資料載入中.....
|
請使用永久網址來引用或連結此文件:
http://libir.tmu.edu.tw/handle/987654321/59468
|
| 題名: | 探討新合成1-benzyl indoles 衍生物 21-900 誘導人類血癌細胞凋亡之體外及體內的作用機轉
21-900, a novel synthetic 1-benzyl indoles derivative, induces cell apoptosis in human leukemia in vitro and in vivo |
| 作者: | 魏于傑
Wei, Yu-Chieh |
| 貢獻者: | 蕭哲志 |
| 關鍵詞: | 去乙醯化酶;微管;細胞凋亡;白血病;癌症
HDAC;microtubule;apoptosis;leukemia;cancer |
| 日期: | 2015-06-23 |
| 上傳時間: | 2020-08-28 15:18:32 (UTC+8) |
| 摘要: | 惡性腫瘤為造成國人死亡的主要原因之一。根據衛生福利部國民健康署統計癌症為十大死因之首有三十二年之久，當癌症無法以外科手術根治時，化學療法和標靶藥物治療即變成重要的治療方法。本研究的目的是探討新合成的雙重標靶pan-HDAC與Tubulin抑制劑21-900小分子藥物對於人類血癌細胞株之抗癌活性，並進一步探討in vitro以及in vivo 的藥理機轉。本實驗一開始先將這系列1-benzyl indoles藥物利用了SRB assay 及MTT assay 作用在人類多種癌細胞株中，結果可以看到 21-900 在多株癌細胞中抑制的效果最好，其中在血癌 (HL-60) 有更好的敏感度。接著利用MTT assay (3-[4,5-dimethythiazol-2-yl]-2,5-diphenyltetrazolium bromide) 的方式來評估21-900對於HL-60以及MOLT-4細胞毒殺的影響，發現在給予21-900 48小時後具有濃度相關性抑制細胞生長作用，其 IC50 分別為133 ± 4 nM及262 ± 14 nM。21-900亦具有抑制 HDACs 活性，其 IC50 為 610 nM。進一步利用 HDAC isoenzyme inhibition assay發現21-900抑制 HDAC6的活性 (IC50 = 64.5 nM) 最高，其對 HDAC1 (IC50 = 573.4 nM) , HDAC2 (IC50 = 1408.1 nM), 和HDAC8 (IC50 = 905.9 nM) 高出10-20倍的抑制能力。接著利用流式細胞儀分析發現，21-900使血癌細胞停滯在G2/M期，並且伴隨著濃度增加促使明顯的Sub G1增加。接著透過細胞外微管聚合實驗及細胞免疫螢光染色，發現21-900的作用類似於vincristine 也會抑制微管聚合，並且使M期指標蛋白MPM2磷酸化增加。另外21-900也會去影響Bcl-2 family 蛋白改變，例如:Bcl-2、Bid及Mcl-1減少，因此影響了粒線體膜通透度改變促使內生性凋亡路徑活化，誘發caspase-3、-7以及PARP的活化，促使呈現濃度相依性之細胞凋亡。接著使用了JNK的抑制劑SP600125發現可以反轉21-900所造成的細胞凋亡現象。最後在小鼠的人類異位移植腫瘤動物模式中證實，21-900可以抑制腫瘤生長且不影響小鼠的體重，腫瘤組織中證實，21-900會活化caspase-3與增加PARP cleavage、增加乙醯化的histone 3和??-tubulin的表現、也增加M期指標蛋白MPM2表現，這些作用的效果與我們在in vitro的結果相符合。結果顯示，21-900在人類白血病細胞株中，具有抑制其HDACs活性且影響了微管的動態平衡造成細胞週期停滯，且能夠使細胞走向凋亡。至今仍尚未有雙重抑制劑在臨床使用，期許21-900能成為治療血癌之候選藥物。
Malignant tumor has been the major cause of death in Taiwan. According to Health Promotion Administration, Ministry of Health and Welfare in Taiwan, cancer has been the first place of ten leading deaths for thirty-two years. When tumors are difficult to be removed by surgery simply, chemotherapies and targeted therapies become important treatments. In order to cure cancer, drug development is an important thing in cancer therapy. The purpose of this study is to evaluate the anti-tumor effects of a novel synthetic HDAC and tubulin dual inhibitor, 21-900, in human leukemia cells, and to further study the mechanism of action in vitro and in vivo. We first determined a series of 1-benzyl indoles as potent anti-cancer drugs which are more cytotoxic to human leukemia HL-60 cells. Among these compounds, 21-900 significant exhibits a cytotoxic effect in human leukemia cancer cell HL-60 and MOLT-4 with an IC50 of 133 ± 4 nM and 262 ± 14 nM, respectively, using the 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. 21-900 also exhibited as a potent HDAC inhibitor with IC50 value of 610 nM. In the HDAC isoenzyme inhibition assay, we found that 21-900 was a pan-HDAC inhibitor but 21-900 displayed selectivity toward HDAC6 (IC50 = 64.5 nM) over HDAC1 (IC50 = 573.4 nM), HDAC2 (IC50 = 1408.1 nM), and HDAC8 (IC50 = 905.9 nM) with 10-20-fold ratio. Cell cycle distribution was determined by flow cytometry, we demonstrated that 21-900 induced arrest of the cell cycle at G2/M phase in HL-60 and MOLT-4 cell line, with the increase of cell distribution at subG1 phase. Then tubulin polymerization assay and immunofluorescence stain suggested that 21-900 was similar to vincristine in causing depolymerization of microtubule. These results showed that 21-900 could affect microtubule dynamic and accompanied by the up-regulation of mitotic marker,MPM2.Further, 21-900 could influenced the Bcl-2 family proteins by decreasing Mcl-1, Bcl-2 and Bid and increasing p-Bcl-2. Moreover, 21-900 induced cell apoptosis in a concentration-dependent manner; it also increased the cleavage of caspase-3 and -7 and poly (ADP-ribose) polymerase (PARP) in HL-60 and MOLT-4 cells Treatment with SP600125 (JNK inhibitor) could reverse 21-900-induced apoptosis.Then 21-900 significantly inhibit HL-60 and MOLT-4 xenograft tumor growth without the loss of body weight in the SCID mice. 21-900 activated caspase-3 and increased the expression of cleavage- PARP, ac-tubulin, ac-H3 and MPM2 in vivo. These data correlated with in vitro results. All these findings suggest that 21-900 could be served as a new candidate in leukemia cancer treatment. |
| 描述: | 碩士
指導教授:蕭哲志
委員:皇甫維君
委員:劉景平
委員:黃聰龍 |
| 資料類型: | thesis |
| 顯示於類別: | [醫學科學研究所] 博碩士論文
|
文件中的檔案:
| 檔案 |
描述 |
大小 | 格式 | 瀏覽次數 |
|---|
| index.html | | 0Kb | HTML | 57 | 檢視/開啟 |
|
在TMUIR中所有的資料項目都受到原著作權保護.
|
