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標題: | 探討天然物穿心蓮內酯對癌細胞之影響 The Effects of A Natural Product Andrographolide in Cancer Cells |
作者: | Ya-Yeh Ho 何雅燁 |
指導教授: | 張震東 |
關鍵字: | 穿心蓮內酯,共價藥物,麥可加成反應,NF-κB,YB-1, Andrographolide,covalent drug,Michael addition,NF-κB;YB-1, |
出版年 : | 2015 |
學位: | 碩士 |
摘要: | 天然藥物源於自然界中存在有藥理活性的天然產物。多種從中草藥萃取出的天然藥物都有抗發炎或抗癌的功效;且大部分的天然物帶有親電子性官能基,可與目標蛋白質共價修飾其親核基側鏈。天然物andrographolide萃取自爵床科穿心蓮 (Andrographis paniculata Nees),其有廣泛性的生藥活性,如抗發炎、抗癌、抗菌等。再者研究報告指出andrographolide以中草藥萃取物或其衍生物均為安全性用藥;其在抗發炎方面功效雖被廣泛探討,然詳細藥理機制尚未明朗。NF-κB轉錄因子在細胞增生與凋亡中,是為重要調控者角色;NF-κB在過度活化的狀態下,會造成慢性發炎而提高癌症風險,且調控多種基因表現,所以此訊號傳導路徑常被視為治療疾病標靶對象。我們分析andrographolide的分子結構並推測其可能為一種共價藥物,藉麥可加成反應方式,andrographolide可被半胱胺酸之側鏈攻擊進行麥可加成反應之位置,與蛋白質形成共價鍵結,進而修飾蛋白質。我們以新的方法學,運用抗andrographolide多株抗體,發現以andrographolide處理細胞後,NF-κB會被andrographolide所修飾。利用子宮頸癌細胞以腫瘤壞死因子 (tumor necrosis factor-α) 來誘導NF-κB活化模式中,我們發現先以andrographolide處理過的細胞,NF-κB從細胞質移轉至細胞核會被抑制。因此,我們推測NF-κB上的某些半胱胺酸側鏈被andrographolide修飾之後,可能會影響NF-κB移轉進核以及活化,也推測其它被修飾的蛋白質也一起影響NF-κB活化。另外,我們也偵測到YB-1亦可被andrographolide所修飾;在肺腺癌細胞中,以順鉑 (cisplatin) 引起基因毒性誘導YB-1活化之模式,發現YB-1進核效果亦受andrographolide所抑制。且以轉化生長因子-β1 (transforming growth factor-β1) 去誘導YB-1表現及上皮間質轉化(epithelial-mesenchymal transition) 之模式,andrographolide能以抑制YB-1表現量且調降上皮間質轉化相關因子表現,得以發現andrographolide有抑制癌細胞移行的潛力而有抗癌功效。 Natural compounds are small molecules that are produced by bio-organisms. Many natural products are endowed with electrophilic functional groups that covalently modify nucleophilic residues in specific protein targets. The natural product, andrographolide, is the major active principle isolated from the plant Andrographis paniculata Nees, has been shown with a broad range of biological activities, such as anti-inflammatory, anticancer, and antibacterial. Moreover, both the use of andrographolide in traditional medicine and toxicity data in animals suggest the safety in andrographolide for diversiform clinical conditions. Although some of its anti-inflammatory effects have been investigated, little is known about the mechanisms underlying the pharmacological action of andrographolide. NF-κB transcription factors are critical regulator of apoptosis and proliferation. NF-κB is frequently constitutively activated in patients with chronic inflammatory conditions such as cancer. Accordingly, NF-κB controls multiple genes involved in human diseases rendering the NF-κB signaling pathway a target for therapy. We analyzed the structure of andrographolide and predicted that andrographolide may be a covalent drug. Andrographolide can be attacked by the cysteine residues of the protein through Michael addition to form the covalent bonds. We developed a new methodology by generating anti-andrographolide polyclonal antibody for target identification. Then, we demonstrated that the NF-κB could be modified by andrographolide after the cells were treated with andrographolide. In the TNF-α induced NF-κB activation model, we found that the NF-κB translocation from the cytoplasm to the nucleus can be inhibited in cervical cancer cells. Therefore, we surmised that some cysteine residues of the NF-κB are crucial for the NF-κB translocation and activation. On the other hand, we also showed that the YB-1 could be modified by andrographolide after the cells were treated with andrographolide. In the cisplatin induced YB-1 activation model, we found that the YB-1 translocation from the cytoplasm to the nucleus can be inhibited in pulmonary adenocarcinoma cells. In the TGF-β1 induced YB-1 activation model, overexpression of YB-1 could be inhibited and EMT-regulating genes could be downregulated, when the cells treated with andrographolide. A potent motility inhibitory effect of andrographolide has been demonstrated in pulmonary adenocarcinoma cells. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/54522 |
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顯示於系所單位: | 生化科學研究所 |
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