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研究生： 吳幸芷
Hsing-Chih Wu
論文名稱： 傳統中草藥爵床純化物爵床素A對肝癌細胞產生細胞自噬的效果
The autophagic effect of Justicidin A purified from traditional Chinese medicine Justicia procumbens in human hepatocellular carcinoma cells
指導教授： 蘇純立
Su, Chun-Li
學位類別： 碩士
Master
系所名稱： 人類發展與家庭學系
Department of Human Development and Family Studies
論文出版年： 2013
畢業學年度： 101
語文別： 中文
論文頁數： 106
中文關鍵詞： 爵床素A 、人類肝癌細胞 、細胞自噬 、細胞凋亡 、Sorafenib
英文關鍵詞： Justicidin A, human hepatocellular carcinoma, autophagy, apoptosis, Sorafenib
論文種類： 學術論文
相關次數： 點閱：160  下載：6 
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    • 中文摘要
    　　行政院衛生署100年統計資料顯示，肝癌在台灣癌症死因中排名第二位，全球癌症死因當中，肝癌排名第三位。目前FDA認可的肝癌標靶藥物Sorafenib並未有效治療肝癌，對正常細胞毒性也很大，病人耐受度較差，而肝癌患者的抗藥性也造成目前的藥物無法有效治療肝癌，因此需要找尋新藥物。爵床素A是傳統中草藥爵床中萃取出來的純化物，我們研究團隊在之前研究中已發現爵床素A對人類正常周邊血細胞的毒性很低，但對於癌細胞，如：大腸癌細胞株以及肝癌細胞株會誘發細胞凋亡，在動物實驗上也發現可抑制腫瘤細胞生長。近期研究發現細胞自噬與細胞凋亡間有許多蛋白會相互影響，細胞自噬可能會促進或抑制細胞凋亡，而影響癌症治療效果。因此，本研究將探討爵床素A對於人類肝癌細胞是否能誘發細胞自噬，並了解細胞自噬與細胞凋亡之間的關係。實驗使用acridine orange染色，以流式細胞儀分析自噬溶酶體產生比例確認自噬現象，發現自噬溶酶體比例與時間及劑量呈正相關。以西方墨點法檢測自噬小體上LC3、p62及溶酶體上LAMP2a蛋白表現量，觀察自噬小體的形成與降解時間點，發現在爵床素A 1 M濃度下作用24小時，LC3-II與LAMP2a的表現量即明顯上升，到96小時p62蛋白表現量與24小時相比明顯降低。利用細胞自噬抑制劑Bafilomycin A1(BAF)前處理抑制細胞自噬，以西方墨點法檢測發現有細胞自噬抑制劑前處理的組別，LC3、p62、LAMP2a表現量較未加抑制劑前處理的組別多，並由共軛焦顯微鏡發現在未加入抑制劑前處理的組別有較多LC3與LAMP2a colocalization的puncta。顯示爵床素A能誘發Hep 3B細胞自噬，並完成細胞自噬流程。為探討細胞自噬與細胞凋亡之間的關聯，利用BAF及細胞凋亡的抑制劑pan-caspase inhibitor zVAD-fmk前處理Hep 3B細胞，以流式細胞儀與西方墨點法分析發現加入BAF前處理的組別，sub-G1比例及活化型caspase 3表現量增加，而有無加入zVAD-fmk前處理不影響LC3-II、p62、LAMP2a的表現量，顯示爵床素A所誘發的細胞自噬會抑制細胞凋亡但細胞凋亡不影響細胞自噬。利用西方墨點法發現爵床素A會誘發Raf/MEK/ERK路徑導致細胞自噬，與抑制class I PI3K/Akt/mTOR路徑以及誘發class III PI3K/Beclin 1路徑則無相關。為觀察爵床素A與臨床抗癌用藥Sorafenib合併效用，以細胞毒殺試驗分析發現Sorafenib在臨床使用之10 M濃度下與0.5 M或1M濃度的爵床素A合併使用皆有加成效應。整體而言，本研究顯示爵床素A在臨床應用上可與細胞自噬抑制劑合併使用來增加抗癌效果，也能增加Sorafenib的用藥敏感性，提升醫療品質。

    Abstract
    The 2011 statistics of Department of Health (DOH) shows that human hepatocellular carcinoma (HCC) is the second death of cancer in Taiwan, and is the third death of cancer in the world. Chemotherapy is the traditional choice for advanced HCC, but the FDA-approved targeted therapy Sorafenib limits the therapeutic effect due to its intolerable side effects to patients. Thus, to develop new drugs for HCC is urgent. Justicidin A (JA) is a purified compound isolated from Justicia procumbens. Our previous published report indicates that JA induced apoptosis of human HCC cells in vitro and in vivo. Recently, many reports indicate that there are interactions between autophagy and apoptosis, and the autophagic effect induced by anticancer drugs may inhibit or promote apoptotic cell death. The present study is proposed to investigate if JA can induce autophagy of HCC and to find out how JA-induced autophagy influences the process of JA-induced apoptosis. When autophagy was induced in cells, acidic vescular organelles (AVOs) produced in cells. By staining cells with acridine orange, induction of autophagy can be investigated. In the present study, JA increased the percentages of AVOs-positive cells in a time- and dose-related manners using flow cytometry. The expressions of LC3-II and LAMP2a increased as the incubation time increased, but that of p62 decreased between 24 to 96 h. To confirm the process of autophagy flux, Hep 3B cells were pretreated with or without autophagy inhibitor Bafilomycin A1 (BAF) and then treated with JA. The increase in protein expression of LC3-II, p62 and LAMP2a using Western blot and the decrease in colocalization of LC3 and LAMP2a puncta using confocal microscopy in BAF-pretreated group confirm that JA not only induced autophagy but also completed the autophagy flux. In order to analyze the interaction between autophagy and apoptosis, Hep 3B cells were pretreated with BAF or pan-caspase inhibitor zVAD-fmk to inhibit autophagy and apoptosis, respectively. In BAF-pretreated Hep 3B cells, JA elevated the percentages of cells at sub-G1 phase and raised the expression of cleaved caspase 3. However, in zVAD-fmk-pretreated Hep 3B cells, no changes in the expression of autophagy-related LC3-II, p62 and LAMP2a proteins was observed, suggesting that using autophagy inhibitor promoted JA-induced apoptosis and JA-induced apoptosis didn’t affect JA-induced autophagy. Moreover, to investigate the molecules involved in JA-induced autophagic pathway, we observed that JA-induced autophagy was via activation of Raf/MEK/ERK pathway, and was not associated with inhibition of class I PI3K/Akt/mTOR or induction of class III PI3K/Beclin 1 pathway by western blot. To examine if addition of JA promotes chemosensitivity of Sorafenib, cell growth inhibition was analyzed using MTT assay. Addictive effect was found when cells were treated with 10 M Sorafenib, the dosage uses for patients, in combination with 0.5 or 1 M JA, suggesting the benefit of using JA in addition to Sorafenib. Taken together, our data suggest that the use of autophagy inhibitor should be taken into consideration to enhance the anticancer effect of JA and addition of JA promotes the chemosensitivity of FDA-approved targeted therapy Sorafenib.

    目錄 第一章　緒論 1 第一節　肝癌 1 一、肝癌的流行與發生 1 二、肝癌治療 2 第二節　爵床素A 5 第三節　計畫性細胞死亡 7 一、細胞凋亡 7 二、細胞自噬 10 第二章　研究目的 14 第三章　材料與方法 16 第一節　實驗藥品與試劑 16 第二節　儀器與實驗耗材 19 第三節　實驗方法 23 一、細胞株繼代培養、解凍及保存 23 二、藥物配製 26 三、細胞存活率分析 26 四、細胞自噬比例分析 27 五、細胞週期比例分析 28 六、西方墨點法 30 七、免疫螢光染色法 36 八、統計分析方法 39 第四章　結果 40 第一節　JA能誘發Hep 3B 細胞自噬現象 40 第二節　抑制細胞自噬能促進JA毒殺Hep 3B細胞及細胞凋亡，而抑制細胞凋亡不影響JA所誘發的細胞自噬 51 第三節　JA所誘發的細胞自噬與MEK/ERK路徑的活化有關 61 第四節　JA與臨床藥物Sorafenib合併使用對於肝癌細胞的影響 72 第五章　討論 77 第六章　結論 85 第七章　參考文獻 86 附錄 103 圖次 Fig. 1 Effect of JA on autophagy of HCC Hep 3B cells. 44 Fig. 2 JA significantly induced LC3-II expression, and the decrease of p62 at 96 h compared to 24 h suggested the process of autophagy flux. 45 Fig. 3 BAF increased JA-induced LC3-II expression in Hep 3B cells. 47 Fig. 4 BAF increased JA-induced p62 expression in Hep 3B cells. 48 Fig. 5 BAF increased JA-induced LAMP2a expression in Hep 3B cells. 49 Fig. 6 BAF dcreased JA-induced colocalization of autophagosomal and lysosomal markers. 50 Fig. 7 BAF increased JA-induced cell growth inhibition in Hep 3B cells. 55 Fig. 8 BAF increased JA-induced sub G1 percentage in Hep 3B cells. 56 Fig. 9 BAF increased JA-induced cleavage caspase 3 expression in Hep 3B cells. 57 Fig. 10 zVAD-fmk decreased JA-induced cleavage caspase 3 expression in Hep 3B cells. 58 Fig. 11 zVAD-fmk didn’t affect JA-induced LC3-II expression in Hep 3B cells. 59 Fig. 12 zVAD-fmk didn’t affect JA-induced p62 expression in Hep 3B cells. 60 Fig. 13 zVAD-fmk didn’t affect JA-induced LAMP2a expression in Hep 3B cells. 61 Fig. 14 Expression of Atg12-Atg5 and Atg5 in JA-treated Hep 3B cells. 65 Fig. 15 Expression of Beclin 1and Class III PI3K in JA-treated Hep 3B cells. 66 Fig. 16 Expression of total mTOR and phospho-mTOR in JA-treated Hep 3B cells. 67 Fig. 17 Expression of total Akt and phospho-Akt in JA-treated Hep 3B cells. 68 Fig. 18 Expression of total class I PI3K and phospho-class I PI3K in JA-treated Hep 3B cells. 69 Fig. 19 Expression of pan-Ras in JA-treated Hep 3B cells. 70 Fig. 20 Expression of total MEK and phospho-MEK in JA-treated Hep 3B cells. 71 Fig. 21 Expression of total ERK and phospho-ERK in JA-treated Hep 3B cells. 72 Fig. 22 Effect of Sorafenib with or without JA on Hep 3B cells viability determined by MTT assay and the interaction of sorafenib and JA determined by the value of q. 76 Fig. 23 Effect of Sorafenib with or without JA on Hep 3B cells viability determined by MTT assay and the interaction of sorafenib and JA determined by the value of q. 77

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