| ObjectiveTo observe the effect of cinobufagin on migration and invasion of esophageal cancer Kyse-520 cells,and to investigate whether the inhibitory effect of cinobufagin on invasion and migration of esophageal cancer Kyse-520cells is related to focal adhesion kinase/phosphatidylinositol 3-kinase/Akt signaling pathway.Methods1.Kyse-520 cells were randomly divided into two groups,control group（0.1%DMSO）and experimental group（0.1,0.1,0.4,1.6,6.4μmol·L-11 of cinobufagin）.The effects of cinobufagin on the proliferation of Kyse-520 cells were detected by CCK-8 assay after treatment of 12 h,24 h and 48 h.2.Kyse-520 cells were randomly divided into two groups,control group（0.1%DMSO）and experimental group（25,50,100 nmol·L-11 of cinobufagin）,and then cells were cultured for 48 h in a real-time cell recorder.The effect of cinobufagin on the"two-dimensional"migration of Kyse-520 cells was detected by scratch healing assay.3.Kyse-520 cells were randomly divided into two groups,control group（0.1%DMSO）and experimental group（25,50,100 nmol·L-11 of cinobufagin）,and then cells were cultured for 24 h.The effects of cinobufagin on the"three-dimensional"migration and invasion of Kyse-520 cells were detected by Transwell assay.4.Kyse-520 cells were randomly divided into two groups,control group（0.1%DMSO）and experimental group（25,50,100 nmol·L-11 of cinobufagin）,and then cells were cultured for 24 h.The expressions of genes mRNA such as focal adhesion kinase,Akt,phosphatase and tensin homolog deleted on chromosome 10,angiogenic factor A,matrix metalloproteinase 2 and matrix metalloproteinase-9 in Kyse-520 cells were analyzed using Real-time fluorescence quantitative PCR assays;5.Kyse-520 cells were randomly divided into two groups,control group（0.1%DMSO）and experimental group（25,50,100 nmol·L-11 of cinobufagin）,and then cells were cultured for 24 h.The expressions of proteins such as focal adhesion kinase,phosphorylated focal adhesion kinase（Try397）,Akt,phospho-Akt（Ser473）,phosphatase and tensin homolog deleted on chromosome 10,angiogenic factor A,matrix metalloproteinase 2 and matrix metalloproteinase 9 in Kyse-520 cells were detected by Western blotting.Results1.CCK-8 results showed that cinobufagin inhibited the proliferation of esophageal cancer cells in a time-and concentration-dependent manner（P<0.05）,that is,the higher the concentration of cinobufagin and the longer treatment time,the lower the cell activity.2.The results of scratch healing assay showed that all concentrations of cinobufagin could significantly reduce the scratch healing area（P<0.01）,and the"wound"enlarged gradually with the prolongation of time and the increase of drug concentration.It suggested that cinobufagin inhibited the"two-dimensional"plane migration of esophageal cancer cells in a concentration-and time-dependent manner.3.The results of Transwell migration assay showed that the number of transmembrane cells decreased significantly with the increase of drug concentration（P<0.05,P<0.01）.It suggested that cinobufagin could significantly inhibit the"three-dimensional"plane migration of esophageal cancer cells,and there was an obvious dose-effect relationship.4.The results of Transwell invasion assay showed that the invasion number after 24 h of 25,50,100 nmol·L-1 cinobufagin treatment was significantly decreased in a concentration-dependent manner（P<0.05,P<0.01）,compared to the control group.It suggested that cinobufagin could significantly inhibit the invasion of esophageal cancer cells.5.The results of Real-time fluorescence quantitative PCR showed that when the concentration of cinobufagin were 50 and 100 nmol·L-1,the expressions of angiogenic factor A,matrix metalloproteinase 2 and matrix metalloproteinase 9 mRNA were significantly down-regulated（P<0.05,P<0.01）and the expression of phosphatase and tensin homolog deleted on chromosome 10 was up-regulated（P<0.01）,which was significantly higher than that in the control group.However,the expressions of focal adhesion kinase and Akt mRNA did not change obviously.6.The results of Western blotting showed that when the concentration of cinobufagin was 50,100 nmol·L-1,the expression levels of angiogenic factor A,matrix metalloproteinase 2,matrix metalloproteinase 9,phosphorylated focal adhesion kinase（Try397）and phospho-Akt（Ser473）were dramatically lower than that of the control group（P<0.05,P<0.01）.Whereas,the expression of phosphatase and tensin homolog deleted on chromosome 10 was significantly increased（P<0 05）,the expression levels of focal adhesion kinase and Akt were not changed between control and all other treatments.Conclusions1.Cinobufagin significantly inhibits the proliferation of esophageal cancer Kyse-520 cells in a time-and concentration-dependent manner.2.Cinobufagin inhibits the invasion and migration of esophageal carcinoma Kyse-520 cells by inducing phosphatase and tensin homolog deleted on chromosome 10 expression,negatively regulating Focal adhesion kinase/Phosphatidylinositol 3-kinase/Akt signaling pathway and down-regulating the expressions of angiogenic factor A,matrix metalloproteinase 2 and matrix metalloproteinase 9. |
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