| OBJECTIVE To observe the effect of Gecko crude peptides(GCPs)on the formation of lymphatic-like structures of human lymphatic endothelial cells(HLECs)on Matrigel,evaluate the ability of tumor-induced lymphangiogenesis and the inhibitory effect of GCPs on lymphangiogenesis induced by hepatocellular carcinoma in co-culture system of HLECs and human hepatoma HepG2 cells,and analyze the effects of GCPs and vascular endothelial growth factor-C(VEGF-C)on the proliferation and migration of HepG2 cells,thus,to investigate the possible mechanism of GCPs inhibiting lymphangiogenesis in hepatocellular carcinoma.METHODS HLECs and HepG2 cells were routinely cultured in vitro,and subsequent experiments with logarithmic growth cells were performed.The total experiment was divided into three parts:(1)The effects of GCPs on HLECs' tube formation capacity and migration,invasion:After HLECs were treated with GCPs at different concentrations(0.04,0.08,0.12,0.16,0.20,0.24,0.28,0.32,0.36,0.40,0.44 mg/mL)respectively,3-(4,5-dimethylthiazol-2yl)-2,5-dip henyltetrazolium bromide(MTT)assay was used to observe the change of proliferation ability.HLECs were treated with GCPs at different concentrations(0.08,0.12,0.18,0.27 mg/mL)for 24 h,tube formation experiments were used to observe the effect of GCPs on the formation of lymphatic-like structures of HLECs on Matrigel in vitro,and to establish a co-culture system of HLECs and HepG2 cells to evaluate the ability of tumor-induced lymphangiogenesis and the inhibitory effect of GCPs on lymphangiogenesis induced by hepatocellular carcinoma.And Western blot was used to analysis the expression of vascular endothelial growth factor receptor 3(VEGFR-3)and stromal cell-derived factor 1(SDF-1)protein.In addition,HLECs were treated with GCPs at different concentrations(0.01,0.02,0.04,0.08 mg/mL)for 24 h,Transwell assay was used to detect the effects of GCPs on the migration and invasion of HLECs.(2)Effects of GCPs on HepG2 cells:a.After HepG2 cells were treated with GCPs at different concentrations(0.1,0.15,0.2,0.25,0.3,0.35,0.4,0.45,0.5,0.6,0.8 mg/mL)respectively,MTT assay was used to observe the change of proliferation ability.HepG2 cells were treated with GCPs at different concentrations(0.1,0.15,0.225,0.3375 mg/mL)for 24 h,apoptosis was detected by flow cytometry.And Hoechst 33258 fluorescence staining was performed to observe nucleus morphological changes of HepG2 cells.Immunohistochemistry,Western blot and Realtime quantitative PCR(qPCR)were used to detect the protein and mRNA levels of VEGF-C and chemokine(C-X-C motif)receptor 4(CXCR4)that regulate lymphangiogenesis.In the meanwhile,Western blot was used to analyze the expression of phosphorylation levels of extracellular regulated protein kinases(ERK1/2),mitogen-activated protein kinase-38(p38 MAPK)and protein kinase B(Akt)proteins,b.HepG2 cells were treated with GCPs at different concentrations(0.025,0.05,0.1,0.15 mg/mL)for 24 h,and Wound-healing assay was performed to observe the influence of GCPs on the wound-healing ability of HepG2 cells.Transwell assays were performed to observe the influence of GCPs on the migration and invasion of HepG2 cells.The expression of matrix metalloprotein 2(MMP-2)and matrix metalloprotein 9(MMP-9)were detected by Western blot assay and qPCR.(3)Effects of VEGF-C overexpression or down-regulation on HepG2 cells:VEGF-C and siVEGF-C were transiently transfected into HepG2 cells,respectively,and to detect the effects of overexpression of VEGF-C and down-regulation of VEGF-C on HepG2 cells with the blank control group,the negative control(empty vector without plasmid DN A)group,and the VEGF-C(2.5 ?g)group/siVEGF-C(2.5?g)group.After transfected 48 h,MTT assays were used to detect the proliferation of HepG2 cells,Hoechst 33258 staining and flow cytometry were used to analyze the apoptosis of HepG2 cells and also detect capacities of cells'migration and invasion,qPCR and Western blot were used to detect the expression levels of relative mRNA and proteins in VEGF-C signaling pathways.RESUITS(1)GCPs inhibited the tube formation capacity and migration,invasion of HLECs:The IC50 values of HLECs treated with GCPs for 24 h,48 h and 72 h were 0.209 mg/mL,0.166 mg/mL and 0.135 mg/mL,respectively.The HLECs were seeded on Matrigel.GCPs were treated for 24 h,the tube formation rates of the GCPs(0.08 mg/mL)group,GCPs(0.12 mg/mL)group,GCPs(0.18 mg/mL)group,and GCPs(0.27 mg/mL)group were 82.66%,40.00%,17.66%,and 2.33%,respectively,compared with the blank control group(100%),the difference was statistically significant.The results showed that GCPs can significantly inhibit the tube-like structure formation of HLECs.The results of coculture system showed that the tube formation ability of HLECs in HepG2 cells group(tube formation rate 100%)was significantly higher than that in the blank control group(tubulation rate was 60.67%),indicating that HepG2 cells can significantly promote the formation of lymphatic vessels in HLECs in vitro.In addition,the tube formation rates of the HepG2 cells + GCPs(0.1 mg/mL)group,HepG2 cells + GCPs(0.15 mg/mL)group,HepG2 cells + GCPs(0.225 mg/mL)group,HepG2 cells + GCPs(0.3375 mg/mL)group were 41.66%,29.000%,16.67%,and 3.32%,respectively,compared with HepG2 cells group,the differences were statistically significant(P<0.001),indicating that GCPs can inhibit the ability of HepG2 cells to promote tube formation in HLECs.Compared with the blank control group,GCPs could significantly inhibit the expressions of VEGFR-3 mRNA and protein,and down-regulate the expression of SDF-1 protein.Transwell assay results showed that the migration inhibition rates of the GCPs(0.01 mg/mL)group,GCPs(0.02 mg/mL)group,GCPs(0.04 mg/mL)group,and GCPs(0.08 mg/mL)group were 34.72%,48.61%,62.50%,and 87.50%,respectively,and the invasion inhibition rates were 8.64%,48.15%,61.73%and 76.54%,respectively.Compared with the blank control group,GCPs can significantly inhibit the migration and invasion of HLECs,the difference was statistically significant(P<0.001).(2)GCPs inhibited the proliferation and migration of HepG2 cells:The IC50 values of HepG2 cells were 0.276 mg/mL,0.203 mg/mL,and 0.165 mg/mL with treated GCPs for 24 h,48 h,and 72 h,respectively.The results of Hoechst 33258 staining showed that the chromatin condensation,cell division and volume decrease of HepG2 cells were observed under fluorescence microscope after treated with GCPs.Flow cytometry showed that the early apoptosis rates of HepG2 cells treated with GCPs at different concentrations were 10.00%± 3.39%13.88%± 2.75%,17.04%± 2.61%,20.05%± 3.27%,respectively,compared with in the blank control group(0.04±0.01%),there was a significant difference(p<0.01).GCPs could remarkably inhibit the expressions of VEGF-C and CXCR4 mRNA and protein,and could also inhibit the phosphorylation of ERK1/2,p38MAPK and Akt protein expression.The scratch test results showed that the scratch healing ability of HepG2 cells in GCPs groups were weakened.Transwell assay results showed that the migration inhibition rates of the GCPs(0.025 mg/mL)group,GCPs(0.05 mg/mL)group,GCPs(0.1 mg/mL)group,and GCPs(0.15 mg/mL)group were 24.740-%,51.71%,60.83%,and 74.14%,respectively,and the invasion inhibition rates were 52.60%,58.33%,63.54%and 74.47%,respectively.Compared with the blank control group,GCPs can significantly inhibit the migration and invasion of HepG2 cells,the difference was statistically significant(P<0.001),and GCPs could remarkably inhibit the expressions of MMP-2 and MMP-9 mRNA and protein.(3)Effects of VEGF-C overexpression or down-regulation on HepG2 cells:a.The effects of VEGF-C on HepG2 cells:HepG2 cells were transiently transfected with VEGF-C.MTT results show that VEGF-C can significantly promote the proliferation of HepG2 cells.The early apoptosis rate was 6.71±1.68%in HepG2 cells after transfected VEGF-C for 48 h,which was significantly different in the blank control group(11.70±2.35%)and in the negative control group(10.10±2.65%)(p<0.01).VEGF-C can induce the expression of Bcl-2 and inhibit the expression of Bax to inhibit apoptosis.Trans well assay results showed that the number of HepG2 cells that migrated through the filter membrane increased significantly in the VEGF-C group compared with the negative control group.In addition,the number of HepG2 cells that invaded the VEGF-C group increased significantly,indicating that VEGF-C can enhance migration and invasion ability of HepG2 cells.GCPs could up-regulate the expressions of CXCR4,MMP-2 and MMP-9 protein,and remarkably increase the expressions of phosphorylated ERK1/2 and p38 MAPK protein,and promote the expression of phosphorylated Akt protein.b.The effects of siVEGF-C on HepG2 cells:HepG2 cells were transiently transfected with siVEGF-C.The results of qPCR and Western blot showed that siVEGF-C1 had obvious interference effect and the transfection efficiency was as high as 80%.The siVEGF-C 1 could significantly inhibit the proliferation of HepG2 cells,and the early apoptotic rates of HepG2 cells in the blank control group,the negative control group,and the siVEGF-C1 group were 8.40%±3.25%,11.10%±2.32%and 20.10%±2.76%,respectively,and the difference between the siVEGF-C 1 group and the blank control group was significant(P<0.05).Western blot results showed that siVEGF-C1 could down-regulate the expression of Bcl-2 protein,whereas it up-regulates the expressions of Bax,caspase-9,and caspase-3 proteins.Transwell assay results showed that the migration inhibition rates of the negative control group and the siVEGF-C 1 group were 13.97%and 77.20%,respectively.In addition,the invasion inhibition rates of the negative control group and the siVEGF-C 1 group were 5.68%and 69.31%,respectively.Thus,down-regulation of VEGF-C can inhibit the migration and invasion of HepG2 cells.And siVEGF-C 1 could down-regulate the mRNA and protein expression levels of CXCR4,MMP-9 and MMP-2 and could significantly reduce phosphorylated ERK1/2,p38 MAPK and Akt proteins expression levels.CONCLUSION(1)HepG2 cells could significantly promote the formation of lymphatic vessels in HLECs in vitro,and GCPs can inhibit the ability of HepG2 cells to promote tube formation in HLECs.(2)GCPs could significantly inhibit the proliferation and migration of HepG2 cells and induce apoptosis,down-regulate the expressions of VEGF-C,CXCR4,p-ERK1/2,p-p38 MAPK and p-Akt.(3)VEGF-C could promote the proliferation and migration of HepG2 cells,and up-regulating the expressions of CXCR4,MMP-2,MMP-9,p-ERK1/2,p-p38 MAPK and p-Akt.It is suggested that VEGF-C is an important factor in promoting lymphangiogenesis in tumors.In summary,GCPs can inhibit the ability of HepG2 cells to promote tube formation in HLECs in vitro,because it can inhibit the proliferation and migration of HepG2 cells,down-regulate the expression of VEGF-C and CXCR4.It was shown that GCPs can play an anti-tumor effect by inhibiting lymphangiogenesis,and its mechanism is to block VEGF-C/VEGFR-3 and SDF-1/CXCR4 pathways and down-regulate the expression of p-ERK1/2,p-p38MAPK and p-Akt. |
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