| 摘要: | 樟芝(學名Antrodia cinnamomea；舊稱Antrodia camphorata)是生長在臺灣深山森林中的一種菇菌，價格非常昂貴，相傳具有解毒及抗癌之功效。我們研究證實樟芝菌絲體及發酵液具有許多生理活性(許游章等人，2002-2006 共計)，包括：(a)樟芝可以保護紅血球產生之過氧化作用。(b)樟芝保護LDL 避免受氧化作用。(c)樟芝發酵液具抗發炎功效。(d)樟芝對腫瘤細胞生長有抑制能力。(e)樟芝可以抑制AAPH 對內皮細胞傷害。近年來保健食品的開發具本土性樟芝甚受注目，樟芝可能具有的生物藥理活性與潛在臨床應用價值。我們將樟芝菌株利用現代生物栽培技術之液態深層發酵培養樟芝，將是最經濟、最符合環保的人工培育法。我們過去研究發現，樟芝會促進雌激素依賴性乳癌細胞(estrogen-responsive MCF-7 breast cancer cells)之細胞凋亡。因此，本研究計畫希望能針對食藥用菇－樟芝對多種人類乳癌細胞株(例如MDA-MB-231, AU565 and BT474)降低癌症發生率及可能分子機制之深入探討；並研究樟芝是否可以輔助治療癌症的可能性。實驗項目包括：第一年計畫：樟芝對人類乳癌細胞COX-2/NFκB 與細胞凋亡之影響及機制探討樟芝對多種人類乳癌細胞株(例如MDA-MB-231, AU565 and BT474 等)：(a)細胞死亡數目(Cell Cytotoxicity) 及型態變化(Morphological changes) 。(b) 染色質濃縮集結(Chromatin condensation)。(c)DNA 階梯形狀光帶(DNA ladder)。(d)以Western blotting 與電泳遷移率變動實驗（Electrophoretic mobility shift assay-EMSA）分析細胞凋亡蛋白質(NF-kB、COX-2、 PARP、Bcl-2、Bax、Cyt c、Caspase-3、Caspase-8 及Caspase-9 等)的表現。第二年計畫：樟芝對人類乳癌細胞訊息傳遞系統(MAPKs)與細胞週期之影響及抗癌機制探討本年度將延續第一年之結果， 利用西方點墨法等方法去檢測腐植酸誘導細胞週期 (Cyclin/cdk/P53/P21/P27 等)、MAPK 訊息傳遞系統(JNK、p38 與ERK pathways)與HER-2/neu 乳癌基因蛋白質活性及表現；找出樟芝發酵液所誘導特定的乳癌細胞凋亡，解開樟芝發酵液對抗乳癌之訊息路徑及分子機制? 第三年計畫：樟芝對乳癌細胞移植裸鼠及輔助抗癌藥物治療之影響及機制探討本年度將延續第一年及第二年結果，將In vitro 之結果應用於動物實驗(In vivo)是必要的。研究方向及架構，有測定器官重量，測定腫瘤大小、腫瘤發生率、腫瘤體積及重量，測定乳腺組織之病理變化，免疫組織化學染色測定細胞凋亡及細胞週期蛋白(cyclin/p53/p21 及Bc1-2/Bax 等)之表現，組織切片測定細胞凋亡(DNA fragementation/TUNEL assay)等。分析比較的項目包括：(1) 實驗動物於樟芝發酵液治療後活存率是否改變(2)比較各組動物腫瘤大小的變化，以確定樟芝發酵液對腫瘤的生長是否有抑制作用(3)觀察不同劑量的樟芝發酵液是否與抑制腫瘤生長的能力呈現正相關性(4)藉由組織病理學的檢查以瞭解不同劑量的樟芝發酵液對體內各組織的傷害。(5) 樟芝發酵液促使活體腫瘤細胞凋亡的探討(Bax 表現及DNA fragementation)。(6) 評估樟芝發酵液在活體內對乳癌細胞週期素蛋白質(cyclin/P53/p21/Bc1-2/Bax 表現)的影響。
Antrodia cinnamomea (Antrodia camphorata) is well known in Taiwan as a traditional Chinese medicine, and it has been shown to exhibit antioxidant and anticancer effects. In our previous study, A. cinnamomea was used for the inhibition of AAPH-induced oxidative hemolysis and lipid/protein peroxidation of normal human erythrocytes. A. cinnamomea in submerged culture protects low-density lipoproteins against oxidative modification and may provide effective protection from atherosclerosis. Moreover, cultured human endothelial cell damage induced by AAPH was suppressed by A. cinnamomea. Our previous results also indicated that A. cinnamomea inhibits LPS induction of cytokine, inducible nitric oxide synthase (iNOS) and COX-2 expression by blocking NF-κB activationin RAW 264.7 macrophages. Interestingly, A. cinnamomea exhibits significant apoptotic cell death against estrogenresponsive MCF-7 cells and premyelocytic leukemia (HL-60) cells. The effects were observed in MCF-7 cells and leukemia HL-60 cells, but not in healthy breast cells (HBL100), erythrocytes, and human umbilical vein endothelial cells. Despite the fact that many tumors initially respond to chemotherapy, breast cancer cells can subsequently survive and gain resistance to the treatment. In this study, the effects of the fermented broth of A. cinnamomea harvested from submerged cultures on human breast cancer cells (MDA-MB-231, AU565 and BT474 etc.,) was investigated, based on the interesting biological activities reported and their potential clinical application. The aim of this study was to investigate：(1) The effects of A. cinnamomea on apoptosis (Morphological changes, chromatin condensation, and DNA laddering etc), apoptotic factors (NF κB, COX-2, P53 and Bcl-2/Bax etc), and mitochondria damage/death receptor pathways (Cytochrome c, PARP, and casapase-3,-8,-9) in MDA-MB-231, AU565 and BT474 cells etc. (2) The effects of A. cinnamomea on cell cylces (Cyclin/cdk/P53/P21/P27)、MAPK pathways (JNK、p38 and ERK) and HER-2/neu expression in cultured breast cancer cells. (3) The effects of A. cinnamomea on tumor growth and apoptotic index in cultured breast cancer cells (such as MDA-MB-231 and MCF-7) in A. cinnamomea-treated female nude mice. Analysis of the study data may provide evidences that A. cinnamomea may be a potential anti-cancer agent for the treatment of breast cancer, and that it may possess anticancer properties potentially valuable for application in drug products. |
