N-sec -butylthioloated β-lactam (NsβL) is a novel beta-lactam antimicrobial with a mechanism of action proposed to inhibit 3-oxoacyl-acyl carrier protein synthase (ACP) III (FabH), resulting in the inhibition of fatty acid synthesis. It has been suggested that NsβL inhibits FabH indirectly by inactivating coenzyme-A (CoA). CoA is an essential cofactor for numerous proteins involved in glycolysis, the citric acid cycle (TCA), and pyruvate metabolism, in addition to fatty acid biosynthesis. This study aimed to determine the effects of NsβL on a diverse array of laboratory and clinical Staphylococcus aureus isolates by analyzing the mode of resistance in spontaneous and adaptive mutant NsβL-resistant mutants. Phenotypic analysis of the mutants was performed, as well as sequence analysis of fabH; along with comparative proteomic analysis of intracellular proteomes. Our results indicate that NsβL resistance is mediated by drastic changes in the cell wall, oxidative stress response, virulence regulation, and those pathways associated with CoA. It is our conclusion that Nsβ L has activity towards CoA, resulting in wide-spread effects on metabolism, virulence factor production, stress response, and antimicrobial resistance.

Ho, Tik Shun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 88-102). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgement --- p.vii / Table of Contents --- p.viii / List of Table --- p.x / List of Figures --- p.xi / List of Abbreviations --- p.xiii / Chapter CHAPTER 1 --- General Introduction --- p.1 / Chapter 1.1 --- Fatty Acid Synthase (FAS) - 7-domain multifunctional enzyme --- p.1 / Chapter 1.1.1 --- Functions --- p.1 / Chapter 1.1.2 --- Structure --- p.2 / Chapter 1.2 --- Fatty Acid biosynthesis reactions --- p.4 / Chapter 1.3 --- Malonyl Coenzyme A - An important mediator in lipogenesis --- p.7 / Chapter 1.4 --- FAS expression in different histotypes --- p.8 / Chapter 1.4.1 --- FAS in normal cells --- p.8 / Chapter 1.4.2 --- FAS in pathological cells --- p.8 / Chapter 1.4.3 --- Tumor-associated FAS (Oncogenic antigen-519) in cancer cells --- p.9 / Chapter 1.5 --- FAS signaling models in breast and prostate cancers --- p.12 / Chapter 1.5.1 --- Association between FAS and PI3K／Akt pathway --- p.12 / Chapter 1.5.2 --- Hypothetical model of FAS hyperactivity in breast and prostate cancer cells --- p.13 / Chapter 1.6 --- FAS inhibition to tackle cancer cell growth --- p.15 / Chapter 1.6.1 --- FAS inhibitors --- p.15 / Chapter 1.6.1.1 --- Cerulenin --- p.16 / Chapter 1.6.1.2 --- C75 --- p.17 / Chapter 1.6.2 --- Small interfering RNA --- p.17 / Chapter 1.7 --- FAS inhibition to enhance chemoresistant cancer cells sensitivity to drugs --- p.19 / Chapter 1.8 --- Hypothesis --- p.20 / Chapter CHAPTER 2 --- Methods and Materials --- p.21 / Chapter 2.1 --- Chemicals and antibodies --- p.21 / Chapter 2.2 --- Cell cultures --- p.21 / Chapter 2.3 --- MTT assay --- p.22 / Chapter 2.4 --- 5-Bromo-2'-deoxyuridine (BrdU)-labeling cell proliferation assay --- p.22 / Chapter 2.5 --- Cytotoxicity detection assay of LDH release --- p.23 / Chapter 2.6 --- DNA flow cytometry --- p.23 / Chapter 2.7 --- Confocal micocropy --- p.24 / Chapter 2.8 --- Immunoblot analysis --- p.24 / Chapter 2.8.1 --- Preparation of protein lysates --- p.24 / Chapter 2.8.2 --- Immunoblotting --- p.25 / Chapter 2.9 --- Caspase inhibitor studies --- p.26 / Chapter 2.10 --- Analysis of mitochondrial membrane potential --- p.26 / Chapter 2.11 --- Determination of caspase activities --- p.27 / Chapter 2.12 --- siRNA transfection --- p.27 / Chapter 2.13 --- Statistical analysis --- p.28 / Chapter CHAPTER 3 --- Results --- p.29 / Chapter 3.1 --- Cytostatic & cytotoxic studies of FAS inhibitors on human cancer cells --- p.29 / Chapter 3.1.1 --- Cerulenin and C75 suppress cell growth of different cancer histotypes --- p.29 / Chapter 3.1.2 --- Cerulenin and C75 suppress cell growth of A-375 dose- and time-dependently --- p.32 / Chapter 3.1.3 --- Cerulenin and C75 exert cytotoxic effect on A-375 but not normal skin HS68 cells --- p.36 / Chapter 3.1.4 --- Cerulenin and C75 arrest cell cycle progression and induce apoptosis with DNA Fragmentation --- p.39 / Chapter 3.2 --- Mechanistic studies of FAS inhibitors in A-375 cells --- p.46 / Chapter 3.2.1 --- Cerulenin and C75 induce caspase-dependent apoptosis --- p.46 / Chapter 3.2.2 --- Cerulenin- and C75-induced apoptosis involve extrinsic death receptor pathway --- p.52 / Chapter 3.2.3 --- Cerulenin- and C75-induced apoptosis involve intrinsic mitochondrial pathway --- p.57 / Chapter 3.2.4 --- Extrinsic death receptor pathway serves as a pioneer and links with intrinsic mitochondrial pathway in cerulenin- and C75-induced apoptosis --- p.65 / Chapter 3.3 --- Small interfering RNA on Fatty Acid Synthase (FAS siRNA) --- p.68 / Chapter 3.3.1 --- FAS siRNA induces PARP cleavage --- p.68 / Chapter 3.3.2 --- FAS siRNA triggers caspase-dependent apoptosis as FAS inhibitors --- p.70 / Chapter CHAPTER 4 --- Discussion --- p.72 / Chapter CHAPTER 5 --- Future Prospect --- p.85 / Chapter CHAPTER 6 --- References --- p.88

Orientadores: Anibal Eugenio Vercesi, Karina Gottardello Zecchin / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-15T13:27:40Z (GMT). No. of bitstreams: 1 Rossato_FrancoAparecido_M.pdf: 961339 bytes, checksum: eb647604d3856b3e12d36a8143d26540 (MD5) Previous issue date: 2010 / Resumo: Ácido graxo sintase (FASN - EC 2.3.1.85) é a enzima responsável pela síntese endógena de ácidos graxos de cadeia longa a partir dos precursores acetil-CoA e malonil-CoA. Diversos estudos mostram que a FASN é altamente expressa em vários tipos de neoplasias malignas humanas, tais como de próstata, mama, melanoma e, em alguns destes tumores, a alta expressão de FASN está associada a um pior prognóstico. O tratamento com inibidores específicos de FASN, como cerulenina, C75 e orlistat, diminui a capacidade de proliferação e induz apoptose em linhagens celulares derivadas de neoplasias malignas de próstata, mama e cólon, porém pouco se sabe sobre os efeitos desses inibidores em células não tumorais. Recentemente mostramos que a inibição de FASN com orlistat reduz a proliferação e induz apoptose em células B16-F10 de melanoma murino (Carvalho et al. 2008). Considerando que (1) pouco é conhecido sobre os efeitos de inibidores de FASN em células "normais", inclusive melanócitos e (2) dados iniciais mostram que o tratamento com orlistat ou cerulenina também induz elevados níveis de apoptose em células "normais", este estudo teve por objetivo principal verificar os mecanismos envolvidos na morte induzida pela inibição da FASN em linhagem celular não-tumorigênica derivada de melanoblastos de camundongos (melan-a). O tratamento in vitro de células melan-a com 5 µg/mL de cerulenina ou com 30 µM de orlistat induziu expressiva porcentagem de apoptose, mas não necrose. As células tratadas também apresentaram redução da proliferação, além de discretas ativação de caspase-3 e liberação de citocromo c. Como o silenciamento de FASN através de RNA de interferência (RNAi) não resultou em apoptose, investigamos o possível envolvimento mitocondrial na morte induzida pelos inibidores de FASN. De fato, o tratamento com cerulenina ou orlistat resultou em diminuição do ??m, além de mais de 50% de inibição da velocidade de respiração das melana no estado de repouso. Paralelamente também foi constatado que esses mesmos inibidores de FASN induzem apoptose e reduzem a proliferação de células derivadas de queratinócitos não tumorigênicos, HaCaT. O presente trabalho mostra, portanto, que os inibidores de FASN, cerulenina e orlistat, apresentam efeitos nocivos sobre células não tumorais, conseqüência da ação sobre a respiração mitocondrial. / Abstract: Fatty acid synthase (FASN - EC 2.3.1.85) is the enzyme responsible for endogenous synthesis of long chain fatty acid palmitate derivate from precursors acetyl-CoA and malonyl-CoA. Studies have shown that FASN is highly expressed in several types of human malignancies, such as prostate, breast, melanoma, and in some of these tumors, high expression of FASN is associated with a poor prognosis. FASN inhibitors, such as cerulenin, C75, and orlistat, decrease cell proliferation and induce apoptosis in prostate, breast, and colon tumor cells lines. Recently we demonstrated that inhibition of FASN with orlistat reduced proliferation and induced apoptosis in cells B16-F10 murine melanoma (Carvalho et al. 2008). Consider that (1) little is known about the effects of FASN inhibitors in normal cells, including melanocytes and (2) previous data show that treatment with orlistat or cerulenin also induces high levels of apoptosis in normal cells, the aim of this study was to analyze the mechanisms involved in FASN inhibitioninduced cell death in cell line derived from non-tumorigenic mice melanoblasts (melana). In vitro treatment of melan-a cells with 5 µg/mL cerulenin or 30 µM orlistat induced a significant percentage of apoptosis, but not necrosis. Treated cells also showed reduced proliferation, and moderate activation of caspase-3 and release of cytochrome c. As FASN silencing through RNA interference (RNAi) did not result in apoptosis, we investigated the possible involvement of mitochondria in FASN inhibition-induced cell death. Cerulenin or orlistat treatment of melan-a cells decreased ?? m and inhibited more than 50% the respiration rate in rest state. We also detected significant apoptosis and reduced proliferation in cells derived from non-tumorigenic keratinocyte, HaCaT, after incubation with the same FASN inhibitors. In conclusion, this study shows that FASN inhibitors, cerulenin and orlistat, have adverse effects on non-tumor cells, as a consequence of direct action on mitochondrial respiration. / Mestrado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Mestre em Fisiopatologia Médica

Orientadores: Anibal Eugênio Vercesi, Karina Gottardelo Zecchin / Texto em português e inglês / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T16:40:04Z (GMT). No. of bitstreams: 1 Rossato_FrancoAparecido_D.pdf: 7526681 bytes, checksum: 3768c017da7bde02ddd95e8e41881438 (MD5) Previous issue date: 2014 / Resumo: Recentemente mostramos que os inibidores da enzima ácido graxo sintase (FASN - EC 2.3.1.85), cerulenina e orlistat, reduzem a proliferação e induzem apoptose em células B16-F10 de melanoma murino via mecanismos mitocondriais. Neste presente estudo investigamos os efeitos desses inibidores de FASN em linhagem celular não-tumorigênica derivada de melanoblastos de camundongos (melan-a). O tratamento in vitro de células melan-a com 5 µg/mL de cerulenina ou com 30 µM de orlistat inibiu a proliferação celular, com acúmulo da proteína supressora de tumor p21WAF1/Cip1, assim como induziu a via intrínseca da apoptose com liberação de citocromo c e ativação de caspases-3 e -9, sem ativação da caspase-8. Os inibidores de FASN não alteram o conteúdo de ácidos graxos livres nas células melan-a, verificados por espectrometria de massas, sugerindo que o tratamento com cerulenina ou orlistat induz apoptose independente da inibição desta enzima. Análise das funções da bioenergética mitocondrial das células melan-a mostraram inibição da respiração, seguido por aumento da produção de superóxido. A inibição da respiração, promovida pelo tratamento com cerulenina ou orlistat, foi restrita à oxidação de substratos ligados a NADH (39,9% DMSO x cerulenina; ou 60,8% EtOH x orlistat) e succinato (45,8% DMSO x cerulenina; ou 51,8% EtOH x orlistat), e não foi significativa quando as mitocôndrias estavam respirando com substrato do complexo IV, N,N,N',N'-tetrametil-p-fenilenodiamina. A proteção conferida pelo sequestrador de radicais livres N-acetil cisteína (NAC) sugere que a disfunção mitocondrial provocada por estes compostos está associada a estresse oxidativo e é provável que seja mediada pela ação de superóxido na cadeia respiratória nos níveis de complexos de I e II. Análise proteômica de mitocôndria dessas células também mostra alterações ligadas ao estresse oxidativo. Nossos dados em conjunto sugerem que cerulenina e orlistat induzem apoptose em células não tumorais como resultado de uma disfunção mitocondrial e de maneira independente de FASN / Abstract: We have previously reported that the fatty acid synthase (FASN) inhibitors, cerulenin or orlistat, induce apoptosis in B16-F10 mouse melanoma cells mediated by mitochondria. Here we investigate the effects of these inhibitors on the non-tumorigenic mouse cell line melan-a. Cerulenin or orlistat treatment decreased cells proliferation, accompanied by increased amounts of the tumor suppressor protein p21WAF1/Cip1, as well as induced apoptosis, but not necrosis, in melan-a cell line. Mitochondrial cytochrome c release and activation of caspases-9 and -3 were detected in melan-a-treated cells. siRNAi for FASN did not culminate in apoptosis, and FASN inhibitors treatment did not alter free fatty acids content in the non-tumorigenic cells, as verified by mass spectrometry, suggesting that cerulenin or orlistat induces apoptosis independent on FASN inhibition. Analysis of energy-linked functions of melan-a mitochondria showed inhibition of respiration followed by large stimulation of superoxide production. Respiratory inhibition after cerulenin or orlistat treatment, respectively, was restricted to the oxidation of NADH-linked substrates (39.9 or 60,8%) and succinate (45.8 or 51.8%) and was not significant when mitochondria were respiring on the complex IV substrate, N,N,N?,N?-tetramethyl-p-phenylendiamine. The protection conferred by the free radical scavenger NAC suggests that the mitochondrial dysfunction caused by these compounds is associated with oxidative stress and is mediated by the action of superoxide on the respiratory chain at the levels of complexes-I and II. Proteomic analysis of mitochondria melan-a cells also indicate major changes linked to oxidative stress. Taken together, the present results show that cerulenin or orlistat induces apoptosis in non-tumorigenic cells via mitochondrial dysfunction, independent on FASN inhibition / Doutorado / Fisiopatologia Médica / Doutor em Ciências