DADS Resist Wnt/β-catenin Pathway Through Mediated By PKCα/RORα In Gastric Cancer MGC803Cells

Objective:To further investigate the effect and mechanism of RORα phosphorylation andthe effect of Wnt/β-catenin pathway mediated by PKCα/RORα in human gastriccancer MGC803cells induced by diallyl disulfide on the basis of previouswork.Observing the the effect of MGC803cells inhibited by diallyl disulfide.Toreveal the molecular mechanism of the RORα as a potential target for DADS andprovide a new direction for the prevention of gastric cancer.Methods:We cultured human gastric MGC803cells in vitro,to detect the proteinexpression of RORα，p RORα，PKCα，p PKCα under the treatment of DADS，TPA(aPKCα activator),Go6976(a PKCα inhibitor),DADS and Go6976respectively.wereexamined by Western blot assay.Co-immunoprecipitation, immunofluorescence wereemployed to confirm RORa binding and localization with PKCα in human gastricMGC803cells which under the treatment of different drugs as before. The proteinexpressions of Wnt/β-catenin pathway molecules Axin、c-Jun、Cyclin D1、c-Myc、β-catenin、p β-catenin with or without drugs treatment as before were tested byWestern blotting assay at last.The molde were establishedly implanting the humangastric carcinoma MGC803cells, which were divited into MGC803、MGC803withDADS treatment、high expression RORα、high expression RORα with DADStreatment、low expression RORα、low expression RORα with DADS treatment sixgroups.Regularly observed the changes of nude mice tumorigenic and the inhibitionrate was calculated in each group. Results:Exposure of human gastric cancer MGC803cells to DADS and TPA(a PKCαactivator) respectively, the protein expression of RORα，p RORα，PKCα，p PKCα weresignificantly upregulated than that without drug treatment(P<0.05),whereas the cellsprotein expression that treatment with Go6976(a PKCα inhibitor) was downregulatedthan without drug treatment(P<0.05).The four protein expression as before with DADSand Go6976combined treatment were upregulated than that only Go6976treatment（P<0.05). Co-immunoprecipitation revealed that DADS and TPA can promote thebinding of PKCα and RORα. Immunofluorescence assay indicated nuclear staining ofRORα，p-RORα，PKCα，p-PKCα in cells with DADS and TPA treatment respectivelywere more than that without drug treatment. whereas the cells nuclear staining thattreatment with Go6976was less than without drug treatment.The nuclear staining incells with DADS and Go6976combined treatment were more than that only Go6976treatment. Western blotting showed that the protein expression of several downstreammolecules of Wnt/β-catenin pathway, such as Axin、c-Jun、Cyclin1、c-Myc weresignificantly downregulated with TPA and DADS treatment respectively,while canupregulated with Go6976treatment. But cells with DADS and TPA treatment can noteffect the protein expression of β-catenin and β-catenin phosphorylation.Wesuccessfully establish of MGC803human gastric cancer cells in nude mice xenograftmodel. The growth curve shows that the tumor volume increases with time, highexpression RORα groups were grew slowly than low expression RORα groups,and thegroups with DADS treatment were significantly grew slowly than that without DADStreatment(P<0.05).Compared with control,the inhibition rate of high expression RORαgroups are significantly high but low expression RORα groups are significantly low(P<0.05),In the light microscope,the inhibition rate of each groups are significantlyhigher with DADS treatment. Cells heteromorphism of low expression RORα groupsare more obvious than controls, high expression RORα groups are decline thancontrols,and the heteromorphism with DADS treatment are obvious than withoutDADS treatment.Immunohistochemistry showed compared with controls,Vimentin、 P53、Ki-67、CD34of high expression RORα groups are significantly low,E-cadherinare significantly high.In controy,Vimentin、P53、Ki-67、CD34of low expression RORαgroups are significantly high,E-cadherin are significantly high,E-cadherin aresignificantly low.Vimentin、P53、Ki-67、CD34with DADS treatment are reduced thanwithout DADS treatment,while E-cadherin are increased.That showed DADS andRORα can inhibits tumor growth.Conclusion:1. The protein expression of RORα and RORα phosphorylation in MGC803cells can upregulated with DADS treatment.2. RORα can phosphorylation by activating PKCα in gastric cells induced bydiallyl disulfide.3.diallyl disulfide can resist Wnt/β-catenin pathway through mediated byPKCα/RORα in gastric cancer MGC803cells.4.diallyl disulfide and RORα can can inhibit MGC803cell tumorigenicitysignificantly.