Influences And Its Molecular Mechanism Study Of Rosmarinic Acid Analogue-11 On Human Gastric Adenocarcinoma Cell Lines

Aim:（1）The inhibitory effects of Rosmarinic Acid and Rosmarinic Acid Analogue-11 on human gastric cancer cells MGC-803 and human gastric cancer cells SGC-7901 were compared in vitro.（2）To investigate the apoptosis and its potential mechanism of human gastric cancer cells MGC-803 and human gastric cancer cells SGC-7901 induced by Rosmarinic Acid Analogue-11.Methods:（1）MTT assay was used to detect the inhibitory effects of Rosmarinic Acid and Rosmarinic Acid Analogue-11 on the proliferation of human gastric cancer cells MGC-803 and human gastric cancer cells SGC-7901respectively.（2）Plate cell cloning and formation experiment was used to observe the effect of Rosmarinic Acid Analogue-11 on the colony formation ability of human gastric cancer cells MGC-803 and human gastric cancer cells SGC-7901.（3）Cell scratch experiment was used to observe the effect of Rosmarinic Acid Analogue-11 on the migration ability of human gastric cancer cells MGC-803and human gastric cancer cells SGC-7901.（4）Hoechst 33258 staining was used to observe the effect of Rosmarinic Acid Analogue-11 on the apoptotic morphological changes of human gastric cancer cells MGC-803 and human gastric cancer cells SGC-7901.（5）Flow Cytometry（FCM）was performed to detect the effects of Rosmarinic Acid Analogue-11 on the early and late apoptosis of human gastric cancer cells MGC-803 and human gastric cancer cells SGC-7901.（6）Fluorescence real-time quantitative PCR（qRT-PCR）was used to detect the effect of Rosmarinic Acid Analogue-11 on the expression levels of human epidermal growth factor receptor EGFR mRNA,apoptosis-related genes Caspase-3 mRNA,Bcl-2 mRNA and Bax mRNA in human gastric cancer cells MGC-803 and human gastric cancer cells SGC-7901.（7）Western blot was used to determine the relative expression levels of Caspase-3,Bcl-2 and Bax in human gastric cancer cells MGC-803 and human gastric cancer cells SGC-7901.（8）Western blot analysed the effect of Rosmarinic Acid Analogue-11 on EGFR and its downstreams Akt/NF-κB pathway and related proteins of MAPK pathway in human gastric cancer cells MGC-803 and human gastric cancer cells SGC-7901.Results:1.The inhibitory effects of Rosmarinic Acid and Rosmarinic Acid Analogue-11 on the proliferation of human gastric cancer cells MGC-803 and human gastric cancer cells SGC-7901:（1）The IC₅₀0 of Rosmarinic Acid on MGC-803 cells at 24 h,36 h and 48 h were 175.792±1.406μmol·L^-1,108.077±0.615μmol·L^-11 and 68.282±1.349μmol·L^-11 respectively.（2）The IC₅₀0 of Rosmarinic Acid on SGC-7901 cells at 24 h,36 h and 48 h were174.302±0.793μmol·L^-1,124.547±2.287μmol·L^-11 and 54.158±2.098μmol·L^-1respectively.（3）The IC₅₀0 of Rosmarinic Acid Analogue-11 on MGC-803 cells at24 h,36 h and 48 h were 72.781±0.975μmol·L^-1,59.979±1.203μmol·L^-11 and40.625±0.623μmol·L^-11 respectively.（4）The IC₅₀0 of Rosmarinic Acid Analogue-11 on SGC-7901 cells at 24 h,36 h and 48 h were 73.299±2.011μmol·L^-1,50.426±1.458μmol·L^-11 and 37.684±0.579μmol·L^-11 respectively.2.The effect of Rosmarinic Acid Analogue-11 on the biological functions of human gastric cancer cells MGC-803 and human gastric cancer cells SGC-7901:（1）The colony formation ability of human gastric cancer cells MGC-803 and human gastric cancer cells SGC-7901 was significantly inhibited by Rosmarinic Acid Analogue-11 in a concentration-dependent manner.（2）According to cell scratch experiments,Rosmarinic Acid Analogue-11 can inhibit the migration ability of human gastric cancer cells MGC-803 and human gastric cancer cells SGC-7901 in a time-concentration dependent manner.（3）The results of Hoechst 33258 staining showed that the nucleus of human gastric cancer cells MGC-803 and human gastric cancer cells SGC-7901 showed typical apoptotic morphological changes after the intervention of Rosmarinic Acid Analogue-11 at different concentrations for 48 h.（4）Flow Cytometry results showed that Rosmarinic Acid Analogue-11 acted on MGC-803 cells for 48 h,and the low concentration group mainly induced late apoptosis of MGC-803cells,while the medium and high concentration groups could induce early apoptosis and late apoptosis of MGC-803 cells.After acting on SGC-7901 cells for 48 h,the low and medium concentration group mainly induced early apoptosis of SGC-7901 cells,while the high concentration group could induce early apoptosis and late apoptosis of SGC-7901 cells.3.Effect of Rosmarinic Acid Analogue-11 on apoptotic mechanism of human gastric cancer cells MGC-803 and human gastric cancer cells SGC-7901:（1）The results of qRT-PCR indicated that the relative expression levels of EGFR gene and Bcl-2 gene decreased,while the relative expression levels of Caspase-3and Bax genes increased（P<0.01）.（2）Western blot results showed that the expressions of pro-apoptotic protein Caspase-3 and Bax were increased,while the expression of anti-apoptotic protein Bcl-2 was significantly decreased（P<0.01）,and showed a dose-response relationship.（3）Western blot results suggested that the relative expressions of pathway proteins EGFR and NF-κB were down-regulated in a concentration-dependent manner（P<0.01）.Compared with the blank group,the relative expression levels of pathway proteins Akt and p-Akt were significantly down-regulated in dose groups.（4）The results of Western blot indicated that the expression levels of JNK and p-JNK in MAPK pathway increased significantly after the Rosmarinic Acid Analogue-11 acted on human gastric cancer cells MGC-803 and human gastric cancer cells SGC-7901 for 48 h,and the relative expression levels of ERK and p-ERK were significantly down-regulated（P<0.01）,showing a dose-response relationship.Conclusion:（1）Comparing IC₅₀0 obtained by Rosmarinic Acid and Rosmarinic Acid Analogue-11 acting on human gastric cancer cells MGC-803and human gastric cancer cells SGC-7901 respectively,it can be shown that Rosmarinic Acid Analogue-11 has more prominent inhibitory effect.（2）Rosmarinic Acid Analogue-11 inhibits the proliferation and migration of human gastric cancer cells MGC-803 and human gastric cancer cells SGC-7901 in a concentration-dependent manner.（3）Rosmarinic Acid Analogue-11 can induce apoptosis in human gastric cancer cells MGC-803 and human gastric cancer cells SGC-7901,which may be related to EGFR and its downstreams Akt/NF-κB pathway and MAPK pathway.