Effects Of Triptolide On Adhesion,Invasion And Metastasis Of SKOV3/DDP Cells And Explored Its Underlying Mechanism

Objective:To observe the effect of TPL on adhesion,invasion and metastasis of cisplatin-resistance human epithelial ovarian cancer cell line SKOV3/DDP,and to explore the mechanism of its action.Methods:Drug concentration groups:(1)blank control group(NC group),(2)DDP group(10?g/mL),(3)TPL group(8ng/mL),(4)combined group(10?g/mL DDP+8ng/mL TPL);the following experiments were carried out.1.Adhesion experiment: 96 well plate in the Matrigel glue,SKOV3 / DDP cells after processing into cells suspension was inoculated into 96 well plates and 37? culture 30 min,observation under inverted microscope after crystal violet staining,cell count,calculate the adhesion rate.2.Invasion assay: Transwell paved on the surface of Matrigel glue,cells were seeded in the Transwell chamber;adding 10%FBS RPMI-1640 Transwell medium in the lower chamber;after cell culture 24 h,observation under inverted microscope after crystal violet staining,calculate the rate of invasion.3.Migration assay: each group cells were inoculated in the Transwell chamber,added with 5% FBS RPMI-1640 medium in the lower chamber of Transwell;after cell culture 24 h,crystal violet staining,the cells were observed by inverted microscope.Determination of metastatic cells in each group on the 570 nm wavelength of the enzyme marker,calculate cell migration rate.4.Wound Healing: take logarithmic phase cells were inoculated in 6-well plates and cultured cells to form a monolayer,starvation culture 24 h,according to the experimental group to join the different intervention,scratch the bottom of the culture plate,after administration,0h and 24 h were observed under the inverted microscope,photos.Use Photoshop Adobe software to measure the scratch area in each picture.Calculated 24 h scratch healing area.5.Western blotting: Detection of drug action in SKOV3/DDP cells after 24 h,than to detect the expression levels of integrin beta 1 and matrix metalloproteinases(MMP-2,MMP-9).Results:1.SKOV3/DDP cells were treated with different drugs,the effect of TPL on cell adhesion of SKOV3/DDP cells: the experimental groups cell adhesion rate are as follows: control group(100%±7.71%)> DDP group(67.73%±10.69%)>TPL group(47.92%±5.54%)> combined group(23.01%±5.04%).There are significant differences between the groups(P < 0.01).The results showed that TPL could inhibit the adhesion of SKOV3/DDP cells,and its inhibition ability is stronger than DDP.The combination of the two is better than the single drug group,indicating that TPL has a sensitization effect on DDP.2.SKOV3/DDP cells were treated with different drugs,the effect of TPL on invasion of SKOV3/DDP cells: the invasion rate of the experimental groups are as follows: control group(100%±11.87%)> DDP group(51.14%±11.92%)>TPL group(14.79%±3.93%)> combined group(3.94%±0.82%).There are significant differences between the groups(P < 0.01).The results showed that TPL could inhibit the invasion of SKOV3/DDP cells,and its inhibition ability is stronger than DDP.The combination of the two is better than the single drug group,indicating that TPL has a sensitization effect on DDP.3.SKOV3/DDP cells were treated with different drugs,the effect of TPL on migration of SKOV3/DDP cells: the cell migration rate of each experimental groups are as follows: control group(100%±19.86%)> DDP group(77.68% ±3.62%)> TPL group(35.83%±1.14%)> combined group(22.69%±2.64%).There are significant differences between the groups(P < 0.05).The results showed that TPL could inhibit the migration of SKOV3/DDP cells,and its inhibition ability is stronger than DDP.The combination of the two is better than the single drug group,indicating that TPL has a sensitization effect on DDP.4.SKOV3/DDP cells were treated with different drugs,the experimental groups 24 h scratch healing area are as follows: control group(66.64±6.04)> DDP group(53.27±6.23)> TPL group(44.22±3.65)> combined group(29.41±10.37).There are significant differences between the groups(P < 0.05).The results showed that the wound healing area of the combined group is the least.Compared with the DDP group and the control group,the differences are statistically significant(P < 0.01).5.SKOV3/DDP cells were treated with different drugs,the relative gray value of the control group,DDP group,TPL group and TPL+DDP group were as follows: MMP-2 : 1.033±0.039 ? 0.975±0.045 ? 0.596±0.013 ? 0.322±0.007;MMP-9 :1.333±0.222?1.007±0.190?0.641±0.152?0.235±0.046;integrin beta 1:0.922±0.103?0.532±0.227?0.376±0.156?0.143±0.130.The protein expression levels of MMP-2,MMP-9 and beta 1-integrin in TPL group were lower than that in DDP group and control group,the difference are statistically significant(P < 0.01).The expression level of each protein in DDP group is lower than that in the control group,and the difference is statistically significant(P < 0.05).The expression level of each protein in the combined group is the lowest,and the differences are statistically significant(P < 0.01).Conclusion:1.TPL can significantly inhibit the adhesion,invasion and metastasis of SKOV3/ DDP cells,and its inhibition ability is stronger than DDP.The combination of the two is better than the single drug group,indicating that TPL has a sensitization effect on DDP.2.TPL may inhibit the adhesion,invasion and metastasis of SKOV3/DDP cells by down regulating the expression of integrin beta 1 and down regulating the expression of matrix metalloproteinase 2 and 9.