Epigenetics override pro-inflammatory PTGS transcriptomic signature towards selective hyperactivation of PGE 2 in colorectal cancer

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al.[Background]: Misregulation of the PTGS (prostaglandin endoperoxide synthase, also known as cyclooxygenase or COX) pathway may lead to the accumulation of pro-inflammatory signals, which constitutes a hallmark of cancer. To get insight into the role of this signaling pathway in colorectal cancer (CRC), we have characterized the transcriptional and epigenetic landscapes of the PTGS pathway genes in normal and cancer cells. [Results]: Data from four independent series of CRC patients (502 tumors including adenomas and carcinomas and 222 adjacent normal tissues) and two series of colon mucosae from 69 healthy donors have been included in the study. Gene expression was analyzed by real-time PCR and Affymetrix U219 arrays. DNA methylation was analyzed by bisulfite sequencing, dissociation curves, and HumanMethylation450K arrays. Most CRC patients show selective transcriptional deregulation of the enzymes involved in the synthesis of prostanoids and their receptors in both tumor and its adjacent mucosa. DNA methylation alterations exclusively affect the tumor tissue (both adenomas and carcinomas), redirecting the transcriptional deregulation to activation of prostaglandin E 2 (PGE 2 ) function and blockade of other biologically active prostaglandins. In particular, PTGIS, PTGER3, PTGFR, and AKR1B1 were hypermethylated in more than 40 % of all analyzed tumors. [Conclusions]: The transcriptional and epigenetic profiling of the PTGS pathway provides important clues on the biology of the tumor and its microenvironment. This analysis renders candidate markers with potential clinical applicability in risk assessment and early diagnosis and for the design of new therapeutic strategies.IC was funded by Fundação para a Ciência e a Tecnologia (SFRH/BD/28464/2006); JC was funded by a FPI fellowship. ADV was supported in part by a contract from the Ministerio de Economía y Competitividad (MINECO) (PTC2011-1091). This work was supported by the MINECO(SAF2011/23638, SAF2014/52492), the Catalan Institute of Oncology and the Instituto de Salud Carlos III (grant PI11-01439, RD12/0042/0019 and CIBERESP CB06/02/2005), the Generalitat de Catalunya (grant 2014SGR647), and the Asociación Española Contra el Cáncer (AECC).Peer Reviewe

Role of Caveolin 1, E-Cadherin, Enolase 2 and PKCalpha on resistance to methotrexate in human HT29 colon cancer cells

<p>Abstract</p> <p>Background</p> <p>Methotrexate is one of the earliest cytotoxic drugs used in cancer therapy, and despite the isolation of multiple other folate antagonists, methotrexate maintains its significant role as a treatment for different types of cancer and other disorders. The usefulness of treatment with methotrexate is limited by the development of drug resistance, which may be acquired through different ways. To get insights into the mechanisms associated with drug resistance and sensitization we performed a functional analysis of genes deregulated in methotrexate resistant cells, either due to its co-amplification with the <it>dhfr </it>gene or as a result of a transcriptome screening using microarrays.</p> <p>Methods</p> <p>Gene expression levels were compared between triplicate samples from either HT29 sensitive cells and resistant to 10^-5M MTX by hybridization to the GeneChip^®HG U133 PLUS 2.0 from Affymetrix. After normalization, a list of 3-fold differentially expressed genes with a p-value < 0.05 including multiple testing correction (Benjamini and Hochberg false discovery rate) was generated. RT-Real-time PCR was used to validate the expression levels of selected genes and copy-number was determined by qPCR. Functional validations were performed either by siRNAs or by transfection of an expression plasmid.</p> <p>Results</p> <p>Genes adjacent to the <it>dhfr locus </it>and included in the 5q14 amplicon were overexpressed in HT29 MTX-resistant cells. Treatment with siRNAs against those genes caused a slight reduction in cell viability in both HT29 sensitive and resistant cells. On the other hand, microarray analysis of HT29 and HT29 MTX resistant cells unveiled overexpression of caveolin 1, enolase 2 and PKCα genes in resistant cells without concomitant copy number gain. siRNAs against these three genes effectively reduced cell viability and caused a decreased MTX resistance capacity. Moreover, overexpression of E-cadherin, which was found underexpressed in MTX-resistant cells, also sensitized the cells toward the chemotherapeutic agent. Combined treatments targeting siRNA inhibition of caveolin 1 and overexpression of E-cadherin markedly reduced cell viability in both sensitive and MTX-resistant HT29 cells.</p> <p>Conclusion</p> <p>We provide functional evidences indicating that caveolin 1 and E-cadherin, deregulated in MTX resistant cells, may play a critical role in cell survival and may constitute potential targets for coadjuvant therapy.</p

Quantification of Unmethylated Alu (QUAlu): a tool to assess global hypomethylation in routine clinical samples

Hypomethylation of DNA is a hallmark of cancer and its analysis as tumor biomarker has been proposed, but its determination in clinical settings is hampered by lack of standardized methodologies. Here, we present QUAlu (Quantification of Unmethylated Alu), a new technique to estimate the Percentage of UnMethylated Alu (PUMA) as a surrogate for global hypomethylation. QUAlu consists in the measurement by qPCR of Alu repeats after digestion of genomic DNA with isoschizomers with differential sensitivity to DNA methylation. QUAlu performance has been evaluated for reproducibility, trueness and specificity, and validated by deep sequencing. As a proof of use, QUAlu has been applied to a broad variety of pathological examination specimens covering five cancer types. Major findings of the preliminary application of QUAlu to clinical samples include: (1) all normal tissues displayed similar PUMA; (2) tumors showed variable PUMA with the highest levels in lung and colon and the lowest in thyroid cancer; (3) stools from colon cancer patients presented higher PUMA than those from control individuals; (4) lung squamous cell carcinomas showed higher PUMA than lung adenocarcinomas, and an increasing hypomethylation trend associated with smoking habits. In conclusion, QUAlu is a simple and robust method to determine Alu hypomethylation in human biospecimens and may be easily implemented in research and clinical settings.RB was supported by a FPI fellowship from Ministerio de Economía y Competitividad. AD-V was supported in part by a contract PTC2011-1091 from Ministerio de Economía y Competitividad. This work was supported by grants from FEDER, the Ministerio de Economía y Competitividad (SAF2011/23638 to MAP), the Instituto de Salud Carlos III (FIS PI11/02421 to JR, FIS PI11/01359 and FIS PI14/00240 to MR, FIS PI14/00308 to MJ, FIS PI12/00511 to MP), and Fundació Olga Torres (to MJ)

Cardiometabolic responses to a battling rope high intensity interval training protocol

Trata de una conferencia invitada que ganó premio a la mejor comunicación científica

Mechanisms of bacterial membrane permeabilization by crotalicidin Ctn and its fragment Ctn 15–34, antimicrobial peptides from rattlesnake venom

© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.Crotalicidin (Ctn), a cathelicidin-related peptide from the venom of a South American rattlesnake, possesses potent antimicrobial, antitumor, and antifungal properties. Previously, we have shown that its C-terminal fragment, Ctn(15-34), retains the antimicrobial and antitumor activities but is less toxic to healthy cells and has improved serum stability. Here, we investigated the mechanisms of action of Ctn and Ctn(15-34) against Gram-negative bacteria. Both peptides were bactericidal, killing ∼90% of Escherichia coli and Pseudomonas aeruginosa cells within 90-120 and 5-30 min, respectively. Studies of ζ potential at the bacterial cell membrane suggested that both peptides accumulate at and neutralize negative charges on the bacterial surface. Flow cytometry experiments confirmed that both peptides permeabilize the bacterial cell membrane but suggested slightly different mechanisms of action. Ctn(15-34) permeabilized the membrane immediately upon addition to the cells, whereas Ctn had a lag phase before inducing membrane damage and exhibited more complex cell-killing activity, probably because of two different modes of membrane permeabilization. Using surface plasmon resonance and leakage assays with model vesicles, we confirmed that Ctn(15-34) binds to and disrupts lipid membranes and also observed that Ctn(15-34) has a preference for vesicles that mimic bacterial or tumor cell membranes. Atomic force microscopy visualized the effect of these peptides on bacterial cells, and confocal microscopy confirmed their localization on the bacterial surface. Our studies shed light onto the antimicrobial mechanisms of Ctn and Ctn(15-34), suggesting Ctn(15-34) as a promising lead for development as an antibacterial/antitumor agent.This work was supported by Spanish Ministry of Economy and Competitiveness (MINECO) Grants SAF2011-24899 and AGL2014-52395-C2, by Fundação para a Ciência e a Tecnologia (FCT, Portugal) Grants PTDC/QEQ-MED/4412/2014, and by EU Marie Skłodowska-Curie Research and Innovation Staff Exchange (RISE) program Grant 644167, 2015–2019.info:eu-repo/semantics/publishedVersio

Strength plus Endurance Training and Individualized Diet Reduce Fat Mass in Overweight Subjects:A Randomized Clinical Trial

Studies with overweight people are a priority in order to observe the effect of the timing of intervention on pre-obesity people. The aim was to compare different physical activity programs plus an individualized hypocaloric diet on body composition in overweight subjects. A randomized controlled clinical trial was carried out in overweight adults with no history of relevant illness. Primary outcome was total fat mass (TFM). Participants were allocated into four activity programs with equal intensity and volume of exercise for 22 weeks: strength training (S), endurance training (E), strength + endurance training (SE), and 'adhering to physical activity recommendations' (C). Participants followed a diet with 25% less energy (50%-55% carbohydrates, 30%-35% fat) measured by accelerometer. Variables were assessed at baseline and at the end of the intervention. Body composition was measured by dual-energy X-ray absorptiometry. One hundred nineteen from 205 subjects were randomized in the four exercise groups (S = 30/E = 30/SE = 30/C = 29) and 84 participants (36 men/48 women) ended the intervention (S = 19/E = 25/SE = 22/C = 18). At the end of the experiment, all groups except C increased their total physical activity (S = 1159 ± 1740; E = 1625 ± 1790; SE = 1699 ± 2516; C = 724 ± 1979 MET-min/week). Using an ANOVA-test, improvements were observed in body weight (S = -4.6 ± 4.5; E = -6.6 ± 4.6; SE = -8.5 ± 2.8; C = -6.1 ± 5.6 kg, p = 0.059) and TFM (S = -4.24 ± 2.02; E = -4.74 ± 2.96; SE = -6.74 ± 3.27; C = -3.94 ± 4.18%; p < 0.05). The main conclusion was that there were no adverse events. Strength and endurance training with a balanced, individualized hypocaloric diet was the most effective at reducing weight loss and fat mass in overweight subjects. Trial registration: NCT01116856

Metabolomic profile of cancer stem cell-derived exosomes from patients with malignant melanoma

Malignant melanoma (MM) is the most aggressive and life-threatening form of skin cancer. It is characterized by an extraordinary metastasis capacity and chemotherapy resistance, mainly due to melanoma cancer stem cells (CSCs). To date, there are no suitable clinical diagnostic, prognostic or predictive biomarkers for this neoplasia. Therefore, there is an urgent need for new MM biomarkers that enable early diagnosis and effective disease monitoring. Exosomes represent a novel source of biomarkers since they can be easily isolated from different body fluids. In this work, a primary patient-derived MM cell line enriched in CSCs was characterized by assessing the expression of specific markers and their stem-like properties. Exosomes derived from CSCs and serums from patients with MM were characterized, and their metabolomic profile was analysed by highresolution mass spectrometry (HRMS) following an untargeted approach and applying univariate and multivariate statistical analyses. The aim of this study was to search potential biomarkers for the diagnosis of this disease. Our results showed significant metabolomic differences in exosomes derived from MM CSCs compared with those from differentiated tumour cells and also in serum-derived exosomes from patients with MM compared to those from healthy controls. Interestingly, we identified similarities between structural lipids differentially expressed in CSC-derived exosomes and those derived from patients with MM such as the glycerophosphocholine PC 16:0/0:0. To our knowledge, this is the first metabolomic-based study aimed at characterizing exosomes derived from melanoma CSCs and patients’ serum in order to identify potential biomarkers for MM diagnosis. We conclude that metabolomic characterization of CSC-derived exosomes sets an open door to the discovery of clinically useful biomarkers in this neoplasia.MICIU FPU15/03682 FPU15/02350Ministerio de Ciencia, Innovación y Universidades (MICIU) MAT2015-62644.C2.2.R RTI2018-101309-BC2Instituto de Salud Carlos III PIE16-00045Junta de Andalucía SOMM17/6109/UGR (UCE-PP2017-3)European Union (EU) SOMM17/6109/UGR (UCE-PP2017-3)Chair 'Doctors Galera-Requena in cancer stem cell research' CMC-CTS963Fundación MEDIN

Single-cell derived clones from human adipose stem cells present different immunomodulatory properties

Human adipose mesenchymal stem cells are a heterogeneous population, where cell cultures derived from single cell-expanded clones present varying degrees of differential plasticity. This work focuses on the immunomodulatory/anti-inflammatory properties of these cells. To this end, 5 single cell clones were isolated (generally called 1.X and 3.X) from 2 volunteers. Regarding the expression level of the lineage-characteristic surface antigens, clones 1.10 and 1.22 expressed the lowest amounts, while clones 3.10 and 3.5 expressed more CD105 than the rest and clone 1.7 expressed higher amounts of CD73 and CD44. Regarding cytokine secretion, all clones were capable of spontaneously releasing high levels of IL-6 and low to moderate levels of IL-8. These differences can be explained in part by the distinct methylation profile exhibited by the clones. Furthermore and after lipopolysaccharide stimulation, clone 3.X produced the highest amounts of pro-inflammatory cytokines such as IL-1β, while clones 1.10 and 1.22 highly expressed IL-4 and IL-5. In co-culture experiments, clones 1.X are altogether more potent inhibitors than clones 3.X for proliferation of total, CD3+T, CD4+T and CD8+T lymphocytes and NK cells. The results of this work indicates that adipose stem cell population is heterogeneous in cytokine production profile, and that isolation, characterization and selection of the appropriate cell clone is a more exact method for the possible treatment of different patients or pathologies.Instituto de Salud Carlos IIIFEDER (PS09/01093), Fundacion Salud 2000-Merck Serono, Generalitat Valenciana (PROMETEO/2012/007) and “Centro de Investigación Biomédica en Red de Fisiopatología de la Obesidad y Nutrición” CIBERobn (CB12/03/30038) to ER, and VALi+d (APOSTD/2012/021) from Generalitat Valenciana to MIA

The relative age effect on physical fitness in preschool children

The aim of the present study was to investigate the existence of a relative age effect (RAE) on physical fitness of preschoolers. Anthropometry and physical fitness were assessed in 3147 children (3–5 years old) using the PREFIT battery. Based on the birth year, participants were divided into 3year groups (3-, 4- and 5-years). Within each year group, 4quarter groups were created: quarter 1, preschoolers born from January to March; quarter 2, from April to June; quarter 3, from July to September; quarter 4, from October to December. The MANCOVA analysis revealed a main effect of year group (Wilks’ λ = 0.383; F10,5996 = 369.64; p < 0.001, ηp 2 = 0.381) and of quarter (Wilks’ λ = 0.874; F15,8276.6 = 27.67; p < 0.001; ηp 2 = 0.044) over the whole battery of tests. To the best of our knowledge, this is the first study to report the existence of RAE at the preschool stage. In general, performance improved as the relative age increased (i.e., those born in quarter 1 performed better than those in the other quarters). Individualization strategies should be addressed within the same academic year not only in elementary or secondary years but also in preschoolers