β‐Phenylethyl isothiocyanate mediated apoptosis: A proteomic investigation of early apoptotic protein changes

Abstract

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.