1. 研究目的与意义
CONTENT: 1. Synthetic an active peptide by solid phase chemical synthesis，sequence of the active peptide is FLPLIAKLAAKCAITKKC2. Identify and screen the peptide by relevant anticancer experiments to observe the antitumor effect of the polypeptide on H157 cells.SIGNIFICANCE:Compared with chemotherapeutic drugs, the antitumor bioactive peptide has the advantages of low toxicity, specificity and being difficult to develop drug resistance. For this reason, we are trying to find novel anti-proliferative active peptides that provide a better choice for the treatment of cancer.
2. 文献综述
Mechanism of Antitumor Active PeptidesAuthor：Wang Rong(School of Pharmacy, Chinese Medicine Resources and Development, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210046, China)The bioactive peptide,with its wide range of sources, has been studied widely in the world[1]. The bioactive peptide has obvious physiological function, such as nerve regulation, hormone action, immune regulation, antithrombotic, antihypertensive, cholesterol-lowering, bacteriostasis, antivirus, anti-cancer, antioxidation and so on.[2] Compared with chemotherapeutic drugs, the antitumor bioactive peptide has the advantages of low toxicity, specificity and being difficult to develop drug resistance[3]. For this reason, it has become a cutting-edge research topic for the discovery and development of anti-tumor drugs. This article mainly introduced constructive effects, the mechanism of antitumor bioactive peptide and forecast the prospect of its development.1 Structural Characteristics of Antitumor Active PeptideThe structure and activity of antitumor peptide are directly related. The cell membrane of normal mammalian is composed of phospholipids and sterols, which is neutral. The surface of the tumor cell membrane was negative because of the overexpression of negative charge molecules such as phosphatidyl serine and O-glycosylated mucin. Cationic amphiphilic peptide (CAP) can bind specifically to the surface of tumor cell membrane, cause cell membrane damage and give rise to cell necrosis and not damage normal tissue cells[4]. Yang Nannan[5] found that The ramifications of Buthus martensii Karsch have very strong antitumor activity and have very high positive charge and amphiphilic group in their molecular structure. Chenmeng[6] and others found that Cyclopeptides containing type D amino acids can enhance antitumor activity in vitro of human liver cancer cells (HepG2), human lung cancer cells(A549) and human cervical cancer cells (Hela). Thymopentin (TP5) is an Immunological activity central fragment of thymopoietin II (located in 32-36). It can be used to treat many malignant tumors, but TP5 has a short half-life(only 30s). The stability, antigenicity and immunogenicity of D-type amino acid substituted peptide were greatly improved, probably because the protease only acted on L-type amino acid but did not degrade on D-type amino acid, which weakened the degradation of the protease in the body, thus protecting its antitumor activity. The alpha-helical structure of peptide chains also affects the antitumor activity of peptide. The alpha-helix is presented in many antitumor peptides and is associated with its specific and widespread killing and toxicity of cancer cells.[7-8]As a result, a growing number of amphiphilic alpha helical antitumor peptides have been designed for research and treatment of cancer.Pic. 1 Structure of amphiphilic alpha helical antitumor peptide[8] 2 Mechanism of Antitumor Active PeptidesAntitumor active peptides act specifically on tumor tissue and interact with signaling molecules associated with cell division, growth or metastasis, thus promoting programmed death, necrosis or inhibition of tumor grows. The antitumor mechanism of active peptide includes inhibiting or interfering tumor DNA synthesis, preventing tumor angiogenesis and metastasis, enhancing immune system function, anti-oxidation and radiation damage. In fact, the mechanism of each anti-tumor active peptide is not a single one, but one or more of them contribute to the destruction of tumor[9].2.1 Enhance Immune SystemSome active peptides can activate the body's immune function against tumor cells, such as stimulating lymphocyte proliferation, enhancing phagocytic activity of macrophages, promoting the production of hemolysin antibodies, and increasing the lethality of NK cells[9-10]. Dongchao[11] reports that active peptides can enhance the anti-tumor function of the immune system by stimulating antitumor immunity, suppressing tumor antigens, and hindering the establishment of tumor neovascularization networks. Thymos in alpha 1 (Tα1) is an immune system-enhancing peptide containing 28 amino acids. Fan Yingzhe[12] has found that it can significantly inhibit the proliferation of human leukocytes (HL-60, K562 and K562 / ADM) and induce apoptosis.2.2 Antioxidation and Radiation DamageRecent studies have found that many antioxidant active peptides can also inhibit tumors and are of great clinical significance in tumor therapy. Leng Bo et al. [13]foundthat clam peptides can activate super-oxide dismutase (SOD), and inhibit the growth and metastasis of human gastric cancer cell BGC-823 by removing active oxygen (ROS), which was involved in cancer cell degeneration. It was found that the chickpea polypeptide CPe could play an anti-tumor role by improving antioxidant capacity.2.3 Interrupting DNA Synthesis in Tumor CellsThe occurrence of tumor is the result of many factors, but the expression and regulation of proto-oncogene should be involved. A variety of bioactive peptides have been found to interfere with or inhibit DNA synthesis of tumor cells by specifically binding tumor-related genes and regulatory factors. The results of William R Gower and others[14] show that the four peptide synthetic hormones inhibit the proliferation of cancer cells by GMP cycle mediated DNA synthesis. Yujie et al. [15] found that Oyster active peptide(BPO) can effectively prevent the transformation of from G1 to S phase, promote the expression of apoptosis gene and inhibit the proliferation of HeLa cells. Ichuan Sheih and others[16] hydrolyzed the essence of chlorella with pepsin, which produced algal protein-rich waste. Antitumor active peptides screened from the waste could block the cell cycle of human gastric adenocarcinoma cells(AGS) in late G1, and cause no toxicity to the WI-38 lung fibroblasts.2.4 Induction of Tumor Cell ApoptosisSome active peptides can directly target the components over-expressed in tumor cells(such as Bcl-2, p53, caspase, telomerase, Bax, etc.) by transmembrane movement and then bind to them specifically, block the cell-related pathway and induce apoptosis[17-18]. Caspase-3 is the most important endcutting enzyme in apoptosis, and it is the effective agent of apoptosis. It can hydrolyze the structure and functional proteins related to cell disintegration directly, thus induce tumor apoptosis and reduce tumor volume. The study of Moon D O[19] showed that bee venom can inhibit proliferation and induce apoptosis of U937 cells in vitro. XIAP is an endogenous cysteine protease inhibitor that is overexpressed in many cancer lesions and can protect tumor cells from apoptosis. Mao H L and others[20] have found that second mitochondria-deactivated activator of caspases can trigger programmed cell death by lowering the XLAP to activate or enhance the activity of caspase-3.2.5 Direct Effects on Tumor Cell Membranes Some polypeptides can target the nonpolar lipid cell membrane of tumor cells, form ion permeation channels, and cause them to depolarize, dissolve irreversibly, and eventually cause cell cracking and death. The host defense peptide[21]is a cationic amphiphilic peptide that has anticancer activity against leukaemia, prostate cancer, ovarian tumors and ascites. Maarja Mae et al.[22] combined the linear breast cancer target peptide CREKA with the transmembrane peptide PVEC to form a new chimeric peptide CREKA-PVEC, which could better transport chemotherapy drugs into cells, and had a significantly better anti-cancer effect than the single anticancer drug(DNA alkylation et al.). Hyun Soo Lee et al.[23] found histone H2A antibacterial peptide derivative buforin Hb (RAGLQFPVG] RLLR]3) induced mitochondrial-dependent cell apoptosis without damage to normal cells by specific action with negative-charged ganglion glycosides on the surface of cancer cells.2.6 Inhibiting Tumor AngiogenesisThe growth, infiltration and metastasis of malignant tumor depend on the ability of tumor angiogenesis. Therefore, some specific molecules in the endothelium of tumor neovascularization can be used as targets of drug action to inhibit tumor proliferation and metastasis. Some bioactive peptides can inhibit the proliferation and migration of capillary endothelial cells and affect the formation of the tumor's vascular endothelium and the newborn cancer cell[24] . The high expression of CD13 in various tumor cells can promote the formation of tumor new blood vessels, thus promote tumor cell proliferation, inhibit tumor cell apoptosis and enhance cell invasiveness[25-26]. Vascular endothelial cells (VECs) are the effect cells of angiogenesis factors and can be used as an important target area for tumor angiotherapy.3 Prospect of Antitumor Active PeptidesThe resources of anti-tumor peptides are huge, and the preparation techniques are diverse. It has become a hot topic in the 21st century. However, there are still many problems in the production and clinical application of anti-tumor peptide. Anti-tumor active peptides have many defects, such as small molecular weight, easy degradation in vivo, short Half-Life, low self-structure instability, low bioavailability, macromolecular peptides may also trigger immune response, which greatly limits the clinical application of anti-tumor active peptides. Therefore, it is necessary to further study the mechanism of the anti-tumor active peptide, improve the stability, solubility and biological potency of the peptide in vivo by molecular modification, slow releaseor development of the corresponding pseudopeptide drugs, and maximize its antitumor effect.References[1] Lapis K. Physiologic and pathophysiologic significance of antimicrobial (host defensive) small peptides. Orv Hetil, 2008, 149(51):2419-2424.[2] Chao J Y, Zhang L. The research progress of biological active peptide [J]. Journal of Jilin Institute of Medicine, 2010, 31 (3)：21-23[3] Jennifer C F. Cancer Immunotherapy[J]. Science,2013,342(C6165):1432-1433.[4] Chow A, Wong A, Francia G. Preclinical analysis of resistance and cross-resistance to low-dose metronomic chemotherapy[J]. Invest New Drugs, 2014 32 (1): 47-59.[5] Yang NN, Storm MB, Mekonnen SM,Svendsen JS,Rekdal O. The effects of shortening lactoferrin derived peptides against tumor cells,bacteria and normal human cells[J]. Journal of Peptide Science, 2004, 10: 37-46[6] Chen M, Qu T, Xu SH. The synthesis and antitumor activity of two Marine cyclic peptide complexes [J]. Chemistry and Biosynthesis, 2012, 29(5): 8-11.[7] Wang KR, Zhang BZ, Zhang W,Yan JX,Li J,Wang R.Antitumor effects,cell selectivity and structure-activity relationship of a novel antimicrobial peptide polybia-MPI[J]. Peptide, 2008, 29(6): 963-968[8] Gao J. Analysis on the bioactivity and structure-activity relationship of the peptides in gaojie and hummus [D]. Tianjin: Tianjin University, 2012.[9] Chernysh S, Irina K, Irina A. Anti-tumor activity of immunomodulatory peptide alloferon-1 in mouse tumor transplantation model[J]. International Immunopharmacology, 2012, 12(1): 312-314.[10] Moradalia M F, Mostafavi H, Ghods S,Hedjaroude GA. Immunomodulating and anticancer agents in the realm of macromycetes fungi(macrofungi)[J]. International Immunopharmacology, 2007, 21(7): 701-724[11] Dong C, Bi LF, Su XL. Bioactive peptide and anti-tumor immunity[J]. Journal of Inner Mongolia Medical College, 2009, 31(3): 252-255.[12] Fan YZ, Chang H, Yu Y. Thymosin alphal suppresses proliferation and induces apoptosis in human leukemia cell lines[J]. Peptides, 2006, 27(9): 2165-2173.[13] Leng B, Liu XD, Chen QX. Inhibitory effects of anticancer peptide from Mercenaria on the BGC-823 cells and several enzymes[J]. FEBS Letters, 2005,(579): 1187-1190.[14] William RG, Brian AV, Abdel AA,Vesely DL. Four peptides decrease human colon adenocarcinoma cell number and DNA synthesis via cyclic GMP[J]. International Journal of Gastrointestinal Cancer, 2005, 36(2): 77-88.[15] Yu J, Yang ZG, Chen MZ. In vitro induction of HeLa cell apoptosis and its mechanism [J]. Food Science, 2012,33(21):290-294.[16] Sheih IC, Fang TJ, Wu TK,Lin PH. Anticancer and antioxidant activities of the peptide fraction from algae protein waste[J]. Journal of Agricultural and Food Chemistry, 2010, 58(2): 1202-1207.[17] Li JT, Zhang Jl, He H,Ma ZL,Nie ZK,Wang ZZ,Xu XG. Apoptosis in human hepatoma HepG2 cells induced by corn peptides and its antituomr efficacy in H22 tumor bearing mice[J]. Food and Chemical Toxicology, 2013, 51: 297-305.[18] Bhutia SK, Maiti TK. Targeting tumors with peptides from natural sources[J].Trends in Biotechnology, 2008, 26(4): 210-217.[19] Moon DO, Park SY, Heo MS, Kim KC,Park C,Ko WS,Choi YH,Kim GY. Key regulators in bee venom-induced apoptosis are Bc-12 and caspase-3 in human leukemic U937 cells through down regulation of ERK and Akt[J]. International Immunopharmacology, 2006, 6(12): 1796-1807.[20] Mao HL, Pang Y, Zhang X,Yang F,Zheng J,Wang Y,Liu P. Smac peptide potentiates TRAIL-or paclitaxel-mediated ovarian cancer cell death in vitro and in vivo[J]. Oncol Pep, 2013, 29(2): 515-522.[21] Riedl S, Zweytick D, Lohner K. Membrane-active host defense peptides-Challenges and perspectives for the development of novel anticancer drugs[J]. Chemistry and Physics of LIpids, 2011, 164: 766-781.[22] Mae M, Myrberg H, EI-Andaloussi S. Design of a tumor homing cell penetrating peptide for drug delivery[J]. International Journal of Peptide Research and Therapeutics, 2009, 15(1): 11-15.[23] Lee HS, Park CB, Kim JM,Jang SA,Park IY,Kim MS,Cho JH,Kim SC. Mechanism of anticancer activity of buforin IIb, a histone H2A-derived peptide[J]. Cancer Letters, 2008, 271: 47-55.[24] Starzec A, Vassy R,Manin A, Lecouvey M,Di Benedetto M,Crpin M,Perret GY. Antiangiogenic and antitumor activities of peptide inhibiting the vascular endothelial growth factor binding to neuropilin-1[J]. Life Sciences, 2006 ,79(25):2370-2381.[25] Wallbrunn A V, Holtke Carsten, Zuhlsdorf Michael, Mesters R,Heindel W, Schfers M,Bremer C. In vivo optical imaging of CD13/APN-expression in tumor xenografts[J]. Journal of Biomedical Optics. 2008, 13(1): 11-19.[26] Terauchi M, Kajiyama H, Shiata K,Ino K,Nawa A,Mizutani S,Kikkawa F.Inhibition of APN/CD13 leads to suppressed progressive potential in ovarian carcinoma cells[J]. BMC Cancer.2007.7(1):1-12.
3. 设计方案和技术路线
Research programmes：First, solid-phase synthesis method was used to repeat the addition of precisely-named amino acids required for synthesis from the oxime end (carboxyl group) to the oxime end (amino group). In order to prevent the occurrence of side reactions, the side faces of the amino acids participating in the reaction are protected by Fmoc, and the C-terminal is released and activated before the reaction. The resin was added to immobilize the polypeptide - FLPLIAKLAAKCAITKKC, which was lyophilized for use. Coupled with mass spectrometry to determine the molecular weight of the polypeptide, plus known sequence information, tentatively verified whether the sequence of the polypeptide meets the requirements. Finally, through the relevant antitumorl experiments, the function of the peptide was identified and screened. The antitumorl effects of the polypeptide on H157 were observed.Technical Route：
4. 工作计划
TIME CONTENT PROGRESS RATE2022.03.03~03.08 Read articles regarding to the experiment and do the presentation completed2022.03.09~03.23 Do the experiment completed2022.03.24~04.04 Read relative articles and write the initiating report and the review paper completed2022.04.05~04.15 Finish the experiment To be completed2022.04.16~05.15 Analyze the experimental data and write the research paper To be completed
5. 难点与创新点
We first synthetic this kind of active peptide by solid phase chemical synthesis, which is a novel peptide that have not been identified yet. This work will finally provide new methods or ideas for the development of antituomr drugs by finding novel bioactive peptides.
以上是毕业论文开题报告，课题毕业论文、任务书、外文翻译、程序设计、图纸设计等资料可联系客服协助查找。
