Two-Dimensional
Trypsin
Protein phosphorylation
Databases
Protein
Phosphotyrosine
Macrophage Migration-Inhibitory Factors
Molecular Biology
Heat-Shock Proteins
Plant Proteins
biology
Chemistry
Molecular biology
Neoplasm Proteins
Up-Regulation
Gene Expression Regulation
Caspases
Spectrometry
Mass
Matrix-Assisted Laser Desorption-Ionization
Isothiocyanate
biology.protein
Tyrosine
Phosphorylation
Isoelectric Focusing
Molecular Chaperones
Signal Transduction
Zdroj: PROTEOMICS. 5:1075-1082
ISSN: 1615-9861
1615-9853
DOI: 10.1002/pmic.200401070
Popis: beta-Phenylethyl isothiocyanate (PEITC) is a promising chemopreventative agent found in abundance in watercress (Rorripa nasturtium aquaticum) as its glucosinolate precursor. In the present investigation, we sought to determine the early changes in protein expression that contribute to the mechanism(s) of PEITC-mediated apoptosis in the human hepatoma HepG2 cell line. Such data may invariably identify new molecular targets of PEITC, contributing to a greater understanding of the mechanism(s) by which isothiocyanates mediate apoptotic cascades. Using two-dimensional difference gel electrophoresis we determined the changes in global protein expression between control (0.01% dimethyl sulfoxide) and PEITC (IC50 approximately 20 microM) treated cells after 3 and 6 h, such time points being used to circumvent the effects of caspase mediate proteolysis. Comparison between PEITC treated cells with their respective controls showed that 17 protein spots were differentially expressed. Fourteen of these spots, representing 9 unique proteins, were successfully identified using matrix-assisted laser desorption / ionization-time of flight (MALDI-TOF) and MALDI tandem time of flight (TOF/TOF) mass spectrometry. We observed significant shifts in isoelectric points on two-dimensional electrophoresis gels in heat shock 27 kDa protein (HSP27), macrophage migration inhibition factor and heterogeneous nuclear ribonucleoprotein K (hnRNP K) indicating that these proteins are probably involved in protein phosphorylation. Indeed, hnRNP K was determined to be phosphorylated on key tyrosine residues as assessed by using antiphosphotyrosine antibodies. In separate experiments we also showed that c-myc is up-regulated in PEITC treated cells, and since hnRNP K is reported to induce overexpression of c-myc, we proposed that PEITC-induced apoptosis may involve a c-myc dependent apoptotic pathway in HepG2 cells.
Databáze: OpenAIRE
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