深層發酵樟芝菌絲體乙醇萃取物對人類肺癌及肝癌細胞生長之影響與其作用機轉之探討

Inhibitory mechanisms of the ethanolic extract of mycelia of Antrodia cinnamomea on the growth of human lung cancer and hepatoma cells

游慧娟(Hui-Chuan Yu)

指導教授 ： 沈立言

國立臺灣大學/生物資源暨農學院/食品科技研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.02003

摘要

樟芝（Antrodia cinnamomea）為台灣特有之真菌類，非常稀少且珍貴，主要原因為其只生長在中高海拔之牛樟樹上且其子實體生長非常緩慢，難以人工培養。在傳統上，主要用於治療腹瀉、高血壓及肝癌等，另已有研究指出樟芝子實體具有抗癌及抗氧化活性。但由於子實體過於昂貴，且目前研究指出液態培養之樟芝菌絲體及發酵濾液具有抗癌、保肝、抗氧化、抗發炎與神經保護等作用，所以目前以發展液態培養之樟芝來取代其子實體之用途。此外，由於目前肺癌及肝癌為分別國人惡性腫瘤之第一、二位，因此本研究乃探討深層培養之樟芝菌絲體乙醇萃取物（ethanolic extract of mycelia of A. cinnamomea，EMA）處理人類肺癌（A549）及肝癌（SK Hep-1及HA 22T/VGH）細胞生長影響及其作用機轉。結果顯示，EMA可抑制A549、SK Hep-1（p53-positve）及 HA 22T/VGH（p53-deletion，HBsAg [+]）細胞生長，在處理48小時後，IC50分別為30.9、48.2及117.1 μg/ml。進一步以流式細胞儀分析EMA對於此三株細胞生長之抑制情形，結果顯示細胞週期分別停滯於S期（A549，40 μg/ml）、G0/G1期（SK Hep-1，20 μg/ml）及S期（HA 22T/VGH，160 μg/ml）。此外，以EMA處理A549 （80 μg/ml）、SK Hep-1 （80 μg/ml）及 HA 22T/VGH（160 μg/ml）24小時後，細胞皆進行細胞凋亡（apoptosis）（sub G1期增加）， DNA電泳結果也顯示DNA呈現片段化（DNA ladder）。此外，以螢光顯微鏡檢測結果顯示，EMA誘導此三株細胞進行細胞凋亡與caspase-3的活化有關。西方墨點法的結果亦顯示以EMA分別處理此三株細胞後，會促使細胞中caspase-3、caspase-9及PARP蛋白的表現量增加，抑制Bcl-2蛋白的含量，使得細胞中的Bcl-2/Bax的比例減少，誘導細胞走向細胞凋亡的現象。綜合以上結果顯示EMA是經由粒線體路徑來調控細胞凋亡相關蛋白之表現，進而誘導此三株細胞進行細胞凋亡。

關鍵字

樟芝 ； 發酵 ； 菌絲體 ； 細胞週期停滯 ； 細胞凋亡 ； 肝癌 ； 肺癌

並列摘要

Antrodia cinnamomea is well-known as a traditional Chinese medicine in Taiwan. Because the growth rate of the natural A. cinnamomea in the wild is very slow, it is difficult to be cultured in green house. It is very expensive to obtain its fruiting bodies. Additionally, it might be a possible and economical way to utilize the submerged fermentation to produce the product of A. cinnamomea with stronger bioactivity. Previous researches have shown A. cinnamomea has many biological activities, such as anti-cancer activity, hepatoprotection, anti-oxidation, anti-inflammation, neuroprotection, and so on. In addition, lung and liver cancer are very common malignant tumors in Taiwan. This study was to investigate the effect of ethanolic extract of mycelia of A. cinnamomea (EMA) in submerged fermentation on the human lung (A549) and liver (SK Hep-1 and HA 22T/VGH) cancer cell lines and their possible mechanisms. The results showed that EMA inhibit the cell viability of A549, SK Hep-1 (p53-positive) and HA 22T/VGH (p53-deletion, HBsAg [+]) for 48hr, and their IC50 value were 30.9、48.2 and 117.1 μg/ml, respectively. The results of cell cycle analysis showed that EMA induced cell cycle arrest in S (A549, 40 μg/ml), G0/G1 (SK Hep-1, 20 μg/ml) and S (HA 22T/VGH, 160 μg/ml) phase. Furthermore, EMA induced apoptosis (accumulation of sub G1 phase) in A549 (80 μg/ml), SK Hep-1 (80 μg/ml) and HA 22T/VGH (160 μg/ml), and the DNA ladder were presented in these three cell lines by DNA electrophoresis. According to the results of fluorescence microscope, it indicated that EMA induced A549, SK Hep-1 and HA 22T/VGH apoptosis through induction of caspase-3 activity. It also indicated that EMA increased caspase-3 and -9 and PARP proteins expression, and decreased the Bcl-2 protein expression and the ratio of Bcl-2/Bax of A549, SK Hep-1 and HA 22T/VGH. In conclusion, EMA could induce these three cell lines apoptosis through mitochondrial pathway by regulating the expression of apoptosis-related proteins.

並列關鍵字

Antrodia cinnamomea ； fermentation ； mycelia ； cell cycle arrest ； apoptosis ； hepatocellular carcinoma ； lung cancer

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