To investigate the effects of autophagy in cisplatin-induced apoptosis of bladder cancer cells.
Bladder cancer cell T24 was regarded as cell model. The transmission electron microscope was employed to detect autophagic vacuoles and the fluorescence microscope to detect the fluorescence accumulation profile of vectors for green fluorescent protein and microtubule associated protein 1 light chain 3 fusion protein (GFP-LC3). Protein immunoblotting was applied to detect the accumulation of LC3-II, thus detecting whether cisplatin could induce the bladder cancer T24 cell autophagy. Moreover, protein immunoblotting was ultilized to detect the autophagic relative signal pathway mammal target of rapamycin (mTOR) and the variation of ribosomal protein S6 kinase (P70S6K) with relative molecular mass 70 000 in downstream as well as the cleavage of apoptosis marker protein poly ADP-ribose polymerase (PARP). Afterwards, with the utilization of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetra zolium, inner salt (MTS), the viability changes in cisplatin-induced bladder cancer cells under the condition of autophagy promoting the presentation or absence of rapamycin were observed. Moreover, RNA interference was also adopted in this experiment to knockdown the LC3 expression.
Compared with the control group, the electron microscope revealed that cisplatin was able to induce plenty of autophagic vacuoles in bladder cancer cells. CFP-LC3 aggregation was viewed by the fluorescence microscope, showing a significant higher in cisplatin group than control group. The results of LC3 detected by protein immunoblotting indicated that the LC3-II content in cisplatin group was significantly enhanced with the prolongation of time and increase of cisplatin concentration. Especially, at the concentration of 50 and 100 μmol/L for 48 hours with cisplatin treatment, the gray value of LC3-II/Actin (%) increased 30 and 44, respectively. Cisplatin treatment inhibited the phosphorylation of mTOR/P70S6K, and its phosphorylated strips were almost completely inhibited in the 100μmol/L for 48 hours with cisplatin treatment. MTS results showed that cisplatin was able to lead to the loss of cell viability, which was 12% and 35% at the concentration of 50 μmol/L, and 100μmol/L for 24 hours with cisplatin treatment. Moreover, the cell viability loss in autophagy-induced rapamycin and cisplatin combined treatment group was bigger than that with single-use cisplatin treatment in control group (F = 74.890, P < 0.01). Besides, RNA interference experiment revealed that knocking down autophagic relative gene LC3 could reduce the PARP cleavage induced by cisplatin and the apoptosis was decreased.
Cisplatin could induce autophagy in bladder cancer cell T24, which promoted cisplatin-induced apoptosis.
Panminerva medica. 2016 Jul 19 [Epub ahead of print]
Zhiqiang Fan, Xuejun Huangfu, Zhonghua Liu
Department of Urology, Henan Provincial People's Hospital, Zhengzhou City, China - .