Novel microtubule-targeting agents, pyrrolo-1,5-benzoxazepines, induce cell cycle arrest and apoptosis in prostate cancer cells

Advanced hormone-refractory prostate cancer is associated with poor prognosis and limited treatment options. Members of the pyrrolo-1,5-benzoxazepine (PBOX) family of compounds exhibit anti-cancer properties in cancer cell lines (including multi-drug resistant cells), ex vivo patient samples and in vivo mouse tumour models with minimal toxicity to normal cells. Recently, they have also been found to possess anti-angiogenic properties in vitro. However, both the apoptotic pathways and the overall extent of the apoptotic response induced by PBOX compounds tend to be cell-type specific. Since the effect of the PBOX compounds on prostate cancer has not yet been elucidated, the purpose of this study was to investigate if PBOX compounds induce anti-proliferative effects on hormone-refractory prostate cancer cells. We examined the effect of two representative PBOX compounds, PBOX-6 and PBOX-15, on the androgen-independent human prostate adenocarcinoma cell line, PC3. PBOX-6 and -15 displayed anti-proliferative effects on PC3 cells, mediated initially through a sustained G2/M arrest. G2/M arrest, illustrated as DNA tetraploidy, was accompanied by microtubule depolymerisation and phosphorylation of anti-apoptotic proteins Bcl-2 and Bcl-xL and the mitotic spindle checkpoint protein BubR1. Phosphorylation of BubR1 is indicative of an active mitotic checkpoint and results in maintenance of cell cycle arrest. G2/M arrest was followed by apoptosis illustrated by DNA hypoploidy and PARP cleavage and was accompanied by degradation of BubR1, Bcl-2 and Bcl-xL. Furthermore, sequential treatment with the CDK1-inhibitor, flavopiridol, synergistically enhanced PBOX-induced apoptosis. In summary, this in vitro study indicates that PBOX compounds may be useful alone or in combination with other agents in the treatment of hormone-refractory prostate cancer.

Novel microtubule-targeting agents, pyrrolo-1,5-benzoxazepines, induce apoptosis in multi-drug-resistant cancer cells

PURPOSE: The development of multi-drug resistance (MDR) due to the expression of members of the ATP binding cassette (ABC) transporter family is a major obstacle in cancer treatment. The broad range of substrate specificities associated with these transporters leads to the efflux of many anti-cancer drugs from tumour cells. Therefore, the development of new chemotherapeutic agents that are not substrates of these transporters is important. We have recently demonstrated that some members of a novel series of pyrrolo-1,5-benzoxazepine (PBOX) compounds are microtubule-depolymerising agents that potently induce apoptosis in several cancer cell lines and impair growth of mouse breast tumours. The aim of this current study was to establish whether PBOXs were capable of inducing apoptosis in cancer cells expressing either P-glycoprotein or breast cancer resistance protein (BCRP), two of the main ABC transporters associated with MDR.

METHODS: We performed in vitro studies to assess the effects of PBOXs on cell proliferation, cell cycle and apoptosis in human cancer cell lines and their drug-resistant substrains expressing either P-glycoprotein or BCRP. In addition, we performed a preliminary molecular docking study to examine interactions between PBOXs and P-glycoprotein.

RESULTS: We established that three representative PBOXs, PBOX-6, -15 and -16 were capable of inducing apoptosis in drug-resistant HL60-MDR1 cells (expressing P-glycoprotein) and HL60-ABCG2 cells (expressing BCRP) with similar potencies as in parental human promyelocytic leukaemia HL60 cells. Likewise, resistance to PBOX-6 and -16 was not evident in P-glycoprotein-expressing A2780-ADR cells in comparison with parent human ovarian carcinoma A2780 cells. Finally, we deduced by molecular docking that PBOX-6 is not likely to form favourable interactions with the substrate binding site of P-glycoprotein.

CONCLUSION: Our results suggest that pro-apoptotic PBOX compounds may be potential candidates for the treatment of P-glycoprotein- or BCRP-associated MDR cancers.

A new microtubule-targeting compound PBOX-15 inhibits T-cell migration via post-translational modifications of tubulin

The ordered, directional migration of T-lymphocytes is a key process during immune surveillance, immune response, and development. A novel series of pyrrolo-1,5-benzoxazepines have been shown to potently induce apoptosis in variety of human chemotherapy resistant cancer cell lines, indicating their potential in the treatment of both solid tumors and tumors derived from the hemopoietic system. Pyrrolobenzoxazepine 4-acetoxy-5-(1-naphtyl)naphtho[2,3-b]pyrrolo[1,2-d][1,4]-oxazepine (PBOX-15) has been shown to depolymerize tubulin in vitro and in the MCF7 breast cancer cell line. We hypothesized that this may suggest a role for this compound in modulating integrin-induced T-cell migration, which is largely dependent on the microtubule dynamics. Experiments were performed using human T lymphoma cell line Hut78 and peripheral blood T-lymphocytes isolated from healthy donors. We observed that human T-lymphocytes exposed to PBOX-15 have severely impaired ability to polarize and migrate in response to the triggering stimulus generated via cross-linking of integrin lymphocyte function associated antigen-1 receptor. Here, we show that PBOX-15 can dramatically impair microtubule network via destabilization of tubulin resulting in complete loss of the motile phenotype of T-cells. We demonstrate that PBOX-15 inhibitory mechanisms involve decreased tubulin polymerization and its post-translational modifications. Novel microtubule-targeting effects of PBOX-15 can possibly open new horizons in the treatment of overactive inflammatory conditions as well as cancer and cancer metastatic spreading.

Free fatty acid receptors:structural models and elucidation of ligand binding interactions

BACKGROUND: The free fatty acid receptors (FFAs), including FFA1 (orphan name: GPR40), FFA2 (GPR43) and FFA3 (GPR41) are G protein-coupled receptors (GPCRs) involved in energy and metabolic homeostasis. Understanding the structural basis of ligand binding at FFAs is an essential step toward designing potent and selective small molecule modulators.

RESULTS: We analyse earlier homology models of FFAs in light of the newly published FFA1 crystal structure co-crystallized with TAK-875, an ago-allosteric ligand, focusing on the architecture of the extracellular binding cavity and agonist-receptor interactions. The previous low-resolution homology models of FFAs were helpful in highlighting the location of the ligand binding site and the key residues for ligand anchoring. However, homology models were not accurate in establishing the nature of all ligand-receptor contacts and the precise ligand-binding mode. From analysis of structural models and mutagenesis, it appears that the position of helices 3, 4 and 5 is crucial in ligand docking. The FFA1-based homology models of FFA2 and FFA3 were constructed and used to compare the FFA subtypes. From docking studies we propose an alternative binding mode for orthosteric agonists at FFA1 and FFA2, involving the interhelical space between helices 4 and 5. This binding mode can explain mutagenesis results for residues at positions 4.56 and 5.42. The novel FFAs structural models highlight higher aromaticity of the FFA2 binding cavity and higher hydrophilicity of the FFA3 binding cavity. The role of the residues at the second extracellular loop used in mutagenesis is reanalysed. The third positively-charged residue in the binding cavity of FFAs, located in helix 2, is identified and predicted to coordinate allosteric modulators.

CONCLUSIONS: The novel structural models of FFAs provide information on specific modes of ligand binding at FFA subtypes and new suggestions for mutagenesis and ligand modification, guiding the development of novel orthosteric and allosteric chemical probes to validate the importance of FFAs in metabolic and inflammatory conditions. Using our FFA homology modelling experience, a strategy to model a GPCR, which is phylogenetically distant from GPCRs with the available crystal structures, is discussed.

Modulation of intracellular calcium homeostasis blocks autophagosome formation

Cellular stress responses often involve elevation of cytosolic calcium levels, and this has been suggested to stimulate autophagy. Here, however, we demonstrated that agents that alter intracellular calcium ion homeostasis and induce ER stress-the calcium ionophore A23187 and the sarco/endoplasmic reticulum Ca (2+)-ATPase inhibitor thapsigargin (TG)-potently inhibit autophagy. This anti-autophagic effect occurred under both nutrient-rich and amino acid starvation conditions, and was reflected by a strong reduction in autophagic degradation of long-lived proteins. Furthermore, we found that the calcium-modulating agents inhibited autophagosome biogenesis at a step after the acquisition of WIPI1, but prior to the closure of the autophagosome. The latter was evident from the virtually complete inability of A23187- or TG-treated cells to sequester cytosolic lactate dehydrogenase. Moreover, we observed a decrease in both the number and size of starvation-induced EGFP-LC3 puncta as well as reduced numbers of mRFP-LC3 puncta in a tandem fluorescent mRFP-EGFP-LC3 cell line. The anti-autophagic effect of A23187 and TG was independent of ER stress, as chemical or siRNA-mediated inhibition of the unfolded protein response did not alter the ability of the calcium modulators to block autophagy. Finally, and remarkably, we found that the anti-autophagic activity of the calcium modulators did not require sustained or bulk changes in cytosolic calcium levels. In conclusion, we propose that local perturbations in intracellular calcium levels can exert inhibitory effects on autophagy at the stage of autophagosome expansion and closure.

The synapsin cycle:a view from the synaptic endocytic zone

Although the synapsin phosphoproteins were discovered more than 30 years ago and are known to play important roles in neurotransmitter release and synaptogenesis, a complete picture of their functions within the nerve terminal is lacking. It has been shown that these proteins play an important role in the clustering of synaptic vesicles (SVs) at active zones and function as modulators of synaptic strength by acting at both pre- and postdocking levels. Recent studies have demonstrated that synapsins migrate to the endocytic zone of central synapses during neurotransmitter release, which suggests that there are additional functions for these proteins in SV recycling.

Molecular pathways: Estrogen pathway in colorectal cancer.

Worldwide, colorectal cancer has a higher incidence rate in men than in women, suggesting a protective role for sex hormones in the development of the disease. Preclinical data support a role for estrogen and its receptors in the initiation and progression of colorectal cancer and establishes that protective effects of estrogen are exerted through ERβ. Hormone replacement therapy (HRT) in postmenopausal women as well as consumption of soy reduces the incidence of colorectal cancer. In the Women's Health Initiative trial, use of HRT in postmenopausal women reduced the risk of colon cancer by 56% [95% confidence interval (CI), 0.38-0.81; P = 0.003]. A recent meta-analysis showed that in women, consumption of soy reduced the risk of colon cancer by 21% (95% CI, 0.03-0.35; P = 0.026). In this review, using the preclinical data, we translate the findings in the clinical trials and observational studies to define the role of estrogen in the prevention of colorectal cancer. We hypothesize that sometime during the tumorigenesis process ERβ expression in colonocytes is lost and the estrogen ligand, HRT, or soy products, exerts its effects through preventing this loss. Thus, in the adenoma-to-carcinoma continuum, timing of HRT is a significant determinant of the observed benefit from this intervention. We further argue that the protective effects of estrogen are limited to certain molecular subtypes. Successful development of estrogen modulators for prevention of colorectal cancer depends on identification of susceptible colorectal cancer population(s). Thus, research to better understand the estrogen pathway is fundamental for clinical delivery of these agents.

Effects of LL-37 Peptide Mimetics on Gingival Fibroblast Function

Host defence peptides, including the cathelicidin LL-37, play an important role in mucosal immunity, functioning as both antimicrobial agents and modulators of the inflammatory response. In the current climate of antibiotic resistance, the idea of using naturally occurring antimicrobial peptides, or their synthetic mimetics, to combat oral infection is particularly appealing. Objectives: The aim of this study was to investigate the effects of parent LL-37, and two peptide mimetics (KR-12 and KE-18), on cytokine expression and response to bacterial challenge by gingival fibroblasts. Methods: KR-12 and KE-18 are peptide mimetics of the biologically active, mid-region sequence of LL-37. The effects of commercially available LL-37, KR-12 and KE-18 on gingival fibroblast response to E coli and P gingivalis LPS challenge, analysed by IL-6 and IL-8 expression, were determined in cell culture by ELISA. The direct effects of each peptide on IL-6, IL-8, CXCL-1 and HGF expression were also determined by ELISA. The MTT assay was used to evaluate peptide effects on fibroblast viability. Results: LL-37 and KE-18, but not KR-12, inhibited LPS induction of inflammatory cytokine expression and directly stimulated CXCL-1 production by fibroblasts. All 3 peptides stimulated production of IL-8 and HGF. Neither LL-37 nor KE-12 affected cell viability, while KE-18, at higher concentrations, induced cell death. Conclusions: Shorter, peptide mimetics of LL-37, in particular KE-18, retain the immunomodulatory effects of the parent molecule and possess excellent potential as therapeutic agents in the treatment of oral infections including periodontal disease.

Aß biology and its contribution to Alzheimer's disease

A doença de Alzheimer (DA) é uma desordem neurodegenerativa progressiva patologicamente caracterizada pela presença de placas de amilóide (placas senis) insolúveis e também pela presença de tranças neurofibrilhares,formadas pela proteína Tau hiperfosforiladada. O principal constituinte das placas senis é o peptídeo beta-amilóide (Ab), que deriva do processamento proteolítico da proteína precursora de amilóide de Alzheimer (APP). Embora Ab exista como um agregado pouco solúvel nas placas senis, ele é secretado pelas células como uma molécula solúvel. O Ab “per se” pode afectar o metabolismo da APP. Alguns autores sugerem que o Ab exerce o seu efeito alterando o processamento ou catabolismo da APP, outros sugerem que ele também induz a transcrição da APP, onde aumentando os níveis da APP pode estar a contribuir para a sua própria produção (mecanismo de “feedback” positivo). Assim sendo, torna-se difícil consolidar todas estas observações e identificar as potenciais funções fisiológicas do Ab “in vivo”, ou as consequências da sua produção. Neste trabalho caracterizaram-se os efeitos do Ab no metabolismo da APP. Os nossos estudos revelaram que um dos mecanismos induzidos pelo Ab é a acumulação intracelular do fragmento neuroprotector sAPP (isAPPa) em estruturas com características vesiculares associadas ao citosqueleto. Estudos adicionais em culturas primárias revelaram que o Ab estava a exercer o seu efeito ao nível da secreção vesicular, provavelmente interferindo com o transporte de APP/sAPP ao longo da rede do citosqueleto. Esta hipótese é sustentada pelo facto do Ab estar a afectar a estabilidade e a polimerização de proteínas envolvidas na dinâmica do citosqueleto. Contrariamente a publicações anteriores o Ab não induziu a transcrição da APP, na verdade em culturas primárias neuronais foi observado uma diminuição nos níveis de expressão da APP. Isto foi acompanhado por um aumento nos fragmentos C-terminais da APP (CTFs) e uma diminuição na localização nuclear do seu domínio intracelular (AICD), sugerindo alterações na sinalização nuclear da APP. O Ab pode afectar outras vias de sinalização, particularmente alterando o balanço entre as actividades das proteínas cinases e fosfatases, o que pode ter consequências para o desenvolvimento da doença. Os dados obtidos indicam que o Ab é capaz de inibir a actividade da proteína fosfatase1, a sua importância numa perspectiva de futuras terapias é discutida. Devido à relevância da agregação do Ab para a sua toxicidade, a formação de complexos com proteínas que promovem a sua desagregação/degradação e o seu efeito no processamento da APP foi avaliado. Na presença destes complexos observou-se uma reversão da acumulação isAPP, demonstrando o potencial terapêutico destas proteínas como moduladores do metabolismo da APP. Este trabalho permitiu compreender melhor os mecanismos envolvidos nos efeitos do Ab no processamento da APP e descobrir algumas moléculas que podem ser relevantes numa perspectiva de diagnóstico e terapia na DA.

Phytochelatin signal tranduction and cadmium chelation in plants

As plantas utilizam diversas estratégias de sinalização para reconhecer e responder aos stresses ambientais. A maioria das vias de transdução de sinais partilham um sinal genérico, normalmente a modulação dos níveis intracelulares de Ca2+. Esta por sua vez pode iniciar uma cascata de fosforilação proteica que finalmente afecta as proteínas directamente envolvidas na protecção celular ou culmina em factores de transcrição que vão determinar a resposta fisiológica ao stresse. A percepção destes sinais e a compreensão de como estes podem activar as respostas adaptativas são factores-chave para a tolerância das plantas a stresses abióticos. Um dos principais stresses abóticos que restrigem o crescimento das plantas é a presença de metais pesados. A produção de fitoquelatinas e a subsequente quelação dos metais é o mecanismo mais conhecido de tolerância ao stresse metálico em plantas. Fitoquelatinas (PCs) são péptidos com grupos tiol que são sintetizados através da transpeptidação da glutationa (GSH), pela acção da enzima fitoquelatina sintase (PCS). No entanto, até ao momento, as vias de sinalização que levam à síntese de fitoquelatinas e à percepção do stresse metálico são pouco compreendidas. Dentro deste contexto, o presente trabalho foi elaborado com o intuito de elucidar a via de sinalização através da qual o cádmio é detectado pelas células vegetais e induz a síntese de PCs. Quase todos, os estudos de stresses abióticos em plantas apontam para o facto de a sua sinalização se basear nos mesmos tipos de sinais moleculares, nomeadamente a sinalização por cálcio, a fosforilação proteica e a indução de espécies reactivas de oxigénio (ROS). Trabalhos recentes sugerem que a sinalização de PCs poderá envolver todos estes parâmetros. Assim, uma primeira abordagem foi efectuada para compreender a síntese de PCs na espécie Arabidopsis thaliana, através da monitorizaçção da actividade de enzimas relacionadas, a γ-EC sintetase, GSH sintetase e a PC sintase (PCS), assim como o tempo necessário para o elongamento das PCs e a sua acumulação. Seguidamente, ao longo deste processo foi analisada a expressão de sinais específicos, associados com sinais de cálcio, fosforilação proteica e sinalização por ROS. A importância destes factores na síntese de PCs foi também avaliada através do uso de moduladores farmacológicos de cálcio e fosfatases proteicas e também pela indução de stresse oxidativo. Os resultados demonstraram novos dados sobre o papel do cálcio e da fosforilação proteica na produção de PCs e na síntese de GSH, revelando que a actvidade da PCS é regulada por fosforilação e que a sinalização de cálcio pode mediar a síntese de GSH. O envolvimento da sinalização de ROS na síntese de GSH, atráves de crosstalk com a sinalização de cálcio também foi proposta. Assim, os resultados aqui apresentados descrevem uma possível via de sinalização de cádmio nas plantas e da indução de fitoquelatinas. Este trabalho poderá ser portanto muito útil na implementação de novas metodologias de agricultura sustentável e práticas de fitorremediação em solos contaminados com metais pesados.

Reconfigurable transmitters for software-defined radios

Flexible radio transmitters based on the Software-Defined Radio (SDR) concept are gaining an increased research importance due to the unparalleled proliferation of new wireless standards operating at different frequencies, using dissimilar coding and modulation schemes, and targeted for different ends. In this new wireless communications paradigm, the physical layer of the radio transmitter must be able to support the simultaneous transmission of multi-band, multi-rate, multi-standard signals, which in practice is very hard or very inefficient to implement using conventional approaches. Nevertheless, the last developments in this field include novel all-digital transmitter architectures where the radio datapath is digital from the baseband up to the RF stage. Such concept has inherent high flexibility and poses an important step towards the development of SDR-based transmitters. However, the truth is that implementing such radio for a real world communications scenario is a challenging task, where a few key limitations are still preventing a wider adoption of this concept. This thesis aims exactly to address some of these limitations by proposing and implementing innovative all-digital transmitter architectures with inherent higher flexibility and integration, and where improving important figures of merit, such as coding efficiency, signal-to-noise ratio, usable bandwidth and in-band and out-of-band noise will also be addressed. In the first part of this thesis, the concept of transmitting RF data using an entirely digital approach based on pulsed modulation is introduced. A comparison between several implementation technologies is also presented, allowing to state that FPGAs provide an interesting compromise between performance, power efficiency and flexibility, thus making them an interesting choice as an enabling technology for pulse-based all-digital transmitters. Following this discussion, the fundamental concepts inherent to pulsed modulators, its key advantages, main limitations and typical enhancements suitable for all-digital transmitters are also presented. The recent advances regarding the two most common classes of pulse modulated transmitters, namely the RF and the baseband-level are introduced, along with several examples of state-of-the-art architectures found on the literature. The core of this dissertation containing the main developments achieved during this PhD work is then presented and discussed. The first key contribution to the state-of-the-art presented here consists in the development of a novel ΣΔ-based all-digital transmitter architecture capable of multiband and multi-standard data transmission in a very flexible and integrated way, where the pulsed RF output operating in the microwave frequency range is generated inside a single FPGA device. A fundamental contribution regarding the simultaneous transmission of multiple RF signals is then introduced by presenting and describing novel all-digital transmitter architectures that take advantage of multi-gigabit data serializers available on current high-end FPGAs in order to transmit in a time-interleaved approach multiple independent RF carriers. Further improvements in this design approach allowed to provide a two-stage up-conversion transmitter architecture enabling the fine frequency tuning of concurrent multichannel multi-standard signals. Finally, further improvements regarding two key limitations inherent to current all-digital transmitter approaches are then addressed, namely the poor coding efficiency and the combined high quality factor and tunability requirements of the RF output filter. The followed design approach based on poliphase multipath circuits allowed to create a new FPGA-embedded agile transmitter architecture that significantly improves important figures of merit, such as coding efficiency and SNR, while maintains the high flexibility that is required for supporting multichannel multimode data transmission.

Estimating the size of the population of persons who inject drugs in the island of Montréal, Canada, using a six-source capture-recapture model

BACKGROUND: To plan and implement services to persons who inject drugs (PWID), knowing their number is essential. For the island of Montréal, Canada, the only estimate, of 11,700 PWID, was obtained in 1996 through a capture-recapture method. Thirteen years later, this study was undertaken to produce a new estimate. METHODS: PWID were defined as individuals aged 14-65 years, having injected recently and living on the island of Montréal. The study period was 07/01/2009 to 06/30/2010. An estimate was produced using a six-source capture-recapture log-linear regression method. The data sources were two epidemiological studies and four drug dependence treatment centres. Model selection was conducted in two steps, the first focusing on interactions between sources and the second, on age group and gender as covariates and as modulators of interactions. RESULTS: A total of 1480 PWID were identified in the six capture sources. They corresponded to 1132 different individuals. Based on the best-fitting model, which included age group and sex as covariates and six two-source interactions (some modulated by age), the estimated population was 3910 PWID (95% confidence intervals (CI): 3180-4900) which represents a prevalence of 2.8 (95% CI: 2.3-3.5) PWID per 1000 persons aged 14-65 years. CONCLUSIONS: The 2009-2010 estimate represents a two-third reduction compared to the one for 1996. The multisource capture-recapture method is useful to produce estimates of the size of the PWID population. It is of particular interest when conducted at regular intervals thus allowing for close monitoring of the injection phenomenon.

Radio over fiber broadband access networks architectures based on wavelength division multiplexing techniques

The recent remarkable growth in bandwidth of both wired optical and wireless access networks supports a burst of new high bandwidth Internet applications such as: peer-topeer file sharing, cloud storage, on-line gaming, video streaming, etc. Within this scenario, the convergence of fixed and wireless access networks offers significant opportunities for network operators to satisfy user demands, and simultaneously reduce the cost of implementing and running separated wireless and wired networks. The integration of wired and wireless network can be accomplished within several scenarios and at several levels. In this thesis we will focus on converged radio over fiber architectures, particularly on two application scenarios: converged optical 60 GHz wireless networks and wireless overlay backhauling over bidirectional colorless wavelength division multiplexing passive optical networks (WDM-PONs). In the first application scenario, optical 60 GHz signal generation using external modulation of an optical carrier by means of lithium niobate (LiNbO3) Mach- Zehnder modulators (MZM) is considered. The performance of different optical modulation techniques, robust against fiber dispersion is assessed and dispersion mitigation strategies are identified. The study is extended to 60 GHz carriers digitally modulated with data and to systems employing subcarrier multiplexed (SCM) mm-wave channels. In the second application scenario, the performance of WDM-PONs employing reflective semiconductor optical amplifiers (RSOAs), transmitting an overlay orthogonal frequency-division multiplexing (OFDM) wireless signal is assessed analytically and experimentally, with the relevant system impairments being identified. It is demonstrated that the intermodulation due to the beating of the baseband signal and wireless signal at the receiver can seriously impair the wireless channel. Performance degradation of the wireless channel caused by the RSOA gain modulation owing to the downstream baseband data is also assessed, and system design guidelines are provided.

How hydrogen peroxide and CXCL8 modulate neutrophil recruitment "in vivo"

Tese de doutoramento, Ciências Biomédicas (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Medicina, 2014

ASYN and tau interaction : new drug target for neurodegenerative diseases

Tese de doutoramento, Bioquímica (Biotecnologia), Universidade de Lisboa, Faculdade de Ciências, 2014