In vivo inhibitory effection of Silibinin on bladder cance proliferation by classic and non-classic apoptosis related signals

Abstract

Objective To investigate the growth inhibition of Silibinin on human bladder cancer cells 5637 subcutaneous tumor and the mechanism of it. Methods The human bladder cancer cells 5637 were cultured in vitro until logarithmic phase, then were inoculated into the right hind dorsal subcutaneous of nude mice to establish the subcutaneous tumor model. Different concentrations of Silibinin were given through the oral method for 32 days, and then executed all the nude mice, removed the tumors and weighed.After that, flow cytometry was used to analyze the tumor cell apoptosis, Western blot and immunohistochemical were engaged to detect the expression of apoptosis related factors Caspase-3, Cleaved PARP, Bax, Bcl-2 and Cytochrome C, also the data were compared and analyzed with statistical methods. Results In fifty nude mice, 42 nude mice were modeled successful, and 40 nude mice were selected into experiment randomly, there was no mice died during the experiment. By measuring the volumes of the tumor, the tumor growth inhibition rates was 10.08%, 47.64%, 63.14%, respectively among the low, medium and high does group(F=52.79, P<0.01). The tumor weights(g) of control, low, medium and high does group was (1.54±0.25), (1.46±0.22), (0.94±0.15) and (0.72±0.16), respectively, and the difference between the three groups was statistically significant(F=794.17, P<0.01). Flow cytometry detected the tumor cells apoptosis rate illustrated that there was significantly difference in tumor cells apoptosis rate among the different does groups(F=794.17, P<0.01). The tumor cells apoptosis rates of control, low, medium and high does group were (6.70±1.34), (8.08±1.19), (26.11±1.91) and (44.87±2.96). Compared with the control group, low dose group had no significant difference in apoptosis rate (P=0.14). All groups’Caspase-3, Cleaved PARP, Bax, Bcl-2 and Cyt-C protein expression were significantly different (P<0.05), but the comparison results in different doses of Silibinin group and with control group were not all P<0.05. Conclusions Silibinin can significantly inhibit bladder cancer growth in vivo, and this effection will more significant accompany with the increase of the Silibinin does.The mechanisim of the inhibition of Silibinin on bladder cancer may associate with caspase-mediated apoptosis pathway, mitochondrial-mediated apoptosis pathway and perhaps also has some relationship with Bcl-2 family proteins down-regulation and the up-regulation of Bax family proteins. Key words: Urinary Bladder Neoplasms; Apoptosis; Silymarin