| Objective:1.To explore the effect of SSPH I on invasion and metastasis and angiogenesis of HCC in vivo and vitro.2.Based on the TGF-β1/Smads signaling pathway to explore the effect and mechanism of SSPH I on regulating EMT against HCC.Methods:1.Study on the effect of SSPH I on against invasion and metastasis in HCC cells by TGF-β1/Smads signaling pathway in vitro.The proliferation of HepG2 and SMMC-7721 cells was detected by MTT assay.Exogenous administration of TGF-β1 to stimulate HepG2 and SMMC-7721 cells,and TGF-β1/Smads signaling pathway inhibitor SIS3 was used as a negative control for observation.Three drug administration groups with different concentration gradients were set up,and the migration and invasion ability of HepG2 and SMMC-7721 cells were detected by woud-healing and transwell assays.Based on the western blot test to detect the expression of Smad7,Smad2/3 and p-Smad2/3 proteins.The expression of TGF-β1,MMP-2 and MMP-9 in the supernatant of HepG2 and SMMC-7721 cells was detected by ELISA assay.2.Study on the effect of SSPH I on anti-invasion and metastasis and angiogenesis in vivo.HepG2 cells were used to establish orthotopic transplantation liver tumor model of nude mice.100 μ L of HepG2 cells were injected into the liver envelope of each nude mouse by surgery.14 days later,nude mice were treated with SSPH I.In the experimental group,SSPH I at doses of 25mg/kg,50mg/kg and 75mg/kg was orally administered to nude mice for 14 consecutive days.Animals in the positive control group were given 2mg/kg cisplatin intraperitoneally every two days.Nude mice in the model group were given oral saline orally every day.The expression levels of MMP-2,MMP-9,VEGF and TGF-β1 proteins in the serum of nude mice were detected by ELISA experiment.H&E staining experiment was used to observe the degree of cancerous damage of liver tissues in nude mice.The expression of TGF-β1,Smad7,MMP-2,MMP-9,CD31,CD34 and VEGF proteins were detected by immunohistochemistry and western blot assays.3.Study on the effect and mechanism of SSPH I on regulating EMT on invasion and metastasis in HCC through TGF-(31/Smads signaling pathway.In vitro experiments,TGF-β1 was used to stimulate HepG2 and SMMC-7721 cells,and TGF-β1/Smads signaling pathway inhibitor SIS3 was used as a negative control for observation.Western blot experiments were used to detect the expression levels of E-cadherin,N-cadherin and Vimentin in HepG2 and SMMC-7721 cells.Based on the orthotopic transplantation liver tumor test in nude mice,the effect of SSPH I on the expression of EMT-marker proteins E-cadherin,N-cadherin and Vimentin proteins,TGF-β1 and Smad7 proteins were detected by immunohistochemistry and western blot experiments.The expression level of TGF-β1 protein in serum of nude mice was detected by ELISA experiment.Results:1.Study on the effect of SSPH I on against invasion and metastasis in HCC cells by TGF-β1/Smads signaling pathway in vitro.SSPH I can effectively inhibit the proliferation of HepG2 and SMMC-7721 cells,the IC50 are 1.49μM and 1.72μM at 24h.Woud-healing and transwell assays showed that,compared with the control group,TGF-β1 significantly increased the cell migration rate and the number of invasive cells.Compared with TGF-β1 group,SSPH I significantly decreased the migration rate and the number of invasive cells.Western blot result showed that compared with the control group,TGF-β1 up-regulated p-Smad2/3 protein and down-regulated Smad7 protein expression,indicating that TGF-β1 can activate the TGF-β1/Smads signaling pathway in cells.After SSPH I administration,the expression of p-Smad2/3 protein in the cells decreased and the expression of Smad7 protein increased,thereby inhibiting the TGF-β1/Smads signaling pathway in the cells.ELISA result showed that TGF-β1 can increase the expression of TGF-β1,MMP-2 and MMP-9 proteins in the cell supernatant.After used SSPH I,TGF-β1,MMP-2 and MMP-9 proteins expression decreased in the supernatant of HepG2 and SMMC-7721 cells.T he results were all concentration-dependent.2.Study on the effect of SSPH I on against invasion and metastasis in HCC by TGF-β1/Smads signaling pathway in vivo.Animal experiment result showed that SSPH I can improve the cancerous damage of liver tissues in nude mice,while reducing the metastasis of spleen and abdominal cavity.The result of H&E staining experiment showed that SSPH I can reduce the cancerous lesions of liver tissues in nude mice.According to the immunohistochemistry and western blot experiments,compared with the model group,SSPH I down-regulated expression of MMP-2,MMP-9,CD31,CD34,VEGF and TGF-β1 proteins,but increased Smad7 protein expression.The result of ELISA experiment showed that,SSPH I inhibited the expression of TGF-β1,MMP-2,MMP-9 and VEGF proteins in the serum of nude mice.3.Study on the effect and mechanism of SSPH I on regulating EMT on invasion and metastasis in HCC through TGF-β1/Smads signaling pathway.Western blot experiment in vitro showed that TGF-β1 can activate the TGF-β1/Smads signaling pathway in HepG2 and SMMC-7721 cells,which showed as up-regulating p-Smad2/3 and down-regulating Smad7.In addition,TGF-β1 promoted the formation of EMT,which showed as the EMT marker protein E-cadherin reduceing,N-cadherin and Vimentin increased.After SSPH I administration,compared with the TGF-β1 group,p-Smad2/3 protein was down-regulated and Smad7 protein was up-regulated,E-cadherin protein expression increased,and N-cadherin and Vimentin protein expression decreased.In the experiment of nude mice orthotopic transplantation liver tumor,according to western blot and immunohistochemistry experiments,we found that SSPH I can significantly inhibit the expression of EMT marker proteins N-cadherin and Vimentin,and promote E-cadherin protein expression.In addition,the result of ELISA experiment showed that SSPH I can effectively down-regulate the expression of TGF-β1 protein in the serum of nude mice.Conclusion:SSPH I can inhibit the invasion and metastasis and angiogenesis in HCC,and its mechanism is related to inhibiting the TGF-β1/Smads signaling pathway and blocking the formation of EMT. |
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