Screening Of Anti Bladder Cancer T24 Cells Activity Of Pomegranate Peel Tannins Main Monomers And Researching On The Mechanism

Objective In this study,human bladder cancer T24 cell line was used to study the anti bladder cancer activity of pomegranate peel tannins,meanwhile the anti cancer effects of the main active monomers of pomegranate peel tannins?Gallic acid,Punicalagin,Ellagic acid and Punicalin?were compared.The best anti bladder cancer monomer of pomegranate peel tannins was screened out and its mechanism was further explored.It provides a theoretical basis for the anti cancer research of tannins and the development of drug form in traditional Chinese medicine for the treatment of bladder cancer in the future,and it is conducive for further development and utilization of tannins.Methods?1?Human bladder cancer T24 cells were cultured in vitro,using CCK-8experiment to detect the inhibition rate of pomegranate peel tannins on T24 cells and to screen the concentration and time of administration.The concentration of pomegranate peel tannins was determined to be 12.5,25 and 50?g·mL^-11 respectively,and the action time was 24 hours according to the results of CCK-8 experiment.The morphological changes of cells and cell nuclei were observed after pomegranate peel tannins treated.Apoptosis rate was detected by flow cytometry after FITC/PI fluorescence stained,and the mitochondrial membrane potential level was detected by JC-1 staining method.?2?CCK-8 experiment was used to compare the anti bladder cancer activity of the main active monomers of pomegranate peel tannins?Gallic acid,Punicalagin,Ellagic acid,and Punicalin?.The results showed that gallic acid had the best anti-bladder cancer activity,so the rest of the experiments were treated with gallic acid.The concentration of gallic acid was determined to be 6.25,12.5 and 25?g·mL^-11 and the action time was 24 hours according to the results of CCK-8 experiment.Cell and nucleus morphological changes were observed after gallic acid treatment.Flow cytometry was used to detect cell apoptosis and cycle distribution.DCFH-DA fluorescent probe was used to detect the level of ROS.JC-1 staining method was used to detect the level of mitochondrial membrane potential.Western blot method and Real-Time PCR method were used to detect the relative expression of the proteins and genes including Bax,Bcl-2,Caspase-3,Cyt-c and P53 in T24 cells after treated with gallic acid?3?Cell scratch test and transwell experiment were used to detect the effects of gallic acid on migration and invasion activity of T24 cells.Western blot method was used to detect the effect of gallic acid on the expression of vascular endothelial growth factor protein in T24 cells.Results?1?The growth and proliferation of T24 cells were inhibited by pomegranate peel tannins in a time and concentration dependent manner.The IC₅₀0 of pomegranate peel tannin treated T24 cells for 24 h,48 h and 72 h were 72.70?g·mL^-1,62.26?g·mL^-11 and 19.64?g·mL^-11 respectively.Pomegranate peel tannins treated T24 cells for 24 h,cell nucleus were shrunk and degenerated,the cells were in an apoptosis state.Flow cytometry confirmed that after 24 hours of stimulation by pomegranate peel tannin,the mitochondrial membrane potential of T24 cells decreased significantly and apoptotic cells increased significantly,mainly inducing early apoptosis.?2?The results of CCK-8 experiment showed the anti bladder cancer T24 cell activities of the main monomers of pomegranate peel tannins from strong to weak was:gallic acid,punicalagin,punicalin,ellagic acid.The IC₅₀0 of gallic acid stimulated T24cells for 24 h was 21.73?g·mL^-1.Flow cytometry analysis showed that gallic acid could block T24 cell cycle in S phase,and the number of early apoptosis cells were increased significantly.After gallic acid stimulated T24 cells for 24 h,we further found that ROS level was increased and mitochondrial membrane potential was decreased in T24 cells.Western blot assay verified the expression of P53,Cyt-c and pro-apoptosis protein Bax in T24 cells were up-regulated in a dose-dependent manner,Caspase-3 protein was activated,while the expression of anti-apoptosis protein Bcl-2was down-regulated and the ratio of Bax/Bcl-2 was up-regulated.Real-Time PCR results showed that gallic acid promoted the expression of P53,Cyt-c,Caspase-3 and Bax mRNA and inhibited the expression of Bcl-2 mRNA in T24 cells.?3?Cell scratch test and transwell experiment verified that gallic acid could inhibit the migration and invasion activity of T24 cells in a concentration dependent manner.Western blot assay showed that the expression of VEGF in T24 cells was significantly decreased after gallic acid stimulation.Conclusion pomegranate peel tannins can inhibit proliferation and induce apoptosis of bladder cancer T24 cell line.Gallic acid is the main monomer of pomegranate peel tannins which has the strongest anti bladder cancer activity.Gallic acid can block T24cell cycle in S phase,inhibit cell proliferation,induce apoptosis,inhibit cancer cell migration and invasion.The apoptosis of T24 cells induced by gallic acid may be related to ROS mediated mitochondrial apoptosis pathway.