Effects Of Tea Polyphenols Combinedwith Epirubicin On T24 Bladder Cancer Cell Line And Its Mechanism

Objective The incidence of bladder cancer ranks the fourth malignancy tumors in America. The proportion of superficial bladder cancer in bladder cancer is 70%. Gold standard of superficial bladder cancer is TUR-BT. While the recurrence after TUR-BT is an important issue to be solved. Bladder irrigation chemotherapy is one of the most key factors for post-TUR-BT recurrence. It is a common use of epirubicin instillation therapy for bladder cancer. Tea polyphenols(TP) is an effective synergist with anticancer drugs for cancer treatment and prevention, we investigate whether the tea polyphenols(TP) in combination with epirubicin(EPI) exhibits a synergistic activity in blocking proliferation and cell apoptosis of human bladder cancer cells T24 in vitro and its mechanism, providing a theoretical basis for combination used in clinical bladder cancer infusion chemotherapy.Methods In vitro cultured human T24 bladder cancer cell lines; MTT assay was applied to investigate the effects of different levels of tea polyphenols or epirubicin on the T24 cells. The experiment was divided into 4 groups: control group(PBS), TP group (88.8 μmol/L),EPI group (4.6 μmol/L), and TP (88.8 μmol/L) plus EPI (4.6 μmol/L) group. The proliferation inhibition rate was assayed by MTT; Annexin V-FITC/PI double staining flow cytometry was used to analyze apoptosis. The ultra-structural cellular changes were observed by transmission electron microscope(TEM); The formation of MAP1LC3-II puncta was examined after transfection of a GFP-LC3 plasmid. The level of LC-3-II, P62, CASP3, PARP proteins were detected by Western blot, respectively.Results Treatment with TP (88.8 μmol/L) and EPI (4.6 μmol/L) leaded to a significant increase of inhibiting rate(P=0.000) and a dramatic improvement in apoptotic changes compared to single EPI or TP group (P=0.000). Autophagy was observed obviously in bladder cancer cells T24 treated with EPI after 8 h. TEM examination revealed the appearance of autophagosome with double-membrane structure. Immunofluorescence showed that EPI strongly augmented the number of cells with increased GFP-LC3 puncta and GFP-LC3 puncta formation within the cell. Western blot showed the expression of LC-3 II was increased in T24 cells after treated with EPI, while the expression of P62 was decreased. TP combined with EPI and single groups both induced the expression of active apoptosis proteins, and the effect was more obvious in TP plus EPI group.Conclusion TP combined with EPI can synergistically inhibit the proliferation and promote cell apoptosis, which may be associated with the reduction of EPI-induced autophagy by TP.