Cinobufacini promotes apoptosis of bladder cancer cells by influencing the expression of autophagy-related genes.

Abstract

The effects of cinobufacini on apoptosis of human bladder cancer T24 cells and the expression of autophagy-related genes and proteins were studied. The human bladder cancer T24 cells were selected, and the inhibitory effect of cinobufacini on the proliferation of human bladder cancer cells was detected by cell viability assay. Flow cytometry and Hoechst staining were used to detect the changes in the apoptosis of bladder cancer cells after being treated with cinobufacini; the changes in the expression levels of human bladder cancer cell apoptosis-related genes and proteins, cleaved caspase-3, Bax, B-cell lymphoma-2 (Bcl-2) and autophagy-related genes and proteins, p62, light chain 3 (LC3) and autophagy-related protein 7 (Atg7) after treatment with cinobufacini were detected by western blot analysis and reverse transcription polymerase chain reaction (RT-PCR). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The results showed that 0.1 mg/ml cinobufacini significantly inhibited the proliferation of human bladder cancer cells cultured in vitro (P<0.01), and it was dose- and time-dependent. Both flow cytometry and Hoechst staining showed that cinobufacini promoted the apoptosis of cells (P<0.01), and the level of cell apoptosis increased with the increase of drug concentration. Both western blot analysis and RT-PCR showed that cinobufacini could decrease the expression level of Bcl-2 in T24 cells (P<0.01), increase the expression levels of Bax and cleaved caspase-3 (P<0.01), increase the ratio of Bax/Bcl-2 (P<0.01), upregulate the expression level of the angiotensin-related protein p62 (P<0.01), reduce the ratio of LC3-II/I (P<0.01) and decrease the expression level of Atg7 (P<0.01). After treatment with rapamycin, the expression levels of Bcl-2, Bax, cleaved caspase-3, autophagy-related genes and proteins, p62, LC3-II/I and Atg7 were similar to those in the control group. Cinobufacini can inhibit the autophagy activation of bladder cancer cells, thus promoting apoptosis of bladder cancer T24 cells and inhibiting the proliferation of T24 cells, which may provide a theoretical basis for the development of new anti-bladder cancer drugs.