Proteomics Study Of MAPK Signaling Pathways Blocked By Drug In Colorectal Cancer Cells

The abnormal activation of mitogen-activated protein kinase (MAPK) signaling pathways is a major cause of uncontrollable proliferation of colorectal cancer. But until now little is known about the targets as a whole in regulating cell proliferation, and the mechanisms is still unclear. In the present study, we examined the change of protein pattern profiling of colon cancer cells with MAPK signaling pathways blocked by drug, and then analyzed the targets of MAPK signaling pathways and revealed the regulation mechanisms for colorectal cancer cells proliferation.MAPK signaling pathways were blocked by PD98059, a specific inhibitor of ERK pathway, and SB203580, a specific inhibitor of p38 pathway. The effects of PD98059 and SB203580 on cell proliferation were assessed by MTT assay. Results showed that PD98059 and SB203580 can inhibit cell proliferation. And the inhibition effects on cell proliferation were in a dose- and time-dependent manner within a certain range of concentration. Results also showed that PD98059 cooperated with SB203580 in inhibiting HT29 cells proliferation.Orthogonal design was used to optimize the treatment condition of PD98059 combined with SB203580. And a treatment for 48h with 25μmol/L PD98059 combined with 20μmol/L SB203580 were decided as the optimization condition for inhibiting HT29 cells proliferation, with a proliferation inhibition rate of (55.96±3.34) %, which was to be undertook in the proteomic analysis of signaling pathway blocked by drug.Flow cytometry analysis is performed to determine the effects of PD98059 and SB203580 on HT29 cells by propidium iodide (PI) staining. The percentage of S phase was decreased and G₀/G₁ phase was increased in HT29 cells treated alone or combinedly by PD98059 and SB203580 for 48h.The two-dimensional electrophoresis (2-DE) maps of HT29 cells before and after MAPK signaling pathways blocked were obtained using 2-DE. And then the protein spots on the maps were matched and analyzed by professional image analyzing software PDQuest. Compared to the control, 11 differentially expressed protein spots were found with PD98059 treatment, and 14 differentially expressed protein spots were found with SB203580 treatment, while 32 protein spots were found to be differentially expressed in PD98059 combined with SB203580 treatment. Results also showed that 3 of these proteins were targets specially regulated by ERK cascade, and 7 protein spots were targets specially regulated by p38 cascade. ERK pathway cooperated with p38 pathways in changing expression pattern of another 10 protein spots.The total 34 differentially expression proteins were indentified through matrix assisted laser desorption/ionization (MALDI)-TOF-TOF tandem mass Spectrometry and bioinformatics. The analysis of the proteins revealed that the differentially expressed proteins include proteasome subunits, translation factors, transcription activation factors, cytoskeletal regulators, molecular chaperones and enzymes involved in cellular synthesis and metabolism.Thus it can be concluded that the HT29 cells proliferation was markedly inhibited when the MAPK signaling pathways were blocked. And the MAPK signaling pathways regulate the proliferation of colorectal cancer cell line HT29 through the controlling cellular processes including proteolysis, translation, transcription and mRNA splicing, dynamic equilibrium of cytoskeleton, protein folding and cellular metabolism.