| Gastric cancer is the third leading malignant tumor in China with more than 400000 newly diagnosed cases,taking up almost 40%of the global cases,however,the overall 5-year survival rate is only 36%in China.As a result,finding new treatment and more sensitive screening methods to improve the prognosis of gastric cancer become a hotspot in this expertise.Cardamonin(CARD),is extracted from Katsumadai seed,which is chemically a chalcone.CARD is found to be multifunctional in our body.Existed studies have proved CARD is valid in anti-inflammation,anti-atherosclerosis and anti-tumor,involved in several signal pathway in the cell.However,there are few studies about CARD in gastric cancer.STAT3 pathway is one of the most fundamental signal pathway in cell,involved in cell immune,proliferation,differentiation and apoptosis.Meanwhile,STAT3 was found to associate with other important signal pathway,especially with epithelium-mesenchymal transitions(EMT).Yet the mechanism of STAT3 in gastric cancer cell EMT is not full illustrated.Our study was aimed to explore the influence of CARD in gastric cancer,mainly about cell proliferation,migration,invasion and apoptosis.Furthermore,we wonder if the regulation of CARD in gastric cancer is by suppressing STAT3 signal pathway.The results of our study may be useful for future study and application of CARD in treatment of gastric cacner.The study was divided into four parts.Part 1 CARD regulates gastric cancer cell proliferation,migration and invasionObjective:According to the existed methods,we tried to extract and purify CARD.Then study the influence of CARD in gastric cancer proliferation,migration and invasionMethods:According to the existed methods,we extracted and purified CARD.Three gastric cancer cell lines were used,AGS,BGC823 and MGC803 in the study.CARD was dissolved in different concentration and them treat with gastric cancer cell lines.MTT,CCK-8,clone formation,transwell and sphere-forming assay were used to study the migration,proliferation,self-renew and invasion of gastric cancer cell.Results:We dissolved CARD in 10、20、30 μmol/L and treated with AGS,BGC823 and MGC803 for 24h and 48h respectively.We found that in 30 μmol/L,treated with 24h,the suppressing effect was most significant in AGS and BGC823,so we used these two cell lines in our further experiments with CARD 30 μmol/L,24h treatment.By conducting MTT,CCK-8 and clone formation assay,we found that CARD can significantly suppress gastric cancer cell proliferation.By wound healing and migration assay,we found that CARD can significantly suppress gastric cancer migration and invasion.By sphere-forming assay,we found that CARD cancer significantly suppress gastric cancer self-renew.Conclusion:CARD can significantly suppress gastric cancer proliferation,self-renew,migration and invasion.Part 2 CARD regulates gastric cancer apoptosis.Objective:To explore the effect of cardamom on apoptosis and cell cycle of gastric cancer cellsMethods:Treated with CARD,we microscopely observe the morphology of gastric cancer.fluorescence staining and flow cytometry were used to analyze cell apoptosis.Western blot assay was used to analyze the expression of Bax,Bcl-2,Caspase-3 and CDK-1,CyclinB1,Cdc25 and p21.Results:After treatment with CARD,gastric cancer cell turned into pathological changes with apoptosis body inside.By fluorescence staining and flow cytometry,we found CARD can significantly promote gastric cancer cell apoptosis.By western blot,we found that CARD can significantly up-regulate the expression of pro-apoptosis protein Bax and down-regulate expression of Bcl-2,Caspase-3 and p21 as well as cell cycle regulatory protein CDK-1,CyclinB1 and Cdc25.Conclusion:Cardamom can significantly promote the apoptosis of gastric cancer cells by regulating apoptotic proteins and cell cycle-related proteins.Part 3 CARD regulate gastric cancer EMTObjective:To evaluate the influence of CARD on gastric cancer cell EMT.Methods:Fluorescence staining and western blot were used to analyzed the expression of E-cadherin,a-SMA,Vimentin,Snail,which are essential proteins in EMT.Results:Treated with CARD,by fluorescence staining,the expression of a-SMA and Vimentin were significantly down-regulated.By Western blot,we found that CARD can significantly up-regulate the expression of E-cadherin while down-regulate the expression of a-SMA,Vimentin and Snail.Conclusion:CARD can significantly suppress gastric cancer EMT.Part 4 CARD regulate gastric cancer migration,invasion,apoptosis and EMT though STAT3Objective:To study whether the regulation of CARD on gastric cancer cell migration,invasion,apoptosis and EMT is through STAT3 signal pathway.Methods:We used over-expression(OE)plasmid to up-regulate the expression of STAT3 in gastric cancer cell.Treated with CARD,we conducted western blot,wound healing,transwell assay to analyzed difference between OE and OE treated with CARD in migration,invasion,EMT protein expression and apoptosis,cell cycle protein expression.Results:After transfection,western blot proved efficacy of OE plasmid.Treated with CARD,the expression of phosphorylated-STAT3(p-STAT3)was down-regulated.By wound healing and transwell assay,we found that CARD can reverse the up-regulating influence of overexpression of STAT3 in gastric cancer;By western blot,we found that CARD can reverse the up-regulating effect of N-cadherin,a-SMA,Vimentin and Snail.E-cadherin was down-regulated after over expression of STAT3,CARD can reverse the expression of E-cadherin.After over expression of STAT3,Bax was up-regulated and Bcl-2,caspase-3 and p21 were down-regulated,treated with CARD,these changes were reversed.After over expression of STAT3,Cdc25,CyclinB1 and CDK-1 were up-regulated,treated with CARD,the expression of these proteins were significantly down-regulated.Conclusion:CARD regulate gastric cancer migration,invasion,apoptosis and EMT though STAT3. |
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