| Abstract: | Part I: 馬栗樹皮素Esculetin（EST）屬於香豆精（coumarin）衍生物的一種多酚類的天然抗氧化物，近代越來越多關於Esculetin在生物及生化方面的活性研究被提出，最近的研究指出，Esculetin能有效的抑制一些人類血癌與乳癌細胞株的增生，然而其中的詳細分子機制仍未明。太平洋紫杉醇 (Paclitaxel)在1967年由 Taxus brevifolia 的樹皮中提煉出來，至今已被廣泛應用在各種癌症治療上，目前如何降低其對人體的毒性又能維持其臨床治療效果，一直是熱門的研究方向。而現在Paclitaxel與其他agents搭配治療有可能加強Paclitaxel的治療效果，減低用藥的劑量，達到副作用及抗藥性降低的功效。本研究就是將Esculetin與Paclitaxel搭配處理下發現，Esculetin可加強Paclitaxel所引起HepG2細胞凋亡的現象，因此再進一步探討其詳細的分子機制。首先發現以Paclitaxel單獨處理HepG2細胞時，會活化其JNK及ERK兩條路徑；在將三種MAPK Kinase 抑制劑(PD98059、SB203580、SP600125)、Esculetin分別與Paclitaxel共同處理下，經由Western blotting及flow cytometry等實驗證實，JNK的活化Paclitaxel誘導HepG2細胞凋亡時所必需，而Paclitaxel附帶活化的ERK pathway，在出現ERK抑制劑 (PD98059、Esculetin)後，則有加強細胞凋亡的現象，顯示Esculetin所加強Paclitaxel引起HepG2細胞凋亡的現象可能來自於Esculetin抑制了ERK這條一直與細胞增生或抗凋亡有關的路徑，而加強Paclitaxel透過JNK活化使細胞走向JNK-dependent apoptosis之路徑。
Part II: 人類經過幾十年來的努力不懈，惡性腫瘤的藥物治療已有了巨大的進步。現代科學，特別是生命科學的迅速發展，正在逐步揭開惡性腫瘤中發生本質的秘密，抗腫瘤藥物研究已進入一個新的階段。目前醫界尋找新藥的迫切性逐漸提高，而且在紫杉醇等抗癌藥物的研究成功，顯示從自然界找尋具新作用機制及獨特化學結構的細胞毒性藥物仍有重要意義。兔兒菜(Ixeris chinensis)是民間普遍使用的藥材，有解熱、鎮痛、消炎、退乳癰、治血癌、肝癌之說，由先前本實驗室已發現兔兒菜的萃取物具有良好的抗氧化能力，而本實驗發現兔兒菜的某些層萃取物對一些癌細胞株具有細胞毒性，再進一步進行成分及活性分析發現，存在於兔兒菜中的類黃酮化合物-Luteolin不但有良好抑制HepG2人類肝癌細胞的增生外，在經由流式細胞儀、DAPI核染分析、DNA片段化分析等證明能誘導細胞凋亡的發生。在利用西方墨點法探討Luteolin誘導HepG2肝癌細胞凋亡時的蛋白發現，除了 CPP32有明顯活化且其受質PARP (poly [ADP-ribose] polymerase)也出現被分解的結果，細胞凋亡之中上游蛋白，如：cytochrome C在細胞質表現上升；Bax，Bak等促進細胞凋亡的成員在粒線體中明顯增加；另外，重要的p53、MAPK kinase蛋白也有活化的現象。綜合以上結果，兔兒菜除了有抗氧化能力之外，還能抑制HepG2肝癌細胞增生，而從兔兒菜鑑定分離出來的Luteolin的抑制效果更佳，甚至還能誘導細胞凋亡，顯示兔兒菜兼具保健與治療的功能，值得進一步開發研究。
[ 英文摘要 ]
Part I: Esculetin (EST) is a phenolic natural antioxidant of coumarin derivative. In modern times, there are more and more researches about the biological and biochemistry activity of Esculetin. Latest finding is Esculetin can inhibit the survival of human lukemia and breast cancer cells. However, the antiproliferation mechanism of Esculetin on cacer is not well understood. Palictaxel was extracted from the bark of Texus brevifolia in 1967 and was applied spreadly on the therapy of cancer. It’s a popular research trend about how to lower the toxicity on humanbody and still keep the clinical therapy effect. The goal to lower the dosage, side effect and drug resistance might be achieved by a combination treatment of Paclitaxel and other agents. In this study we treated Esculetin and Palitaxel together and had a finding that Esculetin can enhance the effect of Paclitaxel-induced apoptosis. Base on the finding, we were going to discuss it’s molecular mechanism. We exposed Paclitaxel alone on HepG2 cell and JNK and ERK pathways were activated. Three kinds of MAPK kinase inhibitors (PD98059, SB203580, SP600125) and Esculetin were added with Palitaxel. This experiment confirmd that activities of JNK was necessary for Palitaxel to induce the apoptosis of HepG2 cell by Western blotting and flow cytometry. Howevr, when ERK inhibitors (PD98059 and Esculetin) was added, the apoptosis enhanced by the decrease of ERK activation. It demonstrated that Esculetin can enhance the effect of Paclitaxel-induced apoptosis in HepG2 cells by inhibiting the antiapoptotic ERK pathway. Therefore JNK-dependent apoptosis could react much completely in the cells.
Part II: After human striving for decades, there is a huge progress on the medicine therapies of maligmant tumor. Modern science develops speedly especially life science which is probing the secret of carcinogen. It sets a milestone for researches of antitumor drug. The success of antitumor drug like Palictaxel reveals the importance to discover new mechanism and unique chemical structure of cytotoxicity drug from the nature. Ixeris chinensis is a kind of popular herb for bleeding, swelling, inflammatory, and pain. Previous study showed Ixeris chinensis to be well antioxidant. In this study, we found the crude extract of Ixeris chinensis having cytoxicity to tumor cell lines. After analyzed the component and activity, we discovered Luteolin a kind of flavonoid inhibited the growth of human hepatoma cell-HepG2. Taking use of flow cytometry, nuclear stain assay, DNA fragmentation assay, we confirmed that Luteolin induced apoptosis. We found that Luteolin induced HepG2 cells apoptosis through increase expression of CPP32 and its substrate, PARP, had been degraded. The upstream proteins of apoptosis like cytochrome C, Bax, Bak increased distinct and p53, MAPK kinase proteins were activated. There was a conclusion that Ixeris chinensis had antioxidant ability and can inhibit the proliferation of HepG2 cells. Luteolin isolated from Ixeris chinensis got better inhibit effect and it can even induce apoptosis. The result showed Ixeris chinensis had helth protection and cure function that is worth advanced development. |
