The Inhibitory Effect And Mechanism Of Rabdosin F On The Growth Of Human Esophageal Cancer Cells EC9706

Background and purposeEsophageal cancer is one of the most common gastrointestinal diseases,and the mortality rate is almost the same as the stomach cancer.Because the treatment effect is poor,the 5 year survival rate is less than 10%,being a serious threat to the human health.Esophageal cancer is divided into esophageal squamous cell carcinoma and esophageal adenocarcinoma,and the majority is esophageal squamous cell carcinoma,the esophageal adenocarcinoma is less.Henan is one of the places of high incidence of esophageal cancer,especially in Linzhou and Anyang.At present,esophageal cancer has no specific treatment,and the treatment options are also associated with the period of the disease.Patients in the early and middle period can be treated with surgical treatment,and patients in the late period can only be treated with chemotherapy,and the effect is not ideal.Rabdosin,belonging to Labiatae,mainly containing two terpenes,three terpenes,lignin and flavonoid compounds.Researches have shown that,Rabdosin A,G and E have significantly inhibition effect on Ehrlich ascites carcinoma(ECA)in vivo,showing the same effect on the tumor.Rabdosin B showed considerable effect on L1210 leukemia mouse model of life extension,but Rabdosin C and D have no anti-tumor effect.At present,there are less researches on Rabdosin F,and we have not find researches of Rabdosin F on the esophageal cancer cells EC9706.This study aims to explore the inhibitory effect and mechanism of rabdosin F on the growth of human esophageal cancer cells EC9706.Materials and methods1.MTT was used to measure the activity of samples The experimental groups were treated with 100,80,50,40,and 30?g/ml of Rabdosin F samples and the control groups with the same volume of complete medium medium.After 24 h,48h and 72 h in the incubator with 37? and 5% CO2,each hole was treated with 20?l MTT.Incubating for another 4 hours,each hole was added with 160?l DMSO,and the absorbance was detected by enzyme marker.2.The effect of the sample on the growth of the cells The experimental groups were treated with 80,50,40,and 30?g/ml of Rabdosin F samples and the control groups with the same volume of complete medium medium.After 72 h in the incubator with 37? and 5% CO2,observe the cell growth and take photos under microscope.3.Cell scratch test The experimental groups were treated with 50,40,and 30?g/ml of Rabdosin F samples and the control groups were not treated with Rabdosin F samples.The positive control groups were added with 50?g/ml cisplatin.After 24 h in the incubator with 37? and 5% CO2,observe the cell growth and take photos and measure the area of each scratch under microscope.4.Apoptosis detection The experimental groups were treated with 80,50,40,and 30?g/ml of Rabdosin F samples and the control groups with the same volume of complete medium medium.After 24 h,48h and 72 h in the incubator with 37? and 5% CO2,the apoptosis rate was detected by flow cytometry.5.Cell cycle detection The experimental groups were treated with 80,50,40,and 30?g/ml of Rabdosin F samples and the control groups with the same volume of complete medium medium.After 24 h,48h and 72 h in the incubator with 37? and 5% CO2,the cell cycle distribution was detected by flow cytometry.6.Effects of different samples on the expression of 5 proteins in EC9706 cells by western blot The experimental groups were added with Rabdosin F samples in accordance with the concentration of 50,40,30?g/ml,and the control groups with the same volume of complete medium medium.After 24 h,48h and 72 h in the incubator with 37 ? and 5% CO2,the related proteins,such as Bax,Bcl-2,Caspase-3,mTOR and P53 were extracted from the cells.Then the protein expression level were detected by Western blot.7.SPSS statistical processing The software SPSS 17.0 was used for statistical processing,and the data were expressed by x ±s,and the experiment was repeated more than three times.Results1.MTT assay results showed that the growth of EC9706 cells was significantly inhibited at 24 h,48h and 72 h,and the IC50 was 46.7?g/ml,25.4?g/ml and 20.6?g/ml respectively,also with the increase of concentration and the extension of time,the inhibitory effect was also enhanced.2.Compared with the control groups,the experimental groups treated with different concentrations of Rabdosin F samples after 72 h showed larger changes.The volume of cells becomes small,the cytoplasm shrinks,and the cells were small granular.3.In the cell scratches test,the scleral area of the cells was about 3.10 × 105cells/ml after incubation with 50?g/ml Rabdosin F,which was significantly higher than that of the control group(2.52 × 105),and the difference was statistically significant(P<0.05).4.The apoptotic rates were 20.20%,21.65% and 53.21% respectively,after 24 h,48h and 72 h with the treatment of Rabdosin F of 50?g/ml,under the same conditions,the proportion of G1 phase cells was 58.53%,57.37% and 52.37% respectively and the proportion of S phase cells was 40.06%,41.16% and 45.62%,respectively.The apoptotic rates were 10.37%,17.84%,53.21% and 86.63% respectively with the increase in concentration when the time was 72 h,and the proportion of G1 phase cells was 59.65%,56.43% and 52.37% respectively,the proportion of S phase cells was 39.71%,42.35% and 45.62% respectively.But there was no significant change in G2 phase cells(P<0.05).5.Effect of Rabdosin F on the expression of 5 proteins in EC9706 cells The gray value of Bax protein were 0.91,1.20 and 1.26,the Caspase-3 protein were 0.77,0.83 and 1.11,the Bcl-2 protein were 1.01,0.95 and 0.92,the mTOR protein were 1.08,0.84 and 0.71,and the P53 protein were 0.94,0.72 and 0.58 respectively with the increase in concentration after 72 h,indicating that it can upregulate the expression of Bax and Caspase-3 proteins and down-regulate the expression of Bcl-2,mTOR and P53 proteins.ConclusionsRabdosin F can up regulate the expression level of Bax,Caspase-3 protein and down regulate the expression level of Bcl-2,mTOR and P53 protein,it can also inhibit the proliferation and migration of esophageal cancer EC9706 cells,block cell growth in S phase,and promote cell apoptosis and necrosis.All of these may be one of the mechanisms of the anti-tumor effect of Rabdosin F.