Dissertations / Theses on the topic 'Anticancer peptides'

Les sphéroïdes multicellulaires tumoraux (SMT) constituent un outil prometteur dans le domaine de l’étude biologique des tumeurs. Le but de la thèse était de développer une technique de la formation de SMT et de démontrer la disponibilité de ces sphéroïdes comme modèle in vitro 3D pour tester l’efficacité de principes actifs anticancéreux ainsi que celle de formulations de délivrance de médicaments. L'effet d’auto-assemblage de cellules induit par une addition des peptides RGD cycliques a été étudié pour 16 lignées cellulaires de différentes origines. Le peptide cyclique RGDfK et sa modification avec le cation triphenylphosphonium (TPP) ont permis de mettre en évidence l’induction de formation de sphéroïdes. Les sphéroïdes ont été employés comme modèles pour évaluer la cytotoxicité de principes actifs antitumoraux (doxorubicine, curcumine, temozolomide) et un certain nombre de formulations nano- et micrométriques (microréservoirs, nano-émulsions et micelles)<br>Multicellular tumor spheroids (MTS) are a promising tool in tumor biology. The aim of the Thesis was to develop a novel highly reproducible technique for MTS formation, and to demonstrate the availability of these spheroids as 3D in vitro model to test anticancer drugs and drug delivery vehicles. Cell self-assembly effect induced by an addition of cyclic RGD-peptides directly to monolayer cultures was studied for 16 cell lines of various origin. Cyclo-RGDfK peptide and its modification with triphenylphosphonium cation (TPP) were found to induce spheroid formation. The spheroids were used as a model to evaluate the cytotoxicity of antitumor drugs (doxorubicin, curcumin, temozolomide) and a number of nano- and micro- formulations (microcontainers, nano-emulsions and micelles)

Les protéines de la famille Bcl-2 sont des régulateurs-clés de l’apoptose (mort cellulaire), qui agissent en contrôlant la perméabilisation de la membrane mitochondriale externe par un processus encore mal connu. La dérégulation des membres de cette famille est souvent associée à la progression tumorale et à la résistance à la chimiothérapie. Notre projet a cherché à éclaircir le mode d’action de ces protéines en se focalisant sur les déterminants structuraux régissant leur interaction avec les membranes biologiques. Les connaissances glanées ont permis (i) de mieux comprendre les déterminants à l’origine de la divergence évolutive entre membres pro- et anti-apoptotiques de la famille Bcl-2 ; (ii) d’ouvrir la voie à la conception de ‘poropeptides’ conçus sur le modèle des hélices d’insertion membranaire des protéines Bcl-2, et qui pourraient être utilisés pour induire l’apoptose de cellules tumorales ou des cellules endothéliales entourant les tumeurs<br>Bcl-2 family proteins, which include pro- and antiapoptotic members, positively or negatively regulate mitochondrial outer membrane permeabilization, i.e. a critical step in apoptosis. Over-expression of pro-survival members is associated with tumor progression and may be responsible for chemotherapy resistance. Detailed understanding of the precise mechanisms by which Bcl-2 family members control apoptosis is therefore of considerable therapeutic interest. The overall aim of our project was to delineate a structure-function relationship of Bcl-2 family proteins with emphasis on their membrane-active domains. This analysis has provided a basis (i) to elucidate the molecular mechanisms by which different Bcl-2 proteins evolved opposite functions ; (i) to develop a new generation of pore-forming peptides targeting the mitochondrial outer membrane that may be used to kill neoplastic or tumor endothelial cells

This thesis is focused on the development of synthetic approaches to obtain new bioactive peptides. The first part deals with the design of new antimicrobial peptide/cell-penetrating peptide conjugates as anticancer agents. Their conjugation enhanced the activity of the antimicrobial peptides against cancer cells while maintained their low toxicity. These compounds are interesting for the design of new anticancer agents. On the second part, a new versatile methodology for the synthesis of natural fengycin derivatives is described. Our strategy represents the first synthetic approach for the total solid-phase synthesis of these cyclic lipodepsipeptides and can be easily adapted to obtain a wide range of analogues.<br>Aquesta tesi doctoral s’ha centrat en el desenvolupament d’estratègies sintètiques útils per a l’obtenció de nous pèptids bioactius. Primerament, s’han dissenyat nous pèptids conjugats antitumorals a través de la unió d’un pèptid antimicrobià i un cell-pentrating peptide. Aquesta conjugació augmenta l’activitat antitumoral del pèptid mantenint la toxicitat baixa. Aquests conjugats són interessants pel desenvolupament de nous agents antitumorals. A continuació, s’ha desenvolupat una metodologia per a la preparació de pèptids cíclics derivats de les fengicines. Aquesta metodologia representa la primera estratègia sintètica descrita per a l’obtenció en fase sòlida d’aquesta família de ciclolipodepsipèptids i pot ser fàcilment adaptada per a l’obtenció d’una àmplia varietat d’anàlegs.

Doctor of Philosophy<br>Department of Chemistry<br>Stefan H. Bossmann<br>Recent development of a variety of superparamagnetic and ferromagnetic iron/iron oxide (Fe/Fe₃O₄) nanoparticles with different surface chemistry have been widely studied for numerous biological applications such as drug delivery, as diagnostics, hyperthermia and magnetic resonance imaging. The wide applications of Fe/Fe₃O₄ nanoparticles are possible since they exhibit favorable properties as high magnetization ability, are smaller than 100 nm in size, they can be coated with several ligands which allow drug delivery at a specific site and are biocompatible. By using Fe/Fe₃O₄ nanoparticles as drug delivery agents treatment costs and side effects can be reduced, however treatment efficacy can be increased. We have demonstrated that Fe/Fe₃O₄ nanoparticles can be utilized in different methods depending on their properties, to be used as therapeutic agents for cancer treatment. In one method we have taken advantage of the Fe/Fe₃O₄ nanoparticles magnetic ability to produce hyperthermia (heat) in cancer cells when subjected to an alternative magnetic field. Here we use the cell based delivery system since the size of the nanoparticles are small they can be taken up by monocyte/ macrophage like cells for systemic transportation to the inflamed cancer cite. The hyperthermia study was conducted in mice with pancreatic cancer. This study demonstrated that the life expectancy of the mice increased by 31%. In the next method we took the advantage of the surface chemistry of the Fe/Fe₃O₄ nanoparticles and changed it with dopamine-peptide and dopamine-thiosemicarbazone ligands. The advantage of the peptide is to deliver the nanoparticle to its target site and the thiosemicarbazone analogue is used as an iron chelator that would initiate apoptosis in cancer cells. This nanoplatform was tested in 4T1 breast cancer cell line and normal fibroblast cell line and demonstrated that it was effective towards the cancer cell line than the normal cell line at a ratio of 5:1 of thiosemicarbazone analogue : dopamine on the nanoparticle. However further studies are needed to be done to clarify the effectiveness of this nanosystem.

The high demand of novel peptide and peptidomimetics based on the amount of genomic and proteomic data need be matched by synthesis and screening. The design and synthesis of peptide and peptidomimetics are so important because the peptide and protein-protein interaction play a key role in molecule recognition and signaling. The modified peptides have better stability and pharmacokinetic properties which may be guided by rational design and molecular modeling. Now many organic and medicinal chemists have chosen peptide and peptidomimetics as potential drug candidates for many targets. In this dissertation, research efforts in design and synthesis of cyclic peptides with stabilized secondary structure have been investigated. Cyclization of linear peptides may restrict the number of available conformations which may improve the affinity attaching to the target. In this study, different beta turn linkers have been designed and synthesized to achieve more stable cyclic peptides with beta-sheet structures. Based on different beta turn linkers, analogs of cyclic peptides have been synthesized and screened. The structure activity relationships (SAR) of these cyclic peptide analogs have been studied. In chapter three, analogs of peptidomimetic inhibitors have been designed and synthesized. These peptide analogs are targetingHuman Rhinovirus (HRV) and Coronavirus (CoV) by inhibiting the cysteine protease. The docking and modeling studies have been shown. The structures of this kind of inhibitors include five fragments. The warhead provides the activity, which can covalently react with the thiol of cysteine protease and permanently eliminate its proteolytic activity. The warhead is linked to a peptide backbone including the other four parts that are designed to position the warhead where it can specially react with the critical thiol of the cysteine protease active site. The side chain of each amino acid has been optimized to achieve better solubility and permeability. We successfully synthesized some compounds with good potency.

Melflufen (melphalan flufenamide, chemical name L-melphalanyl-p-L-fluorophenylalanine ethyl ester hydrochloride, previously called J1) is a derivative of the classical alkylating agent melphalan. Melflufen is potentiated by hydrolytic cleavage by aminopeptidase N (APN), leading to high intracellular concentrations of alkylating moieties and subsequent cell death. Increased APN expression is associated with the malignant phenotype of several human cancers, including acute myeloid leukemia, lymphoma and ovarian cancer, and plays a functional role in tumor angiogenesis. Therefore investigations of melflufen activity in these malignancies as well as detailed studies of inhibition of angiogenesis are interesting. The aim of this project was to investigate the cytotoxic and antiangiogenic effect, in vitro and in vivo, of melflufen, compared to melphalan and other cytotoxic drugs used in the clinic. We showed that melflufen was more effective than its parental drug melphalan in lymphoma, AML and ovarian cancer in cell lines as well as in primary patient samples. An improved in vitro therapeutic index was demonstrated by an increased cytotoxic activity in the patient samples compared to normal peripheral blood mononuclear cells (PBMCs). Furthermore, melflufen in combination with cytarabine was synergistic in an AML cell line in a sequence-dependent manor. Melflufen was shown effective in several animal models using lymphoma, AML and ovarian cell xenografts (single drug or in combination), including an intraperitoneal ovarian xenograft. Finally, we demonstrated that melflufen had antiangiogenic properties in several different models.

Kyoto University (京都大学)<br>0048<br>新制・課程博士<br>博士(農学)<br>甲第11116号<br>農博第1446号<br>新制||農||898(附属図書館)<br>学位論文||H16||N3966(農学部図書室)<br>22666<br>UT51-2004-L913<br>京都大学大学院農学研究科応用生命科学専攻<br>(主査)教授 吉川 正明, 教授 井上 國世, 教授 大東 肇<br>学位規則第4条第1項該当

Cette thèse, nous avons conçu et synthétisé différents copolymères biocompatibles auto-assemblés par copolymérisation par ouverture de cycle (coROP) de trois différents N-hydroxycarboanhydride (NCA) d’alpha-aminoacides, avec du poly(oxyde d'éthylène) (POE-NH2) comme macro-amorceur, formant un bloc hydrophile. Différents degrés de polymérisation du POE ont été utilisés (DP=17,46,114), et la composition du bloc copolypeptidique amphiphile a été ajustée par coROP de l'acide L-glutamique anionique ou de la L-lysine cationique, qui apportent une charge dépendante du pH, et du comonomère L-phénylalanine dont les unités aromatiques apportent des interactions d'empilement pi-pi et servent de réservoir hydrophobe pour l'encapsulation d’un médicament. Différentes morphologies, micelles en forme de bâtonnet, nano-objets de forme ronde et structures vésiculaires, ont été obtenues via différentes techniques d'auto-assemblage (dissolution directe, nano-précipitation, dialyse directe ou hydratation directe dans l'oligo(éthylène glycol)), puis leurs propriétés physico-chimiques détaillées et leur structure ont été étudiées par un panel de méthodes de caractérisation (diffusion de la lumière, des rayons X ou des neutrons, microscopie atomique ou électronique, dichroïsme circulaire...). Les POE-block-copolypeptides à base de poly(acide L-glutamique-co-L-phénylalanine) permettent l'encapsulation et la libération de la curcumine, qui est un médicament antioxydant, anticancéreux et anti-inflammatoire (pléiotrope) très peu soluble dans l'eau, grâce à des interactions hydrophobes avec le bloc copolypeptidique contenant de la L-phénylalanine. Les nano-objets chargés de curcumine ont été formulés par nano-précipitation ou hydratation directe, et leur efficacité a été évaluée préliminairement in vitro par un test de cytotoxicité (MTT) sur des cellules HeLa natives. L'auto-assemblage de copolymères à bloc POE de poly(L-Lysine-co-L-phénylalanine) portant une charge de surface positive à pH neutre a également été étudié dans de l'eau et des solutions tampons de pH avec des techniques de caractérisation similaires. Dans le cadre d'une autre étude, des vésicules complexes polyioniques, également appelées PICosomes dans la littérature, ont été préparées à partir d'une paire de copolymères à bloc POE de charge opposée obtenus dans les chapitres précédents. Les PICosomes ont été caractérisés par la diffusion dynamique de la lumière (DLS), la diffusion de la lumière multi-angles (MALS) et également par la microscopie électronique à transmission (TEM). Dans notre étude, des complexes polyioniques avec de petits ARN interférents (siRNA) ont été préparés et leur activité d'inhibition de gène a été étudiée sur des cellules HeLa-Luc par des essais de bioluminescence in vitro (ces cellules exprimant le gène de la luciférase de la luciole). Dans une dernière partie de la thèse, nous avons synthétisé un autre type de copolymères amphiphiles, à savoir les copolymères séquencés POE-b-PTMC (poly(oxyéthylène-b-poly(triméthylènecarbonate)) qui sont semi-cristallins, biodégradables et biocompatibles. Ensuite, des nanoparticules magnétiques de différents diamètres, recouvertes d'un revêtement hydrophobe, ont été chargées dans leurs membranes afin de préparer des polymérosomes entièrement biodégradables, dotés d'une double réponse magnétique et thermosensible, pour des applications d'administration contrôlée de médicaments<br>In this thesis, we designed and synthesized different biocompatible self-assembling copolymers by ring opening copolymerizaton (coROP) of three different alpha-aminoacid N-hydroxycarboanhydride (NCA), with poly(ethylene oxide) (PEO-NH2) as macro-initiator, forming hydrophilic block. Different degree of polymerization of PEO were used (DP=17,46,114), and amphiphilic co-polypeptide block was composition was tuned by coROP of either anionic L-glutamic acid or cationic L-lysine, which brings a pH dependent charge, and L-phenylalanine comonomer whose aromatic units bring pi-pi stacking interactions and serve as a hydrophobic reservoir for drug encapsulation. Different morphologies, rod-like micelles, round shape nano-objects and vesicular structures, were obtained via different self-assembly techniques (direct dissolution, nanoprecipitation, direct dialysis or direct hydration in oligo(ethylene glycol)), then their detailed physicochemical properties and structure were investigated by a panel of characterization methods (light, X-ray or neutron scattering, atomic or electron microscopy, circular dichroism…). Poly(L-glutamic acid-co-L-phenylalanine) based PEO-block-copolypeptides enable encapsulation and release of curcumin , which is an antioxidant, anticancer and anti-inflammatory (pleiotropic) drug with very low water solubility, thank to hydrophobic interactions with the L-phenylalanine containing copolypeptide block. Curcumin loaded nanoobjects were formulated by nanoprecipitation or direct hydration, and their preliminary efficacy was evaluated in vitro by cytotoxicity assay (MTT) on HeLa wild type cells. Self-assembly of poly(L-Lysine-co-L-phenylalanine) PEO-block-copolymers bearing positive surface charge at neutral pH was also studied in water and pH-buffer solutions with similar characterization techniques. As a further study, polyionic complex vesicles also called PICsomes in literature were prepared from a pair of oppositely charged PEO-block-copolypeptides obtained in previous chapters. PICsomes were characterized by dynamic light scattering (DLS), multi angle light scattering (MALS) and also by transmission electron microscopy (TEM). In our study, small interfering RNA (siRNA) loaded polyionic complexes were prepared and their gene silencing activity was investigated on HeLa-Luc cells by in vitro bioluminescence assays (these cells bearing the lucirefase firefly gene). In a final part of the thesis, we synthesized another type of amphiphilic copolymer, namely PEO-b-PTMC (polyethylene oxide-b-poly(trimethylenecarbonate)) block copolymers which are semi-crystalline, biodegradable and biocompatible. Then hydrophobically-coated magnetic nanoparticles of different diameters were loaded in their membranes in order to prepare fully biodegradable polymersomes featuring a dual magneto and thermo-responsive membrane, for tunable drug delivery applications

Methotrexate (MTX) is an antimetabolite anticancer agent that is used in treatment of multiple cancers, such as acute lymphoblastic leukaemia and osteosarcoma. A lack of selective tumour toxicity is one of the major problems associated with MTX chemotherapy, especially when given at high doses, as in high dose MTX (HDMTX) therapy. MTX causes various toxicity problems including life-threatening nephrotoxicity, haematological toxicity and neurotoxicity. Overcoming this toxicity is of great importance and has been attempted in various ways, not least via the design of prodrugs. The concept of tumour protease, and specifically matrix metalloproteinase (MMP), activated prodrugs was the focus of the work described in this thesis. This concept relies upon attachment of an MMP-sensitive peptide sequence to a specific site in a drug structure, so as to inactive it. The activity of the parent drug is restored once it is activated by the MMPs in the tumour microenvironment. In this work, different MMP-sensitive peptide sequences linked to MTX were synthesised, resulting in 63 MTX prodrugs. The MMP-mediated activation of these conjugates in tumour tissues (specifically HT1080 homogenates) ex vivo was assessed and the results were compared to the activation of these conjugates in various normal tissues specifically liver, kidney and lung. Specific criteria were established for the selection of promising conjugates for more detailed study. From 7 promising compounds, compound 75 was identified as the lead prodrug, demonstrating selective MMP activation, as indicated by inhibition of its activation by broad spectrum MMP inhibitor ilomastat. The pharmacokinetics of compound 75 was studied in tumour (HT1080) xenograft-bearing mice and the results were compared to those obtained from administration of equimolar doses of conventional MTX. Compound 75 led to enhanced tumour concentrations of MTX, with reduced exposure to normal tissues in vivo compared to conventional MTX therapy. Furthermore, the efficacy of equimolar doses of compound 75 and directly dosed MTX in reduction of HT1080 volume were compared. Superior antitumour activity was observed with compound 75 compared to MTX treatment. Compound 75 is the first example of an MMP-activated prodrug to be reported with enhanced therapeutic index, as evidenced by a full in vivo pharmacokinetic analysis and normal tissue metabolism data. The data presented in thesis support the concept of MMP-activated prodrug development, and form a strong foundation upon which to develop a clinicallyuseful MTX prodrug, with the potential to enhance efficacy and reduce toxicity to the patient.

The search for more effective treatment strategies in melanoma led to many new innovative approaches aiming at different molecular targets. Chemotherapy still remains the most effective treatment and many efforts are put in order to improve targeting and delivery of the chemotherapeutic agents. Among these, peptide conjugates of anticancer drugs were designed to increase stability, cell penetration, specificity and accumulation in cancer cells. We as well as others evaluated such a conjugate, termed PSF (L-prolyl-m-L-sarcolysyl-L-p-fluorophenylalanine-ethylester) in terms of its cytotoxicity in vitro and in vivo using a human melanoma tumor as a model, its stability, transport, and metabolisation. <p>By comparing the cytotoxicity of PSF and melphalan towards different cancer primary melanoma cell cultures, we noticed some interesting observations: PSF displayed the same toxicity pattern both in short (2h) and long term (24h) cell exposures whereas melphalan and m-sarcolysin needed long term exposure to reach the same toxicity. This could indicate that PSF very quickly penetrates the cells in accordance with what has been shown with red blood cells (RBCs). PSF has shown a much better and quicker penetration into the cells in vitro as compared to melphalan. <p>In this present work, the cytotoxic effect of PSF was further evaluated in vivo using a standardized nude mice tumor model bearing a human melanoma. First, the acute toxicity in rats and mice and the maximum tolerated dose were determined. After a dose-escalation study one dose was singled out and tested as a single dose and as a fractionated dose. PSF was able to reach the tumor site and a dose-response relationship was observed. The IP administration of fractionated doses of PSF had significantly better effect on tumor growth inhibition, regression and regrowth than single dose administration and this without any evidence for general toxicity monitored by animal weight loss. We also compared the efficacy of PSF to its parent drug m-sarcolysin, melphalan and cyclophosphamide and observed that PSF was much more active than both melphalan and m-sarcolysin at the same molar doses.<p>Body distribution of the 14C-labelled PSF revealed ratios of 2.4 and 1.5 compared to muscle tissue for the two melanoma tumors evaluated with no significant and stable accumulation in any vital organ. The amount of tracer was still high in the blood after 24 hours explaining the high radioactivity in the kidney and partly in the liver. Interestingly, the spleen had an unusual high radioactivity uptake reflecting the exceptional binding of the tracer to blood cells (BC), while the pancreas very high load was an indicator of protease-mediated specific delivery and strongly support our hypothesis elaborated on the basis of in vitro results. <p><p>Our in vitro data point to a particular mechanism of action of PSF based on the transport of PSF through the body by the rapid binding to blood cells and the delivery at the tumor site by the subsequent release of its active metabolites due to cleavage by tumor-associated proteases.<p>Concerning the binding of PSF to membranes and its transport the following observations were made: while PSF was stable in human plasma, it disappeared very quickly in whole blood along with the generation of a main metabolite: m-sarcolysin. The presence of BC membranes was required for both binding and generating the metabolites. Binding to natural or artificial membranes was achieved and only competition with melanoma cells or proteolytic enzymes such as dispase, led to the generation of active metabolites. The different metabolites were isolated using preparative LC and were then identified using Electrospray Ionisation Mass Spectrometry (ESI). Three metabolites, of which m-sarcolysin was the main one, were identified all bearing the chloroethyl alkylating group. <p>Enzymatic catalysis was further supported by a set of experiments where the enzymatic activity was non-specifically and specifically inhibited. In order to look at the effect of extracellular matrix proteases on PSF, three representatives of ECM proteases were incubated with PSF: collagenase A had no effect, but both dispase and trypsine were able to process PSF. <p>The following data indicate the higher processing of PSF in the presence of cells with a higher proteolytic activity and thus the delivery of the blood cell-bound PSF. When comparing BC with melanoma cells (MC), the latter showed a higher ability to bind and process PSF both by membrane-associated and most interestingly soluble proteases. A lot of families of enzymes are reported to be overexpressed by melanoma cells including: metalloproteases, cysteine cathepsins, serine proteases and aminopeptidases. All the melanoma cells and cell lines evaluated were able to generate PSF active metabolites. <p>To identify the families of enzymes expressed on the membrane of melanoma cells that might be involved in the mechanism of action of PSF, we performed 2D-gel electrophoresis on their membrane extracts. The 2D-gels experiments revealed the presence of proteins compatible with enzymes known to be important in melanoma and further work is needed to identify the individual enzymes involved by using mass spectrometry and Western blotting. <p><p>Both our in vitro and in vivo findings strongly suggest that not only melanoma tumor cells and tumor sites but other types of tumors as well may be targets for the toxic activity of PSF owing to their much higher load in proteolytic enzymes that are closely related to their invasive potential. The transport of PSF by the blood cells and the release of its metabolites at the tumor site result in a low amount of drug in its free soluble form within the blood and this may explain the relatively lower side-effects observed. PSF is thus expected to have a much better therapeutic index than conventional alkylating agents. This original mechanism of drug delivery may well be extended to other cancer and non-cancer drugs than alkylating agents.<p><br>Doctorat en Sciences biomédicales et pharmaceutiques<br>info:eu-repo/semantics/nonPublished

Le cancer est la deuxième cause de décès dans le monde. Malgré l'existence des traitements standards, le développement et la recherche de stratégies thérapeutiques innovantes et de médicaments est toujours nécessaire. La combinaison des médicaments induisant la mort cellulaire immunogène (ICD) et l'inhibition des points de contrôle immunitaire (ICB), semble être un protocole prometteur. Dans cette thèse, nous avons démontré que la Lurbinectedine, un inhibiteur de la transcription nouvellement approuvé pour le traitement du cancer du poumon récidivant, déclenche les caractéristiques de l'ICD dans quatre différentes lignées cellulaires humaines et murines in vitro. Vaccinée par des cellules de fibrosarcome traitées par la lurbinectedine, les souris immunocompétentes sont protégées du rechallenge des tumeurs syngéniques. La lurbinectedine limite la croissance du fibrosarcome transplanté d'une manière immunodépendante. Dans les souris, le fibrosarcome murin (MCA205) transplanté et le cancer du sein, induit par des hormones en combinaison avec des cancérigènes, ont été sensibilisés par la lurbinectedine aux deux ICB : PD-1 et CTLA-4. Il convient de noter que la mémoire immunologique à long terme a été générée chez des souris guéries. En outre, nous avons évalué la capacité anticancéreuse de LTX-401, un peptide oncolytique conçu pour l'immunothérapie locale. Les injections intratumorales séquentielles de LTX-401 retardent considérablement la croissance des tumeurs sous-cutanées MCA205 et TC-1 chez un hôte immunocompétent, mais montrent un effet thérapeutique limité sur les tumeurs syngéniques abscopales. Une seule administration de LTX-401 augmente l'efficacité de l'ICB anti-CTLA-4 ou anti-PD-1 + anti-CTLA-4. De plus, le traitement séquentiel avec LTX-401 et les deux ICB présente une immunité antitumorale systémique à la fois contre la tumeur traitée et la tumeur abscopale. En conclusion, la lurbinectedine et le LTX-401 induisent la mort cellulaire immunogène des cellules cancéreuses et renforcent les effets anticancéreux des inhibiteurs de points de contrôle immunitaires. Ces résultats jettent les bases expérimentales de traitements combinés et peuvent faciliter les conceptions d'essais cliniques<br>Cancer is the second leading cause of death worldwide, despite the existence of standard treatment, innovative therapeutic strategies and drugs are still in urgent demand. The combination of immunogenic cell death (ICD) inducing drugs and immune checkpoint blockade (ICB) seems to be a promising modality. In this thesis, we demonstrated Lurbinectedin, a transcription inhibitor newly approved for relapsed lung cancer treatment, triggers hallmarks of ICD in four different human and murine cell lines in vitro. Vaccinated with Lurbinectedin-treated fibrosarcoma cell protects immunocompetent mice from rechallenge with syngeneic tumours. Lurbinectedin restrains transplanted fibrosarcoma growth in an immune dependent manner. Both transplanted MCA205 cancer and hormone/carcinogen induced breast cancer were sensitized by Lurbinectedin to PD-1 and CTLA-4 double ICBs. Of note, long-term immunological memory was generated in cured mice. Further, we evaluated the anticancer capacity of LTX-401, an oncolytic peptide designed for local immunotherapy. Sequential intratumoral injections of LTX-401 dramatically retards subcutaneous MCA205 and TC-1 tumour growth in immunocompetent host, yet shows limited therapeutic effect of anti-CTLA-4 or anti-PD-1/anti-CTLA-4 ICBs. Moreover, sequential LTX-401 treatment with double ICBs exhibits systemic antitumor immunity to both treated and abscopal tumour. In conclusion, lurbinectedin and LTX-401 induce cancer cell immunogenic cell death and enhance the anticancer effects of immune chekcpoint blockade. These results lay the experimental foundation of combination regiments and may facilitate the clinical trial design

Thesis advisor: Udayan Mohanty<br>Computational research can play a crucial component in the discovery of unique biochemical phenomena, from answering fundamental questions about molecular function and structure to the modeling of designed pharmaceuticals to cure many debilitating illnesses. Here computational methods are employed to examine the exquisite role that magnesium ions play in stabilizing ribosomal subunits responsible for protein translation and to analyze the potential of a proposed anticancer drug for a pathway that is impaired in the majority of human cancer cases<br>Thesis (BS) — Boston College, 2011<br>Submitted to: Boston College. College of Arts and Sciences<br>Discipline: College Honors Program<br>Discipline: Chemistry

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2008.<br>Source: Dissertation Abstracts International, Volume: 69-05, Section: B, page: 2686. Adviser: Nicki Engeseth. Includes bibliographical references (leaves 127-135) Available on microfilm from Pro Quest Information and Learning.

The medical field related to bacterial infections and cancer are currently facing currently one of the biggest challenges, mostly due to conventional treatments inefficiency after years of overuse and misuse in clinics. Cases of multi-resistant bacterial infections are increasing every year, according to World Health Organization (WHO), explained by resistant microorganisms’ predominance after antibiotic usage and limited pharmaceutical development of new drugs. As for cancer therapeutics, unspecific treatments that promote severe side effects had another reported consequence, increased cancer resistance, prolonging patients’treatment. As a result, new alternatives are necessary to fight these challenges, such as antimicrobial peptides (AMPs) and anticancer peptides (ACPs). These peptides physical-chemical properties, such as small amino acid sequence, amphipathicity and positive net charge, allow them to act selectively at specific cell membranes, mostly due to electrostatic interactions (cationic vs. anionic membranes). Besides, they can be used against different targets, with reported activity against bacteria, viruses, fungi and cancer cells. In the last case, they are dependent of cancer cell membrane phosphatidylserine (PS) translocation from internal to external membrane leaflet, which increases the negative cell surface charge. Throughout the work here presented, we focused on new AMPs designed according to two different strategies: (i) Pa-MAP 2 and Pa-MAP 1.9, synthetic AMPs redesign from a natural protein from the polar fish Pleuronectes americanus (winter flounder), and (ii) EcAMP1R4, PaDBS1R1 and PaDBS1R6, synthetic peptides designed through a bioinformatics algorithm that considers chemical properties and activity efficiency. In both cases, a multidisciplinary approach was performed, using biophysics and cell biology techniques to study their activity in vitro, using membrane models, bacterial and cancer cell lines, and in vivo infection models. Considering the Pa-MAP peptide family (Pa-MAP 2 and Pa-MAP 1.9), with a minimal inhibitory concentration (MIC) of 3.2 and 6.0 μM against Escherichia coli, respectively, they were shown to be efficient against a multi-resistant strain from a clinical isolates, inclusively with promising results demonstrated with an in vivo infection mice model. Nevertheless, only Pa-MAP 1.9 showed to have dual activity (AMP and ACP), being tested in two different cell lines, HeLa and HCT-166. Despite its efficiency in promoting cancer cell death, Pa-MAP 1.9 showed a different mechanistic behaviour for the cell lines tested, promoting total cell death after 6 h of incubation (IC50 of 51.8 ± 1.23 μM) and membrane homeostasis destabilization. As for the synthetic peptides (EcAMP1R4, PaDBS1R1 and PaDBS1R6), their antimicrobial activity was confirmed in vitro, according to bioinformatics studies, with MIC values against E. coli of 11.7, 1.5 and 8.0 μM, respectively. In vivo studies were also performed for the last two peptides, confirming their potential as future antimicrobial drug molecules. Their dynamics after membrane interaction was likewise studied, either using bacteria cells or lipid vesicles, showing that different biomembrane properties are destabilized, which could determinate AMP efficiency. Structure conversion to a-helix after membrane interaction showed to be the first step for peptide activity. Concluding, the work discussed in this thesis resulted in new peptide molecules that are effective AMPs and, one of them (Pa-MAP 1.9), also as ACP. Their activity was characterized in vitro and in vivo through new approaches, with the objective of identifying new insights that may help in future peptide design. Even with the promising results achieved so far, their potential use in therapeutics need to be further tested, considering their efficiency, but also their applicability, focusing on patients and in the pharmaceutical industry needs.

Os péptidos de defesa do hospedeiro (PDH) são geralmente moléculas curtas, anfipáticas e catiónicas que fazem parte do sistema imune inato da maioria dos organismos multicelulares. Inicialmente descritos como péptidos antimicrobianos (PAMs) devido à interação e destruição de paredes celulares bacterianas que são naturalmente aniónicas, contudo, estudos recentes mostraram que os PDH possuem uma gama de ação mais variada sendo capazes de interagir com células cancerígenas e podendo assim também ser designados de péptidos anticancerígenos (PACs). O aumento da multirresistência a fármacos, juntamente com a baixa taxa de descoberta de novos compostos e o facto de os tratamentos atualmente utilizados na terapêutica de várias doenças poderem ser abrasivos e até prejudiciais aos seres humanos, culminaram na necessidade urgente de estudar novas alternativas como os PDH. Várias espécies de anfíbios Patagónicos adaptaram-se a sobreviver em condições climáticas adversas, habitando zonas áridas ou semiáridas da Patagónia. A pele destes anfíbios produz uma secreção protetora a partir da qual os PDH foram extraídos, identificados e sintetizados nos péptidos PSo-4, PSo-7, PSo2-2, Thau-3 e Ooc-1. O objetivo deste trabalho foi caracterizar as propriedades antimicrobianas e anticancerígenas dos referidos péptidos e a sua estrutura em solução e na presença de vesículas lipídicas. A atividade antimicrobiana bem como a interação com membranas bacterianas foram avaliadas em bactérias Gram-positivas e -negativas por determinação da concentração mínima inibitória e através de microscopia de força atómica. Foram ainda realizados ensaios antiparasitários em formas sanguíneas, tipo selvagem, de Trypanosoma brucei brucei, ao passo que estudos anticancerígenos foram praticados em células Huh-7 de carcinoma hepatocelular humano através de métodos fluorimétricos e de absorção respetivamente. A caracterização da conformação dos péptidos, quando em meio aquoso ou em solução contendo vesículas lipídicas POPC:POPG, foi realizada por ensaios de espectrofluorometria e de dicroísmo circular. PSo-4, PSo-7, Thau-3 e Ooc-1 exibiram atividade antimicrobiana, mas apenas contra E. coli. PSo-4 demonstrou a atividade mais forte enquanto Ooc-1 provou ser bactericida. A análise por AFM apoia a hipótese de que a ação destes péptidos possa ocorrer ao nível da parede celular bacteriana, ou potencialmente interferindo com vias ou alvos intracelulares, culminando na desintegração da célula. Resultados preliminares também indicam que o péptido Ooc-1 exerce efeitos inibitórios no parasita T. b. brucei. Além disso, os péptidos Pso-7, Pso-4 e PSo2-2 revelaram potencial nocivo contra células Huh-7. Estruturalmente, a maioria dos péptidos apresentou propensão à agregação, quando em solução, e estrutura secundária com algum conteúdo de α-hélice, contrariamente a PSo-7 e PSo-4 que mantiveram uma disposição aleatória e baixos níveis de agregação. Tendo em consideração o comportamento e raio de ação demonstrados, é nossa convicção de que estes péptidos seriam dignos candidatos a investigação adicional e possível manipulação a nível da sua sequência, com o fim de aperfeiçoar as capacidades demonstradas de modo a abrir um caminho viável de desenvolvimento farmacológico.<br>Host-defence peptides (HDPs) are usually short, cationic and amphipathic molecules that are part of the innate immune system of most multicellular organisms. Initially described as antimicrobial peptides (AMPs) due to their interaction and destruction of bacterial cell walls which are naturally anionic, however, recent studies have shown that HDPs have a broader range of action being able to interact with cancer cells and may therefore also be referred to as anticancer peptides (ACPs). Increasing worldwide multidrug resistance (MDR) together with the low discovery rate of new drugs and the fact that the current treatments used in the therapy of various diseases can be very harsh and damaging to human beings, culminated in an urgent need to study new alternatives such as HDPs. Several species of Patagonian amphibians have adapted to survive in adverse climatic conditions, inhabiting arid or semi-arid zones of Patagonia. The skin of these amphibians produces a protective secretion from which the HDPs were extracted, identified and synthesized into peptides PSo-4, PSo-7, PSo2-2, Thau-3 and Ooc-1. The aim of this work was to characterize the antimicrobial and anticancer properties of the peptides mentioned as well as their structure in solution and in the presence of lipid vesicles. Antimicrobial activity and interaction with bacterial membranes were assessed in Gram-positive and Gram-negative bacteria by minimal inhibitory concentration determination and atomic force microscopy. In addition, antiparasitic assays were carried out on wild-type bloodstream forms of Trypanosoma brucei brucei while anticancer studies were performed on human hepatocellular carcinoma Huh-7 cells through fluorescence and absorption spectrophotometry methods respectively. The characterization of the peptides conformation when in aqueous medium or in a solution containing POPC:POPG lipid vesicles was done via spectrofluorometric and circular dichroism assays PSo-4, PSo-7, Thau-3 and Ooc-1 exhibited antimicrobial activity but only against the Gram-positive bacteria E. coli. PSo-4 showed the strongest activity while Ooc-1 proved to be bactericidal. Analysis by AFM supports the hypothesis that the action of these peptides may occur either at the bacterial cell wall level or by potentially interfering with intracellular pathways or targets, culminating in cell disintegration. Preliminary results also indicate that peptide Ooc-1 exerts inhibitory effects on the parasite T. brucei brucei. Furthermore, peptides PSo-7, PSo-4, and PSo2-2 revealed deleterious capacity against Huh-7 cells. Structurally most peptides exhibited a propensity for aggregation, when in solution, and presented a secondary structure with some α-helix content, except for PSo-7 and PSo-4 that maintained a random coil arrangement and lower aggregation levels. Considering the behaviour and scope of action demonstrated, we believe these peptides to be worthy candidates for further investigation and possible sequence manipulation to enhance their demonstrated capabilities in order to open the path of pharmacological viability.

Ellipticine (EPT), a natural plant polyphenolic compound, has long been known for its significant anticancer and anti-HIV activities. Recent study on its photophysical properties has revealed that ellipticine has three molecular states: protonated, neutral and crystalline. Further in vitro cytotoxicity tests indicated that protonated ellipticine exhibited much higher anticancer activity than the other two states. To maximize drug therapeutic effect, a small library of ariginine-rich ionic complementary peptides derived from EAK, including EAR8-II, EAR8-a, ELR8-a, and EAR16-II, were investigated as a potential carrier to deliver prescribed protonated ellipticine for treatment of cancer. Fluorescence study demonstrated that all four peptides were able to solubilize and stabilize protonated ellipticine in aqueous solution at 5:1 mass ratio of peptide-to-ellipticine (0.5: 0.1 mg/mL) even upon 4000 times dilution. Physicochemical characteristics of peptides self-assemblies and peptide-ellipticine complexes such as particle size, surface charge, secondary structure and morphology were determined by dynamic light scattering (DLS), zeta potential, circular dichroism (CD) , atomic force microscopy (AFM) and transmission electron microscopy (TEM), respectively. Then the ellipticine maximum suspension was determined by ellipticine UV-absorption. With the help of the peptides and mechanical stirring overtime, up to 100% ellipticine could be uptaken and stabilized in the solution as protonated ellipticine. In vitro cytotoxicity tests indicated that the peptides were demonstrating significant biocompatibility without affecting the survival of two cancer cell lines, human lung carcinoma cell line A549 and breast cancer cell line MCF-7, whereas the complexes with protonated ellipticine were found to show great anticancer activity to the two cancer cell lines. The IC50 values were obtained for each of four different peptide-ellipticine complexes ranged from 0.36±0.12 to 18.90±0.46 μM. It is worth noting that the IC50 value of EAR16-ellipticine complex to MCF-7 was over 50 times higher than that one to A549, which presented that EAR16-ellipticine complex has a selective targeting activity to A549, with the lowest IC50 value of 0.36±0.12 μM among all four complexes. Such a result indicated that this library of novel arginine-rich ionic complementary peptides had a great potential to encapsulate prescribed protonated ellipticine and exhibited an excellent anticancer activity upon serial dilution in aqueous solution. Overall, the charge distribution and increased hydrophobicity of the short (8 amino acids length) peptides seemed not to affect the complex formation and its therapeutic efficacy in vitro; however, the increase in length of the peptides significantly altered the nanostructure of peptides and its complexation with ellipticine, increased the therapeutic efficacy of EAR16-EPT to A549. This work provides essential information for peptide sequence design in the development of self-assembling peptide-based delivery of hydrophobic anticancer drugs.

Self-assembling peptides have emerged as new nanobiomaterials in the areas of nanoscience and biomedical engineering. In this category are self-assembling, ionic-complementary peptides, which contain a repeating charge distribution and alternating hydrophobic and hydrophilic residues in the amino acid sequence, leading to a unique combination of amphiphilicity and ionic complementarity. These peptides can self-assemble into stable nanostructures or macroscopic membranes that can withstand conditions of high temperature, extreme pH, many digesting enzymes and denaturation agents. Moreover, they exhibit good biocompatibility with various cultured mammalian cells, and do not have detectable immune responses when introduced into animals. These properties make them ideal materials for tissue scaffolding, regenerative medicine and drug delivery. This thesis focuses on the utilization of self-assembling peptides for hydrophobic anticancer drug delivery. The hydrophobic anticancer agent ellipticine was selected as a model drug. The studies include: (i) characterization of the photophysical properties of ellipticine in different environments; (ii) study of the formation of peptide-ellipticine complexes and the release kinetics; (iii) investigation of the cellular toxicity of the complexes and ellipticine uptake; (iv) study of the peptide sequence effect on the complex formation and in vitro delivery. Prior to applying ellipticine to the peptide-based delivery system, the fundamental studies on the effect of solution conditions, especially solvent polarity and hydrogen bonding, on the fluorescence of ellipticine were carried out. Ultraviolet (UV) absorption and fluorescence emission of ellipticine were found to be solvent/environment dependent. The absorption and emission maxima shifted to higher wavelengths (red shift) with increased solvent polarity. Large Stokes’ shifts were due to intramolecular charge transfer (ICT), which was enabled by large solvent polarity and hydrogen bonding of ellipticine with the solvents. The photophysical response of ellipticine to changes in solvent polarity and hydrogen bond formation could be used to infer the location of ellipticine in a heterogeneous medium, such as liposomes and cultured cells. EAK16-II, a model self-assembling peptide, was found to be able to stabilize ellipticine in aqueous solution. The equilibration time required to form peptide-ellipticine complex suspensions was found to be peptide concentration-dependent and related to the peptide critical aggregation concentration (CAC, ~0.1 mg/mL). With different combinations of EAK16-II and ellipticine concentrations, two molecular states (protonated or crystalline) of ellipticine could be obtained in the complexes. The release kinetics of ellipticine from the complex into egg phosphatidylcholine (EPC) vesicles (cell membrane mimics) was also affected by the peptide concentration used in the drug formulation. A higher peptide concentration resulted in a faster transfer rate, in relation to the size of the resulting complexes. Subsequent cellular studies on two cancer cell lines, A549 and MCF-7, showed that the complexes with protonated ellipticine were more effective against both cell lines, but their dilutions were not very stable. In addition, it was found that ellipticine uptake in both cell lines was very fast and through direct membrane permeation. Three peptides, EAK16-II, EAK16-IV and EFK16-II, either having a different charge distribution (EAK16-II vs. EAK16-IV) or hydrophobicity (EAK16-II vs. EFK16-II), were tested for the complexation and in vitro delivery of ellipticine. It was found that EAK16-II and EAK16-IV were able to stabilize protonated or crystalline ellipticine depending on the peptide concentration; EFK16-II, on the other hand, could stabilize neutral ellipticine molecules and ellipticine (micro)crystals. The viability results showed that the charge distribution of the peptides seemed not to affect the complex formation and its therapeutic efficacy in vitro; however, the increase in hydrophobicity of the peptides significantly altered the states of stabilized ellipticine and increased the stability of the complexes. This work provides essential information for peptide sequence design in the development of self-assembling peptide-based delivery of hydrophobic anticancer drugs.

Cancer is still one of the major death causes worldwide demanding an urgent search for new therapies that combine selectivity, efficacy and ability to avoid resistance by cancer cells. One of the biggest advances in anticancer therapy is the use of antimicrobial peptides’ (AMPs) as chemotherapy drugs since some of them showed both antimicrobial activity and selective anticancer activity. In this work, the anticancer activity of three different AMPs, pepR, HNP-1 and PvD1, was tested against cancer and non-tumorigenic breast cell lines (MDA-MB-231 and MCF 10A). The cytotoxic activity of each peptide was evaluated by MTT tetrazolium assay and the determination of the half maximal inhibitory concentration (IC50), and showed that pepR and PvD1 act as anticancer peptides (ACPs), able to select between cancer and non-tumorigenic cells, whereas HNP-1 is not a promising ACP once it is not selective, killing both cell lines at the same concentration. Then, zeta-potential was used to evaluate the peptide-cell interaction and its effect in cells’ surface charge. In this case, surface neutralization is not required before cell death, contrary to what happens when these peptides act like AMPs. Finally, according with previous results, PvD1 was chosen as the most promising peptide and, as such, used for imaging assays with atomic force microscopy (AFM). With this technique height profiles and surface roughness were evaluated for both cell lines in absence and presence of three different PvD1 concentrations. It was concluded that, with the increase of peptide concentration there are no significant changes in cell’s height profiles. On the other hand, when analysed separately, nucleus and cytoplasm present surface roughness changes associated to the increasing of PvD1 concentration. Although it is not yet possible to propose a mechanism of action, both pepR and PvD1 are efficient against MDA-MB-231 cells and selective to MCF 10A cells.

Tese de mestrado, Bioquímica (Bioquímica Médica) Universidade de Lisboa, Faculdade de Ciências, 2016<br>O cancro é uma doença que se carateriza pelo crescimento e proliferação descontrolados de células mutadas e constitui uma das principais causas de morte em todo o mundo. O cancro da mama é o mais comum entre o sexo feminino e constitui a principal causa de morte associada ao cancro em mulheres de regiões menos desenvolvidas. Atualmente os procedimentos mais comuns no tratamento do cancro incluem cirurgia, radioterapia ou quimioterapia. No entanto, tanto a radioterapia como a quimioterapia podem causar danos em células e tecidos saudáveis dando origem a um conjunto de efeitos secundários. Adicionalmente, as células de cancro podem também desenvolver resistência aos compostos usados na quimioterapia. Assim, um dos focos da investigação científica na área de combate ao cancro passa pelo desenvolvimento de novos compostos que possam permitir ultrapassar os problemas associados às terapias convencionais. Desde que foi demonstrado que a linfa de alguns insetos, os grânulos de neutrófilos humanos e a pele de sapos continham péptidos capazes de destruir culturas de bactérias, um novo tipo de agentes antimicrobianos tem sido explorado: os péptidos antimicrobianos (AMPs). Os AMPs são péptidos bioativos, que têm entre 11 a 50 resíduos de aminoácidos e possuem carga global positiva, devido à elevada quantidade de resíduos de argininas e lisinas. Estes péptidos podem ser encontrados em microrganismos, plantas e animais, onde atuam como mecanismos de defesa. Neste sentido, os AMPs têm sido explorados como péptidos bioativos no combate a infeções por bactérias, fungos e vírus. O seu mecanismo de ação passa, de uma forma geral, pela disrupção da membrana celular das bactérias, que apresenta um carácter aniónico, o que promove uma interação electroestática com os péptidos catiónicos. Nos últimos anos tem sido proposto que os AMPs podem também ser ativos contra células de cancro, dada a natureza aniónica das membranas destas células, constituindo assim uma alternativa ao desenvolvimento de novos compostos anticancro. Assim, um AMP que apresente atividade anticancro pode também ser classificado como péptido anticancro (ACP). Os ACPs são péptidos catiónicos que têm normalmente menos de 40 resíduos de aminoácidos e apresentam propriedades hidrófobas e catiónicas. A carga global positiva destes péptidos deve-se ao elevado conteúdo em argininas e lisinas. Outra abordagem com vista ao desenvolvimento de novos compostos com atividade contra células de cancro é a otimização de agentes quimioterapêuticos conhecidos. Com base no sucesso terapêutico da cisplatina, um complexo metálico de platina usado no tratamento do cancro, vários complexos com diferentes centros metálicos e ligandos têm sido estudados contra células de cancro. Neste sentido, os complexos de ruténio têm recebido especial atenção uma vez que apresentam menor toxicidade quando comparados com os complexos de platina. Os péptidos translocadores de membranas celulares (CPPs) são péptidos bioativos em membranas com uma composição que varia entre 5 a 30 resíduos de aminoácidos e apresentam um caráter catiónico devido ao elevado conteúdo de resíduos de argininas e lisinas. Estes péptidos apresentam também caraterísticas anfipáticas e a capacidade de translocar membranas celulares. Devido a esta capacidade, estes péptidos têm sido aplicados como sistemas de direcionamento de fármacos (DDS), pelo que podem também ser usados na otimização de agentes quimioterapêuticos. A partilha de caraterísticas estruturais entre AMPs, CPPs e ACPs explica o fato de haver uma relação recíproca entre estas classes de péptidos, uma vez que a ação dos mesmos pressupõe uma primeira interação eletrostática com as membranas celulares, que pode resultar na disrupção membranar ou na translocação da membrana com ação em alvos intracelulares. Neste trabalho de investigação o objetivo foi estudar a atividade anticancro e o modo de ação de um complexo organometálico de ruténio (TM281), assim como dos conjugados que resultam da ligação deste complexo com diferentes péptidos. Esta ligação tem como objetivo promover a internalização do complexo metálico pelas células de cancro, pelo que os péptidos selecionados para ligação ao complexo foram a Tat, pepR, pepM, vCPP 2319 e CrotB que apresentam atividade de CPPs. Numa primeira fase, averiguou-se a atividade do complexo e conjugados contra uma linha de células epiteliais de cancro da mama designada MDA-MB-231, através da determinação dos valores de concentração responsável por 50% de morte celular (IC50). Os resultados obtidos mostram que todos os compostos testados possuem atividade contra as células de cancro MDA-MB-231. No entanto, os conjugados revelaram uma atividade mais elevada quando comparados com o complexo organometálico. Com base nos resultados obtidos foram identificados dois dos conjugados mais ativos, o TM295 e o TM298, com valores de IC50 de 2.42 e 2.70 μM, respetivamente. De modo a completar o estudo, foi testada a atividade anticancro dos péptidos que fazem parte destes conjugados, pepR e vCPP 2319. Os valores de IC50 obtidos foram de 2.55 e 5.21 μM, para o pepR e vCPP 2319, respetivamente, o que mostra que estes péptidos também possuem atividade contra as células de cancro MDA-MB-231. Assim, foi possível concluir que a conjugação do complexo de ruténio com os péptidos pepR e vCPP 2319 resulta em conjugados cuja atividade citotóxica se deve principalmente ao efeito do péptido. Para estudar se a atividade citotóxica do complexo organometálico, dos conjugados TM295 e TM298 e dos péptidos pepR e vCPP 2319 é seletiva para células de cancro, a atividade destes compostos foi testada contra uma linha de células de mama saudáveis. As células saudáveis escolhidas para este estudo são células epiteliais da glândula mamária designadas por MCF 10A. Através da análise dos valores de IC50 obtidos para células de cancro e saudáveis foi possível concluir que todos os compostos atuam preferencialmente em células de cancro. Após avaliação da atividade dos compostos contra células de cancro e saudáveis, realizaram-se estudos sobre o seu mecanismo de ação. De modo a perceber se o mecanismo de ação dos compostos passaria pela ação ao nível na membrana, recorreu-se à técnica de potencial zeta. Os resultados obtidos mostraram que nenhum dos compostos estudados promove alterações significativas na carga superficial de células de cancro ou saudáveis, o que é indicativo que os compostos não ficam retidos na membrana celular e o seu alvo preferencial deve ser intracelular. Assim, recorreu-se à técnica de microscopia de força atómica (AFM), de modo a avaliar o efeito dos compostos na morfologia das células. Nas imagens de altura, erro e projeções tridimensionais (3D) obtidas para as células MDA-MB-231 na presença dos compostos, foi possível observar contração celular, colapso e condensação nuclear e membrana celular intacta. Na presença dos péptidos, foi ainda possível observar fragmentos nucleares nas células de cancro, que poderão constituir corpos apoptóticos. Assim, os resultados obtidos sugerem que os compostos atuam nas células de cancro num alvo intracelular e poderão induzir morte celular por apoptose. Relativamente aos resultados obtidos para as células saudáveis, na presença do complexo organometálico, foi possível observar contração celular, colapso e desorganização nuclear e membrana celular intacta. Na presença do conjugado TM295 e dos péptidos pepR e vCPP 2319 foi possível observar estruturas proeminentes que poderão resultar da fusão dos nucléolos. Na presença dos péptidos foi ainda possível observar contração celular. Estas observações sugerem ação num alvo intracelular e morte celular por apoptose, tal como descrito para as células de cancro. Na presença do conjugado TM298 foi possível constatar a total destruição celular. Os péptidos exibiram um efeito menos agressivo do que os conjugados, uma vez que foi observado um menor grau de destruição celular. A partir das imagens de altura foi possível obter parâmetros, como o perfil de altura das células e a rugosidade da membrana celular. O efeito dos compostos nos perfis de altura das células de cancro revelou-se em concordância com a interpretação das imagens obtidas por AFM, observando-se uma diminuição da altura da célula no caso de colapso nuclear e variações abruptas da altura das células no caso de contração celular. Relativamente às células MCF 10A, perfis de altura irregulares estão em concordância com o elevado grau de destruição celular provocado pelos conjugados. Neste caso, os picos observados que se destacam do restante perfil estão de acordo com as estruturas proeminentes observadas na presença dos péptidos. A análise do efeito na altura máxima das células e na rugosidade da membrana celular apenas foi possível para o TM281 em células de cancro e saudáveis e para o vCPP 2319 em células de cancro. Esta análise acabou por revelar a inexistência de diferenças significativas nestes parâmetros. No entanto, a ausência de uma diminuição significativa da rugosidade da membrana celular na presença do complexo organometálico e do vCPP 2319 pode indicar que o citoesqueleto mantém a sua função de suporte da bicamada lipídica. Com os resultados obtidos neste trabalho foi possível concluir que os conjugados que resultam da ligação dos péptidos ao complexo organometálico de ruténio TM281 apresentam uma elevada atividade anticancro, que supera a atividade do próprio complexo. Em dois dos conjugados mais ativos, TM295 e TM298, os péptidos que os constituem, pepR e vCPP 2319, respetivamente, são os responsáveis pela atividade anticancro. Assim, foi possível concluir que estes péptidos podem não só funcionar como parte integrante dos conjugados, mas também servir de base para uma nova linha de investigação do campo do desenvolvimento de novos ACPs.

碩士<br>東海大學<br>化學系<br>98<br>Id1 proteins, inhibitors of differentiation or DNA binding, act as dominant negative antagonists of the basic helix–loop–helix (bHLH) family of transcription factors, which play an important role in cellular development, proliferation, and differentiation. The mechanism of Id proteins is to antagonize basic helix-loop-helix proteins, they bind as dominant-negative HLH proteins by forming high affinity heterodimers with other bHLH proteins, thereby preventing them from binding to DNA and inhibiting transcription of differentiation associated genes.The goal of this study is to design and synthesize N- or C-terminal deleted analogs of peptide 3C with high affinity for Id1 protein in order to interrupt the interactions among Id1, MyoD, and other bHLH DNA-binding proteins and to inhibit the proliferation of cancer cells. Affinity of each peptide for Id1 HLH domain was determined by surface plasmon resonance (SPR) technology. The secondary structure of each peptide was studied by circular dichroism (CD) spectroscopy. Biological effects of each peptide in breast cancer cell (MCF-7) was analyzed. Results demonstrated that the peptide 3C and peptide 3C-CtD4 showed high affinity for Id1 and the equilibrium dissociation constant (KD) were 3.16 μM and 2.77 μM, respectively. Comparison of the secondary structures of peptides, we found that the ratios of α-helix structure in the deleted peptides were different. Results of MTT assay demonstrated that treatment of MCF-7 with these peptides did not exhibit antiproliferation effects in cancer cells. We suggest that further modifications of amino acid residues in peptides 3C and 3C-CtD4 and further assay of peptides for their effects in other cancer cells may enhance the antiproliferative potency and the specificity of peptides. In summary, peptides 3C and 3C-CtD4 are promising lead compounds for the development of peptidic antagonists or anticancer agents.

An ideal drug delivery system should contain an appropriate therapeutic agent and biocompatible carrier. In this study, we investigated the ability of the all-complementary self-assembling peptide AC8 in stabilizing the anticancer compound and determined the in-vitro therapeutic efficacy of the peptide-mediated anticancer drug delivery. The all-complementary peptide AC8 was designed based on the amino acid pairing principle (AAP), which contains hydrogen bonding, electrostatic, and hydrophobic interaction amino acid pairs. AAP interactions make the peptide capable of self-assembling into β-sheet structure in solution in a concentration dependent manner. Peptide solution concentration is a key parameter in controlling the nanoscale assembling of the peptide. The critical assembly concentration (CAC) of the peptide was found ~ 0.01 mg/ml by several techniques. The all-complementary peptide AC8 was found to be able to stabilize neutral state of hydrophobic anticancer compound ellipticine in aqueous solution. The formation of peptide-ellipticine complex was monitored by fluorescence spectroscopy at different mass ratios of peptide-to-ellipticine. The anticancer activity of the complexes with neutral state of ellipticine was found to show great anticancer activity against two cancer cells lines, A-549 and MCF-7. This peptide-mediated anticancer delivery system showed the induction of apoptosis on cancer cells in vitro by flow Cytometry.

碩士<br>東吳大學<br>微生物學系<br>101<br>In Taiwan, cancer has been reelected to the ten leading causes of death for up to three decades. The death rate increased year by year. Surgical resection and chemotherapy are the major way to cure cancer. But surgical resection has its risks and also can not completely remove the cancer cells. And surgical resection may even result in the transfer of cancer cells. For these reasons, chemotherapy still the major research direction. The anti-microbial activity of antimicrobial peptides as antibiotic alternative medicine is a wide range of research. Some reports have shown that antimicrobial peptides may induce cancer cell death via apoptosis, necrosis and autophagy. Herein, we test the lipopolysaccharide binding peptides with anti-microbial activity to observing it’s cell cytotoxicity and cell cycle effect in five types of cancer cells. Therefor, we try to assess this peptide's anti-cancer ability. The results show that LPSBD0 and LPSBD2 has cytotoxicity to all kind of cancer cell in our research. But the different cancer cells treat with same peptide has different effection. For instance, liver cancer and lung cancer cells are more sensitive to this lipopolysaccharide-binding peptide. Moreover, these lipopolysaccharide-binding peptides will affect the cell cycle of all kinds of cancer cells. And this lipopolysaccharide-binding peptide cause cells to produce large amounts of debris. So, we consider that lipopolysaccharide-binding peptide has the ability to kill cancer cells. Use confocal microscopy to observe treated cells. Show that lipopolysaccharide-binding peptides, LPSBD0 and LPSBD2, are binding on the cell membrane. And different peptide treat with different cell has different affinity. In low concertration, these lipopolysaccharide-binding peptides can disrupt the cell membrane. So, we presume that lipopolysaccharide-binding peptides kill cells by destroy the membrane.

碩士<br>慈濟大學<br>醫學檢驗生物技術學系醫學生物技術碩士班<br>101<br>Many cationic peptides have been reported to show antimicrobial and anticancer activity. The characteristics of the antimicrobial and anticancer peptides are short, cationic, and amphipathic. Bacterial surfaces are anionic which is due to the fact that bacterial cell membranes and cell wall are rich with phosphatidylglycerol, peptidoglycan, and lipopolysaccharide. Outer leaflet of mammalian cell membranes are mainly phosphatidylcholine and sphingomyelin which are neutral charged. Therefore, antimicrobial peptides (AMPs) are strongly selective between bacterial and mammalian cells. Recently, many AMPs have shown to have anticancer activity. This is because the outer leaflets of cancer cell membranes contained larger percentage of phosphatidylserine and O-glycosylated mucins compared to those in normal cells. As a result, the surfaces of cancer cells tend to be negatively charged. This leads to the fact that AMPs can also selectively target and kill cancer cells. This study applied bioinformatic approach to de novo design a peptide sequence for bacteria and cancer cell treatment purposes. A short amino acid sequence (19 amino acids), high positive charge (+9), high percentage of α-helix and good amphipathicity named dN1 was designed. Solid phase peptide synthesis was used to the produce peptide. The peptide was purified by high-performance liquid chromatography and the amino acids sequence of purified peptide was checked by mass spectrometer. This peptide exhibited good bactericidal activity on both Gram positive and negative bacteria. According to AFM imaging, it seems that dN1 has strong ability to cause leakages on E. coli and destroy bonding structures of tightly bonded bacteria. During bactericidal activity, this peptide also induced hydroxyl radical productions in E. coli. To increase the efficiency of dN1, nanodiamond (ND) was considered becoming delivery vector. As dN1 coated on NDs, it presented great effect on bactericidal activity. dN1 was also excellent at cytocidal activity on a cancer cell line, HeLa cells. Hemolysis caused by the peptide at the effective concentrations against bacterial and cancer cells was negligible meaning that this peptide might be of great potential to be a useful drug for treating bacterial infections and cancer cells.

Introduction: Cancer drug delivery has limitations of delivering hydrophobic anticancer drugs and non-targeted adverse effects to normal tissues. This work explores the self-assembling peptide EAK16-II for delivery of hydrophobic anticancer drug ellipticine in human lung carcinoma A549 cells. Hypothesis: Self-assembling peptide based nanoparticles enhance cellular delivery of the hydrophobic anticancer drug ellipticine through an endocytotic mechanism. Methods: EAK16-II and ellipiticine were formulated in water, and size and morphology determined using transmission electron microscopy and dynamic light scattering. The anticancer activity was evaluated by MTS assay. The cellular uptake and subcellular distribution of drug was characterized using endocytotic inhibitors and trackers. Results: EAK16-II can form nanoparticles with ellipticine and has comparable anticancer activity to ellipticine in DMSO. Nano-formulation has enhanced drug uptake through the caveolin dependent pathway. The drug localizes to lysosome and target organelles. Conclusion: EAK16-II forms nanoparticles with the drug and enhances cellular uptake through a caveolin-dependent endocytotic mechanism.

Current diagnostic and therapeutic nanocarriers, including liposomes, micelles, and polymeric- and protein-based nanoparticles, are designed to have key functional properties such as: (i) longevity in the bloodstream, leading to accumulation of therapeutic cargos in neoplastic areas with leaky vasculatures; (ii) targeting of specific pathological sites through surface modification with targeting ligands; (iii) stimuli-responsive characteristics for controlled drug release under specific conditions. While some of these drug delivery systems have advanced into clinical stages, other nanocarriers remain under development to overcome issues with effective delivery such as lack of target-ability and fast clearance from circulation. Self-assembling peptides have recently shown great potential as nanocarrier materials for drug and gene delivery, owing to their safety, efficiency, and targeting capabilities. An amino acid pairing strategy enables us to design self/co-assembling peptides with multiple functionalities to fulfill drug delivery requirements. This thesis focuses on functionalization and characterization of self/co-assembling peptides as nanocarriers for hydrophobic anticancer drug delivery. Diethylene glycol (DEG) conjugation and protein binding are the two modification strategies used in this thesis to impart longevity and target-ability upon the peptide-based delivery system. The studies include: (i) characterization of self-assembling properties of the diethylene glycol (DEG)-conjugated amino acid pairing peptide AAP8, (ii) investigation of the self/co-assembling features of a model ionic-complementary peptide (EAR8-II) in complex with the hydrophobic drug pirarubicin, and the anticancer activity of the complex, (iii) the interactions between peptide-drug complexes and serum proteins from the thermodynamic viewpoint, (iv) quantification of the effect of protein binding to the peptide-based delivery system on immune responses and biocompatibility, and (v) exploration of the targeting capability of albumin-bound peptide-drug complexes towards lung cancer cells. Uncontrollable aggregation of AAP8 was the first issue to address in order to develop a promising platform for the peptide-based delivery system. Diethylene glycol (DEG), a short segment of polyethylene glycol (PEG), was conjugated to AAP8 either at one or both terminals, and then self-assembling and drug encapsulation properties of both functionalized AAP8s were characterized to evaluate the effect of DEG-modification. The results illustrated a significant reduction in uncontrollable aggregation, and the formation of uniform fibular nanostructures. In addition, DEG conjugation provided the peptide with safer features towards immune cells by reducing cellular toxicity to macrophages. Moreover, DEG-functionalization improved hydrophobic drug stabilization, as demonstrated by sustained cytotoxic efficacy against lung carcinoma cells over a relatively long time compared to the non-functionalized AAP8. Protein binding strategy was the second approach to utilize the peptide-based delivery system with more biocompatibility and target-ability features. EAR8-II was studied as a model ionic-complementary peptide with high capability of pirarubicin encapsulation and anticancer activities against different cancer cells. Albumin as a most abundant protein in serum was selected to assess its binding affinity to the delivery system, and evaluate its binding effect on immune responses and anticancer activities. The results showed a central role of albumin in the in vitro delivery of peptide-drug complexes to target lung cancer cells based on the following characteristics: (a) Non-covalent binding of albumin to the complex through hydrogen bonding and Van der Waals interactions. The interaction was confirmed by physicochemical methods such as fluorescence quenching and isothermal titration calorimeter (ITC). (b) Shielding properties of albumin for the complex against macrophages and blood components (erythrocytes and complement protein C5b-9). In the presence of albumin, phagocytosis and cytokine expression level of macrophages and hemolytic activity of the peptide-drug complex reduced significantly due to the smaller particle size of the albumin-bound complexes compared to unprotected ones. (c) Targeting the lung cancer cells, possibly because of the inhibition of the albumin-binding protein SPARC (secreted protein, acidic and rich in cysteine). SPARC is a glycoprotein over expressed in lung cancer cells with high affinity to albumin. The results from in vitro SPARC expression in A549 cells, a type of human non-small cell lung carcinoma (NSCLC), showed a significant drop by the albumin-bound complex at the mRNA level evaluated by qRT-PCR. This effect can be explained by transporting the albumin-bound complex into the cell surface, binding to the SPARC proteins, and so inhibiting the SPARC expressions. This work lays out a foundation for modification and characterization of the self/co-assembly peptide-based nanocarriers for hydrophobic anticancer drug delivery, especially to improve longevity and target-ability properties.

碩士<br>國立清華大學<br>生物科技研究所<br>102<br>Antimicrobial peptides (AMPs) play important roles in the innate immune system of many organisms. Although AMPs have been essentially studied and received increasing attention as potential alternatives against infectious diseases, their use as anticancer peptides (ACPs) in cancer therapy either alone or in combination with other conventional drugs has been considered to be a therapeutic strategy to explore. Recently, We have developed a MD-POPC simulation model to examine the relationship of inserting depth as well as energy statistical calculation between membranes and peptides corresponding to the NMR results. Furthermore, we also proposed a SMD-POPC/POPG simulation model to perform with antimicrobial peptide, WLK and its’ isomers, which design is dependent on the characteristics of helicity and amphipathicity. In bioactivity experiment experiments, the assays of the WLK analogues were showed that WLK11 possess strong antimicrobial, anticancer and low hemolytic activity. Additionally, the biophysical experiments, including circular dichroism and two-dimensional NMR, were used to demonstrate that the stronger antibacterial and salt resistance of WLK11 could be due to the membrane immersion depth. The results of MD simulations demonstrate that the MD statistical calculations of non-bonded energy are positively correlated to the MIC value and cancer cytotoxicity. The assay of antimicrobial and anticancer ability can verify our SMD simulation systems and provide us the in-depth view between the activity and several biophysical properties. To sum up, our work opens a new avenue to develop a potent tool to help us predict not only novel potential AMPs but promising ACPs.

Cationic antimicrobial peptides (CAPs) are small peptides that constitute an important defence against microbial pathogens. Certain CAPs also possess anticancer properties. NRC-03 and NRC-07 are pleurocidins derived from winter and yellowtail flounder, respectively. The purpose of this investigation was to study the anticancer properties of NRC-03 and NRC-07. NRC-03 and NRC-07 killed breast cancer cells, including P-glycoprotein-overexpressing cells, in a time-dependent manner that peaked at 4 h. NRC-03 and NRC-07 lysed breast cancer cells by a mechanism that involved cell binding, mitochondrial destabilization, nuclear localization, and significant membrane damage. Interestingly, NRC-07, but not NRC-03, caused DNA fragmentation. NRC-03 and NRC-07 killed normal human epithelial cells, but did not kill endothelial cells or fibroblasts, or lyse human erythrocytes. NRC-03, and to a lesser extent NRC-07, had chemo-sensitizing properties, suggesting promise for their inclusion in combinational treatment regimens. Importantly, intratumoural injections of NRC-03 or NRC-07 inhibited tumour growth in a mouse model of breast cancer. Fetal bovine serum dose-dependently reduced cell killing by NRC-03. NRC-03 was degraded in human and mouse serum, which limited its potency. NRC-03- and NRC-07-induced cytotoxicity correlated with expression of several different negatively-charged molecules, rationalizing the generation of [D]-NRC-03, which carries the same positive charge as NRC-03, and was more potent but less selective for cancer cells than NRC-03. [D]-NRC-03 was also cytolytic and exhibited in vivo anticancer properties. To further test the clinical potential of NRC-03- and NRC-07-resistant cells were generated. NRC-03 and NRC-07 bound to resistant cells to a lesser extent than parental cells and were phenotypically distinct. Importantly, NRC-03- and NRC-07-resistant cells were killed by chemotherapeutic drugs, as well as [D]-NRC-03. These studies demonstrate that NRC-03, NRC-07, and [D]-NRC-03 are cytolytic peptides that kill breast cancer cells in vitro and in vivo. While more potent than NRC-03, [D]-NRC-03 requires further modification to minimize its toxicity toward normal cells. Although cancer cells may become resistant to NRC-03 and NRC-07 over time, resistant cells are still killed by other cytotoxic drugs, thereby reinforcing the value of adding these peptides to combinational regimens for the treatment of breast cancer.

碩士<br>東海大學<br>化學系<br>105<br>The rapid development of nanotechnology , let it have a chance applied to Biology and Medicine. Recently a great potential of nanodiamond (ND) particles as a multimodal imaging/therapy platform has been demonstrated. Nanodiamonds (NDs) play a key role with their excellent physicochemical properties, including high biocompatibility, physical adsorption and photostabilizing activity. To combined with Biomedical Science development of drug delivery systems may be to facilitate the permeation of drugs provide the best use in medical care. In this study, the use of colon cancer and its EGFR mechanism as the goal, the nanodiamonds as a drug carrier. Here be divided into two parts, synthetic antibody-conjugated nanodiamonds and peptide-conjugated nanodiamonds. Use BCA Protein assay、MALDI-TOF MS and FTIR to identified the effect of nanodiamonds as carrier on HT-29 cells. The results show that antibody-conjugated nanodiamonds can also be successfully associated with HT-29 cells with specific binding without loss activity. The inhibition of HT-29 cells exhibited by peptide 3C-conjugated nanodiamonds is also more effective than peptide 3C. The results show that nanodiamonds as a drug carrier, has a certain potential value.

碩士<br>國防醫學院<br>藥學研究所<br>95<br>Telomerase is the enzymatic activity that maintains the ends of eukaryotic chromosomes. Telomerase activity is detected in most tumor cells whereas it is low or undetectable in most normal somatic cells. The composition of human telomerase consists of two molecules each of TRC (Telomere RNA Component) and TERT (Telomere Reverse Transcriptase). TRC is a single RNA molecule, which provides an AAUCCC (in mammals) as template. TERT is a reverse transcriptase, which is a class of enzyme that creates single stranded DNA using single stranded RNA as a template. Expression of the telomerase catalytic component, the human telomerase reverse transcriptase (hTERT), is believed to be controlled primarily at the level of transcription. Because of this selective expression property of telomerase, it has been touted as a specific target for antitumor chemotherapeutics. In the path, our laboratory investigate telomerase inhibitory, cytotoxicity and the hTERT repressing effects on synthesizing 2,6-diamidoanthraquinones and 1,5-diamidoanthraquinones as compared to their disubstituted homologues. We found that several of the 1,5-diamidoanthraquinones and 2,6-diamidoanthraquinones inhibited telomerase activity effectively with IC50 at the sub-micro to micro molar range and caused acute cytotoxicity to cancer cells with EC50 similar or better than that of mitoxantrone. Therefore, our laboratory synthesis another series of 1,4- 1,5- 2,6- 2,7-diamidoanthraquinone derivatives (peptidyl anthraquinone). By synthesizing a series of 1,4- 1,5- 2,6- 2,7- peptidyl anthraquinone derivatives, we can study human telomerase reverse transcriptase (hTERT), telomeric repeat amplification protocol (TRAP). We use SEAP assay and MTT assay to monitor the hTERT expression; we can evaluate if compounds can inhibit the activity of telomerase by a mechanism consistent with interaction with G-quadruplex structure. On the other hand, some literature points out that aminoanthraquinone can against cancer cell by inhibiting topoisomerase II. The results suggested that these compounds caused multiple effects to cancer cells. Hence we synthesize a series of peptidyl anthraquinone derivatives hope to find some effective compounds.

碩士<br>國立交通大學<br>生物科技學系<br>105<br>Thermostable Direct Hemolysin from Grimontia hollisae (Gh-TDH) has been reported as a pore-forming toxin that results in diverse damage to different kinds of cells. Recently, studies have suggested that TDH is the tetrameric protein by nature, which means it can form pores on cell membranes leading to changing the osmotic pressure, disturbing cell homoeostasis and causing cell death. The N-terminal helix of Gh-TDH has indicated the correlation with binging activity on targeted cell membranes, contributing to hemolytic and cytotoxic activity afterwards. Therefore, the pore-forming toxic activity of Gh-TDH has been adjusted for the requisition of cancer treatment as a protein drug. In order to reduce and avoid ubiquitous toxicity of Gh-TDH, we choose to design several targeted fusion proteins under the Gh-TDH backbone for applying this pore-forming toxin in the treatment of few cancers. In previous research, our group indicated when Gh-TDH conjugated with a peptide at the N-terminal region, resulting in the decrease of hemolytic and cytotoxic activity. Furthermore, construction of mutant Gh-TDHR46E (monomeric form) with a targeting peptide such as epidermal growth factor receptor (EGFR) binding peptide (EB) to obtain a fusion protein, having significant anti-tumor effects on A431 cells1. In my study, there are several Gh-TDH recombinant proteins are designed as the pro-drug by using the concepts of recognition peptide to cell membrane and/or the cleavage sequences for cellular protease activation. The selective target molecules for the designs are MMP-7, Furin and PSA. The recognition peptide is conjugated at the N-terminus of Gh-TDH which might cause toxicity decrease of the designed recombinant protein. Besides, there is a cleavage site at the recognition peptide, which can cleave by proteolytic proteins produced by cancer cells. Next, the recognition peptide contacts with the target cancer cells with the treatment of recombinant protein. After that, the proteolytic proteins secreted from cancer cells interact with recognition peptide resulting in excision at cleavage site. Gh-TDH resumes wild-type activity after peptide-conjugated TDH releases sections cleaved by enzymatic proteins. Afterwards, active Gh-TDH can cause nearby cancer cell death as expected. The protein MMP7-TDHWT is the wild-type Gh-TDH (Gh-TDHWT) conjugates with eight amino acids of MMP-7 recognition and cleavage peptide. MMP7-TDHWT in comparison with Gh-TDHWT shows no significant difference of hemolytic and cytotoxic activity. Furin-PSA-TDHWT which is the Gh-TDHWT conjugates with six amino acids of Furin recognition peptide, and six amino acids of PSA cleavage site peptide. His-Furin-PSA-TDHWT is Furin-PSA-TDHWT conjugates with six His amino acids at the N-terminus. The result of hemolytic activity of His-Furin-PSA-TDHWT is weaker than Gh-TDHWT significantly. And the MTT assay suggests that cytotoxicity of Furin-PSA-TDHWT for LNCaP prostate cancer cells is weaker than Gh-TDHWT. However, the cytotoxicity of His-Furin-PSA-TDHWT is much weaker than Furin-PSA-TDHWT. The conditioned medium of LNCaP cells incubates with His-Furin-PSA-TDHWT for more than three days, which shows the enzymatic activity of PSA. The result indicates releasing of Gh-TDHWT from His-Furin-PSA-TDHWT by enzymatic cleavage.

碩士<br>國立宜蘭大學<br>生物技術與動物科學系生物技術碩士班<br>107<br>According to research statistics, the incidence of gastric cancer is quite high all over the world. The current treatment methods have many side effects, such as nausea, vomiting, nerve damage, etc. In recent years, many cancer cells have gradually developed resistance to chemotherapeutic drugs. However, studies have indicated that anticancer peptides (ACPs) have potential as novel anticancer agents, and can be used in combination with chemotherapeutic drugs. In our previous studies, the self-designed ACPs have good anti-cancer effects on many cancer cell lines, but their toxicity against normal cells is also rather high. Therefore, in the current study we intended to optimize the ACP sequences to obtain derivatives with better anti-cancer selectivity, and further evaluate their combined effects with chemotherapeutic drugs and anti-cancer mechanisms. First, a series of ACPs were designed and synthesized according to helical wheel structure analysis. After the MTT assay (IC50 = 21.13 μM) and the hemolysis test (MHC50 = 148.1 μM), the best selective peptide－Q4-15a-1 was selected for further analysis. The anti-cancer ability was measured by microscopy, wound healing assay, migration assay, and colony formation analysis. The results showed that it has good anticancer ability, and inhibits cancer cell migration and colony formation at low concentration (7 μM and 11 μM). Then, confocal microscopy was applied to monitor the interaction between FITC-labelled Q4-15a-1 and AGS cancer cells. It was found that Q4-15a-1 acts on gastric cancer cells, causes membrane damage and further reaches the nucleus. Proteomic approaches were conducted to verify the changes of protein profiles of AGS cells treated with or without Q4-15a-1 to understand the anti-cancer mechanism. The results revealed that treatment of Q4-15a-1 may affect the integrity, survival, protein synthesis, inflammatory response and other related biological pathways of AGS cancer cells. In addition, when Q4-15a-1 was used in combination with 5-FU or oxaliplatin, the combination index (CI) was less than 1, showing synergistic effects; and the dose-reduction index (DRI) was greater than 1, indicating reduction of drug dosage, respectively. Overall, Q4-15a-1 has quite good cell selectivity and anti-cancer ability. It enters the cancer cell by causing membrane damage, which further affects the nucleus, resulting in several protein changes. And when Q4-15a-1 is used in combination with chemotherapeutic drugs, it exhibits synergistic effects and dose reduction effects. According to the above findings, it shows that Q4-15a-1 has potential to be developed as a novel anti-cancer agent. In the future, we will verify the results of proteomic analysis and explore the correlation between these significantly altered proteins by western blotting to delineate the anti-cancer mechanism. We will also try to generate drug-resistant gastric cancer cell lines to evaluate the anticancer efficacy of the combined preparations.

碩士<br>國立宜蘭大學<br>生物技術研究所碩士班<br>98<br>Resistance is a very disturbing problem during cancer treatment, so the development of new agents with new modes of action attracts a great deal of interest. Recent studies have reported that certain cationic amphipathic antimicrobial peptide (AMPs) have selective toxicity toward various cancer cells. Most antibacterial and anticancer AMPs share a common membranolytic mode of action that results either in the selective disruption of the cancer cell membrane or permeation and swelling of mitochondria. The electrostatic attraction between the negatively charged membrane components of bacterial and cancer cells and AMPs is believed to play a crucial role in the disruption of bacterial and cancer cell membranes. In the current study, we use a series of synthetic AMPs designed by our own laboratory and a C-terminal modified naturally occurring AMP, Pleurocidine-amide (PleA) to test their cytotoxicity toward A549 human lung adenocarcinoma cells. PleA is a 23-mer AMP having selective toxicity toward cancer cells with no hemolytic activity. MTT assay revealed that PleA reduced viability of A549 cells and the IC50 value was 42 μM. In addition, western blot analysis showed that PleA could inhibit autophagy of A549 cells, and induce apoptosis 48-hrs after treatment. Flowcytometic analysis also demonstrated that PleA induced cancer cells to increase their sub-G1 population. Ultimately, confocal microscopy using AO and MDC staining also confirmed that PleA inhibited autophagy and induced apoptosis of A549. Proteomic approaches were also applied to further investigate the anticancer mechanism of PleA against A549 cells. Image analysis of 2-DE gels revealed 10 protein spots with significant differences and 6 of them were identified by LC-ESI-Q-TOF MS/MS to be Phosphoglycerate kinase 1、Poly(rC)-binding protein 2、Retinal dehydrogenase 1、Alpha-enolase Aldose reductase and Partner and localizer of BRCA2. More detailed investigations in the future will help us to know the anticancer mechanism of PleA.

碩士<br>東海大學<br>化學系<br>105<br>Cell signaling is an important physiological function of the cell, which causes the cell to respond to external stimulation. Such as cell growth, division, differentiation, and apoptosis. Protein kinase plays an important role in the signaling pathway. Mitogen-activated protein kinase (MAPK) belongs to serine/threonine protein kinases, it can be transduction into cells to the nucleus, activating transcription factors and regulating gene expression, therefore MAPK making an important target for the development of anticancer agents. MAPK is activated by phosphorylation within its activation segment.It was reported that a phosphorylation-sensitive secondary structure could be formed in a 26-amino-acid (LARVADPDHDHTGFLTEYVATRWYRC-NH2, IDA) long synthetic peptide corresponding to the activation segment of Xenopus MAPK, termed IDA MAPK peptide. The unphosphorylated IDA MAPK peptide can specificly inhibit in vitro MAPK activity. Therefore, we selected this sequence as a template to design a series of peptide analogs IDA-1~3and IDA-1-1~3.For the development of peptidic inhibitors of MAPK pathway as anticancer agents. These peptide analogs were synthesized by solid phase peptide synthesis (SPPS) , followed by reverse phase -high performance liquid chromatography (RP-HPLC) purification and matrix assisted laser desorption ionization -time of flight mass spectrometry (MALDI-TOF MS) characterization of synthetic peptides. Characterized peptides were analyzed for their bioactivitions and cell migration by MTT assay、prestoblue assay and wound healing assay in human colon cancer cells (SW480 and HT-29) .The cell permeability of peptides was analyzed by using confocal microscopy.We used western blotting to observe different properties of pMAPK protein kinase. According to the results, IDA-1-3 exhibited greater anti-proliferative effect in the colon cancer cells. The cell permeability of peptides were evaluated by analyzing cells treated with FITC labeled IDA-1 and FAM labeled IDA-1-3 using confocal microscopy, and the results suggested that peptides into HT-29 cancer cells. However, the results of the western blotting did not show the inhibition of protein expression. The results can be used to explore MAPK-mediated signaling. It is a potential lead peptide for further development of anticancer agents.

碩士<br>國立清華大學<br>分子醫學研究所<br>105<br>In recent years, the development of anticancer peptides attracts more and more attentions and expectations. Anticancer peptides are suggested to have the selective ability to kill cancer cells and minor side effects on normal cells. However, the use of peptides in clinical treatments is still challenging. For example, unmet needs include reducing the side effects of AMP on normal cells and preventing it from proteolysis during transport in human blood. With our efforts, we screened out the peptide 2163 with antimicrobial activity from the genome of Lactobacillus casei strain ATCC 334, and we proved that it can kill the human colon adenocarcinoma cell SW480. In order to optimize the activity to kill cancer cells, we modified the sequence of peptide 2163 of hydrophobic moments and alpha-helix ratios, resulting in the novel peptide KL15. In this study, we applied the CCK8 assay to evaluate cell viability. In 16 hours treatment, the cell-killing activity of KL15 was significantly improved because of the sequence modifications. According to our observations, the peptide 2163 and KL15 have different cell-killing characteristics. KL15 induced cell membrane disruptions in a shorter time compared with peptide 2163. Through conjugating FITC to KL15, we detected the fluorescent signals by confocal microscopy and found that KL15 can enter the cancer cell SW480 in 10 minutes. Also, the survival rate of SW480 may be reduced by 35% in 2 hours indicated by CCK8 assay, and this effect cannot be rescued by treating the caspase inhibitor Z-VAD-FMK. Furthermore, we investigated the cell-death related protein HMGB1 and PARP1 by western blot. HMGB1, the marker for cell necrosis, can be detected in cell culture media in 6 hours. PARP1, the marker for cell apoptosis, didn't show cleavage in the cells. These results show that KL15 rapidly disrupts cell membranes and induces cell necrosis. We infer that KL15 might cause cell membrane disruptions and induce cell necrosis before being proteolysis by proteases in human blood for clinical use.

碩士<br>國立臺灣大學<br>病理學研究所<br>88<br>Nasopharyngeal carcinoma (NPC) is one of the most common cancers amoung Chinese living in southern China, Hong-Kong, Singapore and Taiwan. Our laboratory has established nine NPC cell lines from NPC patients. These cell lines have been used to study various topics in the past several years. The treatment of cancer requires the cooperative efforts of widely divergent medical specialities. In the past, the radiotherapy and surgical removal of NPC tumor masses have been applied for years. Now, new chemo-therapeautic drugs have created important roles of treatment in cancer. In this setting the clinical development of a treatment must be based on a thorough understatnding of the potential for beneficial response and of the pharmacologic, pathologic, toxicological, and physiological knowledge. The genetic instability, heterogeneity, and high mutation rate of cancer cells cause the carcinoma to survive after chemotherapy. The cancer cells might activate their multi-drug resistant potential and resulting in difficulty to treat the tumor. A new solution for multi-drug resistance is the way of anti-angiogenesis. Since the vascular endothelial cells belong to normal cells, there’s no problem with the multi-drug resistance. The present experiment included three anti-cancer drugs to treat NPC cells. (1) AHMA-Ethyl-Carbamate (AHMA-EC): This chemical compound was provided by Dr. Su from Academia Sinica, Taipei. We proved that this drug can kill NPC cells in vitro and in vivo. The anti-cancer phenomenon might be caused through more than one pathways. One possible mechanism was as follow: AHMA-EC bound to DNA and topoisomerase Ⅱ, then the complex inhibited transcription of Topo Ⅱ. Finally, AHMA-EC causes apoptosis. The other pathway was to supress hWee 1, a kinase transcript, resulting in blocking cell cycle at G2/M phase, AHMA-EC may also involve other undefined mechanism to tumor cells. We have also set up a positive control experiment with VP-16 (VepesidTM), an anti-cancer chemo -therapeutic compound which also inhibited Topo II enzyme activity. The result showed that VP-16 also inhibited both Topo II and hWee 1 transcription. In contrast to AHMA-EC, the effective dose of VP-16 was larger than AHMA-EC. In conclusion, AHMA—EC can kill and surpress tumor cell proliferation in the animal model experiment. AHMA-EC is less toxic than VP-16. AHMA-EC can be a potential anti-cancer drug in clinical treatment. (2) Angiostatin: We set up an animal model with NPC tumors to study the anti-cancer effect of angiostatin. Our results show that angiostatin can inhibit human umbilical vascular endothelial cells (HUVEC). In animal model experiments, angiostatin did inhibit the growth of NPC tumor masses on SCID mice. These result proved that the theory of anti-cancer with anti-angiogenesis is effective. For developing new way to anti-tumor, anti-angiogenesis will be a good new method to treat tumor mass. (3) P1: This polypeptide is designed containing arginine-glycine-aspartate sequence. P1 could inhibit tumor partially in animal model. We also found that RGD peptide can inhibit HUVEC. In animal model experiments, P1 could inhibit tumor mass partially. We should study more detail about P1 function to identify its mechanism and to find the way to protect the peptide degraded in vivo.

碩士<br>國立宜蘭大學<br>生物技術與動物科學系生物技術碩士班<br>107<br>In recent years, the effectiveness of chemotherapeutic drugs in cancer treatment has been limited due to the development of cancer drug resistance, so the search for alternative drugs is crucial. Studies have shown that antimicrobial peptides (AMPs), which are rich in positive charge and mostly amphipathic structures, have been shown to have anticancer potential in addition to multi-host defense activities such as antibacterial and immunomodulatory. In our previous studies, we have designed and synthesized a series of AMPs with anti-cancer activity, and have the ability to selectively kill a variety of different cancer cell lines. Among them, AMP Q4a showed good anticancer activity against human colon cancer cell line HCT116. Q4a can rapidly penetrate cell membrane and cause apoptosis of HCT116 cells; however, Q4a is susceptible to protease hydrolysis, which will lead to its limited therapeutic applications in vivo in the future. Therefore, the aim of this study is to evaluate the stability and anticancer activity of the newly designed D-form peptide Q4a (AD-Q4a) and its analogues, and further elucidate its role in the treatment of 5-FU- and oxaliplatin-resistant HCT116 cell line, respectively. Furthermore, AD-Q4a were then combined with the chemotherapeutic drug 5-FU or oxaliplatin to analyze the potency of the drug combination. In all D-form and L-form peptides and their analogues, AD-Q4a not only showed good anticancer activity against HCT116, but also resisted cleavage by trypsin and chymotrypsin for up to 4 hours. After treatment with the above two proteases for 1 hour, it still retains its anticancer activity and exhibits excellent stability. At the same time, AD-Q4a retains its anticancer activity after heating at 100℃ for 60 minutes, but its activity gradually decreased with the increase of serum concentration. In the inhibition zone assay, AD-Q4a also showed good salt ion stability. In addition, we observed that AD-Q4a caused permeabilization of HCT116 cell membrane by confocal microscope. And by using colony formation analysis and scratch-wound assay, we also found that AD-Q4a effectively inhibit proliferation and metastasis of cancer cells in a dose dependent manner. AD-Q4a also exhibits good anticancer activity against 5-FU and oxaliplatin-resistant HCT116 cell lines, and its combination with 5-FU or oxaliplatin showed improved anticancer activity compared to using each chemotherapeutic drug alone, with synergistic and dose reducing effects. In the future, we will further delineate the synergistic anticancer mechanism of the combined preparations in an attempt to develop novel combination preparations against colorectal cancer.

碩士<br>東海大學<br>化學系<br>98<br>Id1 protein is an important factor of tumorigenesis and metastasis. The objective of our study is to suppress the activity of Id1 protein in order to slow down or inhibit the tumorigenesis and metastasis. Our recent study reported in 2010 revealed that peptide 3C, a peptide fragment of MyoD protein, exhibited cytotoxic effect on cancer cells at low concentration. The objective of this thesis is to investigate the relationship between apoptosis and the expression of specific proteins by analyzing the changes of expression levels of specific proteins in cancer cells and peptide-treated cancer cells. Human breast cancer cells (MCF-7 cells) were cultured and treated with peptide 3C for 24 hours and 48 hours, followed by extraction of proteins from cancer cells and measuring the total concentration of cellular proteins. Changes of amounts of specific proteins in cancer cells were analyzed by Western blot and surface plasmon resonance technology. Results obtained by these two methods are correlated, indicating that the changes of expression levels of specific proteins in cancer cells are similar：After MCF-7 cells were treated with peptide 3C, the concentrations of Id1 protein and Bcl-2 protein were decreased, the concentration of Bax protein was increased, and the concentration of β-actin protein remained the same as expected. Comparison of these two methods for quantification of concentrations of specific proteins in cancer cells is useful for the development of novel methods for evaluation of anticancer effects of peptides or drugs. Keywords：Id1 protein, Bcl-2, Bax, β-actin, peptide antagonist, human breast cancer cells, apoptosis, Western blot, surface plasmon resonance.