The Anti-tumor Mechanisms Study Of Toosendanin On Gastric Carcinoma Mediated By MiR-200a/?-catenin Axis

ObjectiveThis study aims to explore the effects of toosendanin?TSN?,an active ingredient of traditional Chinese medicine?TCM?toosendan fruit,on the biological behavior of human gastric carcinoma?GC?cells in vitro and the growth as well as liver metastasis of orthotopic GC in nude mice with splenasthenic syndrome in vivo;This research has illuminated the anti-GC molecular mechanisms of TSN in view of miR-200a/?-catenin axis in order to provide some evidence for future study of anti-GC ingredient of TCM.Methods:?1?The influence of TSN on proliferation of human various GC cell lines was analyzed using MTT assay;Flow cytometry,transwell,wound-healing assay and western blot assay detected the effects of TSN on the cell cycle distribution,early apoptosis,invasion,migration and epithelial-mesenchymal transition?EMT?induced by transforming growth factor?1 in SGC-7901 cells.?2?Lentivirus-mediated?-catenin?CTNNB1?overexpression vector?LV-CTNNB1?was constructed and transfected into SGC-7901 cells;Quantitative Real-time PCR,western blot assay and Cell Immunofluorescence assay examined the level of?-catenin as well as its downstream molecules expression in SGC-7901 cells treated by TSN alone or combined with LV-CTNNB1 transfection;methods for detecting cell activities in vitro mentioned above were used to evaluate the intervention of LV-CTNNB1 transfection in the inhibitory effects of TSN on the biological behavior of SGC-7901 cells.?3?Lentivirus-mediated miR-200a silencing vector?LV-hsa-miR-200a-3p-inhibition?was constructed and transfected into SGC-7901 cells;Quantitative Real-time PCR,western blot assay and Cell Immunofluorescence assay detected the expression level of miR-200a,?-catenin as well as molecules of its signaling in SGC-7901 cells treated by TSN alone or combined with LV-hsa-miR-200a-3p-inhibition transfection;After LV-hsa-miR-200a-3p-inhibition transfection,the effects of TSN on the biological behavior of SGC-7901 cells were also evaluated by the methods for detecting cell activities in vitro mentioned above.?4?Rhabarber and glauber gavage combined with surgery of orthotopic GC transplantation were adopted to establish an orthotopic GC model in nude mice with splenasthenic syndrome.The growth and liver metastasis of orthotopic GC tissues in nude mice treated by intraperitoneal injection of TSN were observed.The effects of TSN adminstration on the expression level of miR-200a and molecules in?-catenin pathway in GC tissues of nude mice were detected by Fluorescence in situ hybridization and Immunohistochemistry.Results:?1?TSN produced marked proliferation inhibition of human various GC cell lines?MGC-803,BGC-823,HGC-27,AGS,SGC-7901,MKN-45?as well as human normal gastric epithelial cells?GES-1?in a time-and dose-dependent manner.The 50%inhibitory concentration(IC₅₀)values of TSN on SGC-7901 cells were the lowest,whereas those on GES-1cells were the highest among all the cell lines?*P<0.05 or**P<0.01?.G1 phase arrest and early apoptosis were induced and capacities of invasion,migration and TGF-?1-induced EMT were suppressed in SGC-7901 cells by TSN.?2?The level of?-catenin expression as well as its downstream molecules?c-Myc,cyclin D1,Bcl-2 and MMP-9?decreased?**P<0.01?and nuclear translocation of?-catenin was inhibited in SGC-7901 cells by TSN.After LV-CTNNB1 transfection,the level of ?-catenin expression increased and inhibitory effects of TSN on the biological behavior of SGC-7901 cells weakened?*P<0.05 or**P<0.01?.?3?The level of miR-200a expression in SGC-7901 cells increased after TSN treatment?**P<0.01?.Inhibitory effects of TSN on?-catenin as well as its downstream molecules?c-Myc,cyclin D1,Bcl-2 and MMP-9?rather than upstream molecules in SGC-7901 cells were diluted by knockdown of miR-200a?*P<0.05 or**P<0.01?.Suppressive effects of TSN on the biological behavior of SGC-7901 cells in vitro also remitted after miR-200a silencing?*P<0.05 or**P<0.01?.?4?TSN could significantly suppress the growth and liver metastasis of orthotopic GC in nude mice with splenasthenic syndrome?**P<0.01?.Overexpression of?-catenin or knockdown of miR-200a in cells weakened the therapeutic effects of TSN?*P<0.05 or**P<0.01?.TSN could enhance the expression of miR-200a and regulate the level of molecules of?-catenin pathway in orthotopic GC tissues.Among these molecules,?-catenin,c-Myc,cyclin D1,Bcl-2 and MMP-9 rather than p-AKT(Ser⁴⁷³),p-ERK1/2(Thr²⁰²/Tyr²⁰⁴)and p-GSK3??Ser⁹?could be further elevated by knockdown of miR-200a.Conclusion:?1?TSN might play anti-tumor effects on gastric carcinoma through inhibiting cell proliferation,invasion,migration and EMT and inducing cell cycle arrest and apoptosis.?2?TSN might suppress the biological behavior of SGC-7901 cells through inactivating?-catenin pathway.?3?MiR-200a/?-catenin axis might participate in the molecular mechanisms in the inhibitory effects of TSN on the biological behavior of SGC-7901 cells in vitro and the growth and liver metastasis of orthotopic GC in nude mice with splenasthenic syndrome in vivo.?4?TSN has significant anti-tumor effects on GC and its molecular mechanisms might be related to the regulation of miR-200a/?-catenin axis.The research has indicated that TSN could become a novel ingredient of traditional Chinese medicine for GC treatment in the future.