- 學位論文
探討單獨使用新穎化學合成物Yao-ram-2-7或合併使用薑黃素對肝癌細胞的抑制並利用連接網路資料庫比對及證實Yao-ram-2-7之生理功能
To study anticancer effect of a novel compound Yao-ram-2-7 in the presence or absence of phytochemical curcumin on human hepatocellular carcinoma cells and to identify other biological function of Yao-ram-2-7 using Connectivity Map
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摘要
肝癌是世界五大癌症之一，因不易早期發現及預後不佳，使得肝 癌在癌症所致之死亡率高居不下。根據行政院衛生署統計，肝癌一直 是國人癌症死因的大宗。治療肝癌方式，以手術和化學治療為主。在 化學治療方面，藥效常局限於抗藥性的產生使藥效減低，因而促使研 發更有效的新穎藥物。在本研究團隊自行合成一系列化合物中經 MTT assay 篩選，發現 Yao-ram-2-7 具有抑制人類肝癌細胞（Hep 3B） 生長的效用。在本研究中，Yao-ram-2-7 與臨床肝癌標靶用藥 Sorafenib 在相同的實驗條件下比較，分別處理 Hep 3B 及人類臍靜脈內皮細胞 （HUVEC）。發現 Yao-ram-2-7 與 Sorafenib 毒殺 Hep 3B 能力相當， 處理 HUVEC 的安全性測試中，Yao-ram-2-7 毒性較 Sorafenib 弱，顯 示 Yao-ram-2-7 可能對人體的副作用較小。利用 propidium iodide 染色分析細胞凋亡現象及以 acridine orange 染色分析細胞自噬現象，發現 Yao-ram-2-7 誘發 Hep 3B 產生細胞凋亡及細胞自噬的比例隨時間和劑 量增加而增加；利用西方墨點法也確定活化態 caspase 3 蛋白及 LC3-II 蛋白的表現增加，證實 Yao-ram-2-7 可誘發 Hep 3B 產生細胞凋亡及 細胞自噬。細胞凋亡實驗結果顯示，相較於 Sorafenib 在高劑量下才 具有毒殺癌細胞能力，Yao-ram-2-7 在較低劑量下即有效果；細胞自 噬實驗發現，Yao-ram-2-7 比 Sorafenib 在較低劑量下即可引發細胞產生自噬現象。因營養素可能具有輔助藥物效用，因此本研究也探討具 有抗癌功用的天然植化素—薑黃素（Curcumin）與 Yao-ram-2-7 或 Sorafenib 合併使用下，是否對抑制 Hep 3B 細胞生長有更好的效果。 MTT assay 實驗結果發現 Hep 3B 在 Yao-ram-2-7 與薑黃素同時處理後，抑制細胞生長效果比單獨處理更佳，具有加乘效應；分析細胞週期的 變化，發現合併使用薑黃素有使 G2/M 期增加的趨勢，使癌細胞分裂 減少。但在肝癌標靶藥物 Sorafenib 與薑黃素合併使用則發現產生拮 抗，因此建議臨床使用 Sorafenib 治療的患者不可食用含有薑黃素的 食物及膳食補充品。另外，本研究透過「連接網路資料庫（Connectivity Map；CMAP）」比對，發現 Yao-ram-2-7 可能與 GSK-3 抑制劑 AR-A014418 作用相似。實驗結果證實 AR-A014418 如 Yao-ram-2-7 皆可誘發 Hep 3B 產生細胞自噬，Yao-ram-2-7 可抑制 phospho-GSK-3 及總 GSK-3蛋白表現，顯示 Yao-ram-2-7 的生醫上的其它應用性。 整體而言，本研究結果發現 Yao-ram-2-7 具有開發為抗癌用藥的潛力， 提供肝癌病患治療上不同選擇；與薑黃素合併使用能增加藥物的敏感 性，使化療藥物發揮更好的效果。
並列摘要
Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide. More than 75% cases of HCC occur in the Asia-Pacific region. High mortality of HCC is due to the difficulty in diagnosis and poor prognosis. Chemotherapy is a traditional choice for inoperable HCC, whereas drug resistant limits the therapeutic effect. Thus, there is an urgent need to develop new potential drugs for HCC. Our research group has synthesized a series of compounds for anti-cancer screening using MTT assay. Yao-ram-2-7 is one of them significantly inhibits the growth of HCC Hep 3B cells. Especially, Yao-ram-2-7 displays less cytotoxicity on normal human umbilical vein endothelial cells than the HCC targeted therapy Sorafenib, suggesting Yao-ram-2-7 is safer than Sorafenib. We further show that Yao-ram-2-7 induces apoptosis of Hep 3B cells in a time- and dose-related manner using propidium iodide staining followed by flow cytometry. Increase of cleavage-caspase 3 expression is observed using Western blotting. Yao-ram-2-7 also induces autophagy of Hep 3B cells characterized by the accumulation of acidic vesicular organelles by flow cytometry after staining the cells with acridine orange. Western blot analysis further observed the conversion of autophagy marker from LC3-I to LC3-II. Compared with Sorafenib, Yao-ram-2-7 induces apoptosis and autophagy at a relatively lower dosage for a shorter period of time. Recently, anticancer and chemopreventive effects of phytochemicals such as curcumin have been suggested. In the present study, combination of Yao-ram-2-7 with curcumin promotes growth inhibition of Hep 3B cells and produces an additivity effect. Cell cycle analysis suggests that the decrease in tumor cell proliferation is due to an increase of G2/M arrest. In contrast, addition of curcumin to Sorafenib displays an antagonism effect, suggesting that patients treated with Sorafenib should avoid food and supplements containing curcumin. In addition, we discover that a GSK-3 inhibitor AR-A014418 and Yao-ram-2-7 have similar biological functions since AR-A014418 alters gene expression of Hep 3B cells similarly to Yao-ram-2-7 by using a bioinformatics database Connectivity Map (CMAP). Western blot and flow cytometric analysis confirm that Yao-ram-2-7 behaves like AR-A014418, inducing autophagy and decreasing protein expression of phospho-GSK-3and total GSK-3 These data demonstrate that query gene expression profiles using CMAP is a useful shortcut to reveal molecular action of a small chemical compound. Taken together, our data suggest chemotherapeutic potential of Yao-ram-2-7 on HCC, and addition of curcumin further promots its chemosensitivity.
並列關鍵字
Yao-ram-2-7 ； human hepatocellular carcinoma ； apoptosis ； autophagy ； curcumin ； Sorafenib ； Connectivity Map參考文獻
Curcumin enhances cytotoxicity of chemotherapeutic agents in prostate cancer
evaluating the effectiveness of drug combinations and the revision of Burgi's
Abounit, K., Scarabelli, T. M., & McCauley, R. B. (2012). Autophagy in mammalian
cells. World J Biol Chem, 3(1), 1-6.
Design and synthesis of novel platelet fibrinogen receptor antagonists with
延伸閱讀
- 鄭弘萱（2009）。探討乙醯基半胱胺酸對薑黃素抑制A549肺癌細胞中終端酶活性之影響〔碩士論文，中山醫學大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0003-3007200914253400
- 陳聖美（2005）。對乙醯氨基酚與薑黃素使用於肝腫瘤細胞Hep G2之細胞毒性研究〔碩士論文，亞洲大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0118-0807200916271325
- 周鳳英、戚謹文、衛元耀、蔣淑娟、謝明學、雷永耀（1997）。Ho-166-Acetylacetonate-Lipiodol核醫藥物研制及其做爲肝癌體內輻射治療之細胞實驗評估。放射治療與腫瘤學，4(3)，185-193。https://doi.org/10.6316/TRO/199704(3)185
- 陳尚球、陳懋良、魏如東（1956）。Studies on the Intake, Blood Level and Urinary Excretion of Vitamin A, Carotene, Thiamine, Riboflavin and Ascorbic Acid in Chinese Men. I. Dietary Survey and Plasma Levels of Vitamin A and Carotene。化學，(45年2)，67-74。https://doi.org/10.6623/chem.1956010
- 黃中岳（2012）。1. The effect of aqueous extract of Prunella vulgaris in suppressing invasion and migration in human non-small cell lung cancer cells. 2. Identification of novel small chemicals with autophagic clearance of polyglutamine aggregation in human neuroblastoma cells.〔碩士論文，國立臺灣師範大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0021-1610201315295460