鴻喜菇蛋白質對人類肝癌細胞 (HepG2) 生長抑制及 細胞週期停滯之研究

Growth Inhibition and Cell Cycle Arrest in Human Hepatoma HepG2 Cells by Protein HM-3 from Hypsizigus marmoreus

張雅筑(Ya-Chu Chang)

指導教授 ： 吳瑞碧

國立臺灣大學/生物資源暨農學院/食品科技研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.01642

摘要

肝癌為近年來國人癌症死因中第二位，目前常用的治療方法有手術切除、肝臟移植、栓塞治療以及化學藥物治療等，但皆有其侷限性或副作用；因此，由天然物開發有效之抗癌藥物已成為重要的課題。鴻喜菇 (Hypsizigus marmoreus) 為低熱量、低脂肪且營養價值高的食用菇，目前研究結果顯示其具有抗腫瘤、抗真菌、抗氧化等功能。本研究將鴻喜菇以 40~80% 飽和度之硫酸銨沉澱得蛋白質粗萃物，再以陰離子交換樹脂 DEAE-Sepharose CL-6B 經連續鹽梯度或階梯鹽梯度溶離收集樣品。以 0~1 M連續鹽梯度所得樣品 HM-3 及階梯梯度 0.2 與 0.4 M 鹽濃度溶離之樣品 ( fraction Ⅰ、Ⅱ) 對 HepG2 進行生長抑制測試，經 50 µg/ml 之 HM-3 處理 12 及 24 小時得到的生長抑制率分別為 87.41% 及 88.25%，且生長抑制具有濃度效應。以 50 µg/ml 之 fraction Ⅰ 對 HepG2 處理 12 及 24 hr 的抑制率分別為 24.41% 與 19.44%，而 fraction Ⅱ 則無抑制效果。階梯梯度樣品中以 fraction Ⅰ 抑制效果為佳，但仍較 HM-3 差，故後續探討生長抑制機制皆以 HM-3 為樣品。由流式細胞儀分析細胞週期結果顯示，以 HM-3 處理 24小時，會使 HepG2 出現細胞週期停滯 (cell cycle arrest)，當濃度由 0 提升至 200 µg/ml，G1 phase 比例由 54.35% 提高至 69.08%，但並無細胞凋亡之現象。以瓊脂糖膠體電泳檢測 DNA 完整性，也未觀察到細胞凋亡特徵之ㄧ的 DNA 片段化。利用西方墨點法檢測 G1 細胞週期停滯相關蛋白 p53、p21、p27、cyclin A2、cyclin D1、cyclin E1 及 CDK2 之表現，發現隨處理濃度提高，p53 及 p21 表現量皆有增加的現象，p21 表現量與控制組相比具顯著差異。cyclin E1 表現量與控制組相比有顯著下降的現象，而 p27、cyclin A2、cyclin D1 及 CDK2 表現量則無明顯變化。根據以上結果推測，HM-3 能抑制 HepG2 生長，並可能透過活化 p53 與 p21 而抑制 cyclin E1 之表現，使其細胞週期停留在 G1 phase。

關鍵字

肝癌 ； 鴻喜菇 ； 細胞週期停滯 ； 細胞凋亡

並列摘要

The mortality rate of hepatocellular carcinoma in all cancer mortality ranks second in Taiwan. There are some common treatments against this disease such as surgical resection, liver transplantation, embolization and chemotherapy. Although these treatments have been carried out for many years, they still have limitations and side effects. Therefore, exploration of effective chemotherapeutic agents from natural food is urgently needed. Hypsizigus marmoreus is a low-calorie, low-fat and high protein concent mushroom. A number of bioactive molecules including antitumor, antifungal and antioxidant substances have been identified from this mushroom. In this research, proteins in H. marmoreus were precipitated by 40~80% saturated ammonium sulfate. DEAE-Sepharose CL-6B ion exchange chromatography was used subsequently, and eluted by 0~1 M NaCl continuous or stepwise gradient. Fraction isolated by continuous gradient named HM-3, and eluted by 0.2 or 0.4 M NaCl stepwise gradient named fraction Ⅰ or Ⅱ. HM-3 at a concentration of 50 µg/ml strongly inhibited the growth of HepG2 cells by 87.41% and 88.25% for 12 and 24 hr respectively. HM-3 inhibited the growth of HepG2 cells in a concentration-dependent manner. Cells were incubated with 50 µg/ml fractionⅠ or Ⅱ for 12 and 24 hr, and results showed that fractionⅠinhibited the growth of HepG2 cells by 24.41% and 19.44%. Fraction Ⅱ had no obvious growth inhibition. Above results indicated that HM-3 had better growth inhibition than others, so we chose HM-3 to investigate the mechanism of growth inhibition. G1 phase arrest was observed by flow cytometry when cells were incubated with HM-3 for 24 hr. G1 phase percentage ranged from 54.35% to 69.08% when concentration increased to 200 µg/ml from 0 µg/ml, but no apoptosis was observed. DNA ladder was not observed by agarose gel electrophoresis. Western blotting analysis showed the expression of p53 and p21 increased in HepG2 cells with the increasing concentration of HM-3. Obvious decrease in cyclin E1 was observed and no apparent change in p27, cyclin A2, cyclin D1 and CDK2. According to these results, HM-3 might induce growth inhibition by activating p53 and p21, then inactivating cyclin E1, causing G1 phase cell cycle arrest in HepG2 cells.

並列關鍵字

hepatocellular carcinoma ； Hypsizigus marmoreus ； cell cycle arrest ； apoptosis

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