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

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.

Differentially expressed genes of HepG2 cells treated with gecko polypeptide mixture

OBEJECTIVE Gecko has been clinically used in China for many years.It has been proved that the gecko polypeptide mixture(GPM) extracted from gecko could inhibit the growth of multiple types of tumor cells.In order to investigate the possible anti-tumor molecular mechanisms of GPM,we used RNA-seq technology to identify the differentially expressed genes of human hepatocellular carci.noma(HCC) HepG2 cells treated with or without GPM.METHODS The HepG2 cells were treated with different concentration of GPM(0,0.1,0.2,0.3,0.4 mg·mL-1) for 6 h,12 h and 24 h,respectively.MTT assay was used to detect the viability of HepG2 cells.DAPI fluorescence staining was performed to observe nucleus morphological changes of HepG2 cells.Western blot analysis was applied to observe the expres.sion of apoptosis-related proteins and endoplasmic reticulum stress(ERs)-related proteins in HepG2 cells.Flow cytometry was also applied to detect reactive oxygen species(ROS) generation.In this report,we showed that GPM could induce HepG2 cells apoptosis and influence HepG2 cells proliferation in a dose-dependent manner.We applied many analysis methods,including differentially expressed genes analysis,Gene Ontology(GO) enrichment analysis,KEGG pathway enrichment analysis,protein-protein interaction network analysis to screen out possible molecular mechanisms.RESULTS ER-nucleus signaling pathway,cellular response to stress and apoptotic processes were identified the potential anti-cancer molecular biological process of GPM.GPM may also induce apoptosis in HepG2 cells via endoplasmic reticulum stress pathway.The GPM could induce ROS generation and up-regulate ERs-related proteins.CONCLUSION The present study revealed the potential anti-tumor mechanism of GPM.

Differentially expressed genes of HepG2 cells treated with gecko polypeptide mixture

OBJECTIVE In order to investigate the possible anti-tumor molecular mechanisms of gecko polypeptide mixture(GPM).METHODS RNA-seq technology was used to identify the differentially expressed genes of human hepatocellular carcinoma(HCC)HepG2 cells treated with or without GPM.The HepG2 cells were treated with different concentration of GPM(0,0.1,0.2,0.3,0.4 mg·mL^(-1))for 6 h,12 h and 24 h,respectively.MTT assay was used to detect the viability of HepG2 cells.DAPI fluorescence staining was performed to observe nucleus morphological changes of HepG2 cells.Western blot analysis was applied to observe the expression of apoptosis-related proteins in HepG2 cells.RESULTS The results showed that GPM could induce HepG2 cells apoptosis and influence HepG2 cells proliferation in a dose-dependent manner.We applied many analysis methods,including differentially expressed genes analysis,Gene Ontology(GO)enrichment analysis,KEGG pathway enrichment analysis,protein-protein interaction network analysis to screen out possible molecular mechanisms.ER-nucleus signaling pathway,cellular response to stress and apoptotic processes were identified the potential anti-cancer molecular biological process of GPM.GPM may also induce apoptosis in HepG2 cells via endoplasmic reticulum stress pathway.The mechanism is closely related to ERs,which might be beneficial for clinical therapy of HCC.CONCLUSION GPM can inhibit cells proliferation and induce apoptosis in HepG2 cells.The gene expression profile of GPM in HepG2 cells was obtained.The present study revealed the potential anti-tumor mechanism of GPM.