Search results for: MAPK

Authors: Carmela Nardelli, Laura Iaffaldano, Valentina Capobianco, Antonietta Tafuto, Maddalena Ferrigno, Angela Capone, Giuseppe Maria Maruotti, Maddalena Raia, Rosa Di Noto, Luigi Del Vecchio, Pasquale Martinelli, Lucio Pastore, Lucia Sacchetti

Abstract:

Maternal obesity and nutrient excess in utero increase the risk of future metabolic diseases in the adult life. The mechanisms underlying this process are probably based on genetic, epigenetic alterations and changes in foetal nutrient supply. In mammals, the placenta is the main interface between foetus and mother, it regulates intrauterine development, modulates adaptive responses to sub optimal in uterus conditions and it is also an important source of human amniotic mesenchymal stem cells (hA-MSCs). We previously highlighted a specific microRNA (miRNA) profiling in amnion from obese (Ob) pregnant women, here we compared the miRNA expression profile of hA-MSCs isolated from (Ob) and control (Co) women, aimed to search for any alterations in metabolic pathways that could predispose the new-born to the obese phenotype. Methods: We isolated, at delivery, hA-MSCs from amnion of 16 Ob- and 7 Co-women with pre-pregnancy body mass index (mean/SEM) 40.3/1.8 and 22.4/1.0 kg/m2, respectively. hA-MSCs were phenotyped by flow cytometry. Globally, 384 miRNAs were evaluated by the TaqMan Array Human MicroRNA Panel v 1.0 (Applied Biosystems). By the TargetScan program we selected the target genes of the miRNAs differently expressed in Ob- vs Co-hA-MSCs; further, by KEGG database, we selected the statistical significant biological pathways. Results: The immunophenotype characterization confirmed the mesenchymal origin of the isolated hA-MSCs. A large percentage of the tested miRNAs, about 61.4% (232/378), was expressed in hA-MSCs, whereas 38.6% (146/378) was not. Most of the expressed miRNAs (89.2%, 207/232) did not differ between Ob- and Co-hA-MSCs and were not further investigated. Conversely, 4.8% of miRNAs (11/232) was higher and 6.0% (14/232) was lower in Ob- vs Co-hA-MSCs. Interestingly, 7/232 miRNAs were obesity-specific, being expressed only in hA-MSCs isolated from obese women. Bioinformatics showed that these miRNAs significantly regulated (P<0.001) genes belonging to several metabolic pathways, i.e. MAPK signalling, actin cytoskeleton, focal adhesion, axon guidance, insulin signaling, etc. Conclusions: Our preliminary data highlight an altered miRNA profile in Ob- vs Co-hA-MSCs and suggest that an epigenetic miRNA-based mechanism of gene regulation could affect pathways involved in placental growth and function, thereby potentially increasing the newborn’s risk of metabolic diseases in the adult life.

Keywords: hA-MSCs, obesity, miRNA, biosystem

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Authors: Nouha Bouayed Abdelmoula, Rim Louati, Nawel Abdellaoui, Balkiss Abdelmoula, Oldez Kaabi, Walid Smaoui, Samir Aloulou

Abstract:

Background and Aims: Cardiofaciocutaneous syndrome (CFC) is an autosomal dominant disorder with the vast majority of cases arising by a new mutation of BRAF, MEK1, MEK2, or rarely, KRAS genes. Here, we report a rare Tunisian case of CFC syndrome for whom we identify SOS1 mutation. Methods: Genomic DNA was obtained from peripheral blood collected in an EDTA tube and extracted from leukocytes using the phenol/chloroform method according to standard protocols. High resolution melting (HRM) analysis for screening of mutations in the entire coding sequence of PTPN11 was conducted first. Then, HRM assays to look for hot spot mutations coding regions of the other genes of the RAS-MAPK pathway (RAt Sarcoma viral oncogene homolog Mitogen-Activated Protein Kinases Pathway): SOS1, SHOC2, KRAS, RAF1, KRAS, NRAS, CBL, BRAF, MEK1, MEK2, HRAS, and RIT1, were applied. Results: Heterozygous SOS1 point mutation clustered in exon 10, which encodes for the PH domain of SOS1, was identified: c.1655 G > A. The patient was a 9-year-old female born from a consanguineous couple. She exhibited pulmonic valvular stenosis as congenital heart disease. She had facial features and other malformations of Noonan syndrome, including macrocephaly, hypertelorism, ptosis, downslanting palpebral fissures, sparse eyebrows, a short and broad nose with upturned tip, low-set ears, high forehead commonly associated with bitemporal narrowing and prominent supraorbital ridges, short and/or webbed neck and short stature. However, the phenotype is also suggestive of CFC syndrome with the presence of more severe ectodermal abnormalities, including curly hair, keloid scars, hyperkeratotic skin, deep plantar creases, and delayed permanent dentition with agenesis of the right maxillary first molar. Moreover, the familial history of the patient revealed recurrent brain malignancies in the paternal family and epileptic disease in the maternal family. Conclusions: This case report of an overlapping RASopathy associated with SOS1 mutation and familial history of brain tumorigenesis is exceptional. The evidence suggests that RASopathies are truly cancer-prone syndromes, but the magnitude of the cancer risk and the types of cancer partially overlap.

Keywords: cardiofaciocutaneous syndrome, CFC, SOS1, brain cancer, germline mutation

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Authors: Ankan Roy, Niharika, Samir Kumar Patra

Abstract:

Anomalous nexus of complex topological assemblies and spatiotemporal epigenetic choreography at chromosomal territory may forms the most sophisticated regulatory layer of gene expression in cancer. Colon cancer is one of the leading malignant neoplasms of the lower gastrointestinal tract worldwide. There is still a paucity of information about the complex molecular mechanisms of colonic cancerogenesis. Bioinformatics prediction and analysis helps to identify essential genes and significant pathways for monitoring and conquering this deadly disease. The present study investigates and explores potential hub genes as biomarkers and effective therapeutic targets for colon cancer treatment. Colon cancer patient sample containing gene expression profile datasets, such as GSE44076, GSE20916, and GSE37364 were downloaded from Gene Expression Omnibus (GEO) database and thoroughly screened using the GEO2R tool and Funrich software to find out common 2 differentially expressed genes (DEGs). Other approaches, including Gene Ontology (GO) and KEGG pathway analysis, Protein-Protein Interaction (PPI) network construction and hub gene investigation, Overall Survival (OS) analysis, gene correlation analysis, methylation pattern analysis, and hub gene-Transcription factors regulatory network construction, were performed and validated using various bioinformatics tool. Initially, we identified 166 DEGs, including 68 up-regulated and 98 down-regulated genes. Up-regulated genes are mainly associated with the Cytokine-cytokine receptor interaction, IL17 signaling pathway, ECM-receptor interaction, Focal adhesion and PI3K-Akt pathway. Downregulated genes are enriched in metabolic pathways, retinol metabolism, Steroid hormone biosynthesis, and bile secretion. From the protein-protein interaction network, thirty hub genes with high connectivity are selected using the MCODE and cytoHubba plugin. Survival analysis, expression validation, correlation analysis, and methylation pattern analysis were further verified using TCGA data. Finally, we predicted COL1A1, COL1A2, COL4A1, SPP1, SPARC, and THBS2 as potential master regulators in colonic cancerogenesis. Moreover, our experimental data highlights that disruption of lipid raft and RAS/MAPK signaling cascade affects this gene hub at mRNA level. We identified COL1A1, COL1A2, COL4A1, SPP1, SPARC, and THBS2 as determinant hub genes in colon cancer progression. They can be considered as biomarkers for diagnosis and promising therapeutic targets in colon cancer treatment. Additionally, our experimental data advertise that signaling pathway act as connecting link between membrane hub and gene hub.

Keywords: hub genes, colon cancer, DNA methylation, epigenetic engineering, bioinformatic predictions

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Authors: Kuladip Jana

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Benzo(a)pyrene [B(a)P] is an environmental toxicant present mostly in cigarette smoke and car exhaust, is an aryl hydrocarbon receptor (AhR) ligand that exerts its toxic effects on both male and female reproductive systems. In this study, the effect of B(a)P at different doses (0.1, 0.25, 0.5, 1 and 5 mg /kg body weight) was studied on male reproductive system of rat. A significant decrease in cauda epididymal sperm count and motility along with the presence of sperm head abnormalities and altered epididymal and testicular histology were documented following B(a)P treatment. B(a)P treatment resulted apoptotic sperm cells as observed by TUNEL and Annexin V-PI assay with increased ROS, altered sperm mitochondrial membrane potential (ΔΨm) with a simultaneous decrease in the activity of antioxidant enzymes and GSH status. TUNEL positive apoptotic cells also observed in testis as well as isolated germ and Leydig cells following B(a)P exposure. Western Blot analysis revealed the activation of p38MAPK, cytosolic translocation of cytochrome-c, up-regulation of Bax and inducible nitric oxide synthase (iNOS) with cleavage of PARP and down-regulation of BCl2 in testis upon B(a)P treatment. The protein and mRNA levels of testicular key steroidogenesis regulatory proteins like StAR, cytochrome P450 IIA1 (CYPIIA1), 3β HSD, 17β HSD showed a significant decrease in a dose dependent manner while an increase in the expression of cytochrome P450 1A1 (CYP1A1), Aryl hydrocarbon Receptor (AhR), active caspase- 9 and caspase- 3 following B(a)P exposure. We conclude that exposure of benzo(a)pyrene caused testicular gamatogenic and steroidogenic disorders by induction of oxidative stress, inhibition of StAR and other steroidogenic enzymes along with activation of p38MAPK and initiated caspase-3 mediated germ and Leydig cell apoptosis.The possible protective role of naturally occurring phytochemicals against B(a)P induced testicular toxicity needs immediate consideration. Curcumin and resveratrol separately were found to protect against B(a)P induced germ cell apoptosis, and their combinatorial effect was more significant. Our present study in isolated testicular germ cell population from adult male Wistar rats, highlighted their synergistic protective effect against B(a)P induced germ cell apoptosis. Curcumin-resveratrol co-treatment decreased the expression of pro-apoptotic proteins like cleaved caspase 3,8,9, cleaved PARP, Apaf1, FasL, tBid. Curcumin-resveratrol co-treatment decreased Bax/Bcl2 ratio, mitochondria to cytosolic translocation of cytochrome c and activated the survival protein Akt. Curcumin-resveratrol decreased the expression of p53 dependent apoptotic genes like Fas, FasL, Bax, Bcl2, Apaf1.Curcumin-resveratrol co-treatment thus prevented B(a)P induced germ cell apoptosis. B(a)P induced testicular ROS generation and oxidative stress were significantly ameliorated with curcumin and resveratrol. Curcumin-resveratrol co-treatment prevented B(a)P induced nuclear translocation of AhR and CYP1A1 production. The combinatorial treatment significantly inhibited B(a)P induced ERK 1/2, p38 MAPK and JNK 1/2 activation. B(a)P treatment increased the expression of p53 and its phosphorylation (p53 ser 15). Curcumin-resveratrol co-treatment significantly decreased p53 level and its phosphorylation (p53 ser 15). The study concludes that curcumin-resveratrol synergistically modulated MAPKs and p53, prevented oxidative stress, regulated the expression of pro and anti-apoptotic proteins as well as the proteins involved in B(a)P metabolism thus protected germ cells from B(a)P induced apoptosis.

Keywords: benzo(a)pyrene, germ cell, apoptosis, oxidative stress, resveratrol, curcumin

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Authors: Aastha Arora, Vikas Bhuria, Saurabh Singh, Uma Pathak, Shweta Mathur, Puja P. Hazari, Rajat Sandhir, Ravi Soni, Anant N. Bhatt, Bilikere S. Dwarakanath

Abstract:

Radiotherapy is an effective curative and palliative option for patients with thoracic malignancies. However, lung injury, comprising of pneumonitis and fibrosis, remains a significant clin¬ical complication of thoracic radiation, thus making it a dose-limiting factor. Also, injury to the lung is often reported as part of multi-organ failure in victims of accidental radiation exposures. Radiation induced inflammatory response in the lung, characterized by leukocyte infiltration and vascular changes, is an important contributing factor for the injury. Therefore, countermeasure agents to attenuate radiation induced inflammatory response are considered as an important approach to prevent chronic lung damage. Although Amifostine, the widely used, FDA approved radio-protector, has been found to reduce the radiation induced pneumonitis during radiation therapy of non-small cell lung carcinoma, its application during mass and field exposure is limited due to associated toxicity and ineffectiveness with the oral administration. The amifostine analogue (DRDE-30) overcomes this limitation as it is orally effective in reducing the mortality of whole body irradiated mice. The current study was undertaken to investigate the potential of DRDE-30 to ameliorate radiation induced lung damage. DRDE-30 was administered intra-peritoneally, 30 minutes prior to 13.5 Gy thoracic (60Co-gamma) radiation in C57BL/6 mice. Broncheo- alveolar lavage fluid (BALF) and lung tissues were harvested at 12 and 24 weeks post irradiation for studying inflammatory and fibrotic markers. Lactate dehydrogenase (LDH) leakage, leukocyte count and protein content in BALF were used as parameters to evaluate lung vascular permeability. Inflammatory cell signaling (p38 phosphorylation) and anti-oxidant status (MnSOD and Catalase level) was assessed by Western blot, while X-ray CT scan, H & E staining and trichrome staining were done to study the lung architecture and collagen deposition. Irradiation of the lung increased the total protein content, LDH leakage and total leukocyte count in the BALF, reflecting endothelial barrier dysfunction. These disruptive effects were significantly abolished by DRDE-30, which appear to be linked to the DRDE-30 mediated abrogation of activation of the redox-sensitive pro- inflammatory signaling cascade, the MAPK pathway. Concurrent administration of DRDE-30 with radiation inhibited radiation-induced oxidative stress by strengthening the anti-oxidant defense system and abrogated p38 mitogen-activated protein kinase activation, which was associated with reduced vascular leak and macrophage recruitment to the lungs. Histopathological examination (by H & E staining) of the lung showed radiation-induced inflammation of the lungs, characterized by cellular infiltration, interstitial oedema, alveolar wall thickening, perivascular fibrosis and obstruction of alveolar spaces, which were all reduced by pre-administration of DRDE-30. Structural analysis with X-ray CT indicated lung architecture (linked to the degree of opacity) comparable to un-irradiated mice that correlated well with the lung morphology and reduced collagen deposition. Reduction in the radiation-induced inflammation and fibrosis brought about by DRDE-30 resulted in a profound increase in animal survival (72 % in the combination vs 24% with radiation) observed at the end of 24 weeks following irradiation. These findings establish the potential of the Amifostine analogue, DRDE-30, in reducing radiation induced pulmonary injury by attenuating the inflammatory and fibrotic responses.

Keywords: amifostine, fibrosis, inflammation, lung injury radiation

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