蘋果多酚抑制膀胱泌尿上皮癌細胞的機制探討

惡性腫瘤為台灣十大死亡原因之首，其中膀胱癌是泌尿系統常見的癌症，在目前有效的治療下，仍有很高的復發率。近年來許多研究趨向以天然萃取物來抑制或是延緩癌症的發生，所以天然萃取物在抑癌作用機制之探討引起學者廣泛的興趣。在之前研究證實類黃酮對於一些惡性腫瘤具有治療的潛力。蘋果多酚內含有許多種類黃酮與酚酸衍生物，目前許多文獻指出蘋果多酚具有抗癌、抗發炎、抗氧化…等生理活性。本研究想要了解蘋果多酚對於膀胱癌之影響。由於膀胱癌90%以上都是泌尿上皮細胞癌，本篇採用人類泌尿上皮細胞癌細胞株(TSGH-8301)進行研究。在細胞毒性測定 (MTT assay) 中 發現蘋果多酚對於TSGH-8301具有細胞毒性，且呈現劑量依存現象(dose- dependent)。從細胞週期分析發現，蘋果多酚除了可以造成細胞週期停滯在G2/M期，處理高濃度的蘋果多酚會使癌細胞走向細胞凋亡，利用西方墨點法進一步的分析，蘋果多酚會造成G2/M期調控蛋白Cdc2與Cyclin B1減少，使細胞週期停滯。當細胞凋亡過程粒線體膜電位降低會使通透性改變釋放出細胞凋亡相關蛋白，apoptosis induce factor (AIF) 就是其中一種。在處理高濃度的蘋果多酚除了能顯著增加AIF蛋白，也會影響Bax/Bcl-2蛋白的比值使細胞走向細胞凋亡。使用粒線體膜電位染劑JC-1，結果發現處理蘋果多酚會降低粒線體膜電位。除此之外將TSGH-8301細胞注射到裸鼠大腿外側，以管餵餵食蘋果多酚。結果也發現蘋果多酚可以減少腫瘤的大小。以上的實驗都顯示蘋果多酚可透過讓細胞週期停滯或細胞凋亡，達到抑制癌症的效果。 動物實驗採用5週Fischer-344 母鼠給予10週0.05%OH-BBN (N-Butyl-N- (4-hydroxybutyl) nitrosamine) 誘導實驗性膀胱癌動物，在處理10週OH-BBN後開始以管餵餵食0.2或0.5g/kg蘋果多酚，在犧牲前使用非侵入型的超音波系統觀察膀胱內部病變的情況。由結果發現處理OH-BBN會造成膀胱內部出現許多不規則的增生，動物犧牲後將膀胱秤重也發現餵食蘋果多酚組別重量與單純處理OH-BBN相比重量有顯著的減少。觀察膀胱組織切片，發現處理OH-BBN組別其黏膜層 (mucosal layer) 細胞層較多，餵食蘋果多酚能降低細胞層數。進一步免疫化學染色觀察膀胱組織Ki-67蛋白的表現，Ki-67蛋白是一種細胞增生的指標，結果發現餵食蘋果多酚確實能降低OH-BBN所誘導Ki-67蛋白。利用西方墨點法做進一步確認，結果發現OH-BBN會增加PCNA蛋白表現，處理蘋果多酚後會降低PCNA蛋白表現，這結果與免疫化學染色結果相同。以上的動物實驗顯示蘋果多酚對於OH-BBN所誘導的實驗性膀胱癌具有抑癌潛力。綜合上述研究結果，我們的實驗證實蘋果多酚使人類膀胱癌細胞株TSGH-8031的細胞週期，發生G2/M arrest，並促成經由內在路徑(internal pathway)細胞凋亡的發生，達到抑制膀胱癌的生長。在OH-BBN誘導嚙齒目動物產生膀胱癌的動物模式中，經由超音波影像及組織切片免疫染色及蛋白表現證實蘋果多酚確實可抑制膀胱癌的生長。

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.