利用細胞株探討樟芝對人類膀胱癌之療效與其作 用機轉 膀胱癌是一種和環境或職業因素有關的惡性腫瘤，其中以移行性惡性細胞腫瘤 (Transitional Cell Carcinoma) 最為常見。在男性泌尿生殖系統的癌症死亡原因中居第二大位，而在台灣目前位居第六位， 而且也有逐年上升的趨勢。 樟芝 (Antrodia camphorata) 是一種獨特而且具有許多醫療用途的菇菌植物，只生長在台灣特有的牛樟 樹上，因此屬於稀少且昂貴的保育類植物之一。長久以來，曾經被用來當做藥物中毒、腹瀉、腹痛、 高血壓及肝癌等的治療處方。基於近年來包括中草藥治療的另類輔助療法 (CAM, The Complementary and Alternative Medicine) 逐漸興起，因此，我們嘗試探討樟芝是否具有抑制病理上惡性程度不同的膀 胱癌細胞生長之作用，並進一步希望了解其作用機制，以作為臨床上使用的參考。藉由三株分化程度 不同的膀胱癌細胞株 (RT4, TSGH-8301, 及 T24) 作為實驗組以及另一株老鼠胚胎纖維母細胞 (MEF) 作 為正常細胞對照組，在加入樟芝子實體 (fruit body) 粗萃取物處理後的 24 至 72 小時，利用 MTT 呈色試 劑分析，檢測其細胞存活率；以流式細胞儀分析細胞週期的分佈, 並以西方墨點法針對其細胞週期相 關的蛋白質的表現作分析；最後，則以一些和癌症轉移有關的實驗做功能性的探討。由初步的實驗結 果顯示，不論是淺層侵犯性的膀胱癌細胞 ( 例如 RT4) ，或是深層侵犯性的膀胱癌細胞 ( 例如 T24) ，樟 芝皆有抑制其生長的作用，惟其作用機制則因細胞不同而有所差別。 RT4 細胞經 100 ? 慊 /mL 樟芝粗萃 取物處理 24 小時後，和 G1 週期相關的 p21 蛋白質表現明顯增加，同時，磷酯化的 Rb 蛋白質表現則逐漸 減弱，此種變化，由於並無 p53 蛋白質的參與，應屬於細胞複製性的靜止現象 (replicative senescence) ； 另一方面， TSGH-8301 和 T24 細胞株以 50 μg/mL 樟芝粗萃取物處理 48 小時後，其和 G2 週期相關的 Cdc2 及 cyclin B 蛋白質表現均逐漸減弱，且其移轉能力也被抑制，但正常的 MEF 細胞則未受樟芝粗萃取物 之影響。 總而言之，對於復發性高或惡性轉移能力高的膀胱癌細胞，樟芝皆可能有其臨床醫療上之價值。

Bladder cancer has been cited to result from the neoplastic lesion with environmental and/or occupational factors identified as causatives. Among which the transitional cell carcinoma (TCC) is the most common. The mortality of TCC has now become the second top among the cancers of male urinary system, and overall, it is the top sixth cancer mortality in Taiwan, and still continuing to increase. Antrodia camphorata (Polyporaceae) (AC), a unique mushroom that grows merely on the inner bark of a characteristic species of camphor tree called Cinnamomum kanehirai, is a very rare and valuable ecologically protected plant. Since long ago, it has been adopted for treatment of intoxication, diarrhea, abdominal pain, hypertension, and hepatoma. Recently the development of the so-called the Complementary and Alternative Medicine (CAM) has strongly attracted us to investigate the potential of anti-bladder cancer of AC. Tests were performed to check whether AC is capable of inhibiting the growth of bladder cancers of different stages, in addition, to understand the action mechanism of AC to ensure an effective and safe clinical use in the future. In this present study, we used three bladder cancer cell lines (RT4, TSGH-8301, and T24) and the mouse embryonic fibroblast cells (MEF) as the control cells. After treatment with the crude extracts obtained from the fruit bodies of AC for 24 and 48 h respectively, the cell viability was examined by MTT assay. Flow cytometric and Western blot analyses were conducted to study the cell population during the cell cycle and the expression of cell-cycle-related proteins. Migration capability of cancer cells affected by AC treatment was also determined by wound scratch and transwell analyses. Results from this study indicate that AC is capable of inhibiting all type of bladder carcinoma cells, despite of lower (e.g. RT4) or higher (e.g. T24) invasiveness. However, the action mechanisms are different among these cells. After treated with crude extract of AC (ACCE) at a concentration of 100 μg/mL for 24 h, the expression of G1 phase associated p21 protein was significantly up-regulated in RT4 cells In the meanwhile, the phosphorylated Rb was steadily down regulated in this high invasive cells. Being not participated by the protein p53, such a phenomenon was ascribed due to a phenomenon called replicative senescence. In contrast, after treated with ACCE at a concentration of 50 μg/mL for 48 h, Cdc2 and cyclin B, the proteins associated with the G2 phase in cells TSGH-8301 and T24, were all gradually downregulated. At the same time, the migration capability of these low invasive cells was completely suppressed. As a control, the MEF cells were totally unaffected. In conclusion, the mushroom Antrodia camphorata might be effective for inhibiting all bladder cancer cells despite of their invasiveness, implicating its potential clinical application. Characterization of Therapeutic Potential and Action Mechanism of Antrodia camphorata on Human Bladder Cancers through Cancer Cell Lines