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OBJECTIVE To explore the role of gecko crude peptides(GCPs)in the proliferation,apoptosis,migration and lymphangiogenesis of human hepatocellular carcinoma cells(Hep G2)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 si RNA-VEGF-C on Hep G2 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 m RNA expressions of vascular endothelial 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 m RNA 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 si RNA-VEGF-C inhibited Hep G2 proliferation,invasion and migration,and the most obvious inhibitory effect was both synergistic effects.Thus,GCPs suppressed HLECs proliferation,migration and tubelike structure formationin a dose-dependent manner,and had inhibitory effect of tumor-induced lymphangiogenesis in vitro.Additionally,we found that GCPs and si RNA-VEGF-C decreased the expressions of MMP-2,MMP-9,VEGF-C,CXCR4,phospho-ERK1/2,phospho-P38,phospho-JNK and PI3K in Hep G2 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 si RNA-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.
OBJECTIVE To explore the role of gecko crude peptides (GCPs) in the proliferation, apoptosis, migration and lymphangiogenesis of human hepatocellular carcinoma cells (Hep G2) 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 si RNA-VEGF-C on Hep G2 cells, Hoechst 33258 staining and flow cytometry were performed to analyze cycle and apoptosis. migration and invasion ability of cells were assayed by transwell chamber experiment and wound-healing assay. The protein and m RNA expressions of vascular endothelial 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) threonine kinase (Akt) and phosphatidylinositol-3-kinase (PI3K) were detec ted by western blot. The anti-lymphangiogenesis effect of GCPs on the HLECs was analyzed using an in vitro tube-formation assay. The protein and m RNA 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 si RNA-VEGF-C inhibited Hep G2 proliferation, invasion and migration, and the most significant inhibitory effect was both synergistic effects.Thus, GCPs suppressed HLECs proliferation, migration and tubelike structure formation in a dose-dependent manner, and had inhibitory effect of tumor-induced lymphangiogenesis in vitro. Additionally, we found that GCPs and si RNA-VEGF-C decreased the expressions of MMP- 9, VEGF-C, CXCR4, phospho-ERK1 / 2, phospho-P38, phospho-JNK and PI3K in Hep G2 cells. Moreover, GCPs had a dose-dependent depressive effect of the expressions of VEGFR-3, SDF- 1 in HLECs . CONCLUSION The low expression of VEGF-C mediated by si RNA-VEGF-C and GCPs inhibit tumor proliferation, invasion and migration by 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.