Study On The Mechanism Of Casticin In Inhibiting The Migration And Invasion Of Human Gastric Cancer Cells

Objective:Casticin of Fructus Viticis has been found to have a property of suppressing the proliferation of multiple tumor cells.This study aimed to investigate the effect of Casticin on the migration and invasion of gastric cancer cells and explore its underlying molecular mechanism.Thus,the results would provide fundamental insights into the anti-tumor mechanism of Casticin.Methods:1)Effect of Casticins on gastric cancer cells.Different concentrations of Casticin were added into in-vitro cultured AGS and NCI-N87 cells.Then CCK-8(cell-counting kit-8)and colony formation assays were conducted.Transwell Assay and Scratch Assay were used to examine the migration and invasion capabilities of cells,respectively.Flow cytometry and DAPI were used to analyze the cell cycles and detect the cell apoptosis,respectively.The expression of MMP-2 and MMP-9 were examined using qRT-PCR and WB,and gelatinase activity was determined by gelatin zymography.2)Study of the molecular mechanism of Casticin in inhibiting the migration and invasion of gastric cancer cells.After being treated with Casticin,RECK expression of AGS and NCI-N87 cells were examined using qRT-PCR and WB,and the effect of Casticin on RECK methylation state was examined by BSP experiment.After transfection of siRECK and RECK-OV into gastric cancer cells,qRT-PCR and WB were used to examine the changes of MMP-2 and MMP-9 expression,in order to clarify the relationship between RECK and MMP-2/-9 in gastric cancer cells.After PP2A inhibitors(LB-100)and PP2A activators(DES)treatments on gastric cancer cells,the expression of RECK?MMP-2 and MMP-9 in these cells were examined using qRT-PCR and WB,and the change in RECK methylation state was examined using BSP.After being treated with LB-100 and DES on gastric cancer cells,WB was used to examine the expressions of DNMT1,MEK,and p-MEK,in order to elucidate the signaling pathway relevant of PP2A activation/inhibition causing change in RECK expression.qRT-PCR and WB were used to examine the effects of Casticin on the expressions of PP2A,DNMT1,MEK,and p-MEK.Furthermore,the gastric cancer cells were treated with PP2A inhibitor followed by Casticin,and the impact of Casticin on PP2A expression and its downstream signal molecules was verified through response verification test.3)Establishing tumor xenograft models in nude mice to evaluate the anti-tumor effect of Casticin.AGS and NCI-N87 cells were respectively transplanted into nude mice,and then administrated with different concentrations of Casticin with control group established.During this progression,volume of mice tumor was intermittently recorded.Changes in expressions of MMP-2/9,PP2A and RECK were determined through immunohistochemical staining(IHC)and WB.Results:1)Effect of Casticin on gastric cancer cells.Based on the results of CCK-8,colony formation assays,flow cytometry,DAPI,scratch assay,Transwell assay,qRT-PCR,WB and gelatin zymography,it was found that different concentrations of Casticin could significantly inhibit the proliferation,apoptosis,migration and invasion of gastric cancer cells,as well as the expression and activity of MMP-2/9,in a concentration-dependent manner.While,it was also found that the effect of Casticin on human gastric smooth muscle cells was little(HGSMC)2)Molecular mechanism of Casticin in inhibiting the migration and invasion of gastric cancer cells.Based on the results of qRT-PCR,WB,and methylation experiment,it was found that Casticin inhibited MEK activation and DNMT1 expression by promoting PP2A expression.It was also found that Casticin suppressed MMP-2/9 expression by upgrading RECK expression and attenuated RECK methylation.Finally,it reduced the migratory and invasive abilities of cells.3)Establishing human gastric cancer xenograft models in nude mice to evaluate the anti-cancer effect of Casticin.The volume of transplanted tumors after being treated with Casticin was significantly smaller than the control group.Based on IHC results,a significantly lower expression of MMP-2/9 was found in tumor tissue treated with Casticin in a concentration-dependent manner.In contrast,a higher expression of PP2A and RECK were found.Based on WB examination,it was found that expression of MMP-2/9 proteins in tumor tissue treated with Casticin was significantly downregulated,while expressions of PP2A and RECK were significantly upregulated.Conclusions:1)Casticin can effectively suppress proliferation,migration,and invasion capabilities of gastric cancer cells.2)Casticin inhibits the MEK activation and DNMT1 expression by promoting PP2A expression,resulting in a decrease of MMP-2/9 expression by increasing RECK expression,and ultimately inhibits the invasion and metastasis of gastric cancer cells3)Casticin can significantly inhibit the subcutaneous tumor growth in xenografted mice.A significantly lower expression of MMP-2/9 was found in tumor tissue treated with Casticin,and Casticin further promoted the expressions of PP2A and RECK in tumor tissue.