Sustained inhibition of deacetylases is required for the antitumor activity of the histone deactylase inhibitors panobinostat and vorinostat in models of colorectal cancer

Despite compelling preclinical data in colorectal cancer (CRC), the efficacy of HDACIs has been disappointing in the clinic. The goal of this study was to evaluate the effectiveness of vorinostat and panobinostat in a dose- and exposure-dependent manner in order to better understand the dynamics of drug action and antitumor efficacy. In a standard 72 h drug exposure MTS assay, notable concentration-dependent antiproliferative effects were observed in the IC50 range of 1.2-2.8 μmol/L for vorinostat and 5.1-17.5 nmol/L for panobinostat. However, shorter clinically relevant exposures of 3 or 6 h failed to elicit any significant growth inhibition and in most cases a >24 h exposure to vorinostat or panobinostat was required to induce a sigmoidal dose-response. Similar results were observed in colony formation assays where ≥ 24 h of exposure was required to effectively reduce colony formation. Induction of acetyl-H3, acetyl-H4 and p21 by vorinostat were transient and rapidly reversed within 12 h of drug removal. In contrast, panobinostat-induced acetyl-H3, acetyl-H4, and p21 persisted for 48 h after an initial 3 h exposure. Treatment of HCT116 xenografts with panobinostat induced significant increases in acetyl-H3 and downregulation of thymidylate synthase after treatment. Although HDACIs exert both potent growth inhibition and cytotoxic effects when CRC cells were exposed to drug for ≥ 24 h, these cells demonstrate an inherent ability to survive HDACI concentrations and exposure times that exceed those clinically achievable. Continued efforts to develop novel HDACIs with improved pharmacokinetics/phamacodynamics, enhanced intratumoral delivery and class/isoform-specificity are needed to improve the therapeutic potential of HDACIs and HDACI-based combination regimens in solid tumors.

DNA microarray profiling of genes differentially regulated by the histone deacetylase inhibitors vorinostat and LBH589 in colon cancer cell lines

BACKGROUND: Despite the significant progress made in colon cancer chemotherapy, advanced disease remains largely incurable and novel efficacious chemotherapies are urgently needed. Histone deacetylase inhibitors (HDACi) represent a novel class of agents which have demonstrated promising preclinical activity and are undergoing clinical evaluation in colon cancer. The goal of this study was to identify genes in colon cancer cells that are differentially regulated by two clinically advanced hydroxamic acid HDACi, vorinostat and LBH589 to provide rationale for novel drug combination partners and identify a core set of HDACi-regulated genes.

METHODS: HCT116 and HT29 colon cancer cells were treated with LBH589 or vorinostat and growth inhibition, acetylation status and apoptosis were analyzed in response to treatment using MTS, Western blotting and flow cytometric analyses. In addition, gene expression was analyzed using the Illumina Human-6 V2 BeadChip array and Ingenuity Pathway Analysis.

RESULTS: Treatment with either vorinostat or LBH589 rapidly induced histone acetylation, cell cycle arrest and inhibited the growth of both HCT116 and HT29 cells. Bioinformatic analysis of the microarray profiling revealed significant similarity in the genes altered in expression following treatment with the two HDACi tested within each cell line. However, analysis of genes that were altered in expression in the HCT116 and HT29 cells revealed cell-line-specific responses to HDACi treatment. In addition a core cassette of 11 genes modulated by both vorinostat and LBH589 were identified in both colon cancer cell lines analyzed.

CONCLUSION: This study identified HDACi-induced alterations in critical genes involved in nucleotide metabolism, angiogenesis, mitosis and cell survival which may represent potential intervention points for novel therapeutic combinations in colon cancer. This information will assist in the identification of novel pathways and targets that are modulated by HDACi, providing much-needed information on HDACi mechanism of action and providing rationale for novel drug combination partners. We identified a core signature of 11 genes which were modulated by both vorinostat and LBH589 in a similar manner in both cell lines. These core genes will assist in the development and validation of a common gene set which may represent a molecular signature of HDAC inhibition in colon cancer.

Integration of copy number and transcriptomics provides risk stratification in prostate cancer: A discovery and validation cohort study

BACKGROUND: Understanding the heterogeneous genotypes and phenotypes of prostate cancer is fundamental to improving the way we treat this disease. As yet, there are no validated descriptions of prostate cancer subgroups derived from integrated genomics linked with clinical outcome.

METHODS: In a study of 482 tumour, benign and germline samples from 259 men with primary prostate cancer, we used integrative analysis of copy number alterations (CNA) and array transcriptomics to identify genomic loci that affect expression levels of mRNA in an expression quantitative trait loci (eQTL) approach, to stratify patients into subgroups that we then associated with future clinical behaviour, and compared with either CNA or transcriptomics alone.

FINDINGS: We identified five separate patient subgroups with distinct genomic alterations and expression profiles based on 100 discriminating genes in our separate discovery and validation sets of 125 and 103 men. These subgroups were able to consistently predict biochemical relapse (p = 0.0017 and p = 0.016 respectively) and were further validated in a third cohort with long-term follow-up (p = 0.027). We show the relative contributions of gene expression and copy number data on phenotype, and demonstrate the improved power gained from integrative analyses. We confirm alterations in six genes previously associated with prostate cancer (MAP3K7, MELK, RCBTB2, ELAC2, TPD52, ZBTB4), and also identify 94 genes not previously linked to prostate cancer progression that would not have been detected using either transcript or copy number data alone. We confirm a number of previously published molecular changes associated with high risk disease, including MYC amplification, and NKX3-1, RB1 and PTEN deletions, as well as over-expression of PCA3 and AMACR, and loss of MSMB in tumour tissue. A subset of the 100 genes outperforms established clinical predictors of poor prognosis (PSA, Gleason score), as well as previously published gene signatures (p = 0.0001). We further show how our molecular profiles can be used for the early detection of aggressive cases in a clinical setting, and inform treatment decisions.

INTERPRETATION: For the first time in prostate cancer this study demonstrates the importance of integrated genomic analyses incorporating both benign and tumour tissue data in identifying molecular alterations leading to the generation of robust gene sets that are predictive of clinical outcome in independent patient cohorts.

Does childhood bullying lead to the development of psychotic symptoms? A meta-analysis and review of prospective studies

Purpose: Researchers have demonstrated associations between trauma and psychosis. Childhood trauma, in particular, appears to be an important determinant. Recently, bullying has become considered a traumatic experience in its own right. This review aims to analyse research with prospective designs, which will enable conclusions about whether or not bullying causes psychosis.

Methods: A systematic review of the literature was carried out independently by two reviewers. Eligibility and quality assessment criteria were applied. A meta-analysis and narrative synthesis were then completed.

Results: Ten studies met inclusion criteria. Four used data from the same large database, and were combined as one. The majority provided confirmation that bullying appears to cause later development of psychosis. A meta-analysis yielded an unadjusted odds ratio (OR) of 2.148 [95% confidence interval (CI) 1.140–4.044].

Conclusions: The studies reviewed here suggest that bullying does predict the later development of psychotic symptoms. What is lacking from the literature is adequate investigation into other potential mediating factors. The current review highlights the significant role of bullying within this complex interaction. Potential mediating variables are explored, including a dose–response effect for the severity and frequency of victimization. Suggestions for targeting intervention are also suggested alongside clinical implications and recommendations for future research.

Health care professionals’ experience, understanding and perception of need of advanced cancer patients with cachexia and their families: The benefits of a dedicated clinic.

Background: Cachexia has been defined as an on-going loss of skeletal muscle mass that cannot be fully reversed by conventional nutritional support. It can be found in up to 80% of patients with advanced cancer and has profound psycho-social consequences for patients and their families. There is a paucity of studies examining the role and experience of healthcare professionals in relation to cachexia and existing studies suggest that professional staff have limited understanding and do not intervene effectively.
Aim: To identify barriers and facilitators to good practice in cachexia care in order to inform future developments in service provision.
Design: An exploratory qualitative study was conducted employing semi-structured interviews with a range of healthcare professionals recruited purposefully from an Australian hospital. Interviews were conducted in private rooms within the hospital.
Setting/participants: A range of healthcare professionals responsible for cancer care were recruited from a large Australian teaching hospital.
Results: Interviews were conducted with 8 healthcare professionals responsible for delivering cancer care. Four themes were identified: formal and informal education, knowledge and understanding, truth telling in cachexia and palliative care, and, a multi-disciplinary approach. Findings show how improved knowledge and understanding across a staff body can lead to improved staff confidence and a willingness to address cancer cachexia and its consequences with patients and their families.
Conclusion: Comparison with previous studies illustrates the importance of improving knowledge and understanding about cachexia and how this can contribute to staff having the skills and experience necessary to address cachexia and provide an improved care experience for patients and carers.

Self and identity in women with symptoms of borderline personality: A qualitative study

Identity disturbance has been suggested to be a core feature of borderline personality disorder (BPD). However, there is little known about the identity of individuals with symptoms of BPD from the participant’s perspective. This study availed of in-depth lightly structured life story interviews with five female participants. Thematic analysis was utilized to derive three themes of identity: connection, distance between us, and hurt and healing. Results provided support for multiple and flexible conceptualizations of identity in comparison to the idea of a unitary self/identity. Results also suggested that participants were able to establish differing connections to others ranging from disconnection to intimacy and care. Participants reported that their identities were impacted upon by historical and current family/relationship dysfunction, but life stories also illustrated the positive impact of healing relationship experiences. Findings provide support for psychological theories that consider a multiple and relational self/identity and the empowerment of healthy aspects of the self in BPD recovery. Studies that assess the association between insight and change may further our knowledge into this complex population.

Statin use after diagnosis of breast cancer and survival: a population-based cohort study

BACKGROUND: Preclinical studies have shown that statins, particularly simvastatin, can prevent growth in breast cancer cell lines and animal models. We investigated whether statins used after breast cancer diagnosis reduced the risk of breast cancer-specific, or all-cause, mortality in a large cohort of breast cancer patients.

METHODS: A cohort of 17,880 breast cancer patients, newly diagnosed between 1998 and 2009, was identified from English cancer registries (from the National Cancer Data Repository). This cohort was linked to the UK Clinical Practice Research Datalink, providing prescription records, and to the Office of National Statistics mortality data (up to 2013), identifying 3694 deaths, including 1469 deaths attributable to breast cancer. Unadjusted and adjusted hazard ratios (HRs) for breast cancer-specific, and all-cause, mortality in statin users after breast cancer diagnosis were calculated using time-dependent Cox regression models. Sensitivity analyses were conducted using multiple imputation methods, propensity score methods and a case-control approach.

RESULTS: There was some evidence that statin use after a diagnosis of breast cancer had reduced mortality due to breast cancer and all causes (fully adjusted HR = 0.84 [95% confidence interval = 0.68-1.04] and 0.84 [0.72-0.97], respectively). These associations were more marked for simvastatin 0.79 (0.63-1.00) and 0.81 (0.70-0.95), respectively.

CONCLUSIONS: In this large population-based breast cancer cohort, there was some evidence of reduced mortality in statin users after breast cancer diagnosis. However, these associations were weak in magnitude and were attenuated in some sensitivity analyses.

Digoxin use after diagnosis of colorectal cancer and survival: a population-based cohort study.

BACKGROUND:

Digoxin has been shown to affect a number of pathways that are of relevance to cancer, and its use has been associated with increased risks of breast and uterus cancer and, more recently, a 40% increase in colorectal cancer risk. These findings raise questions about the safety of digoxin use in colorectal cancer patients, and, therefore, we investigated whether digoxin use after colorectal cancer diagnosis increased the risk of colorectal cancer-specific mortality.

METHODS:

A cohort of 10,357 colorectal cancer patients newly diagnosed from 1998 to 2009 was identified from English cancer registries and linked to the UK Clinical Practice Research Datalink (to provide digoxin and other prescription records) and to the Office of National Statistics mortality data (to identify 2,724 colorectal cancer-specific deaths). Using time-dependent Cox regression models, unadjusted and adjusted HRs and 95% confidence intervals (CI) were calculated for the association between postdiagnostic exposure to digoxin and colorectal cancer-specific mortality.

RESULTS:

Overall, 682 (6%) colorectal cancer patients used digoxin after diagnosis. Digoxin use was associated with a small increase in colorectal cancer-specific mortality before adjustment (HR, 1.25; 95% CI, 1.07-1.46), but after adjustment for confounders, the association was attenuated (adjusted HR, 1.10; 95% CI, 0.91-1.34) and there was no evidence of a dose response.

CONCLUSIONS:

In this large population-based colorectal cancer cohort, there was little evidence of an increase in colorectal cancer-specific mortality with digoxin use after diagnosis.

IMPACT:

These results provide some reassurance that digoxin use is safe in colorectal cancer patients.

Synthesis and biological activities of polyphenolic hybrids - evaluation of anti-cancer, anti-inflammatory and antioxidant activities

Os compostos polifenólicos constituem uma classe de metabolitos secundários de plantas, mas existe também uma enorme quantidade de derivados sintéticos ou semi-sintéticos contendo múltiplas unidades fenólicas. Estes compostos apresentam importantes características biológicas, que dependem das suas estruturas básicas. Certos derivados desta família de compostos, tais como flavonoides, cromonas e cumarinas contribuem para os benefícios da dieta humana, e partilham o núcleo de benzopiran-(2 e 4)-ona ou benzofuran-3-ona. A presente dissertação inclui uma introdução geral e três capítulos que descrevem as novas rotas sintéticas estabelecidas para a preparação de novos híbridos de diversos compostos polifenólicos, assim como a sua elucidação estrutural e termina com a presentação dos resultados da avaliação biológica desses mesmos compostos. No segundo capítulo discute-se a preparação de híbridos de pirimidina- e imidazolidina-polifenóis, especialmente a síntese diastereoseletiva de novos híbridos benzofuran-3-ona-hidantoína e derivados de uracilo. A rota sintética envolve a ação de carbodiimidas sobre os ácidos cromona-(2- e 3)-carboxílicos num só passo ou em dois passos sequenciais, catalisada por uma base orgânica ou inorgânica. O terceiro capítulo descreve reações do tipo adições conjugadas 1,4 - hetero-ciclisações em cascata de compostos 1,3-dicarbonílicos em ácido cromona-3-carboxílico catalisadas por uma base orgânica, que originaram novas cromonas, cromanonas e flavonas polissubstituídas. As bispiranonas [bispiran-2 e 4)-onas] foram elaboradas numa reacção de acoplamento da 4-hidroxicumarina ou da lactona do ácido triacético com o ácido cromona-3-carboxílico ou precursores formil-funcionalizados (ω-formil-2’-hydroxy acetofenonas e cromona-3-carbaldeídos) utilizando organocatálise básica. Finalmente, alargou-se o estudo das adições conjugadas 1,4 para uma variedade de 4-hidroxipiran-2-onas e cetonas α,β-insaturadas para originar novos análogos de warfarina. Obteve-se uma variedade de estruturas complexas por hibridação das unidades de 4-hidroxicumarina ou da lactona do ácido triacético com os novos derivados de cromonas polissubstituídas. Todos as reações foram executadas em condições suaves e ambientalmente favoráveis, utilizando a 4-pirrolidinopiridina como organocatalisador básico. As estruturas dos novos híbridos polifenólicos foram caracterizados por técnicas espectroscópicas de alta resolução, incluindo espectroscopia de ressonância magnética nuclear (1D e 2D) e por difractometria de raios-X, que nos permitiram resolver o complexidade estrutural dos compostos sintetizados. O quarto capítulo apresenta os resultados da avaliação biológica obtidos com os híbridos polifenólicos sintetizados neste trabalho, mostrando a possibilidade de seu envolvimento na terapia do cancro. A maioria dos compostos foram avaliados quanto ao seu efeito sobre a citotoxicidade e proliferação de células leucémicas e ao seu envolvimento na regulação de via pró-inflamatória NF-kB, na qual, os híbridos de biscumarinas exibiram actividades elevadas (IC50 = 6-19 μM para inibição de NF-kB depois de 8 horas de incubação e IC50 = 15-39 μM para efeitos citotóxicos em células cancerosas, após 24 horas de incubação). Uma inibição moderada das enzimas HDAC e Cdc25 foi induzida pelos derivados de benzofuran-3-ona-hidantoína. Catorze dos novos derivados polifenólicos polissubstituídos, tendo como estrutura básica a benzopiran-4-ona, foram avaliados pela sua actividade quimiopreventiva do cancro mediada pela indução de sinalização citoprotectora Nrf2 (fator 2 relacionado com o fator nuclear da proteína E2) e capacidade para inibir a proliferação das células de cancro da mama. Os derivados da classe das cromanonas foram identificados como os indutores mais potentes da actividade Nrf2. As concentrações necessárias para aumentar a actividade de luciferase em 10 vezes (C10) foram de 2,8-21,3 μM. Todos os novos híbridos polifenólicos que apresentam atividade citotóxica e anti-proliferativa não afectam o crescimento de células saudáveis periféricas do sangue (PBMC) (IC50 > 50 μM), indicando a sua seletividade para as células cancerosas e sugerindo que alguns deles são estruturalmente interessantes para posteriores análises. A avaliação da atividade antioxidante utilizando os testes do radical livre DPPH e o poder redutor do ião férrico FRAP foram realizados em algumas estruturas híbridas polifenólicas.

Metabolic signature of lung cancer: a metabolomic study of human tissues and biofluids

This thesis reports the application of metabolomics to human tissues and biofluids (blood plasma and urine) to unveil the metabolic signature of primary lung cancer. In Chapter 1, a brief introduction on lung cancer epidemiology and pathogenesis, together with a review of the main metabolic dysregulations known to be associated with cancer, is presented. The metabolomics approach is also described, addressing the analytical and statistical methods employed, as well as the current state of the art on its application to clinical lung cancer studies. Chapter 2 provides the experimental details of this work, in regard to the subjects enrolled, sample collection and analysis, and data processing. In Chapter 3, the metabolic characterization of intact lung tissues (from 56 patients) by proton High Resolution Magic Angle Spinning (HRMAS) Nuclear Magnetic Resonance (NMR) spectroscopy is described. After careful assessment of acquisition conditions and thorough spectral assignment (over 50 metabolites identified), the metabolic profiles of tumour and adjacent control tissues were compared through multivariate analysis. The two tissue classes could be discriminated with 97% accuracy, with 13 metabolites significantly accounting for this discrimination: glucose and acetate (depleted in tumours), together with lactate, alanine, glutamate, GSH, taurine, creatine, phosphocholine, glycerophosphocholine, phosphoethanolamine, uracil nucleotides and peptides (increased in tumours). Some of these variations corroborated typical features of cancer metabolism (e.g., upregulated glycolysis and glutaminolysis), while others suggested less known pathways (e.g., antioxidant protection, protein degradation) to play important roles. Another major and novel finding described in this chapter was the dependence of this metabolic signature on tumour histological subtype. While main alterations in adenocarcinomas (AdC) related to phospholipid and protein metabolisms, squamous cell carcinomas (SqCC) were found to have stronger glycolytic and glutaminolytic profiles, making it possible to build a valid classification model to discriminate these two subtypes. Chapter 4 reports the NMR metabolomic study of blood plasma from over 100 patients and near 100 healthy controls, the multivariate model built having afforded a classification rate of 87%. The two groups were found to differ significantly in the levels of lactate, pyruvate, acetoacetate, LDL+VLDL lipoproteins and glycoproteins (increased in patients), together with glutamine, histidine, valine, methanol, HDL lipoproteins and two unassigned compounds (decreased in patients). Interestingly, these variations were detected from initial disease stages and the magnitude of some of them depended on the histological type, although not allowing AdC vs. SqCC discrimination. Moreover, it is shown in this chapter that age mismatch between control and cancer groups could not be ruled out as a possible confounding factor, and exploratory external validation afforded a classification rate of 85%. The NMR profiling of urine from lung cancer patients and healthy controls is presented in Chapter 5. Compared to plasma, the classification model built with urinary profiles resulted in a superior classification rate (97%). After careful assessment of possible bias from gender, age and smoking habits, a set of 19 metabolites was proposed to be cancer-related (out of which 3 were unknowns and 6 were partially identified as N-acetylated metabolites). As for plasma, these variations were detected regardless of disease stage and showed some dependency on histological subtype, the AdC vs. SqCC model built showing modest predictive power. In addition, preliminary external validation of the urine-based classification model afforded 100% sensitivity and 90% specificity, which are exciting results in terms of potential for future clinical application. Chapter 6 describes the analysis of urine from a subset of patients by a different profiling technique, namely, Ultra-Performance Liquid Chromatography coupled to Mass Spectrometry (UPLC-MS). Although the identification of discriminant metabolites was very limited, multivariate models showed high classification rate and predictive power, thus reinforcing the value of urine in the context of lung cancer diagnosis. Finally, the main conclusions of this thesis are presented in Chapter 7, highlighting the potential of integrated metabolomics of tissues and biofluids to improve current understanding of lung cancer altered metabolism and to reveal new marker profiles with diagnostic value.

Are older people prescribed antidepressants on the basis of fewer symptoms of depression, and for longer periods of time? A survey of 1825 New Zealanders

Aims: To determine whether older people are prescribed antidepressants at lower levels of depression and with fewer symptoms, and whether they are more likely to engage in chronic usage. Methods: An online survey about experiences with, and opinions about, depression and antidepressants, was completed by 1,825 New Zealand adults who had been prescribed antidepressants in the preceding five years. Results: Participants over 55 were prescribed antidepressants with significantly fewer symptoms and were significantly less likely to meet DSM criteria for depression. They were also significantly more likely to have used the drugs for three years and still be using them. Conclusions: Prescribing physicians and their older patients might benefit from discussing the pros and cons of antidepressants (including the additional risk factors with this age group) and the alternatives; and, if prescription does occur, careful monitoring to avoid unnecessary, potentially damaging, long-term use is recommended.

Enoxacin inhibits growth of prostate cancer cells and effectively restores microRNA processing

Prostate cancer (PCa) is one of the most incident malignancies worldwide. Although efficient therapy is available for early-stage PCa, treatment of advanced disease is mainly ineffective and remains a clinical challenge. microRNA (miRNA) dysregulation is associated with PCa development and progression. In fact, several studies have reported a widespread downregulation of miRNAs in PCa, which highlights the importance of studying compounds capable of restoring the global miRNA expression. The main aim of this study was to define the usefulness of enoxacin as an anti-tumoral agent in PCa, due to its ability to induce miRNA biogenesis in a TRBP-mediated manner. Using a panel of five PCa cell lines, we observed that all of them were wild type for the TARBP2 gene and expressed TRBP protein. Furthermore, primary prostate carcinomas displayed normal levels of TRBP protein. Remarkably, enoxacin was able to decrease cell viability, induce apoptosis, cause cell cycle arrest, and inhibit the invasiveness of cell lines. Enoxacin was also effective in restoring the global expression of miRNAs. This study is the first to show that PCa cells are highly responsive to the anti-tumoral effects of enoxacin. Therefore, enoxacin constitutes a promising therapeutic agent for PCa.

Feasibility and acceptability of an exergame intervention for schizophrenia

Objectives To evaluate the feasibility and acceptability of an exergame intervention as a tool to promote physical activity in outpatients with schizophrenia. Design Feasibility/Acceptability Study and Quasi-Experimental Trial. Method Sixteen outpatients with schizophrenia received treatment as usual and they all completed an 8-week exergame intervention using Microsoft Kinect® (20 min sessions, biweekly). Participants completed pre and post treatment assessments regarding functional mobility (Timed Up and Go Test), functional fitness performance (Senior Fitness Test), motor neurological soft signs (Brief Motor Scale), hand grip strength (digital dynamometer), static balance (force plate), speed of processing (Trail Making Test), schizophrenia-related symptoms (Positive and Negative Syndrome Scale) and functioning (Personal and Social Performance Scale). The EG group completed an acceptability questionnaire after the intervention. Results Attrition rate was 18.75% and 69.23% of the participants completed the intervention within the proposed schedule. Baseline clinical traits were not related to game performance indicators. Over 90% of the participants rated the intervention as satisfactory and interactive. Most participants (76.9%) agreed that this intervention promotes healthier lifestyles and is an acceptable alternative to perform physical activity. Repeated-measures MANOVA analyses found no significant multivariate effects for combined outcomes. Conclusion This study established the feasibility and acceptability of an exergame intervention for outpatients with schizophrenia. The intervention proved to be an appealing alternative to physical activity. Future trials should include larger sample sizes, explore patients' adherence to home-based exergames and consider greater intervention dosage (length, session duration, and/or frequency) in order to achieve potential effects.