WBP2 promotes BTRC mRNA stability to drive migration and invasion in triple-negative breast cancer via NF-κB activation.
WW-domain-binding protein 2 (WBP2) is an oncogene that drives breast carcinogenesis through regulating Wnt, estrogen receptor (ER), and Hippo signaling. Recent studies have identified neoteric modes of action of WBP2 other than its widely recognized function as a transcriptional coactivator. Here, we identified a previously unexplored role of WBP2 in inflammatory signaling in breast cancer via an integrated proteogenomic analysis of The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA BRCA) dataset. WBP2 was shown to enhance the migration and invasion in triple-negative breast cancer (TNBC) cells especially under tumor necrosis factor alpha (TNF-α) stimulation. Molecularly, WBP2 potentiates TNF-α-induced nuclear factor kappa B (NF-κB) transcriptional activity and nuclear localization through aggrandizing ubiquitin-mediated proteasomal degradation of its upstream inhibitor, NF-κB inhibitor alpha (NFKBIA; also known as IκBα). We further demonstrate that WBP2 induces mRNA stability of beta-transducin repeat-containing E3 ubiquitin protein ligase (BTRC), which targets IκBα for ubiquitination and degradation. Disruption of IκBα rescued the impaired migratory and invasive phenotypes in WBP2-silenced cells, while loss of BTRC ameliorated WBP2-driven migration and invasion. Clinically, the WBP2-BTRC-IκBα signaling axis correlates with poorer prognosis in breast cancer patients. Our findings reveal a pivotal mechanism of WBP2 in modulating BTRC-IκBα-NF-κB pathway to promote TNBC aggressiveness.
Publication Date: 2021-07-02
Journal: Molecular oncology
IκBα targeting promotes oxidative stress-dependent cell death.
Oxidative stress is a hallmark of many cancers. The increment in reactive oxygen species (ROS), resulting from an increased mitochondrial respiration, is the major cause of oxidative stress. Cell fate is known to be intricately linked to the amount of ROS produced. The direct generation of ROS is also one of the mechanisms exploited by common anticancer therapies, such as chemotherapy.
We assessed the role of NFKBIA with various approaches, including in silico analyses, RNA-silencing and xenotransplantation. Western blot analyses, immunohistochemistry and RT-qPCR were used to detect the expression of specific proteins and genes. Immunoprecipitation and pull-down experiments were used to evaluate protein-protein interactions.
Here, by using an in silico approach, following the identification of NFKBIA (the gene encoding IκBα) amplification in various cancers, we described an inverse correlation between IκBα, oxidative metabolism, and ROS production in lung cancer. Furthermore, we showed that novel IκBα targeting compounds combined with cisplatin treatment promote an increase in ROS beyond the tolerated threshold, thus causing death by oxytosis.
NFKBIA amplification and IκBα overexpression identify a unique cancer subtype associated with specific expression profile and metabolic signatures. Through p65-NFKB regulation, IκBα overexpression favors metabolic rewiring of cancer cells and distinct susceptibility to cisplatin. Lastly, we have developed a novel approach to disrupt IκBα/p65 interaction, restoring p65-mediated apoptotic responses to cisplatin due to mitochondria deregulation and ROS-production.
Publication Date: 2021-04-18
Journal: Journal of experimental & clinical cancer research : CR
Low-Dose Coconut Oil Supplementation Induces Hypothalamic Inflammation, Behavioral Dysfunction, and Metabolic Damage in Healthy Mice.
Coconut oil (CO) diets remain controversial due to the possible association with metabolic disorder and obesity. This study investigates the metabolic effects of a low amount of CO supplementation.
Swiss male mice are assigned to be supplemented orally during 8 weeks with 300 µL of water for the control group (CV), 100 or 300 µL of CO (CO100 and CO300) and 100 or 300 µL of soybean oil (SO; SO100 and SO300). CO led to anxious behavior, increase in body weight gain, and adiposity. In the hypothalamus, CO and SO increase cytokines expression and pJNK, pNFKB, and TLR4 levels. Nevertheless, the adipose tissue presented increases macrophage infiltration, TNF-α and IL-6 after CO and SO consumption. IL-1B and CCL2 expression, pJNK and pNFKB levels increase only in CO300. In the hepatic tissue, CO increases TNF-α and chemokines expression. Neuronal cell line (mHypoA-2/29) exposed to serum from CO and SO mice shows increased NFKB migration to the nucleus, TNF-α, and NFKBia expression, but are prevented by inhibitor of TLR4 (TAK-242).
These results show that a low-dose CO changes the behavioral pattern, induces inflammatory pathway activation, TLR4 expression in healthy mice, and stimulates the pro-inflammatory response through a TLR4-mediated mechanism.
Publication Date: 2021-03-03
Journal: Molecular nutrition & food research
Association of -94 ATTG insertion/deletion NFkB1 and c.*126G>A NFkBIA genetic polymorphisms with oxidative and nitrosative stress biomarkers in Brazilian subjects with Parkinson's Disease.
Parkinson's disease (PD) is a complex neurodegenerative disorder, resulting dopaminergic neuronal cell death in the substantia nigra. The disease is characterized by major motor impairment, being bradykinesia, rest tremor, rigidity and loss of postural reflexes the most common, while autonomic dysfunctions, sleep disturbances and psychiatric disorders are some of the wide range of non-motor symptoms. Several processes have been identified to be associated with disease development, such as mitochondrial dysfunction, oxidative/nitrosative stress and neuroinflammation. NF-κB is an important transcription factor that regulates several inflammatory elements and pathways, and polymorphisms on NFKB1 and NFKBIA genes can potentially influence redox balance towards a pro-oxidative frame, modulating disease progression. Evaluation of these polymorphisms in the redox status of PD subjects could provide new insights on the pathogenesis of this disorder. The study aimed to test associations of -94 in./del ATTG NFKB1 (rs28362491) and c.*126G > A NFKBIA (rs696) polymorphisms with PD development, and to test the influence of both polymorphisms on oxidative/nitrosative stress (OS/NS) parameters. A total of 110 Brazilian individuals were enrolled, being 55 subjects recruited from University Hospital of Londrina as the PD group, and 55 subjects matched for age, sex and ethnicity composed the healthy control (HC) group. NFkB1 and NFkBIA polymorphisms were genotyped by PCR-RFLP. Lipid hydroperoxides (LOOH), nitric oxide metabolites (NOx), advanced oxidation protein products (AOPP), sulfhydryl groups (SH), total radical trapping antioxidant parameter (TRAP) and paraoxonase-1 activity (PON-1) were assessed. Despite no association of polymorphisms on disease development was observed, in PD subjects the NFKB1 del/del genotype was associated with higher levels of LOOH, while NFkBIA GA and AA genotypes were associated with higher NOx levels, suggesting that NFkB plays a role in PD susceptbility. In conclusion, the prospect of genetic polymorphisms of elements involved in inflammation and OS/NS might be a new approach to unravel PD etiology.
Publication Date: 2020-11-09
Journal: Neuroscience letters
Progesterone receptor membrane component 1 leads to erlotinib resistance, initiating crosstalk of Wnt/β-catenin and NF-κB pathways, in lung adenocarcinoma cells.
In non-small-cell lung cancer, mutation of epidermal growth factor receptor (EGFR) stimulates cell proliferation and survival. EGFR tyrosine kinase inhibitors (EGFR-TKIs) such as erlotinib are used as first-line therapy with drastic and immediate effectiveness. However, the disease eventually progresses in most cases within a few years due to the development of drug resistance. Here, we explored the role of progesterone membrane component 1 (PGRMC1) in acquired resistance to erlotinib and addressed the molecular mechanism of EGFR-TKI resistance induced by PGRMC1. The erlotinib-sensitive cell line PC9 (derived from non-small-cell lung cancer) and the erlotinib-resistant cell line PC9/ER were used. In proteomic and immunoblotting analyses, the PGRMC1 level was higher in PC9/ER cells than in PC9 cells. WST-8 assay revealed that inhibition of PGRMC1 by siRNA or AG-205, which alters the spectroscopic properties of the PGRMC1-heme complex, in PC9/ER cells increased the sensitivity to erlotinib, and overexpression of PGRMC1 in PC9 cells reduced their susceptibility to erlotinib. In the presence of erlotinib, immunoprecipitation assay showed that AG-205 suppressed the interaction between EGFR and PGRMC1 in PC9/ER cells. AG-205 decreased the expression of β-catenin, accompanied by up-regulation of IκBα (also known as NFKBIA). Furthermore, AG-205 reduced the expression of β-TrCP (also known as BTRC), suggesting that PGRMC1 enhanced the crosstalk between NF-κB (also known as NFKB) signaling and Wnt/β-catenin signaling in an erlotinib-dependent manner. Finally, treatment with the Wnt/β-catenin inhibitor XAV939 enhanced the sensitivity of PC9/ER cells to erlotinib. These results suggest that PGRMC1 conferred resistance to erlotinib through binding with EGFR in PC9/ER cells, initiating crosstalk between the Wnt/β-catenin and NF-κB pathways.
Publication Date: 2020-03-18
Journal: Scientific reports
Polymorphisms in the NFkB, TNF-alpha, IL-1beta, and IL-18 pathways are associated with response to anti-TNF therapy in Danish patients with inflammatory bowel disease.
Anti-tumor necrosis factor-α (TNF-α) is used for the treatment of severe cases of IBD, including Crohn's disease (CD) and ulcerative colitis (UC). However, one-third of the patients do not respond to the treatment. We have previously investigated whether single nucleotide polymorphisms (SNPs) in genes involved in inflammation were associated with response to anti-TNF therapy among patients with CD or UC.
A new cohort of patients was established for replication of the previous findings and to identify new SNPs associated with anti-TNF response.
Fifty-three SNPs assessed previously in cohort 1 (482 CD and 256 UC patients) were genotyped in cohort 2 (587 CD and 458 UC patients). The results were analysed using logistic regression (adjusted for age and gender).
Ten SNPs were associated with anti-TNF response either among patients with CD (TNFRSF1A(rs4149570) (OR: 1.92, 95% CI: 1.02-3.60, P = 0.04), IL18(rs187238) (OR: 1.35, 95% CI: 1.00-1.82, P = 0.05), and JAK2(rs12343867) (OR: 1.35, 95% CI: 1.02-1.78, P = 0.03)), UC (TLR2(rs11938228) (OR: 0.55, 95% CI: 0.33-0.92, P = 0.02), TLR4(rs5030728) (OR: 2.23, 95% CI: 1.24-4.01, P = 0.01) and (rs1554973) (OR: 0.49, 95% CI: 0.27-0.90, P = 0.02), NFKBIA(rs696) (OR: 1.45, 95% CI: 1.06-2.00, P = 0.02), and NLRP3(rs4612666) (OR: 0.63, 95% CI: 0.44-0.91, P = 0.01)) or in the combined cohort of patient with CD and UC (IBD) (TLR4(rs5030728) (OR: 1.46, 95% CI: 1.01-2.11, P = 0.04) and (rs1554973)(OR: 0.80, 95% CI: 0.65-0.98, P = 0.03), NFKBIA(rs696) (OR: 1.25, 95% CI: 1.01-1.54, P = 0.04), NLRP3(rs4612666) (OR: 0.73, 95% CI: 0.57-0.95, P = 0.02), IL1RN(rs4251961) (OR: 0.81, 95% CI: 0.66-1.00, P = 0.05), IL18(rs1946518) (OR: 1.24, 95% CI: 1.01-1.53, P = 0.04), and JAK2(rs12343867) (OR: 1.24, 95% CI: 1.01-1.53, P = 0.04)).
The results support that polymorphisms in genes involved in the regulation of the NFκB pathway (TLR2, TLR4, and NFKBIA), the TNF-α signalling pathway (TNFRSF1A), and other cytokine pathways (NLRP3, IL1RN, IL18, and JAK2) were associated with response to anti-TNF therapy. Our multi-SNP model predicted response rate of more than 82% (in 9% of the CD patients) and 75% (in 15% of the UC patients), compared to 71% and 64% in all CD and UC patients, respectively. More studies are warranted to predict response for use in the clinic.
Publication Date: 2019-02-28
Journal: Alimentary pharmacology & therapeutics
TRIM59 regulates autophagy through modulating both the transcription and the ubiquitination of BECN1.
Macroautophagy/autophagy is a multistep cellular process that sequesters cytoplasmic components for lysosomal degradation. BECN1/Beclin1 is a central protein that assembles cofactors for the formation of a BECN1-PIK3C3-PIK3R4 complex to trigger the autophagy protein cascade. Discovering the regulators of BECN1 is important for understanding the mechanism of autophagy induction. Here, we demonstrate that TRIM59, a tripartite motif protein, plays an important role in autophagy regulation in non-small cell lung cancer (NSCLC). On the one hand, TRIM59 regulates the transcription of BECN1 through negatively modulating the NFKB pathway. On the other hand, TRIM59 regulates TRAF6 induced K63-linked ubiquitination of BECN1, thus affecting the formation of the BECN1-PIK3C3 complex. We further demonstrate that TRIM59 can mediate K48-linked ubiquitination of TRAF6 and promote the proteasomal degradation of TRAF6. Taken together, our findings reveal novel dual roles for TRIM59 in autophagy regulation by affecting both the transcription and the ubiquitination of BECN1. Abbreviations: ACTB: actin beta; BECN1: beclin 1; CHX: cycloheximide; CQ: chloroquine; GFP: green fluorescent protein; HA: haemagglutinin tag; His: polyhistidine tag; LC3B: microtubule associated protein 1 light chain 3 beta; NFKB: nuclear factor kappa B; NFKBIA: NFKB inhibitor alpha; NSCLC: non-small cell lung cancer; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; RELA: RELA proto-oncogene, NF-kB subunit; SQSTM1: sequestosome 1; tGFP: Turbo green fluorescent protein; TRAF6: TNF receptor associated factor 6; TRIM59: tripartite motif containing 59; B: ubiquitin.
Publication Date: 2018-09-21
Single nucleotide polymorphism rs696 in miR449a binding site of NFKBIA gene is correlated with risk of colorectal cancer.
In present study we have elucidated the role of 2758 A>G (rs696), in the recognition site of miR449a in the 3' UTR of NFKB inhibitor alpha (NFKBIA) gene, in development of sporadic colorectal cancer.
Colorectal cancer (CRC) is rated as second cause of cancer death. Genetic determinants are considered as driving forces in development of sporadic CRC. Single nucleotide polymorphisms (SNPs), are attributed as the main genetic factor in cancers susceptibility. MicroRNAs, are key players in post-translational gene regulation by binding to their specific recognition sequences located at 3' untranslated region (UTR) of mRNAs.
A case-control study using 143 CRC patients and 137 noncancerous counterparts were undertaken in order to determine rs696 genotypes using polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) method.
There was a significant difference for the genotype frequencies of rs696 between patients and controls. The frequencies of GG, AG, AA genotypes in the control group were 38.7, 45.3, and 16.1 %, respectively, and the genotype frequencies in case group were 19.6, 40.6, and 39.9 %, respectively.
Our results suggest significant correlation between rs696 polymorphism and colorectal cancer risk.
Publication Date: 2018-03-23
Journal: Gastroenterology and hepatology from bed to bench
Increased mitochondrial fission promotes autophagy and hepatocellular carcinoma cell survival through the ROS-modulated coordinated regulation of the NFKB and TP53 pathways.
Mitochondrial morphology is dynamically remodeled by fusion and fission in cells, and dysregulation of this process is closely implicated in tumorigenesis. However, the mechanism by which mitochondrial dynamics influence cancer cell survival is considerably less clear, especially in hepatocellular carcinoma (HCC). In this study, we systematically investigated the alteration of mitochondrial dynamics and its functional role in the regulation of autophagy and HCC cell survival. Furthermore, the underlying molecular mechanisms and therapeutic application were explored in depth. Mitochondrial fission was frequently upregulated in HCC tissues mainly due to an elevated expression ratio of DNM1L to MFN1, which significantly contributed to poor prognosis of HCC patients. Increased mitochondrial fission by forced expression of DNM1L or knockdown of MFN1 promoted the survival of HCC cells both in vitro and in vivo mainly by facilitating autophagy and inhibiting mitochondria-dependent apoptosis. We further demonstrated that the survival-promoting role of increased mitochondrial fission was mediated via elevated ROS production and subsequent activation of AKT, which facilitated MDM2-mediated TP53 degradation, and NFKBIA- and IKK-mediated transcriptional activity of NFKB in HCC cells. Also, a crosstalk between TP53 and NFKB pathways was involved in the regulation of mitochondrial fission-mediated cell survival. Moreover, treatment with mitochondrial division inhibitor-1 significantly suppressed tumor growth in an in vivo xenograft nude mice model. Our findings demonstrate that increased mitochondrial fission plays a critical role in regulation of HCC cell survival, which provides a strong evidence for this process as drug target in HCC treatment.
Publication Date: 2016-04-29
Maternal consumption of organic trace minerals alters calf systemic and neutrophil mRNA and microRNA indicators of inflammation and oxidative stress.
Organic trace mineral (ORG) supplementation to dairy cows in substitution of sulfate (INO) sources has been associated with improvement in immune function during stressful states such as the peripartal period. However, the effect of supplemental ORG during pregnancy on the neonatal calf is unknown. Therefore, our aim was to investigate the effects of ORG supplementation during late pregnancy on the immune system and growth of the neonatal calf. Of specific interest was the evaluation of inflammation-related microRNA (miRNA) and target gene expression in blood neutrophils as indicators of possible nutritional programming. Forty multiparous cows were supplemented for 30d prepartum with 40 mg/kg of Zn, 20 mg/kg of Mn, 5 mg/kg of Cu, and 1mg/kg of Co from either organic (ORG) or sulfate (INO) sources (total diet contained supplemental 75 mg/kg of Zn, 65 mg/kg of Mn, 11 mg/kg of Cu, and 1 mg/kg of Co, and additional Zn, Mn, and Co provided by sulfates), and a subset of calves (n=8/treatment) was used for blood immunometabolic marker and polymorphonuclear leukocyte (PMNL) gene and miRNA expression analyses. Samples were collected at birth (before colostrum feeding), 1d (24 h after colostrum intake), and 7 and 21d of age. Data were analyzed as a factorial design with the PROC MIXED procedure of SAS. No differences were detected in BW, but maternal ORG tended to increase calf withers height. Calves from INO-fed cows had greater concentrations of blood glucose, GOT, paraoxonase, myeloperoxidase, and reactive oxygen metabolites. Antioxidant capacity also was greater in INO calves. The PMNL expression of toll-like receptor pathway genes indicated a pro-inflammatory state in INO calves, with greater expression of the inflammatory mediators MYD88, IRAK1, TRAF6, NFKB, and NFKBIA. The lower expression of miR-155 and miR-125b in ORG calves indicated the potential for maternal organic trace minerals in regulating the PMNL inflammatory response at least via alterations in mRNA and miRNA expression. Overall, these results indicate that maternal nutrition with organic trace minerals could alter the neonatal innate immune response at least in part via changes in gene and miRNA expression. Further studies involving inflammatory challenges during the neonatal period should be performed to determine the functional benefit of maternal organic trace minerals on the neonatal immune response.
Publication Date: 2015-09-01
Journal: Journal of dairy science
Integrated genomic approaches identify major pathways and upstream regulators in late onset Alzheimer's disease.
Previous studies have evaluated gene expression in Alzheimer's disease (AD) brains to identify mechanistic processes, but have been limited by the size of the datasets studied. Here we have implemented a novel meta-analysis approach to identify differentially expressed genes (DEGs) in published datasets comprising 450 late onset AD (LOAD) brains and 212 controls. We found 3124 DEGs, many of which were highly correlated with Braak stage and cerebral atrophy. Pathway Analysis revealed the most perturbed pathways to be (a) nitric oxide and reactive oxygen species in macrophages (NOROS), (b) NFkB and (c) mitochondrial dysfunction. NOROS was also up-regulated, and mitochondrial dysfunction down-regulated, in healthy ageing subjects. Upstream regulator analysis predicted the TLR4 ligands, STAT3 and NFKBIA, for activated pathways and RICTOR for mitochondrial genes. Protein-protein interaction network analysis emphasised the role of NFKB; identified a key interaction of CLU with complement; and linked TYROBP, TREM2 and DOK3 to modulation of LPS signalling through TLR4 and to phosphatidylinositol metabolism. We suggest that NEUROD6, ZCCHC17, PPEF1 and MANBAL are potentially implicated in LOAD, with predicted links to calcium signalling and protein mannosylation. Our study demonstrates a highly injurious combination of TLR4-mediated NFKB signalling, NOROS inflammatory pathway activation, and mitochondrial dysfunction in LOAD.
Publication Date: 2015-07-24
Journal: Scientific reports
Dexamethasone acutely down-regulates genes involved in steroidogenesis in stallion testes.
In rodents, livestock and primate species, a single dose of the synthetic glucocorticoid dexamethasone acutely lowers testosterone biosynthesis. To determine the mechanism of decreased testosterone biosynthesis, stallions were treated with 0.1mg/kg dexamethasone 12h prior to castration. Dexamethasone decreased serum concentrations of testosterone by 60% compared to saline-treated control stallions. Transcriptome analyses (microarrays, northern blots and quantitative PCR) of testes discovered that dexamethasone treatment decreased concentrations of glucocorticoid receptor alpha (NR3C1), alpha actinin 4 (ACTN4), luteinizing hormone receptor (LHCGR), squalene epoxidase (SQLE), 24-dehydrocholesterol reductase (DHCR24), glutathione S-transferase A3 (GSTA3) and aromatase (CYP19A1) mRNAs. Dexamethasone increased concentrations of NFkB inhibitor A (NFKBIA) mRNA in testes. SQLE, DHCR24 and GSTA3 mRNAs were predominantly expressed by Leydig cells. In man and livestock, the GSTA3 protein provides a major 3-ketosteroid isomerase activity: conversion of Δ(5)-androstenedione to Δ(4)-androstenedione, the immediate precursor of testosterone. Consistent with the decrease in GSTA3 mRNA, dexamethasone decreased the 3-ketosteroid isomerase activity in testicular extracts. In conclusion, dexamethasone acutely decreased the expression of genes involved in hormone signaling (NR3C1, ACTN4 and LHCGR), cholesterol synthesis (SQLE and DHCR24) and steroidogenesis (GSTA3 and CYP19A1) along with testosterone production. This is the first report of dexamethasone down-regulating expression of the GSTA3 gene and a very late step in testosterone biosynthesis. Elucidation of the molecular mechanisms involved may lead to new approaches to modulate androgen regulation of the physiology of humans and livestock in health and disease.
Publication Date: 2014-07-11
Journal: The Journal of steroid biochemistry and molecular biology
Polymorphism of the NFKB1 affects the serum inflammatory levels of IL-6 in Hashimoto thyroiditis in a Turkish population.
Hashimoto thyroiditis (HT) is a chronic inflammatory autoimmune disease of thyroid gland affected by interaction of multiple genes and various cytokines. Variants in the genes coding for the NFKB and IKB proteins can be potentially involved in the development of the inflammatory diseases. NFKB, a key transcription factor of the regulation of immune responses, is interesting candidate for association studies about autoimmune disorder. The aim of the present study was to investigate the relationship between NFKB1 and NFKBIA (NFKB1 inhibitor gene) polymorphisms, and the risk of HT in a Turkish Population in the context of IL-6 serum levels which may contribute to susceptibility to the disease. We analyzed the distribution of NFKB1-94ins/del ATTG and NFKBIA 3'UTR A→G polymorphisms using PCR-RFLP method and IL-6 serum levels using ELISA method in 120 HT patients and 190 healthy controls in Turkish population. Although, there was no statistical significant difference in distribution of the genotypes and alleles of NFKB1-94ins/del ATTG or NFKBIA 3'UTR A→G polymorphisms in patients and control subjects as single, ins/ins/GG combined genotype had protective effect on the disease when compared to ins/ins/AG combined genotype as combined genotypes of both polymorphisms. In addition to this finding, IL-6 serum levels in HT patients with del/del genotype were significantly higher than in patients with del/ins genotype (p<0.001). According to the combined genotype analysis of NFKB1-94ins/del ATTG and NFKBIA 3'UTR A→G polymorphisms, IL-6 levels were also higher in patients with del/del genotype when at least one G allele existing (p=0.007). Therefore, our findings suggest that the functional promoter NFKB1-94ins/del ATTG polymorphism was significantly associated with population HT disease through acting by directly modulating IL-6 serum levels.
Publication Date: 2014-04-08
Transcriptomic analysis of the effect of ifosfamide on MDCK cells cultivated in microfluidic biochips.
We investigated the behavior of renal cells cultivated in microfluidic biochips when exposed to 50 μM of ifosfamide, an antineoplastic drug treatment. The microarray analysis revealed that ifosfamide had any effect in Petri conditions. The microfluidic biochips induced an early inflammatory response in the MDCK in the untreated cells. This was attributed to cells adapting to the dynamics and micro environment created by the biochips. This led to modulations in the mitochondria dysfunction pathway, the Nrf-2 and oxidative stress pathways and some related cancer genes. When exposed to 50 μM of ifosfamide, we detected a modulation of the pathways related to the cancer and inflammation in the MDCK cultivated in the biochips via modulation of the ATM, p53, MAP Kinase, Nrf-2 and NFKB signaling. In addition, the genes identified and related proteins affected by the ifosfamide treatment in the biochips such as TXNRD1, HSP40 (DNAJB4 and DNAJB9), HSP70 (HSPA9), p21 (CDKN1A), TP53, IKBalpha (NFKBIA) are reported to be the molecular targets in cancer therapy. We also found that the integrin pathway was perturbed with the ifosfamide treatment. Finally, the MYC proto-oncogene appeared to be a potential bridge between the integrin signaling and the anti-inflammatory response.
Publication Date: 2012-05-15
Complement C5A regulates prolabor mediators in human placenta.
Human preterm and term parturition is associated with inflammatory cascades in the uteroplacental unit. Activation of the complement cascade releases potent proinflammatory mediators, including the anaphylatoxin C5a, which exerts its biological effects through its receptors, C5AR (also known as CD88) and C5L2, official symbol GPR77. To date, there are few data available on the role of C5a and CD88 in human pregnancy, so the aim of this study was to determine the effect of C5a and CD88 on some key inflammatory pathways involved in human parturition. Placental tissue samples were obtained from normal pregnancies at the time of Cesarean section. Human placental and fetal membranes were incubated in the absence (basal control) or presence of 0.5 μg/ml (~60 nM) human recombinant C5a for 24 h. Concentrations of proinflammatory cytokines, prostaglandins, and 8-isoprostane (a marker of oxidative stress) were quantified by ELISA and secretory matrix metalloproteinases (MMPs) activity by zymography. NFKB DNA binding activity and NFKBIA (IkappaB-alpha; inhibitor of NFKB) protein degradation were analyzed by ELISA and Western blotting, respectively. In the presence of C5a, proinflammatory cytokines (IL6 and IL8), cyclooxygenase (COX)-2; official symbol PTGS2) expression, and subsequent prostaglandin (PGE(2) and PGF(2alpha)), MMP9 enzyme production, and NFKB DNA activation were all significantly increased. The C5a-induced prolabor responses were significantly reduced by treatment with the selective CD88 antagonist PMX53 and the NFKB inhibitor BAY 11-7082. We conclude that C5a upregulates prolabor mediators in human gestational tissues via CD88-mediated NFKB activation.
Publication Date: 2012-03-24
Journal: Biology of reproduction
Activation of cyclic AMP signaling leads to different pathway alterations in lesions of the adrenal cortex caused by germline PRKAR1A defects versus those due to somatic GNAS mutations.
The overwhelming majority of benign lesions of the adrenal cortex leading to Cushing syndrome are linked to one or another abnormality of the cAMP or protein kinase pathway. PRKAR1A-inactivating mutations are responsible for primary pigmented nodular adrenocortical disease, whereas somatic GNAS activating mutations cause macronodular disease in the context of McCune-Albright syndrome, ACTH-independent macronodular hyperplasia, and, rarely, cortisol-producing adenomas.
The whole-genome expression profile (WGEP) of normal (pooled) adrenals, PRKAR1A- (3) and GNAS-mutant (3) was studied. Quantitative RT-PCR and Western blot were used to validate WGEP findings.
MAPK and p53 signaling pathways were highly overexpressed in all lesions against normal tissue. GNAS-mutant tissues were significantly enriched for extracellular matrix receptor interaction and focal adhesion pathways when compared with PRKAR1A-mutant (fold enrichment 3.5, P < 0.0001 and 2.1, P < 0.002, respectively). NFKB, NFKBIA, and TNFRSF1A were higher in GNAS-mutant tumors (P < 0.05). Genes related to the Wnt signaling pathway (CCND1, CTNNB1, LEF1, LRP5, WISP1, and WNT3) were overexpressed in PRKAR1A-mutant lesions.
WGEP analysis revealed that not all cAMP activation is the same: adrenal lesions harboring PRKAR1A or GNAS mutations share the downstream activation of certain oncogenic signals (such as MAPK and some cell cycle genes) but differ substantially in their effects on others.
Publication Date: 2012-01-20
Journal: The Journal of clinical endocrinology and metabolism
Molecular basis for defining the pineal gland and pinealocytes as targets for tumor necrosis factor.
The pineal gland, the gland that translates darkness into an endocrine signal by releasing melatonin at night, is now considered a key player in the mounting of an innate immune response. Tumor necrosis factor (TNF), the first pro-inflammatory cytokine to be released by an inflammatory response, suppresses the translation of the key enzyme of melatonin synthesis (arylalkylamine-N-acetyltransferase, Aanat). Here, we show that TNF receptors of the subtype 1 (TNF-R1) are expressed by astrocytes, microglia, and pinealocytes. We also show that the TNF signaling reduces the level of inhibitory nuclear factor kappa B protein subtype A (NFKBIA), leading to the nuclear translocation of two NFKB dimers, p50/p50, and p50/RelA. The lack of a transactivating domain in the p50/p50 dimer suggests that this dimer is responsible for the repression of Aanat transcription. Meanwhile, p50/RelA promotes the expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide, which inhibits adrenergically induced melatonin production. Together, these data provide a mechanistic basis for considering pinealocytes a target of TNF and reinforce the idea that the suppression of pineal melatonin is one of the mechanisms involved in mounting an innate immune response.
Publication Date: 2011-01-01
Journal: Frontiers in endocrinology
Activation of the transcription factor, nuclear factor kappa-B, during the estrous cycle and early pregnancy in the pig.
Establishment and maintenance of pregnancy in the pig involves intricate communication between the developing conceptuses and the maternal endometrium. This process occurs during trophoblast elongation which is spaciotemporally associated with conceptus synthesis and release of IL1B concomitant with pregnancy-specific endometrial up-regulation of IL-1 receptors, providing the potential for activation of the transcription factor, NFKB. The objective of the current investigation was to determine changes in expression and cellular localization of NFKB and associated factors during the estrous cycle and early pregnancy in the pig. In situ hybridization was used to localize changes in PGR, ESR1, and TNFRSF11A during the peri-implantation period. Quantitative RT-PCR was utilized to demonstrate gene expression changes for NFKB1, RELA, TNFRSF11A, TLR4, NFKBIA and NFKBIB. Transcription factor ELISA demonstrated an overall increase in RELA during the peri-implantation period in both cyclic and pregnant gilts. While the presence of TNFSF11A and TLR4 were both detected, TLR4 expression changes were temporally associated with NFKB expression and activation. Collectively, these data demonstrate that NFKB activation may occur during the period of uterine receptivity in both the cyclic and pregnant endometrium.
Publication Date: 2010-04-30
Journal: Reproductive biology and endocrinology : RB&E
Nuclear factor kappaB pathway and interleukin-6 are affected in eutopic endometrium of women with endometriosis.
In order to investigate the role of the nuclear factor kappaB (NFKB) pathway on gene expression in the eutopic endometrium in endometriosis, and in particular of interleukin-6 (IL6), we evaluated RELA, IkappaB kinase (CHUK), NFKBIA and IL6 expressions and NFKB DNA binding in eutopic endometrium from women with endometriosis. Eutopic endometrium was obtained from 37 women with endometriosis and 42 fertile women during laparoscopy. We analysed RELA, CHUK, NFKBIA and IL6 mRNA levels (RT-PCR); RELA, CHUK and NFKBIA proteins and p-NFKBIA/NFKBIA ratio (western blot); and NFKB binding (DNA shift assay) and IL6 concentration (ELISA) in endometrial explants. Our results indicate that mRNA and cytoplasmic proteins of RELA and CHUK exhibit constant levels in normal endometrium during the menstrual cycle. A dramatic increase (P<0.05) in NFKBIA mRNA expression, RELA nuclear presence and the mRNA and the protein of IL6 during late secretory phase was also observed in this tissue. By contrast, in eutopic endometrium from endometriosis patients, a decrease (P<0.05) in IL6 mRNA and protein (61%), NFKBIA mRNA (46%), p-NFKBIA/NFKBIA ratio (42%), RELA nuclear stromal (68%) and CHUK (48%) proteins were found exclusively during the late secretory phase compared with normal endometrium. In conclusion, the canonical activation of NFKB pathway is deregulated and may have reduced transcriptional function affecting NFKBIA and IL6 expression, genes related local proinflammatory processes. These molecular alterations observed during the late secretory phase in eutopic endometrium from endometriosis patients constitute a NFKB system dysfunction, suggesting that NFKB could be an important factor in endometriosis aetiology.
Publication Date: 2009-01-09
Journal: Reproduction (Cambridge, England)
TRAIL and Taurolidine induce apoptosis and decrease proliferation in human fibrosarcoma.
Disseminated soft tissue sarcoma still represents a therapeutic dilemma because effective cytostatics are missing. Therefore we tested TRAIL and Tarolidine (TRD), two substances with apoptogenic properties on human fibrosarcoma (HT1080).
Viability, apoptosis and necrosis were visualized by TUNEL-Assay and quantitated by FACS analysis (Propidiumiodide/AnnexinV staining). Gene expression was analysed by RNA-Microarray and the results validated for selected genes by rtPCR. Protein level changes were documented by Western Blot analysis. NFKB activity was analysed by ELISA and proliferation assays (BrdU) were performed.
The single substances TRAIL and TRD induced apoptotic cell death and decreased proliferation in HT1080 cells significantly. Gene expression of several genes related to apoptotic pathways (TRAIL: ARHGDIA, NFKBIA, TNFAIP3; TRD: HSPA1A/B, NFKBIA, GADD45A, SGK, JUN, MAP3K14) was changed. The combination of TRD and TRAIL significantly increased apoptotic cell death compared to the single substances and lead to expression changes in a variety of genes (HSPA1A/B, NFKBIA, PPP1R15A, GADD45A, AXL, SGK, DUSP1, JUN, IRF1, MYC, BAG5, BIRC3). NFKB activity assay revealed an antipodal regulation of the several subunits of NFKB by TRD and TRD+TRAIL compared to TRAIL alone.
TRD and TRAIL are effective to induce apoptosis and decrease proliferation in human fibrosarcoma. A variety of genes seems to be involved, pointing to the NFKB pathway as key regulator in TRD/TRAIL-mediated apoptosis.
Publication Date: 2008-12-17
Journal: Journal of experimental & clinical cancer research : CR