Development of Pleiotropic TrkB and 5-HT4 Receptor Ligands as Neuroprotective Agents.

One common event that is the most detrimental in neurodegenerative disorders, even though they have a complex pathogenesis, is the increased rate of neuronal death. Endogenous neurotrophins consist of the major neuroprotective factors, while brain-derived neurotrophic factor (BDNF) and its high-affinity tyrosine kinase receptor TrkB are described in a number of studies for their important neuronal effects. Normal function of this receptor is crucial for neuronal survival, differentiation, and synaptic function. However, studies have shown that besides direct activation, the TrkB receptor can be transactivated via GPCRs. It has been proven that activation of the 5-HT4 receptor and transactivation of the TrkB receptor have a positive influence on neuronal differentiation (total dendritic length, number of primary dendrites, and branching index). Because of that and based on the main structural characteristics of LM22A-4, a known activator of the TrkB receptor, and RS67333, a partial 5-HT4 receptor agonist, we have designed and synthesized a small data set of novel compounds with potential dual activities in order to not only prevent neuronal death, but also to induce neuronal differentiation in neurodegenerative disorders.

Effect of somatostatin on human retinal pigment epithelial cells permeability.

PURPOSE: To assess the effect of somatostatin (SST) on the permeability of human retinal pigment epithelial cells. METHODS: We conducted two experiments, exposing cells from human-fetal retinal pigment epithelium (hfRPE) cultures to vascular endothelial growth factor (VEGF), with or without SST pretreatment, in one, and to hypoxic conditions, again with or without SST pretreatment, in the other. The paracellular permeability of hfRPE was assessed by measuring transepithelial electrical resistance (TER) and fluorescein isothiocyanate-sodium (FITC-sodium) flux. Immunochemistry analysis was used to assess the expression of occludin and Zonula occludens-1(ZO-1). RESULTS: Both VEGF and hypoxia increased permeability of the hfRPE, as measured by TER and tracer flux, and decreased occludin and ZO-1staining, as measured by immunochemistry. Pretreatment of cultures with SST partially counteracted these effects. CONCLUSIONS: Somatostatin may play a role in the regulation of permeability across retinal pigment epithelium. It may act as an anti-permeability factor in the retina through the enhancement of tight junction function.

[Promoting directives of the Quality Law of the Spanish National Health System: Computer-interpretable clinical practice guidelines]. / Impulsando las directrices de la Ley de Calidad del SNS: modelos computacionales de guías de práctica clínica.

The Cohesion and Quality Act of the National Health System promotes the use of new technologies to make it possible for health professionals put the scientific evidence into practice. In order to do this, there are technological tools, known as of computer-interpretable guidelines, which can help achieve this goal from an innovation perspective. They can be adopted using an iterative process, having a great initial potential as an educational tool, of quality and safety of the patient, in the decision making and, optionally, can be integrated with the electronic medical history, once they are rigorously validated. This article presents updates on these tools, reviews international projects, and personal experiences in which they have demonstrated their value, and highlights the advantages, risks, and limitations they present from a clinical point of view.

Antioxidant, anticancer activities and mechanistic studies of the flavone glycoside diosmin and its oxidovanadium(IV) complex. Interactions with bovine serum albumin.

The natural antioxidant flavonoid diosmin, found in citric fruits, showed low antioxidant properties among other flavonoids due to its structural characteristics and low cytotoxicity against lung (A549) and breast (T47D, SKBR3 and MDAMB231) cancer cell lines. The anticancer behavior has been improved by the metal complex generated with the flavonoid and the oxidovanadium(IV) ion. This new complex, [VO(dios)(OH)3]Na5·6H2O (VOdios), has been synthesized and characterized both in solid and solution states. The interaction of the metal ion through the sugar moiety of diosmin precluded the improvement of the antioxidant effects. However, the cell-killing effects tested in human lung A549 and breast T47D, SKBR3 and MDAMB231 cancer cell lines, were enhanced by complexation. The anti-proliferative effects on the human lung cancer cell line were accompanied by cellular ROS generation and an increase in cytoplasm condensation. The breast cancer cell lines did not produce caspase3/7 activation, mitochondrial potential reduction and ROS generation. Therefore, a non-apoptotic form of cell death in a caspase- and oxidative stress-independent manner has been proposed. The protein binding ability has been monitored by the quenching of tryptophan emission in the presence of the compounds using bovine serum albumin (BSA) as a model protein. Both compounds could be distributed and transported in vivo and the complex displayed stronger binding affinity and higher contributions to the hydrogen bond and van der Waals forces.

Inhibition of the metastatic progression of breast and colorectal cancer in vitro and in vivo in murine model by the oxidovanadium(IV) complex with luteolin.

The anticancer and antimetastatic behavior of the flavonoid luteolin and its oxidovanadium(IV) complex [VO(lut)(H2O)2]Na·3H2O (VOlut) has been investigated. Considering that the complex displayed strong anticancer activity on MDAMB231 human breast cancer cell line we herein determined through in vitro assays that the complex would probably reduce breast cancer cell metastasis in a higher extent than the natural antioxidant. In the CT26 colon cancer cell line a stronger anticancer effect has also been determined for the complex (IC50 0.9µM) and in addition it did not exert toxic effects on normal colon epithelial cells at concentrations up to 10µM. Working with a murine model of highly aggressive, orthotopic colon cancer model (CT26 cancer cell lines) it has been determined that the complex might prevent metastatic dissemination of the colon cancer cells to the liver. The flavonoid luteolin also exerted anticancer effects (at a low degree, IC50 5.9µM) on CT26 cell line and produced a 24% reduction of colon cancer liver metastasis.

Evaluation of a New Immunochromatographic Test Using Recombinant Antigen B8/1 for Diagnosis of Cystic Echinococcosis.

Diagnosis of cystic echinococcosis (CE) is based on the identification of the cyst(s) by imaging, using immunodiagnostic tests mainly as complementary tools in clinical settings. Among the antigens used for immunodiagnosis, previous studies described a good performance of the recombinant antigen B8/1 (rAgB) in an enzyme-linked immunosorbent assay (ELISA) format; however, in remote parts of areas where the disease is endemic, the implementation of an ELISA is difficult, so a more simple, rapid, and reliable method such as the immunochromatographic test (ICT) is required. In this study, using a set of 50 serum samples from patients with surgically confirmed CE, we compared the performance of an ICT and that of an ELISA using the rAgB. The overall sensitivities of ICT and ELISA were not statistically different (78% versus 72%; P = 0.36). The overall agreement between both tests was moderate (κ = 0.41; P < 0.01). Concordance between ICT and ELISA was substantial or almost perfect for patients with liver involvement (κ = 0.65; P < 0.001) and patients with more than one hydatid cyst (κ = 0.82; P < 0.001), respectively. Moreover, specificity analysis using a total of 88 serum samples from healthy individuals (n = 20) and patients (n = 68) with other parasitic infections revealed that ICT had a specificity of 89.8%. ICT and ELISA had similar performance for the detection of specific antibodies to E. granulosus, and ICT had a high specificity, opening the possibility of using ICT as a screening tool in rural settings.

Serum concentrations of mast cell tryptase are reduced in heavy drinkers.

BACKGROUND: Baseline serum tryptase concentrations are commonly used in clinical practice as a marker of the body's mast cell burden. This study aimed to investigate serum tryptase concentrations in heavy drinkers. METHODS: Serum tryptase concentrations were determined in 126 heavy drinkers (75% males, median age 47 years) who were admitted to the hospital because of alcohol withdrawal syndrome (n = 60), general symptoms with abnormalities on biochemical tests that indicated acute liver disease (n = 19), complications of advanced liver disease (n = 33), and miscellaneous reasons (n = 14). Results were compared with those of 70 healthy controls (66% males, median age 40 years). RESULTS: Serum tryptase concentrations were lower in heavy drinkers than in healthy controls (median 2.23 µg/l vs. median 3.25 µg/l, p < 0.001). Ten heavy drinkers (7.9%) had undetectable (<1 µg/l) serum tryptase levels versus none of the healthy controls (p = 0.01). The association of low tryptase levels with heavy drinking was independent of age, gender, and smoking status. Among heavy drinkers, the lowest tryptase concentrations were observed in patients with alcohol withdrawal syndrome and patients with general symptoms with abnormalities on biochemical tests that indicated acute liver disease. Furthermore, serum tryptase concentrations were negatively correlated with markers of acute liver damage or alcohol consumption (serum aspartate aminotransferase and gamma-glutamyl transferase). Atopy (skin prick test positivity) was not associated with serum tryptase concentrations in heavy drinkers. CONCLUSIONS: Serum concentrations of mast cell tryptase are lower in heavy drinkers than in healthy controls.

Metastatic lesions with and without interleukin-18-dependent genes in advanced-stage melanoma patients.

IL-18 is an immune-stimulating cytokine that promotes experimental melanoma metastasis via vascular endothelial growth factor (VEGF)-induced very late antigen (VLA)-4. We studied genes associated with the ability of melanoma cells to allow metastasis under IL-18 effects, and we verified their expression in metastatic lesions from patients with melanoma. Human melanoma cell lines with and without the IL-18 receptor (IL-18R)/VEGF/VLA-4-expressing phenotype were identified, and their metastatic potential was studied in nude mice. RNA from untreated and IL-18-treated melanoma phenotypes was hybridized to a cDNA microarray, and their signature genes were studied. RNA from primary and metastatic lesions from patients with melanoma was hybridized to a cDNA microarray to identify lesions with the transcript patterns of melanoma cells with and without the IL-18R/VEGF/VLA-4 phenotype. IL-18R/VEGF/VLA-4-expressing A375 and 1182 melanoma cells produced a higher metastasis number than 526 and 624.28 melanoma cells, not using this prometastatic pathway. Melanoma cells with and without the IL-18R/VEGF/VLA-4 phenotype had distinct transcript patterns. However, the type I transcriptional cluster, including cutaneous and lymph node metastases, but not the type II cluster, not including cutaneous metastases, had signature genes from IL-18-treated melanoma cells with, but not without, the IL-18R/VEGF/VLA-4 phenotype. Metastatic melanoma lesions with and without IL-18-dependent genes were identified, suggesting that melanoma metastasis developed via inflammation-dependent and inflammation-independent mechanisms. Signature genes from melanomas with and without the IL-18R/VEGF/VLA-4 phenotype may serve as diagnostic biomarkers of melanoma predisposition to prometastatic effects of IL-18.

Zooplankton vulnerability to glyphosate exacerbated by global change.

Anthropogenic activities generate a severe footprint at a global scale. Intensive agriculture is a global change driver that affects aquatic systems due to the discharge of pollutants. This situation can be modified or aggravated by other aspects, such as the disturbance history and other global change factors. Following our study line, it is necessary to evaluate how the disturbance history combined with temperature changes can affect the functioning of aquatic systems. The objectives of this study were divided into two phases. The objectives of phase 1 were to induce vulnerability in Daphnia magna populations through a disturbance history based on sublethal glyphosate concentration exposure under different temperature conditions (20 °C and 25 °C). In phase 2, vulnerability was assessed through the exposure to subsequent stressors (starvation, increased salinity and paracetamol) combined with changes in temperature. During the glyphosate exposure period in phase 1, differences were observed in the D. magna populations with respect to temperature, with lower abundance at 25 °C than at 20 °C. However, no differences were observed in abundance regarding glyphosate treatment. The results obtained in phase 2 with the new stressors combined with temperature changes in both directions, revealed stronger effects in vulnerable populations than in control populations. In addition, the temperature changes modulated the effects in the starvation and increased salinity tests. Agrochemical sublethal concentrations induce vulnerability in D. magna populations and inflicted temperature changes can act as a modulating factor for this vulnerability, showing the complexity in assessing the responses under the multiple scenarios associated with global change.

Comprehensive characterization of the neurogenic and neuroprotective action of a novel TrkB agonist using mouse and human stem cell models of Alzheimer's disease.

BACKGROUND: Neural stem cell (NSC) proliferation and differentiation in the mammalian brain decreases to minimal levels postnatally. Nevertheless, neurogenic niches persist in the adult cortex and hippocampus in rodents, primates and humans, with adult NSC differentiation sharing key regulatory mechanisms with development. Adult neurogenesis impairments have been linked to Alzheimer's disease (AD) pathology. Addressing these impairments by using neurotrophic factors is a promising new avenue for therapeutic intervention based on neurogenesis. However, this possibility has been hindered by technical difficulties of using in-vivo models to conduct screens, including working with scarce NSCs in the adult brain and differences between human and mouse models or ethical limitations. METHODS: Here, we use a combination of mouse and human stem cell models for comprehensive in-vitro characterization of a novel neurogenic compound, focusing on the brain-derived neurotrophic factor (BDNF) pathway. The ability of ENT-A011, a steroidal dehydroepiandrosterone derivative, to activate the tyrosine receptor kinase B (TrkB) receptor was tested through western blotting in NIH-3T3 cells and its neurogenic and neuroprotective action were assessed through proliferation, cell death and Amyloid-ß (Aß) toxicity assays in mouse primary adult hippocampal NSCs, mouse embryonic cortical NSCs and neural progenitor cells (NPCs) differentiated from three human induced pluripotent stem cell lines from healthy and AD donors. RNA-seq profiling was used to assess if the compound acts through the same gene network as BDNF in human NPCs. RESULTS: ENT-A011 was able to increase proliferation of mouse primary adult hippocampal NSCs and embryonic cortical NSCs, in the absence of EGF/FGF, while reducing Aß-induced cell death, acting selectively through TrkB activation. The compound was able to increase astrocytic gene markers involved in NSC maintenance, protect hippocampal neurons from Αß toxicity and prevent synapse loss after Aß treatment. ENT-A011 successfully induces proliferation and prevents cell death after Aß toxicity in human NPCs, acting through a core gene network shared with BDNF as shown through RNA-seq. CONCLUSIONS: Our work characterizes a novel BDNF mimetic with preferable pharmacological properties and neurogenic and neuroprotective actions in Alzheimer's disease via stem cell-based screening, demonstrating the promise of stem cell systems for short-listing competitive candidates for further testing.

Consequences of Amyloid-ß Deficiency for the Liver.

The hepatic content of amyloid beta (Aß) decreases drastically in human and rodent cirrhosis highlighting the importance of understanding the consequences of Aß deficiency in the liver. This is especially relevant in view of recent advances in anti-Aß therapies for Alzheimer's disease (AD). Here, it is shown that partial hepatic loss of Aß in transgenic AD mice immunized with Aß antibody 3D6 and its absence in amyloid precursor protein (APP) knockout mice (APP-KO), as well as in human liver spheroids with APP knockdown upregulates classical hallmarks of fibrosis, smooth muscle alpha-actin, and collagen type I. Aß absence in APP-KO and deficiency in immunized mice lead to strong activation of transforming growth factor-ß (TGFß), alpha secretases, NOTCH pathway, inflammation, decreased permeability of liver sinusoids, and epithelial-mesenchymal transition. Inversely, increased systemic and intrahepatic levels of Aß42 in transgenic AD mice and neprilysin inhibitor LBQ657-treated wild-type mice protect the liver against carbon tetrachloride (CCl4)-induced injury. Transcriptomic analysis of CCl4-treated transgenic AD mouse livers uncovers the regulatory effects of Aß42 on mitochondrial function, lipid metabolism, and its onco-suppressive effects accompanied by reduced synthesis of extracellular matrix proteins. Combined, these data reveal Aß as an indispensable regulator of cell-cell interactions in healthy liver and a powerful protector against liver fibrosis.

Sublethal exposure to agrochemicals impairs zooplankton ability to face future global change challenges.

Intensive agriculture is characterized by the application of multiple stressors that damage aquatic systems. Currently, ecotoxicological evaluations are considered insufficient to understand the environmental risks of stressor mixtures and their consequences in ecosystems. In addition, future global change scenarios could alter the predicted effects of agrochemicals in aquatic systems based on single exposures, making it necessary to consider the history of environmental disturbances that may result in vulnerability to subsequent environmental changes. The objectives of this study were to induce disturbance histories by exposure to sublethal glyphosate concentrations in microcosms and to assess whether this disturbance results in vulnerable populations using Daphnia magna as the target species. Populations were considered vulnerable if their sensitivity to new stressors (inanition, temperature and salinity) was higher than that of undisturbed populations. To induce disturbance history, microcosm aquatic communities (two cladocerans and one microalgae) were exposed to two glyphosate sublethal concentrations (below the No Observed Effect Concentration, NOEC values for D. magna, 0.1 and 1 mg L-1) in microcosms under controlled conditions in a culture chamber for 48 days. After this period, no significant differences were observed with respect to the control microcosms in the selected parameters (cladoceran abundance, microalgae cell abundance, microalgae colony formation, pH and dissolved oxygen). To test vulnerability, our target D. magna populations, which were previously exposed to different glyphosate treatments, were subjected to inanition, elevated temperature and salinity. Our results showed that D. magna populations with disturbance history performed worse in all the scenarios compared to the populations from undisturbed conditions. These results underscore the need to study how environmental disturbance history influences population responses to new and future stressors. Moreover, our findings raise concern regarding the sublethal effects of pesticides on aquatic populations.

Design, synthesis and biological characterization of novel activators of the TrkB neurotrophin receptor.

Numerous studies have been published about the implication of the neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor TrkB in the pathogenesis of several neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, Multiple Sclerosis and motor neuron disease. BDNF activates the TrkB receptor with high potency and specificity, promoting neuronal survival, differentiation and synaptic plasticity. Based on the main structural characteristics of LM22A-4, a previously published small molecule that acts as activator of the TrkB receptor, we have designed and synthesized a small data set of compounds. The lead idea for the design of the new compounds was to modify the third position of the LM22A-4, by introducing different substitutions in order to obtain compounds which will have not only better physicochemical properties but selective activity as well. ADME and toxicity profiles of molecules have been evaluated as well as their biological properties through the TrkB receptor and affinity to promote neurite differentiation.

Correlation between Positive Orientation and Control of Anger, Anxiety and Depression in Nursing Students in Poland, Spain and Slovakia during the COVID-19 Pandemic.

(1) This study analysed the relationship between positive orientation and subjective control of anger, anxiety and depression in nursing students in Poland, Spain and Slovakia during the pandemic. (2) The survey was carried out by the diagnostic survey method in a group of 756 nursing students in Poland, Spain and Slovakia. The empirical data were gathered using an original survey questionnaire, the Positive Orientation Scale and the Courtauld Emotional Control Scale. (3) It was shown that the positive orientation level in Polish students was significantly lower than in students in Spain (p < 0.0001) and Slovakia (p < 0.0001). Low results for positive orientation were obtained in nearly half (47.18%) of the respondents in the Polish sample, whereas it was 34.18% and 31.18% in the Spanish and Slovak samples, respectively (p < 0.0001). A positive orientation was the most important predictor of emotional control among the nursing students at all the sites (p < 0.0001). (4) Positive orientation has been shown to have a significant impact on emotional control in nursing students during the pandemic. Therefore, it is important to carefully monitor students' mental health during the pandemic to determine the demand for psychological and emotional support.

Validation of a Nursing Workload Measurement Scale, Based on the Classification of Nursing Interventions, for Adult Hospitalization Units.

We conducted validation of a scale to measure nursing workloads, previously designed using NIC interventions within the four nursing functions (patient care, teaching, management, and research). METHODS: This is an analytical, descriptive, prospective, and observational study using qualitative methodology (focus groups and in-depth interviews) with a quantitative and qualitative section (committee of experts and real application of the scale through a validation pilot and with multicentric application, including hospitalization units of internal medicine and surgery of four hospitals). Qualitative analysis was performed with Atlas.ti8 and quantitative analysis with R. RESULTS: Qualitatively, all the participants agreed on the need to measure workloads in all nursing functions with standardized terminology. The expert committee found greater relevance (91.67%) in "prevention" and "health education" as well as consistency with the construct and adequate wording in 99% of the selected items. In the pilot test and multicenter application, the nurses spent more time on the caring dimension, in the morning shift, and on the items "self-care", "medication", "health education", "care of invasive procedures", "wounds care", "comfort", and "fluid therapy". Cronbach's alpha 0.727, composite reliability 0.685, AVE 0.099, and omega coefficient 0.704 were all acceptable. Construct validity: KMO 0.5 and Bartlett's test were significant. CONCLUSIONS: The scale can be considered valid to measure nursing workloads, both qualitatively in obtaining the consensus of experts and health personnel and quantitatively, with acceptable reliability and validity superior to other similar scales.

Multicenter application of a nursing workload measurement scale in adult hospitalization units.

Objectives: The study aimed to the multicenter application of a nursing workload measurement scale in the internal medicine and surgery adults hospitalization units. Methods: The study design was a multicenter, observational, and descriptive study. A multicenter application of the MIDENF® nursing workload measurement scale was carried out, which consists of 21 items, and covers the four nursing functions (patient care items, teaching, manager, and researcher), in units of hospitalization of adults of internal medicine and surgery of four different hospitals. Each item contains one or more of the nursing interventions of Nursing Interventions Classification (NIC) and has an assigned time, after comparing the real time it takes to perform each intervention with the North American Nursing Diagnosis Association (NANDA) standardized time. The study was carried out during nine months of the year 2020, measuring two days each month in the three work shifts (morning, evening, and night) to all patients admitted on the days of measurement in the indicated units. Results: The descriptive and inferential analysis of 11,756 completed scales, 5,695 in general surgery and 6,061 in internal medicine, showed a greater care load for the two units during the morning shift (227,034 min in general surgery, 261,835 min in internal medicine), especially in the items of "self-care", "medication", "common invasive procedures", "fluid therapy", and "patient and family support", while the managerial function was similar during the three work shifts in the two units studied, getting values between 57,348 and 62,901 min. In the analysis by shift and unit, statistical significance was obtained both for the total workload and the four nursing functions(P < 0.001). Conclusions: It is shown that the use of validated scales with the standardized language of nursing functions, adapted to the units, provides objective information to adjust the nursing staff to the real situation of care in any hospital and unit where it is applied, improving quality and patient safety.

Relationship between Job Satisfaction and Workload of Nurses in Adult Inpatient Units.

Among Nursing and Psychology professionals, the job satisfaction of those in Adult Inpatient Units is analyzed, with a new scale to measure nursing workloads validated. Objective: The objective of this study was to relate nursing workloads to professional job satisfaction. Methods: This is an observational, analytical, descriptive, concurrent and quantitative study, which used the Overall Job Satisfaction scale and subscales therein, to identify global satisfaction-intrinsic or related to motivational factors and extrinsic or associated with hygienic factors-in nursing professionals (n = 104) from eight Inpatient Units of Internal Medicine and Surgery, in four hospital centers, to describe job satisfaction in the professionals studied and to find statistically significant associations between job satisfaction and workload (measured with the scale MIDENF®) in the inpatient units where they work. Results: There were higher levels of satisfaction in the variables "relationship with immediate boss" and "relationship with fellow workers", and lower levels in "relationship with senior management" and "organizational system of the unit". In the inferential analysis, the scores were 75.63 for overall satisfaction, 35.28 for intrinsic satisfaction, and 40.36 for extrinsic satisfaction. Conclusions: There is a close relationship between workload and job satisfaction, showing more dissatisfaction regarding organizational aspects and professional recognition.

Analysis of midwives' situation and the need to measure their workloads.

OBJECTIVE: to analyze midwives' employment situation of midwives and detect their workload measurement needs. METHODS: a mixed methodology (quantitative and qualitative), observational, descriptive and cross-sectional study. Two phases were carried out. The first methodological phase consisted of conducting semi-structured individual interviews. The second methodological phase analyzed the Nursing Intervention Classification (NIC) interventions that midwives perform in the labor room during daily practice. RESULTS: 90.3% of midwives have work overload, since for 80.6% the midwife-pregnant mother ratio is not well established, since the mean execution time of a Nursing Intervention Classification (NIC) intervention is 20 minutes. For this reason, 100% of the sample considers the development of workload measurement tools to be highly useful. CONCLUSIONS: the need to generate an instrument that considers the diversity of Nursing Intervention Classification (NIC) interventions that develop within the labor room is ratified.

Resveratrol prevents inflammation-dependent hepatic melanoma metastasis by inhibiting the secretion and effects of interleukin-18.

BACKGROUND: Implantation and growth of metastatic cancer cells at distant organs is promoted by inflammation-dependent mechanisms. A hepatic melanoma metastasis model where a majority of metastases are generated via interleukin-18-dependent mechanisms was used to test whether anti-inflammatory properties of resveratrol can interfere with mechanisms of metastasis. METHODS: Two experimental treatment schedules were used: 1) Mice received one daily oral dose of 1 mg/kg resveratrol after cancer cell injection and the metastasis number and volume were determined on day 12. 2) Mice received one daily oral dose of 1 mg/kg resveratrol along the 5 days prior to the injection of cancer cells and both interleukin-18 (IL-18) concentration in the hepatic blood and microvascular retention of luciferase-transfected B16M cells were determined on the 18th hour. In vitro, primary cultured hepatic sinusoidal endothelial cells were treated with B16M-conditioned medium to mimic their in vivo activation by tumor-derived factors and the effect of resveratrol on IL-18 secretion, on vascular cell adhesion molecule-1 (VCAM-1) expression and on tumor cell adhesion were studied. The effect of resveratrol on melanoma cell activation by IL-18 was also studied. RESULTS: Resveratrol remarkably inhibited hepatic retention and metastatic growth of melanoma cells by 50% and 75%, respectively. The mechanism involved IL-18 blockade at three levels: First, resveratrol prevented IL-18 augmentation in the blood of melanoma cell-infiltrated livers. Second, resveratrol inhibited IL-18-dependent expression of VCAM-1 by tumor-activated hepatic sinusoidal endothelium, preventing melanoma cell adhesion to the microvasculature. Third, resveratrol inhibited adhesion- and proliferation-stimulating effects of IL-18 on metastatic melanoma cells through hydrogen peroxide-dependent nuclear factor-kappaB translocation blockade on these cells. CONCLUSIONS: These results demonstrate multiple sites for therapeutic intervention using resveratrol within the prometastatic microenvironment generated by tumor-induced hepatic IL-18, and suggest a remarkable effect of resveratrol in the prevention of inflammation-dependent melanoma metastasis in the liver.

Vascular endothelial growth factor regulates melanoma cell adhesion and growth in the bone marrow microenvironment via tumor cyclooxygenase-2.

BACKGROUND: Human melanoma frequently colonizes bone marrow (BM) since its earliest stage of systemic dissemination, prior to clinical metastasis occurrence. However, how melanoma cell adhesion and proliferation mechanisms are regulated within bone marrow stromal cell (BMSC) microenvironment remain unclear. Consistent with the prometastatic role of inflammatory and angiogenic factors, several studies have reported elevated levels of cyclooxygenase-2 (COX-2) in melanoma although its pathogenic role in bone marrow melanoma metastasis is unknown. METHODS: Herein we analyzed the effect of cyclooxygenase-2 (COX-2) inhibitor celecoxib in a model of generalized BM dissemination of left cardiac ventricle-injected B16 melanoma (B16M) cells into healthy and bacterial endotoxin lipopolysaccharide (LPS)-pretreated mice to induce inflammation. In addition, B16M and human A375 melanoma (A375M) cells were exposed to conditioned media from basal and LPS-treated primary cultured murine and human BMSCs, and the contribution of COX-2 to the adhesion and proliferation of melanoma cells was also studied. RESULTS: Mice given one single intravenous injection of LPS 6 hour prior to cancer cells significantly increased B16M metastasis in BM compared to untreated mice; however, administration of oral celecoxib reduced BM metastasis incidence and volume in healthy mice, and almost completely abrogated LPS-dependent melanoma metastases. In vitro, untreated and LPS-treated murine and human BMSC-conditioned medium (CM) increased VCAM-1-dependent BMSC adherence and proliferation of B16M and A375M cells, respectively, as compared to basal medium-treated melanoma cells. Addition of celecoxib to both B16M and A375M cells abolished adhesion and proliferation increments induced by BMSC-CM. TNFα and VEGF secretion increased in the supernatant of LPS-treated BMSCs; however, anti-VEGF neutralizing antibodies added to B16M and A375M cells prior to LPS-treated BMSC-CM resulted in a complete abrogation of both adhesion- and proliferation-stimulating effect of BMSC on melanoma cells. Conversely, recombinant VEGF increased adherence to BMSC and proliferation of both B16M and A375M cells, compared to basal medium-treated cells, while addition of celecoxib neutralized VEGF effects on melanoma. Recombinant TNFα induced B16M production of VEGF via COX-2-dependent mechanism. Moreover, exogenous PGE2 also increased B16M cell adhesion to immobilized recombinant VCAM-1. CONCLUSIONS: We demonstrate the contribution of VEGF-induced tumor COX-2 to the regulation of adhesion- and proliferation-stimulating effects of TNFα, from endotoxin-activated bone marrow stromal cells, on VLA-4-expressing melanoma cells. These data suggest COX-2 neutralization as a potential anti-metastatic therapy in melanoma patients at high risk of systemic and bone dissemination due to intercurrent infectious and inflammatory diseases.