A comparative study on the three quantitative methods frequently used in proteomics, 2D DIGE (difference gel electrophoresis), cICAT (cleavable isotope-coded affinity tags) and iTRAQ (isobaric tags for relative and absolute quantification), was carried out. DIGE and cICAT are familiar techniques used in gel- and LC-based quantitative proteomics, respectively. iTRAQ is a new LC-based technique which is gradually gaining in popularity. A systematic comparison among these quantitative methods has not been reported. In this study, we conducted well-designed comparisons using a six-protein mixture, a reconstituted protein mixture (BSA spiked into human plasma devoid of six abundant proteins), and complex HCT-116 cell lysates as the samples. All three techniques yielded quantitative results with reasonable accuracy when the six-protein or the reconstituted protein mixture was used. In DIGE, accurate quantification was sometimes compromised due to comigration or partial comigration of proteins. The iTRAQ method is more susceptible to errors in precursor ion isolation, which could be manifested with increasing sample complexity. The quantification sensitivity of each method was estimated by the number of peptides detected for each protein. In this regard, the global-tagging iTRAQ technique was more sensitive than the cysteine-specific cICAT method, which in turn was as sensitive as, if not more sensitive than, the DIGE technique. Protein profiling on HCT-116 and HCT-116 p53 -/- cell lysates displayed limited overlapping among proteins identified by the three methods, suggesting the complementary nature of these methods.

Abstract6-Gingerol, a natural product of ginger, has been known to possess anti-tumorigenic and proapoptotic activities. However, the mechanisms by which it prevents cancer are not well understood in human colorectal cancer. Cyclin D1 is a proto-oncogene that is overexpressed in many cancers and plays a role in cell proliferation through activation by β-catenin signaling. Nonsteroidal antiinflammatory drug (NSAID)-activated gene-1 (NAG-1) is a cytokine associated with pro-apoptotic and anti-tumorigenic properties. In the present study, we examined whether 6-gingerol influences cyclin D1 and NAG-1 expression and determined the mechanisms by which 6-gingerol affects the growth of human colorectal cancer cells in vitro. 6-Gingerol treatment suppressed cell proliferation and induced apoptosis and G 1 cell cycle arrest. Subsequently, 6-gingerol suppressed cyclin D1 expression and induced NAG-1 expression. Cyclin D1 suppression was related to inhibition of β-catenin translocation and cyclin D1 proteolysis. Furthermore, experiments using inhibitors and siRNA transfection confirm the involvement of the PKCε and glycogen synthase kinase (GSK)-3β pathways in 6-gingerol-induced NAG-1 expression. The results suggest that 6-gingerol stimulates apoptosis through upregulation of NAG-1 and G 1 cell cycle arrest through downregulation of cyclin D1. Multiple mechanisms appear to be involved in 6-gingerol action, including protein degradation as well as β-catenin, PKCε, and GSK-3β pathways.

Troglitazone (TGZ) is a peroxisome proliferator-activated receptor ␥ (PPAR␥) ligand that has pro-apoptotic activity in human colon cancer. Although TGZ binds to PPAR␥ transcription factors as an agonist, emerging evidence suggests that TGZ acts independently of PPAR␥ in many functions, including apoptosis. Early growth response-1 (Egr-1) transcription factor has been linked to apoptosis and shown to be activated by extracellular signal-regulated kinase (ERK). We investigated whether TGZ-induced apoptosis may be related to Egr-1 induction, because TGZ has been known to induce ERK activity. Our results show that Egr-1 is induced dramatically by TGZ but not by other PPAR␥ ligands. TGZ affects Egr-1 induction at least by two mechanisms; TGZ increases Egr-1 promoter activity by 2-fold and prolongs Egr-1 mRNA stability by 3-fold. Inhibition of ERK phosphorylation in HCT-116 cells abolishes the Egr-1 induction by TGZ, suggesting its ERK-dependent manner. Further, the TGZ-induced Egr-1 expression results in increased promoter activity using a reporter system containing four copies of Egr-1 binding sites, and TGZ induces Egr-1 binding activity to Egr-1 consensus sites as assessed by gel shift assay. In addition, TGZ induces ERK-dependent phosphorylation of PPAR␥, resulting in the down-regulation of PPAR␥ activity. The fact that TGZ-induced apoptosis is accompanied by the biosynthesis of Egr-1 suggests that Egr-1 plays a pivotal role in TGZ-induced apoptosis in HCT-116 cells. Our results suggest that Egr-1 induction is a unique property of TGZ compared with other PPAR␥ ligands and is independent of PPAR␥ activation. Thus, the up-regulation of Egr-1 may provide an explanation for the anti-tumorigenic properties of TGZ.