Applephenols induce cell cycle arrest at G2/M phase and apoptosis in bladder urothelial carcinoma

• Main Authors: Yu-Lin Kao, 高育琳
• Other Authors: 楊順發 教授
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/88162795976854281044

博士 === 中山醫學大學 === 醫學研究所 === 103 === Cancers are still the leading cause of death in Taiwan. Bladder cancer is the most common cancer in urological system. High recurrence rate of bladder cancer still bothers clinicians who try any efforts to overcome this situation. In recent years, trends of many researches focus on the effect of natural extracts to prevent or inhibit the developments of malignancy, therefore the mechanism of natural extract on cancer inhibition arouse the researchers’ widespread interest. Flavonoid phytochemicals were studied on prevention or therapy in human malignancies including bladder cancer. Apple polyphenols (AP) were reported to possess anti-cancer, anti-oxidation, and anti-inflammation. In this study, we aimed to detect the effect of AP in human bladder urothelial cell carcinoma (UCC). We firstly used MTT assay to evaluate the cytotoxicity in the TSGH-8301 cells. The results showed AP potently caused cell death in a dose-dependent manner. In flow cytometric analysis, the results showed treating with low doses of AP can induce significant cell cycle arrest at G2/M phase, and high doses of AP increases the subG1 ratio in cell cycle. Furthermore, we used Western blot to analyze the relative regulatory proteins in G2/M-phase and apoptosis. The results showed AP decreases the protein levels of cyclin B1 and cdc2 to cause cell cycle arrest. On the other side, the pathway of AP inducing apoptosis might involve increasing the apoptosis induce factor (AIF) protein expression and the ratio of Bax/Bcl2 protein expression. In addition to in vitro assay, TSGH-8301 cells were xenografted into nude mice to confirm the antitumor efficacy of AP in bladder carcinoma. The result also showed that AP markedly reduces tumor size in TSGH-8301 cells-xenografted tumor tissues. In conclusion, the results show AP possesses the potentiality in antitumor via mediating the cell cycle arrest and causing apoptosis in human bladder transitional cell carcinoma. In the animal study, we aimed to detect the effect of AP in an experimental model of urinary bladder cancer. Female Fischer-344 rat was given 0.05% N-butyl-N- (4-hydroxybutyl) nitrosamine (OH-BBN) in drinking water for 10 weeks to induce bladder cancer. After the final OH-BBN treatment for one week, different concentrations of 0.2 and 0.5g/kg of AP were administrated every day until this study terminated. Before sacrificing the rats, we used Vevo® visualsonics system to evaluate the possible abnormality of bladder. The results showed OH-BBN induced severe hyperplasia of bladder as compared with non-OH-BBN treated. After rats sacrificed, we found the concentrations of 0.2 and 0.5g/kg of AP can reduce the weight gain of OH-BBN-induced bladders by 73.6% and 86.9% respectively. In histopathological examination, phyllodes papilloma were found in the OH-BBN-induced urinary bladder, and the formation of papilloma was reduced in AP-treated experimental urinary bladders. We further analyzed the relative regulatory proteins of cell proliferation or apoptosis. The results showed OH-BBN increased the protein levels of PCNA to cause cell proliferation and reduced the protein levels of cleavage Poly(ADP-ribose) polymerase (PARP) as well as the previous studies to decelerate the process of apoptosis. Interestingly, AP possessed the ability to decrease the protein levels of PCNA and increase the level of PARP. In conclusion, the results show that AP possesses the potential effect to inhibit the development of OH-BBN induced papilloma in experimental bladders.