Effects Of DADS On Proliferation, Migration And Invasion In LIMK1Overexpression MGC803Cells

Objective: Our previous studies reported that diallyl disulfide (DADS) inhibitedproliferation, migration and invasion in human Gastric cancer MGC803cells viadown-regulation of LIMK1, LIMK1silenced increases the inhibition effects of DADSon invasion of MGC803cells. In this study, we will investigate the effects of DADSon migration and invasion in LIMK1overexpression MGC803cells by constructingLIMK1overexpression MGC803cells.Methods:The eukaryotic expression vector encoding human LIMK1gene sequence(code the LIMK1protein) was constructed, and transfected it into MGC803cells toestablish a stable cell line, which stably overexpressed LIMK1protein. RT-PCR andWestern blotting assay were performed to detect the expressions of LIMK1mRNAand protein level. The effect of LIMK1overexpression on proliferation, migration andinvasion of MGC803cells with or without DADS treatment were detected by MTT,flow cytometry, wound healing, transwell invasion chamber assays and nude micexenograft experiments.Results1. The successful construction of LIMK1/MGC803cells with stably overexpressedLIMK1.Restriction endonuclease digestion and sequencing showed that the eukaryoticexpression vectors of human LIMK1(pIRES2-EGFP/LIMK1eukaryotic expressionvector) were successfully constructed. The pIRES2-EGFP/LIMK1eukaryoticexpression vector was transfected into MGC803cells. RT-PCR and Western blottingassays showed that the expressions of LIMK1mRNA and protein were stablyincreased in the LIMK1/MGC803cells, and suggested that LIMK1/MGC803cellswith stably overexpressed LIMK1was successfully constructed.2.Effect of LIMK1overexpression on the proliferation, migration and invasion inMGC803cells. MTT and flow cytometry assays showed that the proliferation capability ofLIMK1/MGC803were increased comparing with MGC803cells andpIRES2-EGFP/MGC803(P<0.05). DADS inhibited cellular proliferation ofLIMK1/MGC803cells and MGC803cells, and induced G2/M arrest (P<0.05).LIMK1silenced decreased the proliferation capability of miRi-LIMK1/MGC803cells(P<0.05).Wound healing and Transwell invasion chamber assays showed that themigration and invasion of LIMK1/MGC803were significantly increased comparingwith MGC803cells and pIRES2-EGFP/MGC803. DADS inhibited the migration andinvasion in MGC803cells and LIMK1/MGC803cells.3. Effect of DADS and LIMK1overexpression regulated expression of LIMK1,cofilin1and cofilin1phosphorylation.Western blot and RT-PCR showed that the protein and expressions of LIMK1were increased in LIMK1/MGC803cells comparing with MGC803cells andpIRES2-EGFP/MGC803(P<0.05), cofilin1were not affected in LIMK1/MGC803cells (P>0.05). Whereas the cofilin1phosphorylation level were increased inLIMK1/MGC803cells (P<0.05).4.Effect of DADS and LIMK1on MGC803cells in nude mice xenograft experiments.Nude mice xenograft tumor model was successfully established, Nude micexenograft experiments showed that the volume and weight of xenograft tumor ofLIMK1/MGC803cells groups were significantly lager than MGC803cells groups andmiRi-LIMK1/MGC803cells groups (P<0.05). DADS significantly inhibited thegrowth of nude mice xenograft tumor (P<0.05).Conclusion1. Overexpression of LIMK1can promote the proliferation, migration and invasion ofMGC803cells.2. The inhibition effects of DADS on the proliferation, migration and invasion ofMGC803cells were weakened by the LIMK1overexpression.3. LIMK1overexpression can up-regulates the cofilin1phosphorylation level. 4. DADS can inhabited MGC803cells tumorigenicity in vivo. LIMK1overexpressioncan promoted MGC803cells tumorigenicity in vivo, and LIMK1silenced caninhabited MGC803cells tumorigenicity in vivo.