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

• Main Authors: Tsai, Tsung Lin, 蔡宗霖
• Other Authors: Lin, Thy Hou
• Format: Others
• Language: en_US
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/16030813155780757769

博士 === 國立清華大學 === 分子醫學研究所 === 103 === 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.