| Objective:Celastrol was co-cultured with human bladder cancer T24 to observe its effects on T24 cell proliferation,apoptosis,metastasis,and invasion activity.At the same time,the effect of Celastrol on Wnt/β-catenin signaling pathway-related factors in T24 cell was explored.Methods:Different concentrations of Celastrol(0 μ,1 μM,2 μM,4 μM)were co-cultured with human bladder cancer T24 cells for 24 h,48 h,and 72 h.MTT assay was used to detect the effects of Celastrol on cell proliferation.After co-culturing different concentrations of Celastrol(0 μM,1 μM,2 μM,4 μM)with human bladder cancer T24 cells for 24 h,Annexin V-FITC/PI combined with flow cytometry was used to detect apoptosis,cell scratch test was used to detect cell migration ability,Transwell chamber test was used to detect the invasion ability.Western blot was used to detect the effects of Celastrol on Wnt/β-catenin signaling pathway key factorsβ-catenin,Glycogen synthase kinase-3β(GSK-3β)and Dishebelled1(Dv1)in T24 cells,as well as the protein expression of target downstream genes of the Wnt/β-catenin signaling pathway such as c-myc and cyclin D1,which can promote the proliferation of cancer cells,survivin,which can inhibit cancer cell apoptosis,Vimentin,matrix metalloprotein-9(MMP-9),and α-Smooth Muscle Actin(α-SMA)protein expression.Reverse transcription-polymerase chain reaction(RT-PCR)was used to detect oepithelial cell markers cadherin-E(E-cadherin)and tight junction protein-1(Zona occluden-1,Zo-1)mRNA expression.Results:1.After co-culturing different concentrations of Celastrol(0 μM,1 μM,2 μM,4μM)with human bladder cancer T24 cells for 24 h,48 h,and 72 h,the cell proliferation rate decreased with the increase in drug concentration.The difference was statistically significant(F(24 h)=4.361,P=0.031;F(48 h)=10.821,P=0.014;F(72 h)=11.356,P=0.000),suggesting Celastrol can inhibit the proliferative activity of human bladder cancer T24 cells,and this inhibitory effect increases with the increase of drug dose.2.After different concentrations of Celastrol(0 μM,1 μM,2 μM,4 μM)were co-cultured with human bladder cancer T24 cells for 24 h,the apoptosis rates were(4.6±0.67)%,(15.8±1.7))%,(26.3±2.66)%and(41.73±3.04)%,respectively.With the increase of drug concentration,the apoptosis rate increased,and the difference was statistically significant(F=6.410,P=0.000).It is suggested that Celastrol can promote the apoptosis of human bladder cancer T24 cells,and this inhibitory effect is dose-dependent in a certain range.3.After co-culturing the cells with different concentrations of Celastrol(0μM,1μM,2μM,4μM)for 24 h,the cell migration rates of the cells were:(93.90±5.84)%,(67.26±6.10)%,(38.22±4.20)%,and(19.88±2.31)%,the cell migration rate was statistically significant between the drug concentration groups(F=9.510,P<0.001).It is suggested that Celastrol can inhibit the migration ability of human bladder cancer T24 cells,and this inhibitory effect is dose-dependent.4.After co-culturing the cells with different concentrations of Celastrol(0 μM,1μM,2 μM,4 μM)and cells for 24 h,the cells in the 0 μM,1 μM,2 μM,and 2μM group passed through the basement membrane cells of the cell after 24 h were(842.3±52.27),(704.2±47.10),(504.3±32.78),and(207.3±28.01),and the number of cells was statistically significant between each drug concentration group(F=11.256,P<0.001).It is suggested that triptolide can inhibit the invasion ability of human bladder cancer T24 cells,and this inhibitory effect is dose-dependent.5.After co-cultured with different concentrations of Celastrol(0 μM,1 μM,2μM,4 μM)and cells for 24 h,β-catenin protein expression in human bladder cancer T24 cells was 0.92±0.12,0.78±0.09,0.42±0.05 and 0.26±0.03,with the increase of drug concentration,the expression of β-catenin protein decreased,and the difference between groups was statistically significant(F=12.410,P<0.001);the expression of GSK-3β protein was 0.31±0.06,0.56±0.12,0.89±0.13,and 1.06±0.21,with the increase of drug concentration,the expression of GSK-3β protein increased,and the differences between groups were statistically significant(F=9.642,P<0.001);The protein expression levels of Dvl were 1.15±0.31,0.90±0.10,0.73±0.07,and 0.48±0.05.As the drug concentration increased,the Dvl protein expression decreased,and the differences between groups were statistically significant(F=9.642,P<0.001).It is suggested that Celastrol may inhibit Wnt/β-catenin signaling pathway activity in human bladder cancer T24 cells.6.After co-culturing the cells with different concentrations of Celastrol(0 μM,1μM,2 μM,4 μM)for 24 hours,the E-cadherin mRNA expression in the cells was 0.07±0.01,0.25±0.06,0.47±0.07 and 0.78±0.10,the difference between the groups was statistically significant(F=11.513,P<0.001);Zo-1 mRNA expression was 0.10±0.02,0.45±0.07,0.56±0.09,and 0.88±0.12,respectively,the difference between the groups was statistical significance(F=10,315,P<0.001),with the increase of drug concentration,the expression of endothelial cell markers in human bladder cancer T24 cells increased.7.After co-culturing the cells with different concentrations of Celastrol(0 μM,1μM,2 μM,4 μM)for 24 hours,the expression of Vimentin in the cells was 0.93±0.11,0.81±0.06,0.47±0.08,and 0.32±0.05,the difference between groups was statistically significant(F=5.042,P=0.016);MMP-9 expression was 0.74±0.12,0.65±0.03,0.41±0.04,and 0.28±0.05,respectively,and the difference between groups was statistically significant(F=8.972,P<0.001);MMP-9 expressions were 1.19±0.09,0.82±0.11,0.57±0.08,and 0.26±0.07,respectively,and the differences between groups were statistically significant(F=14.351,P<0.001).It is suggested that Celastrol can inhibit the epithelial-mesenchymal transition of human bladder cancer T24 cells.Conclusions:1.Celastrol can inhibit the proliferative activity of human bladder cancer T24 cells,promote its apoptosis,and at the same time inhibit its ability to migrate and invade.2.Celastrol may regulate the biological function of human bladder cancer T24 cells by inhibiting the activity of Wnt/β-catenin signaling pathway,and then suppressing its downstream target genes,such as C-myc,Survivin,cyclin D1,and E-cadherin,Zo-1,Vimentin,MMP-9 and α-SMA. |
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