白芍及没食子酸經由細胞週期停滯及細胞凋亡抑制膀胱癌生長

根據世界衛生組織報導，全球男性死於膀胱癌的人數為125,000；女性為54,000。目前使用的抗癌藥不但副作用大且具有抗藥性。傳統的中草藥一直被用來治療癌症。其具有活性物質成份，已有許多研究證實傳統中草藥可以治療惡性腫瘤。白芍 ( Radix Paeoniae Alba；RPA)為傳統中草藥，主要治療癌症。我們以HPLC分析白芍成份其含有多酚成份-没食子酸。我們研究顯示白芍 ( RPA)與沒食子酸 ( GA)可以抑制膀胱癌細胞TSGH-8301生長及OH-BBN誘導的膀胱癌生長。RPA及GA引起細胞凋亡及細胞週期停滯。其造成細胞凋亡機制是透過caspase的活化，因為caspase抑制劑降低細胞凋亡。RPA及GA引起G2/M細胞週期調控蛋白降低包括cdc2、cyclin B1、cdc25B及cdc25C。我們亦證明RPA及GA抑制細胞生長主要透過活化Cip1/p21及phosphor-chk2蛋白表現。更深入探討GA促進phospho-cdc25C ( Ser-216)，其與14-3-3β結合，而由細胞核移動至細胞質。活化ATM/chk2可磷酸化cdc25C，其可能是GA造成DNA損傷，而活化H2A.X ( Ser-139)。接下來以OH-BBN誘導膀胱癌動物模式，大白鼠飲水中加入0.05 % OH-BBN持續8週，之後餵食RPA ( 0.5 or 1 g/kg)及GA ( 50 mg/kg or 150 mg/kg)至26週。結果顯示RPA及GA可以抑制OH-BBN引起的papillary/ nodular dysplasia。分析膀胱組織，結果顯示飲水加入RPA的老鼠，明顯降低Bcl2、 PCNA與cyclin D1蛋白表現，增加phospho-chk2 ( Thr-68)、Bax和Cip1/p21的表現。我們的實驗結果證明RPA和GA可能可以發展成為治療膀胱癌的藥物。關鍵字：白芍、沒食子酸、細胞週期、細胞凋亡、膀胱癌。

The World Health Report attributes an estimated 125,000 death in males and 54,000 deaths in females worldwide as a result of urothelial carcinoma. While many anticancer agents have been used, the side effects and resistance are serious problems. Traditional herbal medicines have been used since ancient times to treat malignancies. There has been increasing interest in the biological activity of traditional medicines, and numerous studies report their clinical benefit for cancer. Radix Paeoniae Alba (RPA), is a traditional Chinese medicines, has been shown to treat malignancies. We assayed the components of RPA via determination of HPLC. The components belong to phenolic compounds- gallic acid (GA). We report that RPA and GA significantly inhibit growth of human bladder transitional carcinoma cells (TSGH-8301) and bladder cancer induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (OH-BBN). We observed that RPA and GA treatment of cell resulted induction of apoptosis and arrest in G2/M phase. This induction of apoptosis seems to be mediated via activation of caspase because N-benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluromethylketone significantly reduced apoptosis induced by RPA and GA. Treatment of TSGH-8301 cells with RPA and GA resulted in G2-M phase cell cycle arrest that was associated with a marked decline in protein levels of G2-M regulatory proteins including cyclin-dependent kinase 1 (cdc2), cyclin B1, cell division cycle 25B (Cdc25B) and Cdc25C. We also reported that RPA- and GA-mediated growth inhibition of TSGH-8301 cells was correlated with activation of Cip1/p21 and checkpoint kinase 2 (Chk2). Additional mechanistic studies showed that GA induces phosphorylation of Cdc25C at Ser-216. This mechanism leads to its translocation from nucleus to cytoplasm resulting in an increase binding with 14-3-3β. When treated with GA, phosphorylated Cdc25C can be activated by ataxia telangiectasia mutated (ATM)-checkpoint kinase 2 (Chk2). It might be a DNA damage response proved by Ser-139 phosphorylation of histine H2A.X. Herein, we further evaluated urinary bladder cancer by RPA and GA in a model of bladder cancer induced by OH-BBN. RPA and GA strongly arrest OH-BBN-induced tumor progression at the stage of mucosal dysplasia with a striking reduction in papillary nodular dysplasia. Analysis of tumors from RPA-treated rats showed significant decrease in the expression of Bcl2, cyclin D1, and PCNA, and increase in the expression of p-Chk2 (Thr-68), Bax, and Cip1/p21. Our data suggest that RPA could be developed as an agent against bladder cancer. Key words: Radix Paeoniae Alba, gallic acid, cell cycle, apoptosis, OH-BBN, Chk2, ATM.