TRAP220 is modulated by the antineoplastic agent 6-Mercaptopurine, and mediates the activation of the NR4A subgroup of nuclear receptors

The NR4A1-3 (Nur77, NURR1 and NOR-1) subfamily of nuclear hormone receptors (NRs) has been implicated in Parkinson's disease, schizophrenia, manic depression, atherogenesis, Alzheimer's disease, rheumatoid arthritis, cancer and apoptosis. This has driven investigations into the mechanism of action, and the identification of small molecule regulators, that may provide the platform for pharmaceutical and therapeutic exploitation. Recently, we found that the purine antimetabolite 6-Mercaptopurine (6-MP), which is widely used as an anti-neoplastic and anti-inflammatory drug, modulated the NR4A1-3 subfamily. Interestingly, the agonist-mediated activation did not involve modulation of primary coactivators' (e.g. p300 and SRC-2/GRIP-1) activity and/or recruitment. However, the role of the subsequently recruited coactivators, for example CARM-1 and TRAP220, in 6-MP-mediated activation of the NR4A1-3 subfamily remains obscure. In this study we demonstrate that 6-MP modulates the activity of the coactivator TRAP220 in a dose-dependent manner. Moreover, we demonstrate that TRAP220 potentiates NOR-1-mediated transactivation, and interacts with the NR4A1-3 subgroup in an AF-1-dependent manner in a cellular context. The region of TRAP220 that mediated 6-MP activation and NR4A interaction was delimited to amino acids 1-800, and operates independently of the critical PKC and PKA phosphorylation sites. Interestingly, TRAP220 expression does not increase the relative induction by 6-MP, however the absolute level of NOR-1-mediated trans-activation is increased. This study demonstrates that 6-MP modulates the activity of the NR4A subgroup, and the coactivator TRAP220.

Nausea, Vomiting And Quality Of Life Of Patients With Cancer Undergoing Antineoplastic Treatment: An Evaluation By Pharmacists.

This study aims to evaluate the frequency and severity of nausea and vomiting using two different instruments and relate them to quality of life (QOL) in patients with cancer receiving antineoplastic treatment. Severity of chemotherapy-induced nausea and vomiting (CINV) was measured by Common Terminology Criteria for Adverse Events (CTCAE) and a numerical scale. QOL was assessed using the Functional Assessment of Cancer Therapy-General questionnaire. Of the 50 patients studied, 60.0% reported nausea (40.0% CTCAE grade 1; 66.7% moderate intensity on numerical scale) and 30.0% reported vomiting (46.7% CTCAE grades 1 and 2, each; 66.7% moderate intensity on numerical scale). CINV did not influence overall QOL. The frequency of CINV was high. There was no association between nausea/vomiting and overall QOL.

Cyclophosphamide levels in sites of preparation and administration of antineoplastic drugs

The extensive use of antineoplastic agents in chemotherapy may be at risk to health care workers involved in the preparation and administration of these drugs. In this study cyclophosphamide, a drug classified as a human carcinogen, was quantified by adapting a previous analytical method using gas chromatography coupled to mass spectrometry (GC-MS) after solid phase extraction with diatomaceous earth. The drug was measured by analysis in surfaces (wipe samples) and gloves, collected from four different hospitals, before and after the practice of cleaning procedures, and the use of a closed-system device for the preparation and administration. Validation results were satisfactory and cyclophosphamide levels ranging from below the quantification limit to 141000 ng. Our findings demonstrated that surfaces and materials contamination was found in all hospitals during the traditional open technique for preparation and administration of cyclophosphamide and a significant reduction in contamination when a closed-system device was used. However, some values were considered unexpected, especially those obtained from samples collected after the cleaning surfaces.

Antineoplastic drugs contamination of workplace surfaces in two Portuguese hospitals

Despite the classification as known or suspected human carcinogens, by the International Agency for Research on Cancer, the antineoplastic drugs are extensively used in cancer treatment due to their specificity and efficacy. As human carcinogens, these drugs represent a serious threat to the healthcare workers involved in their preparation and administration. This work aims to contribute to better characterize the occupational exposure of healthcare professionals to antineoplastic drugs, by assessing workplace surfaces contamination of pharmacy and administration units of two Portuguese hospitals. Surface contamination was assessed by the determination of cyclophosphamide, 5-fluorouracil, and paclitaxel. These three drugs were used as surrogate markers for surfaces contamination by cytotoxic drugs. Wipe samples were taken and analyzed by HPLCDAD. From the total of 327 analyzed samples, in 121 (37%) was possible to detect and quantify at least one drug. Additionally, 28 samples (8.6 %) indicate contamination by more than one antineoplastic drug, mainly in the administration unit, in both hospitals. Considering the findings in both hospitals, specific measures should be taken, particularly those related with the promotion of good practices and safety procedures and also routine monitoring of surfaces contamination in order to guarantee the appliance of safety measures.

Genotoxic assessment in different exposure groups working with antineoplastic agents

Antioneoplastic drugs are widely used in treatment of cancer, and several studies suggest acute and long-term effects associated to antineoplastic drug exposures, namely associating workplace exposure with health effects. Cytokinesis blocked micronucleus (CBMN) assay is one promising short-term genotoxicity assays for human risk assessment and their combination is recommended to monitor populations chronically exposed to genotoxic agents. The aim of this investigation is the genotoxicity assessment in different professionals that handle cytostatics drugs. This research is case-control blinded study constituted by 46 non-exposed subjects and 44 workers that handle antineoplastic drugs, such as pharmacists, pharmacy technicians, and nurses. It was found statistically significant increases in the genotoxicity biomarkers in exposed comparising with controls (p<0.05). The findings address the need for regular biomonitoring of personnel occupationally exposed to these drugs, confirming to an enhanced health risk assessment.

Molecular pathways involved in the antineoplastic effects of calcitriol on insulinoma cells.

We have previously reported that in tumorigenic pancreatic beta-cells, calcitriol exerts a potent antitumorigenic effect by inducing apoptosis, cell growth inhibition, and reduction of solid beta-cell tumors. Here we have studied the molecular pathways involved in the antineoplastic activity of calcitriol on mouse insulinoma beta TC(3) cells, mouse insulinoma beta TC expressing or not expressing the oncogene p53, and beta TC-tet cells overexpressing or not the antiapoptotic gene Bcl2. Our results indicate that calcitriol-induced apoptosis was dependent on the function of p53 and was associated with a biphasic increase in protein levels of transcription factor nuclear factor-kappa B. Calcitriol decreased cell viability by about 40% in p53-retaining beta TC and in beta TC(3) cells; in contrast, beta TC p53(-/-) cells were only minimally affected. Calcitriol-induced cell death was regulated by members of the Bcl-2 family of apoptosis regulatory proteins, as shown by calcitriol-induced up-regulation of proapoptotic Bax and Bak and the lack of calcitriol-induced cytotoxicity in Bcl-2-overexpressing insulinoma cells. Moreover, calcitriol-mediated arrest of beta TC(3) cells in the G(1) phase of the cell cycle was associated with the abnormal expression of p21 and G(2)/M-specific cyclin B2 genes and involved the DNA damage-inducible factor GADD45. Finally, in beta TC(3) cells, calcitriol modulated the expression of IGF-I and IGF-II genes. In conclusion, these findings contribute to the understanding of the antitumorigenic effects of calcitriol on tumorigenic pancreatic beta-cells and further support the rationale of its utilization in the treatment of patients with malignant insulinomas.

Tumor microenvironment and immune effects of antineoplastic therapy in lymphoproliferative syndromes.

Lymphomas represent a wide group of heterogenic diseases with different biological and clinical behavior. The underlying microenvironment-specific composition seems to play an essential role in this scenario, harboring the ability to develop successful immune responses or, on the contrary, leading to immune evasion and even promotion of tumor growth. Depending on surrounding lymphoid infiltrates, lymphomas may have different prognosis. Moreover, recent evidences have emerged that confer a significant impact of main lymphoma's treatment over microenvironment, with clinical consequences. In this review, we summarize these concepts from a pathological and clinical perspective. Also, the state of the art of lymphoma's anti-idiotype vaccine development is revised, highlighting the situations where this strategy has proven to be successful and eventual clues to obtain better results in the future.

Nosocomial nontyphoidal salmonellosis after antineoplastic chemotherapy : reactivation of asymptomatic colonization ?

Abstract An increased frequency of nontyphoidal salmonellosis is well established in cancer patients, but it is unclear whether this represents increased susceptibility to exogenous infection or opportunistic, endogenous reactivation of asymptomatic carriage. In a retrospective study, a simple case definition was used to identify the probable presence of reactivation salmonellosis in five cancer patients between 1996 and 2002. Reactivation salmonellosis was defined as the development of nosocomial diarrhea >72 h after admission and following the administration of antineoplastic chemotherapy in an HIV-seronegative cancer patient who was asymptomatic on admission, in the absence of epidemiological evidence of a nosocomial outbreak. Primary salmonellosis associated with unrecognized nosocomial transmission or community acquisition and an unusually prolonged incubation period could not entirely be ruled out. During the same time period, another opportunistic infection, Pneumocystis pneumonia, was diagnosed in six cancer patients. Presumably, asymptomatic intestinal Salmonella colonization was converted to invasive infection by chemotherapy-associated intestinal mucosal damage and altered innate immune mechanisms. According to published guidelines, stool specimens from patients hospitalized for longer than 72 h should be rejected unless the patient is neutropenic or ≥65 years old with significant comorbidity. However, in this study neutropenia was present in only one patient, and four patients were <65 years old. Guidelines should thus be revised in order not to reject stool culture specimens from such patients. In cancer patients, nosocomial salmonellosis can Occur as a chemo-therapy-triggered opportunistic reactivation infection that may be similar in frequency to Pneumocystis pneumonia.

Identification of the biosynthetic gene cluster for the Pseudomonas aeruginosa antimetabolite L-2-amino-4-methoxy-trans-3-butenoic acid.

L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) is a potent antibiotic and toxin produced by Pseudomonas aeruginosa. Using a novel biochemical assay combined with site-directed mutagenesis in strain PAO1, we have identified a five-gene cluster specifying AMB biosynthesis, probably involving a thiotemplate mechanism. Overexpression of this cluster in strain PA7, a natural AMB-negative isolate, led to AMB overproduction.

Interactions between cells and functionalized nanoparticles

In this present thesis Superparamagnetic Iron Oxide Nanoparticles (SPIONs) with 9 nm in diameter were selected as nanocarriers in order to study their potential application as drug delivery systems. Therefore the aim of the study was to demonstrate the proof of concept by establishing an efficient system of drug delivery, which would be a valuable tool in biomedical applications, such as the treatement of cancer, by reducing the side effects due to administration of a high concentration of therapeutic agents. As demonstrated in a previous study, the uptake of SPIONs by tumoral human cells was enhanced by the presence of amino groups on their surface. The stabilization of SPIONs were then performed and optimized by the coating of poly(vinylalcohol) and poly(vinylalcohol/vinylamine). Such nanoparticles were known as aminoPVA-SPIONs. The toxicity and the inflammatory reaction of aminoPVA-SPIONs were evaluated in order to establish their potentiel use in the human body. The results demonstrated that the human cells were able to invaginate aminoPVA-SPIONS without revealing any toxicity and inflammatory reaction. The analysis by transmission electron microscopy (TEM), scanning electron microscopy (SEM), cryo-TEM, confocal microscopy and histological staining (i.e. Prussian Blue) showed that the iron oxide core of SPIONs were located in the cytoplasm of cells and concentrated in vesicles. The evaluation of the mechanism of uptake of aminoPVA-SPIONs revealed that their uptake by monolayer cell culture was performed via an active mechanism, which was achieved by a clathrin-mediated endocytosis. Consequently, it was suggested that aminoPVA-SPIONs were good candidates as nanocarriers in drug delivery systems, which were able to reach the cytoplasm of cells. Their incubation with three-dimensional models mimicing tissues, such as differentiated rat brain cell-derived aggregates and spheroids, revealed that aminoPVA-SPIONs were able to invade into deep cell layers according to the stage of growth of these models. In the view of these promising results, drug-SPIONs were prepared by the functionalization of aminoPVA-SPIONs via a biological labile chemical bond by one of these three antineoplastic agents, which are widely used in clinical practice: 5-fluorourdine (Fur) (an antimetabolite), or camptothecin (CPT) (a topoisomerase inhibitor) or doxorubicin (DOX) (an anthracycline which interfere with DNA). The results shown that drug-SPIONs were internalized by human melanoma cells, as it was expected due the previous results with aminoPVA-SPIONs, and in addition they were active as anticancer agents, suggesting the efficient release of the drug from the drug-SPIONs. The results with CPT-SPIONs were the most promising, whereas DOX- SPIONs did not demonstrate a prononced activity of DOX. In conclusion, the results demonstrated that functionalized iron oxide nanoparticles are a promising tool in order to deliver therapeutic agents. - Dans le cadre de ce travail de thèse, les nanoparticules superparamagnétiques d'oxyde de fer (SPIONs) ayant un diamètre de 9 nm ont été choisies, afin d'étudier leur éventuelle utilisation dans un système de délivrance d'agents thérapeutiques. Ainsi le but de la thèse est de démontrer la faisabilité de fabriquer un système efficace de délivrance d'agents thérapeutiques, qui serait un outil intéressant dans le cadre d'une utilisation biomédicale, par exemple lors du traitement du cancer, qui pourrait réduire les effets secondaires provoqués par le dosage trop élevé de médicaments. Comme il a été démontré dans une précédente étude, l'invagination des SPIONs par des cellules humaines cancéreuses est améliorée par la présence de groupes fonctionnels amino à leur surface. La stabilisation des SPIONs est ainsi effectuée et optimisée par l'enrobage de poly(vinylalcool) et de (poly(vinylalcool/vinylamine), qui sont connues sous le nom de aminoPVA-SPIONs. La toxicité et la réaction inflammatoire des aminoPVA-SPIONs ont été évaluées dans le but de déterminer leur potentielle utilisation dans le corps humain. Les résultats démontrèrent que les cellules humaines sont capables d'invaginer les aminoPVAS-SPIONs sans induire une réaction toxique ou inflammatoire. L'analyse par la microscopie électronique en transmission électronique (TEM), la microscopie électronique à balayage (SEM), le cryo-microscopie électronique (SEM), la microscopie confocale et la coloration histologique (par ex, le bleu de Prusse) a montré que l'oxyde de fer des SPIONs est localisé dans le cytoplasme des cellules et est concentré dans des vesicules. L'évaluation du méchanisme d'invagination des aminoPVA-SPIONs ont révélé que leur invagination par des monocultures de cellules est effectué par un méchanisme actif, contrôlé par une endocytose induite par les clathrins. Par conséquent, les aminoPVA-SPIONs sont de bons candidats en tant que transporteurs (nanocamers) dans un système de délivrance d'agents thérapeuthique, capable d'atteindre le cytoplasme des cellules. Leur incubation avec des modèles tridimenstionnels imitant les tissues, tels que les aggrégats de cellules de cerveau différenciées et les sphéroïdes, a montré que les aminoPVA-SPIONs sont capable de pénétrer dans les couches profondes des modèles, selon l'état d'avancement de leur croissance. En vue de ces résultats prometteurs, les drug-SPIONs ont été préparés en fonctionalisant les aminoPVA-SPIONs par le biai d'une liaison chimique labile par un des trois agents thérapeutiques, déjà utilisé en pratique : 5-fluorourdine (Fur) (un antimétabolite), or camptothecin (CPT) (un inhibiteur de la topoisomerase) or doxorubicin (DOX) (un anthracycline qui interfère avec le DNA). Les résultats ont montré que les drug-SPIONs sont capable d'être internalisés par les mélanomes, comme il a été attendu d'après les résultats obtenus précédemment avec les aminoPVA-SPIONs, et de plus, les drug-SPIONs sont actifs, ce qui suggère un relargage efficace de l'agent thérapeutique du drug-SPIONs. Les résultats obtenus avec les CPT-SPIONs sont les plus prometteurs, tandis que ceux avec les DOX-SPIONs, ce n'est pas le cas, dont l'activité thérapeutique de DOX n'a pas été aussi efficace. En conclusion, les résultats ont pu démontrer que les nanoparticules d'oxyde de fer fonctionnalisées sont un outil prometteur dans la délivrance d'agents thérapeutiques.

The Pseudomonas aeruginosa antimetabolite L-2-amino-4-methoxy-trans-3-butenoic acid inhibits growth of Erwinia amylovora and acts as a seed germination-arrest factor

The Pseudomonas aeruginosa antimetabolite L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) shares biological activities with 4-formylaminooxyvinylglycine, a related molecule produced by Pseudomonas fluorescens WH6. We found that culture filtrates of a P.aeruginosa strain overproducing AMB weakly interfered with seed germination of the grassy weed Poa annua and strongly inhibited growth of Erwinia amylovora, the causal agent of the devastating orchard crop disease known as fire blight. AMB was active against a 4-formylaminooxyvinylglycine-resistant isolate of E.amylovora, suggesting that the molecular targets of the two oxyvinylglycines in Erwinia do not, or not entirely, overlap. The AMB biosynthesis and transport genes were shown to be organized in two separate transcriptional units, ambA and ambBCDE, which were successfully expressed from IPTG-inducible tac promoters in the heterologous host P.fluorescens CHA0. Engineered AMB production enabled this model biocontrol strain to become inhibitory against E.amylovora and to weakly interfere with the germination of several graminaceous seeds. We conclude that AMB production requires no additional genes besides ambABCDE and we speculate that their expression in marketed fire blight biocontrol strains could potentially contribute to disease control.

Nosocomial nontyphoidal salmonellosis after antineoplastic chemotherapy: reactivation of asymptomatic colonization?

An increased frequency of nontyphoidal salmonellosis is well established in cancer patients, but it is unclear whether this represents increased susceptibility to exogenous infection or opportunistic, endogenous reactivation of asymptomatic carriage. In a retrospective study, a simple case definition was used to identify the probable presence of reactivation salmonellosis in five cancer patients between 1996 and 2002. Reactivation salmonellosis was defined as the development of nosocomial diarrhea >72 h after admission and following the administration of antineoplastic chemotherapy in an HIV-seronegative cancer patient who was asymptomatic on admission, in the absence of epidemiological evidence of a nosocomial outbreak. Primary salmonellosis associated with unrecognized nosocomial transmission or community acquisition and an unusually prolonged incubation period could not entirely be ruled out. During the same time period, another opportunistic infection, Pneumocystis pneumonia, was diagnosed in six cancer patients. Presumably, asymptomatic intestinal Salmonella colonization was converted to invasive infection by chemotherapy-associated intestinal mucosal damage and altered innate immune mechanisms. According to published guidelines, stool specimens from patients hospitalized for longer than 72 h should be rejected unless the patient is neutropenic or >or=65 years old with significant comorbidity. However, in this study neutropenia was present in only one patient, and four patients were <65 years old. Guidelines should thus be revised in order not to reject stool culture specimens from such patients. In cancer patients, nosocomial salmonellosis can occur as a chemotherapy-triggered opportunistic reactivation infection that may be similar in frequency to Pneumocystis pneumonia.

The Pseudomonas aeruginosa antimetabolite L -2-amino-4-methoxy-trans-3-butenoic acid (AMB) is made from glutamate and two alanine residues via a thiotemplate-linked tripeptide precursor.

The Pseudomonas aeruginosa toxin L-2-amino-4-methoxy-trans-3-butenoic acid (AMB) is a non-proteinogenic amino acid which is toxic for prokaryotes and eukaryotes. Production of AMB requires a five-gene cluster encoding a putative LysE-type transporter (AmbA), two non-ribosomal peptide synthetases (AmbB and AmbE), and two iron(II)/α-ketoglutarate-dependent oxygenases (AmbC and AmbD). Bioinformatics analysis predicts one thiolation (T) domain for AmbB and two T domains (T1 and T2) for AmbE, suggesting that AMB is generated by a processing step from a precursor tripeptide assembled on a thiotemplate. Using a combination of ATP-PPi exchange assays, aminoacylation assays, and mass spectrometry-based analysis of enzyme-bound substrates and pathway intermediates, the AmbB substrate was identified to be L-alanine (L-Ala), while the T1 and T2 domains of AmbE were loaded with L-glutamate (L-Glu) and L-Ala, respectively. Loading of L-Ala at T2 of AmbE occurred only in the presence of AmbB, indicative of a trans loading mechanism. In vitro assays performed with AmbB and AmbE revealed the dipeptide L-Glu-L-Ala at T1 and the tripeptide L-Ala-L-Glu-L-Ala attached at T2. When AmbC and AmbD were included in the assay, these peptides were no longer detected. Instead, an L-Ala-AMB-L-Ala tripeptide was found at T2. These data are in agreement with a biosynthetic model in which L-Glu is converted into AMB by the action of AmbC, AmbD, and tailoring domains of AmbE. The importance of the flanking L-Ala residues in the precursor tripeptide is discussed.

Preliminary Study on Safety Conditions in Health Workers Exposed to Antineoplastic Drugs

A study was done to establish work practices and preventive measures for nurses handling antineoplasticdrugs (AND) and to determine the risk of developing AND-related symptoms. A descriptive cross sectional study was made. Workers from 5 health centers in Valencia, Venezuela, were selected. Demography, occupational and clinical history, shift, work practices, safety precautions, antineoplastic drugs used, residues disposal and life styles were obtained via a questionnaire. Most prevalent symptoms were adjusted for age, shift, and smoking. Age was significant for cough and dizziness; smoking was significant for abdomipo seleccionado de trabajadores que manejan FAN en cinco centros de salud de la ciudad de Valencia, Venezuela, con el propósito de evaluar de forma preliminar su riesgo potencial de desarrollar signos-síntomas derivados de este oficio; a su vez, establecer si existe la necesidad de continuar con una evaluación más profunda que permita hacer recomendaciones concluyentes para evitar sus efectos adversos. Metodología La población estuvo constituida por el personal de las unidades de oncología de cinco centros de salud de la ciudad de Valencia, Venezuela. La muestra la conformaron veinte trabajadores que pertenecen a dichos centros. La participación fue voluntaria y mediante firma de una carta de consentimiento. Recolección de datos Se realizó una entrevista a cada participante con información que hizo referencia a datos demográficos, historia ocupacional, turnos de trabajo, entrenamiento en medidas de seguridad para el manejo de este tipo de fármacos, actividades que realizan, uso de equipos de protección personal (EPP), sitio de manipulación de nal pain and shift was significant for nausea and cough. Nauseas were the most prevalent symptom (55%). Dizziness was directly associated with use of gowns and inversely half-face respirator. None of the studied centers had satisfactory working conditions. A follow up study should be made including physical exam and environmental and biological monitoring.