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.