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The Effects and the Mechanism of a bFGF Binding Peptide on bFGF-stimulated K562 Cells

Author: JiaXingLong
Tutor: WuXiaoPing
School: Jinan University
Course: Immunology
Keywords: bFGF binding peptide cell proliferation cell signal pathway proteomics
CLC: R329
Type: Master's thesis
Year: 2011
Downloads: 39
Quote: 0
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Abstract


Aim:To explore the effect of a novel bFGF-binding peptide previously obtained by screening from the phage display random heptapeptide library on the proliferation of human chronic myeloid leukemia cells (K652) induced by basic fibroblast growth factor.Methods:An inverted microscope was applied to observe the cytotoxic effect of P7 on K652 cells. The influences of P7 at different concentrations on the proliferation of K562 cells stimulated with or without bFGF were detected by MTT assay, and the effect of P7 on cell cycle distribution was analyzed by Flow cytometry. Western blot was used to check the effect of P7 on bFGF-induced activation of ERK1/2 and MEK in K562 cells. Atomic force microscope was used to check the change of the surface ultrastructure of bFGF-stimulated K562 cells caused by P7. Two-dimensional electrophoresis and mass spectrometry technology were applied to analyze P7 specific differentially expressed proteins, and the result of the proteomics was verified by western blot.Results:No significant cell morphology change was observed in K562 cells treated with P7. MTT assay showed that P7 could inhibit cell proliferation induced by bFGF in a dose-dependent manner while P7 alone had no significant influence on the growth of K562 cells. Flow cytometry analysis indicated that P7 decreased S-phase cell ratios and caused G0/G1 phage arrest in bFGF-stimulated K562 cells. Western blot analysis showed that P7 could reduce bFGF-induced ERK1/2 and MEK phosphorylation in K562 cells. Atomic force microscopy analysis showed that bFGF played a proliferation effect on K562 cells by increasing the roughness of cell surface, and P7 inhibited cell proliferation induced by bFGF via decreasing the roughness of cell surface. High resolution and good reproducibility protein profiles were obtained, and fourteen of P7 specific differentially expressed proteins were identified via two-dimensional electrophoresis and mass spectrometry technology, such as PA2G4, EIF3I, PSME2, et al. The expression levels of nine proteins were decreased by bFGF stimulation alone and increased by addition of P7, whereas the other five were upregulated by bFGF treatment alone and downregulated by addition of P7. The protein levels of PA2G4, one of the differentially expressed proteins, was further confirmed by western blot. Conclusion:The novel bFGF-binding peptide P7 previously obtained by screening had inhibitory effect on the proliferation of K562 cells induced by bFGF possibly via cell cycle arrest at the G0/G1 phase, down-regulation of ERK1/2 and MEK activation in MAPK pathway, decrease of cell surface ultrastructure, and influence on the expression of some proteins related to the regulation of cell proliferation.

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