Gecko crude peptides inhibit migration and lymphangiogenesis by down regulating the expression of VEGF-C in human hepatocellular carcinoma cells and human lymphatic endothelial cells / 中国药理学与毒理学杂志

ABSTRACT

OBJECTIVE To explore the role of gecko crude peptides (GCPs) in the proliferation, apoptosis, migration and lymphangiogenesis of human hepatocellular carcinoma cells (HepG2) and human lymphaticendothelial cells (HLECs) in vitro. METHODS The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the anti- proliferative effect of GCPs and siRNA-VEGF-C on HepG2 cells, Hoechst 33258 staining and flow cytometry were performed to analyze cycle and apoptosis. The migration and invasion ability of cells were assayed by transwell chamber experiment and wound-healing assay. The protein and mRNA expressions of vascular endo?thelial growth factor-C (VEGF-C) and CXC chemokine receptor-4 (CXCR4) were detected by q-PCR, immunofluorescence, Western blot. The protein expressions of the extracellular signal regulated kinase (ERKI/2), c-Jun N-terminal kinase (JNK), p38-mitogen activated protein kinases (p38 MAPK), serine/threonine kinase (Akt) and phosphatidylinositol- 3- kinase (PI3K) were detected by western blot. The anti-lymphangiogenesis effect of GCPs on the HLECs was analyzed using an in vitro tube-formation assay. The protein and mRNA expressions of vascular endothelial growth factor receptor-3 (VEGFR-3) and stromal cell-derived factor-1 (SDF-1) were detected by q-PCR, Western blot. RESULTS GCPs and siRNA-VEGF-C inhibited HepG2 proliferation, invasion and migration, and the most obvious inhibitory effect was both synergistic effects. Thus, GCPs suppressed HLECs proliferation, migration and tube-like structure formationin a dose- dependent manner, and had inhibitory effect of tumor- induced lymphangiogenesis in vitro. Additionally, we found that GCPs and siRNA- VEGF- C decreased the expressions of MMP-2, MMP-9, VEGF-C, CXCR4, phospho-ERK1/2, phospho-P38, phospho-JNK and PI3K in HepG2 cells. Moreover, GCPs had a dose-dependent depressive effecton the expressions of VEGFR- 3, SDF- 1 in HLECs. CONCLUSION The low expression of VEGF- C mediated by siRNA-VEGF-C and GCPs inhibit tumor proliferation, invasion and migrationby suppressing the MAPK signaling pathway through reduced levels of VEGF-C, and GCPs inhibit tumor lymphangiogenesis by suppressing the CXCR4/SDF-1 signaling pathway through suppressed VEGF-C/VEGFR-3.