The endogenous cannabinoid system in the gut of patients with inflammatory bowel disease

Activation of cannabinoid receptors (CBs) by endocannabinoids impacts on a number of gastrointestinal functions. Recent data indicate that CB1 agonists improve 2,4-dinitrobenzene sulfonic acid-induced colitis in mice, thus suggesting a role for the endocannabinoid agonist anandamide (AEA) in protecting the gut against inflammation. We here examined the gut endocannabinoid system in inflammatory bowel disease (IBD) patients, and investigated the ex vivo and in vitro effects of the non-hydrolysable AEA analog methanandamide (MAEA) on the mucosal proinflammatory response. The content of AEA, but not of 2-arachidonoyl-glycerol and N-palmitoylethanolamine, was significantly lower in inflamed than uninflamed IBD mucosa, and this was paralleled by lower activity of the AEA-synthesizing enzyme N-acyl-phosphatidylethanolamine-specific phospholipase D and higher activity of the AEA-degrading enzyme fatty acid amide hydrolase. MAEA significantly downregulated interferon-γ and tumor necrosis factor-α secretion by both organ culture biopsies and lamina propria mononuclear cells. Although these results are promising, further studies are needed to determine the role of cannabinoid pathways in gut inflammation. © 2011 Society for Mucosal Immunology

Identification of CDKs as Novel Targets of Aspirin and Its Metabolites: A Potential Role in Cancer Prevention

Background:The pursuit of drugs that inhibit cyclin-dependent kinases (CDKs) has been an intense area of research for more than 15 years. Till date, although multiple CDK inhibitors have been identified and few are undergoing clinical trials, only two synthetic drugs have been approved by Food and Drug Administration (FDA) for use in the treatment of cancer. These two drugs are mainly used for the treatment of metastatic breast cancer in combination with other drugs; however they have toxicity associated with their use and extends patients life not more than 24 months. Therefore, there is an urgent need for developing newer drugs that are more safe and efficacious. Uncontrolled cell proliferation is a hallmark of cancer. In mammalian cells, cell cycle is controlled by the sequential activation of cyclin dependent kinases (CDKs). Four CDKs (CDKs 1, 2, 4 and 6) and their activating cyclins (A, B, D and E), play key roles in cell cycle progression. It has been established that CDK4,6/cyclin D and CDK2/cyclin E/A promote the passage through G1 and S phases, whereas CDK1/cyclin B regulates the transition through the late G2 and mitosis. In addition, specific proteins classified as CDK inhibitors capable of binding to cyclin/CDK complexes to inhibit their enzyme activity also play a significant role in regulating cell cycle. While, expression and activity of cyclins and CDKs are tightly regulated in normal cells, they are often deregulated in cancer cells through frequent overexpression and frequent inactivation. Studies carried in the past 2 decades have clearly established that regular aspirin use for 5 – 10 years decreases the cancers of the epithelial tissues particularly the cancers of the colon. This evidence came from numerous epidemiological studies, clinical trials, in-vitro cell culture experiments as well as experiments in animal models. Despite its potential role in cancer prevention, it is not clear precisely how aspirin exerts its chemopreventative effects in epithelial tissues. In this context, multiple targets and signaling pathways have been identified; however a unifying mechanism has not been identified till date. The objective of this dissertation is to investigate the novel mechanisms by which aspirin prevents the occurrences of cancer and discover newer protein targets that maybe responsible for mediating its chemopreventative actions. Understanding aspirin-mediated chemopreventive mechanism and pinpointing its direct cellular targets is of high value, if it is to be used as a prophylactic drug. We hypothesized that aspirin and/or its primary metabolite salicylic acid may target cell cycle regulatory proteins modulating their level as well as functions. To address this, numerous biochemical, molecular biological studies were carried out in multiple cancer cell lines along with molecular docking studies to determine the interactions between aspirin/salicylic acid with CDKs and cyclins. The studies carried out during the course of this dissertation work have established that aspirin, salicylic acid and salicylic acid metabolites and derivatives target all more 4 members of CDK family namely CDKs 1, 2, 4 and 6, the major findings of which are detailed below.Results: Major finding 1: Our studies demonstrate that both aspirin and its primary metabolite, salicylic acid, decreased cyclin A2, B1, D3, CDKs 1, 2, 4 and 6 protein levels in a diverse panel of cancer cell lines. The decrease in cyclin A2 and cyclin B1 levels as well as CDK1 and CDK2 protein levels were associated with a corresponding decrease in the levels of messenger RNAs, suggesting that both aspirin salicylic acid regulate their expression at both transcriptional and post translational levels. Aspirin and salicylic acid also increased the levels of CDK inhibitors namely p21 and p27. The decrease in cyclin A2 and cyclin B1 protein levels appears to be mediated through 26S proteasomes. Major Finding 2: Through biochemical and molecular modeling studies we showed that salicylic acid directly binds to CDK2. Molecular docking studies identified Asp145 and Lys33 as the potential sites of salicylic acid interactions with CDK2. Extension of these studies showed that salicylic acid also binds to CDK1 using Asp146 and Lys33. Despite salicylic acid interacting with CDK 1 and 2 via interactions using amino acids in the active site of the enzyme, inhibition of the enzyme activity was not observed. Major Finding 3: We investigated the ability of salicylic acid metabolites 2,3- dihydroxy benzoic acid (2,3-DHBA) and 2,5-dihydroxy benzoic acid (2,5-DHBA) known to be generated by cytochrome p450 metabolism to CDK enzyme activity. In-vitro CDK assays showed that both metabolites inhibited CDK1 enzyme activity. Interestingly several derivatives 2,4-dihydroxybenzoic acid (2,4, DHBA), 2,6-dihydroxybenzoic acid (2,6- DHBA) and 2,4,6-trihydroxybenzoic acid (2,4,6-THBA) also inhibited CDK1 enzyme activity. 2,3-DHBA and 2,6-DHBA did not inhibit CDK2 and 4; however, both inhibited CDK-6 activity. Interestingly, 2,4,6-THBA was highly effective in inhibiting CDK1, 2, 4 and 6 activity. Molecular docking showed that these compounds potentially interact with CDK1. Immunoblotting experiments showed that aspirin acetylated CDK1, and preincubation with salicylic acid and its derivatives prevented aspirin-mediated CDK1 acetylation, which supports the data obtained from molecular docking studies.Conclusion: We identified CDK1 and 2 as salicylic acid binding proteins. In addition, we have demonstrated the interactions of salicylic acid metabolites and derivatives with CDKs. We suggest that intracellularly generated salicylic acid metabolites through CYP450 enzymes within the colonic epithelial cells may be responsible to the preferential chemopreventive effect of aspirin against CRC through inhibition of CDKs. This novel hypothesis and mechanism of action in aspirin’s chemopreventive effects opens a new area for future research. In addition, structural modification to salicylic acid derivatives may prove useful in the development of a novel CDK inhibitors in cancer prevention and treatment

Cysteine, a facilitator of hypoxia adaptation and a promoter of drug-resistance : a new route to better diagnose and treat ovarian cancer patients

Ovarian cancer is the third most common gynaecologic malignancy and the main cause of death from gynaecologic cancer. Despite in the last 30 years an improvement of the overall survival of ovarian cancer patients has been observed, an increased cure rate was not registered. The high mortality associated with ovarian cancer is mainly due to a late diagnosis and resistance to treatment, barring ovarian cancer cure. Given the lack of a specific therapy against ovarian cancer, the treatment depends essentially on the use of generalised and comprehensive cytotoxic drugs, most of them belonging to the group of alkylating/oxidative agents, as platinum salts. Chemotherapy imposes high selection pressures in cancer cells, which may affect their evolutionary trajectories, selecting the chemoresistant ones, which will continue the progression and relapse of the disease, contributing to morbidity and mortality. In recent years, cancer metabolism has acquired a central position in oncobiology, being the metabolic remodelling a requirement for tumour progression, allowing cancer cells to respond to the selective Pressures of the microenvironment, such as hypoxia and cytotoxic drugs. These selective pressures promote cell death in non-adapted cells and positively select cells that exhibit growth advantage that will further sustain cancer progression and metastasis. Endogenous metabolism also limits drugs response. A role of cysteine in cancer by contributing for H2S generation and as a precursor of the antioxidant glutathione (GSH), were already reported. GSH has a crucial role as an antioxidant and also as a detoxifying system allowing the physiological maintenance of metabolic pathways, being intimately associated with chemoresistance. H2S is involved in several biological processes, acting also as an antioxidant and being associated with cancer progression and chemoresistance. As a solid tumour grows, such as an ovarian tumour, due to inefficient vascularisation, cancer cells are exposed to regions of hypoxia, known as a boost factor for tumour progression, metastasis and resistance to therapy. The present thesis aimed to clarify the relevance of cysteine metabolism in ovarian cancer cells adaptability to both hypoxia and platinum salts (carboplatin). These stressful conditions impose strong evolutionary selection pressure on cancer cells. Our results have provided evidence that cysteine metabolism has a role in ovarian cancer cells fast response and adaptation to hypoxic conditions that, in turn, are capable of driving chemoresistance. Moreover, cysteine showed to present a widespread protective effect against both hypoxia and carboplatininduced death among ovarian cancer cell lines. Importantly, our findings were also supported in a clinical context, as an overall increase of thiols concentration was found in serum from patients with ovarian neoplasms, regardless malignancy. Strikingly, the free levels of cysteine together with protein-Scysteinylation levels were able to distinguish patients with malignant tumours from patients with benign tumours and also from healthy individuals, supporting that the levels of cysteine and protein-S-cysteinylation can be putative biomarkers for ovarian cancer early diagnosis. Cysteine was also the prevalent thiol and Scysteinylation was the most abundant form of S-thiolated proteins in the ascitic fluid from patients with advanced disease. The ascitic fluid is an important compartment of the ovarian cancer cells microenvironment, supporting a clinical relevance of cysteine also in the progression of the disease. Given the protective effect of cysteine in hypoxic ovarian cancer cells, we also aimed to address the possible mechanisms by which cysteine would be beneficial under hypoxia. Our results have supported a role of a higher thiols turnover in the adaptation to this environment, especially in ES2 cells. Moreover, results have also supported a role of cysteine in energy production mediated by the xc- system, that requires cysteine metabolism instead of H2S per se. However, the direct role of cysteine in ATP production is still uncertain as this amino acid can have an indirect contribution to ATP synthesis driven by an increased GSH content, allowing the redox equilibrium crucial for the overall cellular metabolism. Strikingly, 1H-NMR results have suggested that cysteine impacted profoundly ES2 cells metabolism under hypoxia, allowing a metabolic reprogramming, that probably underlies ES2 cells adaptation to hypoxia. Taken together, this thesis has shed light on new paths for ovarian cancer screening, diagnosis, prognosis and therapy, where cysteine metabolic profile might allow the design of new and useful approaches to fight this disease, thus overcoming its poor prognosis.Apesar de todo o progresso na prevenção e no desenvolvimento de novas abordagens terapêuticas, o cancro é a segunda principal causa de morte a nível mundial. Cancro refere-se a um conjunto de doenças complexas, sendo que existem mais de 200 diferentes tipos de cancro. No entanto, foi proposto que as alterações fisiopatológicas implicadas na transformação maligna são comuns à maioria dos tumores humanos: auto-suficiência em sinais estimuladores de proliferação celular (mitogénese), insensibilidade a sinais que inibem a proliferação, evasão da morte celular programada, capacidade de replicação ilimitada, indução de angiogénese, capacidade de invasão de tecidos e de metastização, reprogramação do metabolismo energético e evasão ao sistema imunitário. Tanto a incidência como a mortalidade por cancro têm vindo a aumentar, não só devido ao envelhecimento e crescimento populacionais mas também devido ao aumento da exposição a factores de risco tais como o tabaco e obesidade. O cancro do ovário não é exceção a este cenário, sendo o terceiro tipo de cancro ginecológico mais comum e a principal causa de morte de cancro ginecológico. Cancro do ovário define um conjunto de neoplasias distintas, sendo os carcinomas (neoplasia malignas com origem epithelial) o grupo dominante. Apesar de nos últimos 30 anos se ter observado um aumento da sobrevivência de pacientes com esta doença, não se observou um aumento na taxa de cura. O mau prognóstico associado ao cancro do ovário deve-se essencialmente a um diagnóstico tardio e à emergência de resistência à terapia convencional, dificultando o tratamento da doença. O tratamento do cancro do ovário envolve geralmente cirurgia citorredutora e terapia combinada de sais de platina e de taxanos, ambos agentes oxidativos. Estes fármacos impõem pressões seletivas muito fortes nas células neoplásicas, seleccionando células resistentes responsáveis pela progressão e recidiva da doença. A quimioresistência contribui assim para uma grande morbilidade e mortalidade da doença. Recentemente, o metabolismo do cancro adquiriu uma relevância central em oncobiologia, sendo a remodelação metabólica uma característica necessária para a progressão tumoral, permitindo que as células cancerígenas respondam e se adaptem a inúmeras pressões seletivas diferentes impostas pelo microambiente tumoral, tais como hipóxia, acidez e presença de fármacos citotóxicos. O papel da cisteína em cancro já foi demonstrado devido ao seu envolvimento como percursor de dois compostos essenciais: o glutatião (GSH) e o sulfureto de hidrogénio (H2S). O GSH é o tiol não proteico mais abundante em células de mamífero, desempenhando diversas funções biológicas tais como uma função antioxidante, e destoxificante, constituindo um mecanismo de quimioresistência em vários tipos de tumores. Por sua vez, o H2S está também envolvido em vários processos biológicos, tendo também um papel antioxidante e estando também associado a progressão tumoral e quimioresistência. Durante o crescimento dos tumores sólidos, como os tumores de ovário, as células cancerígenas são sujeitas a gradientes de hipóxia, devido a uma vascularização ineficiente. A hipóxia exerce uma forte pressão seletiva nas células cancerígenas, estando relacionada com a progressão tumoral, metastização e resistência à quimioterapia. Na presente tese, o nosso objectivo principal foi clarificar a relevância do metabolismo da cisteína na capacidade de adaptação de células de carcinomas do ovário a condições de hipóxia e à presença de sais de platina (carboplatina), duas condições que impõem uma forte pressão seletiva nas células cancerígenas. Para isto, recorremos essencialmente a duas linhas celulares de cancro de ovário de dois tipos histológicos diferentes: uma linha de carcinoma seroso de alto grau (HG-OSC) – OVCAR3 e uma linha de carcinoma de células claras (OCCC) – ES2. É importante referir que o tipo histológico carcinoma seroso de alto grau (HGOSC) é o mais frequente, enquanto que o OCCC, apesar de menos frequente, é geralmente resistente à terapia convencional. Adicionalmente, recorremos a uma linha celular de carcinoma do ovário cujo tipo histológico é desconhecido (A2780 sensíveis/parentais e A2780 resistentes a cisplatina) e a outra linha de HG-OSC (OVCAR8). Recorremos ainda a amostras de soro de mulheres com tumores de ovário benignos e malignos, assim como a amostras de soro de mulheres saudáveis, a amostras de carcinomas de ovário de diferentes tipos histológicos e a amostras de líquido ascítico de mulheres com cancro de ovário. No primeiro capítulo de resultados experimentais começámos por investigar qual o efeito da seleção de células ES2 e OVCAR3 em condições de normóxia e em hipóxia na capacidade destas se adaptarem à carboplatina, pretendendo ainda verificar qual o papel da cisteína na resposta a estes dois ambientes hipóxia/carboplatina. Os nossos resultados mostraram que a cisteína facilita a adaptação a condições de hipóxia que, por sua vez, é capaz de induzir quimioresistência. No segundo capítulo de resultados experimentais pretendemos aprofundar o papel da cisteína na proteção de células de carcinoma do ovário em condições de hipóxia e na presença de carboplatina. Pretendemos ainda verificar se a cisteína apresenta relevância em contexto clínico, averiguando a possibilidade para o seu uso em diagnóstico e como ferramenta de prognóstico da doença. Os nossos resultados indicaram que a cisteína tem um papel protetor amplamente distribuído por diferentes linhas de cancro de ovário, tanto em condições de hipóxia, como na presença de carboplatina. Nas células ES2, os dados sugeriram que as dinâmicas de síntese e degradação de tióis estão subjacentes ao efeito protetor da cisteína em condições de hipóxia. Relativamente ao seu papel em contexto clínico, através da quantificação de tióis em soros de mulheres saudáveis e em mulheres com tumores de ovário benignos e malignos, verificámos que os níveis totais (cisteína livre e proteínas cisteinilada) conjuntamente com os níveis livre totais de cisteína foram capazes de distinguir estes três grupos de mulheres, sugerindo assim que este tiol poderá ser usado como um marcador de diagnóstico precoce da doença. Em amostras de tumores, verificámos uma tendência (não significativa) para níveis de cisteína mais altos no tipo histológico OCCC e também em HG-OSC apenas após quimioterapia, reforçando um papel importante da cisteína no tipo histológico OCCC e na aquisição de quimioresistência. Para além disto, através da análise do conteúdo de tióis em líquidos ascíticos de pacientes com carcinoma de ovário em estadios avançados da doença, verificámos ainda que a cisteína foi o tiol prevalente encontrado nesta importante fracção do microambiente tumoral, suportando um papel relevante deste tiol também na progressão da doença. No terceiro capítulo referente a dados experimentais, pretendemos clarificar o mecanismo por detrás do papel protetor da cisteína em condições de hipóxia. Assim, para além das dinâmicas de síntese e degradação de GSH, pretendemos explorar qual o papel do H2S na adaptação das células de cancro de ovário a condições de hipóxia. Os nossos resultados sugeriram um papel importante da cisteína na produção de ATP, mediado pelo sistema de transporte xc- , que requer o metabolismo da cisteína, não sendo o H2S por si suficiente para aumentar a produção de ATP em condições de hipóxia. No entanto, um papel direto da cisteína na produção de ATP é ainda incerto, uma vez que a contribuição deste tiol no metabolismo energético celular poderá ser devido a uma contribuição indireta, conduzido por um aumento dos níveis de GSH, permitindo assim um equilíbrio redox, crucial para o metabolismo celular. Através de 1H-RMN (espectroscopia por ressonância magnética nuclear) verificámos que a cisteína teve um impacto profundo no metabolismo das células ES2, permitindo uma remodelação metabólica em condições de hipóxia, o que poderá estar subjacente à adaptação destas células nestas condições. De um modo geral, este trabalho permitiu aumentar o conhecimento e conduzir a novas abordagens para o diagnóstico, prognóstico e terapia do cancro de ovário através do metabolismo da cisteína. Isto poderá permitir a implementação de novas abordagens de rastreio, diagnóstico e tratamento da doença, permitindo assim superar a quimioresistência e o mau prognósticos associados ao cancro do ovário

Understanding Cofactor F420 dependent mechanism(s) in the activation of bicycle nitroimidazoles and in the physiology of Mycobacterium tuberculosis

Ph.DDOCTOR OF PHILOSOPH