篩選抗菌胜肽 m2163 和 m2386 自 Lactobacillus casei ATCC 334 或一些有機藥物並應用在抑制人類大腸癌細胞生長和人類巨噬細胞移動抑制因子

Screening AMP m2163 and m2386 from Lactobacillus casei ATCC 334 or some drugs for studying the antiproliferation against a human colorectal cancer cell and the inhibitory effect on human MIF factor

蔡宗霖(Tsai, Tsung Lin)

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/博士(2015年)

摘要

在現代的社會中癌症的發生與治療仍然是難解之謎，為了能夠早日克服這個難關，許多新的研究與方法都在不斷的推陳出新。每個方法上都有其優劣，而在本研究室之中，我們投身於癌症的藥物開發研究上，也希望能早一步對於新的癌症藥物開發上有所貢獻。所以我們針對兩個較新興領域抗癌藥物研究上下功夫，其一是蛋白藥物的開發，我們針對天然的乳酸菌所帶有的抗菌胜肽蛋白基因進行更一步的研究並且將其應用在癌細胞的毒殺作用研究，並證明其對於腸癌細胞株有效，在未來或許能經由餵食乳酸菌的方式達到消化道相關癌症的預防與抑制。其二是利用電腦輔助藥物設計來縮減新藥物尋找的時間，針對一個廣泛性的癌症因子MIF進行虛擬藥物篩選並經由試管實驗證明篩選到了幾個能夠有不錯抑制效果的新結構分子，這個系統希望能夠在尋找新藥物或藥物設計上能夠有所助益。所以接下來的章節中將分別針對這兩個方法進行介紹，並在後續的內容中分成兩個方向為主的說明。
(1)自1945年以來發現抗菌胜肽的抗微生物效用後。到目前為止，一系列的研究也顯示某些抗菌胜肽除了具有抗微生物效用外也同時具有對於真核癌細胞的毒殺作用。本實驗室之前自乾酪乳桿菌屬ATCC334中發現了兩種新型抗菌胜肽命名為m2163和m2386也證實了能夠抑制其他細菌生長的能力。在本篇研究中，我們利用人類大腸癌細胞株SW480測試這些抗菌胜肽的抗癌能力。我們在培養基中添加這兩種胜並肽利用MTT法測試其細胞毒殺IC50劑量以及細胞週期分析和AnnexinV/PI雙重染色用於顯示癌細胞死亡的情況。經由定量PCR和西方點墨法分別分析細胞RNA和蛋白質層級細胞死亡相關基因表現。流式細胞儀和共軛焦顯微鏡分析結果顯示兩種胜肽在不同的時間點聚集在細胞不同的相對位置。實驗結果顯示，兩種胜肽的半有效細胞生長抑制劑量IC50約為40 μg/ml。在細胞死亡相關基因表現部分，Fas和TRAILR1等死亡受體表現量有所上升。兩種胜肽也能夠促進一些粒腺體內和外的細胞凋亡通路相關蛋白活化，如Smac和caspase3等的表達。FITC螢光標定的胜肽在細胞位置中分析顯示，一開始，m2163和m2386能夠附著在細胞膜上，並隨著時間逐漸穿過細胞膜進入到細胞質中。本篇研究結果顯示，抗菌胜肽m2163和m2386擁有對人類大腸癌細胞株SW480的抗癌能力，並可以跨越細胞膜進入細胞，對細胞造成進一步的影響，包括細胞死亡相關蛋白的激活。
(2)巨噬細胞移動抑制因子（MIF）屬於一種能夠自分泌和旁分泌作用的細胞因子，MIF已被證實與人體的發炎反應、自身免疫現象、病原體感染和癌症等相關。臨床數據表明，抑制MIF和其互變異構酶的活性區域在治療MIF相關的疾病模型上是很有效的。本實驗室利用虛擬篩選從資料庫中搜索得到一些化合物並經由試管實驗測試證明能夠抑制MIF的互變異構酶活性。藉由一個利用X-ray 繞射定義出立體3D結構的MIF蛋白和AutoDock4.2 分子對接軟體。計算出有機分子與MIF蛋白結合能量後進行排序，篩選出了17個具有活性潛力的化合物。實驗室中最後使用這17個化合物用於試管實驗測試對MIF互變異構酶的半有效抑制活性值IC50，IC50值的測定同時使用人類單核細胞THP-1的蛋白和純化重組MIF蛋白進行。實驗結果顯示，17個測試的其中3個化合物稱為 ZINC02693801 ， ZINC00141102 和ZINC12368346有著較佳能夠抑制MIF互變異構酶活性。所測定的IC50比另一個目前已知的MIF的互變異構酶活性抑制劑ISO-1還要好。此外，這三個化合物分子的結構和目前其他已知的MIF抑制物有所不同，具有進一步修飾和發展新藥的潛力。

關鍵字

癌細胞 ； 篩選 ； 抗菌胜肽 ； 乳酸菌

並列摘要

In modern society, the occurrence and treatment of cancer disease is still the difficult problem.In order to overcome this difficulty as soon as possible, many newly research and methods are in constant innovation andeach method has its advantages and disadvantages. In our laboratory,we engaged in the study of cancer treatment and hoped able to contribute ondevelop of newly cancer drugs. We focused on the anti-cancer drug development in two relatively new research areas, one of which was the development of protein drugs.We found some antimicrobial peptides fromnatural Lactobacillus and applied on cancer cytotoxicity studies.The result proved the effect on colon cancer cell line growth inhibition and might be able to achieve the prevention and suppression of gastrointestinal related cancers by feedingLactobacilluscarriedthese peptides. The second project was to utilize computer-aided drug design to reduce the seeking time forsearching new anti-cancer drugs.The virtual drug screening focus on an extensive tumor progress related factor MIF. Several new structure compounds were screened and thein vitroexperiments proved their inhibitory effect on MIF protein.This system able to apply on searching new drugs andhelpful ofnew drug design. Therefore, the next section described separately for these two approaching methods, and the following results divided in two chapters for clear described.
(1) Since 1945 the finding of antimicrobial activity of antimicrobial peptides (AMPs), a series of studies on the toxicity toward eukaryotic cancer cellhad been conducted. Previously, two novel AMPs named m2163 and m2386 identified from Lactobacillus (L.)caseiATCC 334 had revealed their antimicrobial ability by our laboratory. In this study, we tested the anti-cancer ability of these peptides on human colorectal cancer cell line SW480. The anti-proliferation IC50 defined of both peptides by MTT assay. Cell cycle analysis and AnnexinV/PI double staining showed the cell death subpopulation. Cell death correlated protein regulation analyzed by qPCR and western blot for RNA and protein level respectively. FACS and confocal microscopy revealed the cellular location of peptides at different time point.Both peptides had about 40 μg/ml IC50 for cell growth inhibition. Cell death receptor expression including Fas and TRAILR1 were activated. Furthermore, both peptides increased some of the internal mitochondria and external cytosol apoptosis pathway related proteins such as Smac and caspase 3 expression. The FITC-conjugated peptide location revealed that m2163 and m2386 peptide attached on the cell membrane and could penetrate into the cell cytoplasm at late time. The antimicrobial peptides m2163 and m2386 had the anti-cancer ability on human colorectal cancer cell line SW480 and could cross cell membrane, therefore causing downstream effects including cell death pathway activation.
(2) Macrophage migration inhibitory factor (MIF) is an autocrine- and paracrine-acting cytokine that is involved in several inflammatory, autoimmune, infectious, and oncogenic diseases. Clinical data revealed that inhibition of MIF, especially its tautomerase activity, with small compounds being beneficial in some disease models. A virtual screening (VS) experiment is conducted for searching some active compounds from the ZINC database to inhibit the tautomerase activity site of MIF. By using an x-ray–determined MIF structure as template and AutoDock4.2 molecular docking program, we screened out some 17 possible compounds for ranking by docking energy. In vitro experiments for these 17 compoundsinhibition for measuring their inhibitory activity IC50 against the MIF tautomerase. The IC50 measured using both human monocytic THP-1 cell lysate and purified recombinant human MIF protein. We found that the IC50 oftop three searched compounds (namely, ZINC02693801, ZINC00141102, and ZINC12368346) had better inhibitor activity than that determined for ISO-1, a known MIF tautomerase inhibitor and standard used throughout our VS experiment. Moreover, the scaffolds of most of our searched active compounds also quite different from those published drugs previously and showed the potential for further modification and development of new drugs.

並列關鍵字

cancer cell ； screening ； bacteriocin ； Lactobacillus

參考文獻

1 NissenMeyer, J. & Nes, I. F. Ribosomally synthesized antimicrobial peptides: Their function, structure, biogenesis, and mechanism of action. Arch Microbiol167, 67-77, doi:DOI 10.1007/s002030050418 (1997).

2 Nes, I. F., Yoon, S. S. & Diep, D. B. Ribosomally synthesiszed antimicrobial peptides (Bacteriocins) in lactic acid bacteria: A review. Food Sci Biotechnol16, 675-690 (2007).

3 Hammami, R., Fernandez, B., Lacroix, C. & Fliss, I. Anti-infective properties of bacteriocins: an update. Cellular and molecular life sciences : CMLS70, 2947-2967, doi:10.1007/s00018-012-1202-3 (2013).

4 Kjos, M., Nes, I. F. & Diep, D. B. Class II one-peptide bacteriocins target a phylogenetically defined subgroup of mannose phosphotransferase systems on sensitive cells. Microbiology155, 2949-2961, doi:10.1099/mic.0.030015-0 (2009).

5 Gardiner, G. E. et al. Fate of the two-component lantibiotic lacticin 3147 in the gastrointestinal tract. Appl Environ Microbiol73, 7103-7109, doi:10.1128/AEM.01117-07 (2007).