穿膜性抗菌胜肽KL15對抑制大腸癌細胞株增生之探討

Anti-proliferative effect on acolon adenocarcinoma cell line exerted by amembrane disrupting antimicrobial peptide KL15

陳又菁(Yu-Ching Chen)

指導教授 ： 林志侯

國立清華大學/生命科學院/分子醫學研究所/碩士(2014年)

摘要

近年來，抗菌胜肽相關的研究逐漸受到重視，除了其抗菌能力外，在癌症治療上也相當的有潛力。過去幾年來，本實驗室從乳酸桿菌Lactobacillus casei ATCC 334中篩選出數個具抗癌潛力之抗菌胜肽包含m2163及m2386。本篇論文中，有兩個研究方向同時進行。一方面，為了設計出更強的具抗癌潛力之抗菌胜肽，我們根據文獻中整理出的原則修改m2163及m2386，並做初步測試。另一方面，我們把重點放在一個稍早從m2163修改而來的短胜肽穿膜蛋白KL15之定性及細胞增生研究。
KL15是個具抗癌活性之抗菌胜肽。在細胞毒性測試中，以KL15投藥後，兩個癌細胞株- SW480及Caco-2 (人類大腸癌細胞株)相較於非癌細胞株- H184B5F5/M10 (人類乳腺表皮細胞株)存活率下降得更明顯；前者之IC50約在50 μg/ml (26.3 μM)，而後者則是在150 μg/ml。以KL15對SW480細胞投藥之後，以DNA、細胞型態及膜通透性、可能的細胞死亡路徑，三個層面觀察細胞的改變。並且，結合共軛焦顯微鏡的觀察，我們得知隨著投藥時間增加，KL15會因膜通透性增加而穿膜進入細胞。整體的結果意味著KL15造成的細胞膜受損，在細胞死亡中扮演重要的角色，而細胞死亡的路徑可能與細胞壞死有關。然而，其中的死亡機制仍尚未釐清，有待日後研究。

關鍵字

抗癌之抗菌胜肽 ； 穿膜,擾膜 ； 小胜肽(蛋白)藥 ； 胜肽KL15 ； 掃描式電子顯微鏡 ； 共軛焦顯微鏡

並列摘要

Recent research has paid increasing attention to antimicrobial peptides (AMPs), not only for their antimicrobial activity, but also for their potential of therapeutic application in cancer therapy. In the past few years, our lab has found several novel bacteriocins including m2163 and m2386 with anticancer potential from Lactobacillus casei ATCC 334. In this thesis, two directions of research were simultaneously processed. On one hand, to design stronger anticancer peptides, several modifications of both m2163 and m2386 were made following the principle, and went through preliminary tests. On the other hand, we focused on characterization and the anti-proliferative study of the membrane disrupting peptide KL15, a short peptide which was earlier modified from m2163.
KL15 is an AMP with anticancer activity. In the cytotoxicity assay, both cancerous- human colon adenocarcinoma cell lines, SW480 and Caco-2, showed a more obvious decrease on survival rate than non-cancerous- human mammary epithelial cell line, H184B5F5/M10; IC50 of SW480 and Caco-2 are around 50 μg/ml (26.3 μM), while IC50 of H184B5F5 is at 150 μg/ml. SW480 cell changes after treated with KL15 were observed in three aspects- DNA, membrane permeability and morphology, and possible cell death pathway. Also, combined with confocal microscope, we knew that KL15 would enter the cell as membrane permeability increase in the later stage. The overall results implied that the cell membrane disruption causing by KL15 played a crucial role in cell death, moreover, the concerning cell death pathway may be necrosis. However, the underlying mechanism is not yet clear, and is still under investigation.

並列關鍵字

anticancer antimicrobial peptide (AMP) ； membrane disruption ； small peptide (protein) drug ； peptide KL15 ； SEM ； confocal microscope

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