P65 Subunit Research Articles

Role of p65 NF-κB, caspase-3 activities and VEGF gene polymorphisms on the development of preterm labor in women with placental dysfunction

The objective: to study the activity of the p65 subunit of the nuclear factor – NF-κB and effector caspase-3, as well as the presence of a single nucleotide polymorphism of the VEGF gene (936C/T) in pregnant women with placental disorders and various clinical types of the course of premature labor (with premature rupture of the membranes and without it).Materials and methods. At the clinical base of the Department of Obstetrics and Gynecology No. 1 of O. O. Bogomolets National Medical University, which is located in the non-commercial enterprise “Perinatal Center of Kyiv”, during 2019–2022, a case-control study was conducted with the involvement of 90 pregnant women: 60 women of the main group with placental disorders and the development of spontaneous premature labor in the period of 24–34 weeks of gestation (I group – 30 women with premature labor and premature rupture of membranes, II group – 30 women with premature rupture of the membranes) and 30 women of control group (CG) with term normal delivery and the physiological course of the pregnancy.In pregnant women, the number of total and activated fractions of the p65 subunit of nuclear factor NF-κB and caspase-3 was determined by enzyme-linked immunosorbent assay (ELISA) in placenta lysates with further calculation of their activity based on these data, as well as the presence of a single nucleotide polymorphism of the VEGF gene (936C/T) using the polymerase chain reaction.Results. In women with placental disorders and preterm birth a significantly higher values of fraction content and activity of p65 NF-κB and caspase-3 were found, with some peculiarities within the groups compared to the control group. Women of the I group were characterized by a higher activity of p65-subunit of nuclear factor (I group — 61.6 % with 95 % CI 59.7–64.2; II group — 33.8 pg/ml with 95 % CI 31.2-35.2; CG — 27.3 pg/ml, 95 % CI 26.4–28.6; p<0.05). Pregnant women of the II group had higher values of caspase-3 activity (II group — 59.2 % with 95 % CI 57.4–59.8, I group — 39.5 % with 95 % CI 38.5–40.5, CG — 31.2 %, 95 % CI 30.4–31.9; p<0.01). It was established that the presence of the T allele at position 936 of VEGF gene polymorphism is a risk factor for the development of placental disorders with the development of premature labor, the rate of this allele in the main group was 11.7 % versus 1.7 % – in the control cohort (p<0.05).Conclusions. Pregnant women with placental disorders and the development of premature birth are characterized by an increased activity of the p65 subunit of nuclear factor κB and caspase-3 compared to the control group: in women without premature rupture of the fetal membranes, a significant 2.2-fold increase in p65 NF-κB activity was found, and caspase-3 activity – by 1.3 times; in the group of pregnant women with preterm premature rupture of membranes the level of caspase-3 activity exceeded the control group by 1.9 times, and the level of p65-subunit of nuclear factor activity — by 1.2 times (p<0.01 for all groups).936(C/T) single nucleotide polymorphism of the vascular endothelial growth factor gene was determined significantly more often in women with placental disorders and preterm birth (11.7 % in the study group versus 1.7 % in CG, p<0.05), the T-allele on the VEGF gene carriers may be associated with the development of these pregnancy complications. However, a study on a larger sample of women is needed to obtain definitive results.

Smilax china L. Polyphenols Improves Insulin Resistance and Obesity in High-fat Diet-induced Mice Through IRS/AKT-AMPK and NF-κB Signaling Pathways.

Smilax china L. is an important herb used in traditional Chinese medicine. In this study, the mechanism of Smilax china L. polyphenols (SCP) on insulin resistance and anti-obesity in mice induced by a high-fat diet (HFD) was investigated. Fifty female mice were randomly divided into five groups: control, HFD and low, medium, and high doses of SCP for 70 d. SCP significantly decreased intraperitoneal adipose tissue index, body weight gain, liver lipids, and serum inflammatory factor levels. Blood glucose and insulin concentrations, as well as insulin resistance index in SCP, were significantly lower than those in HFD. In addition, SCP markedly up-regulated the gene expression of glucose transporter 4 (GLUT4), insulin receptor substrate 1 (IRS1), insulin receptor substrate 2 (IRS2), serine-threonine kinase (AKT), Acyl-CoA oxidase (ACO), and protein kinase A (PKA), and down-regulated the expression of mammalian target of rapamycin complex 1 (mTORC1), sterol-responsive element-binding protein-1c (SREBP1c), fatty acid synthase (FAS), 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGCR), and forkhead box protein O1 (FOXO1). SCP significantly increased the protein expression of AKT, GLUT4, AMP-activated protein kinase (AMPK), phosphorylated-AMPK (p-AMPK), phosphorylated-AKT (p-AKT), and uncoupling protein 1 (UCP-1), and decreased the expression of SREBP1c, FAS, HMGCR, phosphorylation of IKBα (p-IKBα), and nuclear factor kappa B subunit p65 (P65) in the liver. Overall, SCP effectively reduced HFD-induced insulin resistance and obesity in mice, partly through NF-κB and IRS/AKT-AMPK signaling pathways to regulate inflammatory factors. Therefore, SCP may improve lifestyle diseases.

The Atypical MAP Kinase MAPK15 Is Required for Lung Adenocarcinoma Metastasis via Its Interaction with NF-κB p50 Subunit and Transcriptional Regulation of Prostaglandin E2 Receptor EP3 Subtype

Studying the relatively underexplored atypical MAP Kinase MAPK15 on cancer progression/patient outcomes and its potential transcriptional regulation of downstream genes would be highly valuable for the diagnosis, prognosis, and potential oncotherapy of malignant tumors such as lung adenocarcinoma (LUAD). Here, the expression of MAPK15 in LUAD was detected by immunohistochemistry and its correlation with clinical parameters such as lymph node metastasis and clinical stage was analyzed. The correlation between the prostaglandin E2 receptor EP3 subtype (EP3) and MAPK15 expression in LUAD tissues was examined, and the transcriptional regulation of EP3 and cell migration by MAPK15 in LUAD cell lines were studied using the luciferase reporter assay, immunoblot analysis, qRT-PCR, and transwell assay. We found that MAPK15 is highly expressed in LUAD with lymph node metastasis. In addition, EP3 is positively correlated with the expression of MAPK15 in LUAD tissues, and we confirmed that MAPK15 transcriptionally regulates the expression of EP3. Upon the knockdown of MAPK15, the expression of EP3 was down-regulated and the cell migration ability was decreased in vitro; similarly, the mesenteric metastasis ability of the MAPK15 knockdown cells was inhibited in in vivo animal experiments. Mechanistically, we demonstrate for the first time that MAPK15 interacts with NF-κB p50 and enters the nucleus, and NF-κB p50 binds to the EP3 promoter and transcriptionally regulates the expression of EP3. Taken together, we show that a novel atypical MAPK and NF-κB subunit interaction promotes LUAD cell migration through transcriptional regulation of EP3, and higher MAPK15 level is associated with lymph node metastasis in patients with LUAD.

Long-term exercise preserves pancreatic islet structure and β-cell mass through attenuation of islet inflammation and fibrosis.

Islet fibrosis is associated with the disruption of islet structure and contributes to β-cell dysfunction, playing an essential role in the pathogenesis of type 2 diabetes. Physical exercise has been shown to attenuate fibrosis in various organs; however, the effect of exercise on islet fibrosis has not been defined. Male Sprague-Dawley rats were divided into four groups: normal diet sedentary [N-Sed], normal diet + exercise [N-Ex], high-fat diet sedentary [H-Sed], and high-fat diet + exercise [H-Ex]. After 60 weeks of exercise, 4452 islets from Masson-stained slides were analyzed. Exercise led to a 68% and 45% reduction in islet fibrosis in the normal and high-fat diet groups and was correlated with a lower serum blood glucose. Fibrotic islets were characterized by irregular shapes and substantial loss of β-cell mass, which were significantly reduced in the exercise groups. Remarkably, the islets from exercised rats at week 60 were morphologically comparable to those of sedentary rats at 26 weeks. In addition, the protein and RNA levels of collagen and fibronectin, and the protein levels of hydroxyproline in the islets were also attenuated by exercise. This was accompanied by a significant reduction in inflammatory markers in the circulation Interleukin-1 beta (IL-1β)] and pancreas [IL-1β, Tumor Necrosis Factor-alpha, Transforming Growth Factor-β, and Phosphorylated Nuclear Factor Kappa-B p65subunit], lower macrophage infiltration, and stellate cell activation in the islets of exercised rats. In conclusion, we have demonstrated that long-term exercise preserves pancreatic islet structure and β-cell mass through anti-inflammatory and anti-fibrotic actions, suggesting additional rationales for the success of exercise training in the prevention and treatment of type 2 diabetes that should be further explored.

(S)-2-(Cyclobutylamino)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)isonicotinamide Attenuates RANKL-Induced Osteoclast Differentiation by Inhibiting NF-κB Nuclear Translocation.

Osteoporosis is a common skeletal disease; however, effective pharmacological treatments still need to be discovered. This study aimed to identify new drug candidates for the treatment of osteoporosis. Here, we investigated the effect of EPZ compounds, protein arginine methyltransferase 5 (PRMT5) inhibitors, on RANKL-induced osteoclast differentiation via molecular mechanisms by in vitro experiments. EPZ015866 attenuated RANKL-induced osteoclast differentiation, and its inhibitory effect was more significant than EPZ015666. EPZ015866 suppressed the F-actin ring formation and bone resorption during osteoclastogenesis. In addition, EPZ015866 significantly decreased the protein expression of Cathepsin K, NFATc1, and PU.1 compared with the EPZ015666 group. Both EPZ compounds inhibited the nuclear translocation of NF-κB by inhibiting the dimethylation of the p65 subunit, which eventually prevented osteoclast differentiation and bone resorption. Hence, EPZ015866 may be a potential drug candidate for the treatment of osteoporosis.

Whole-Transcriptome Analysis Highlights Adenylyl Cyclase Toxins-Derived Modulation of NF-κB and ERK1/2 Pathways in Macrophages.

Edema toxin (ET), one of the main toxic factors of Bacillus anthracis (B. anthracis), is a kind of potent adenylate cyclase (AC). B. anthracis has adapted to resist macrophage microbicidal mechanisms in part by secreting ET. To date, there is limited information on the pathogenic mechanisms used by ET to manipulate macrophage function, especially at the transcriptome level. We used RNA sequencing to study transcriptional changes in RAW264.7 cells treated with ET. We aimed to identify molecular events associated with the establishment of infection and followed changes in cellular proteins. Our results indicate that ET inhibited TNF-α expression in the RAW264.7 mouse macrophage cell line by activating the cAMP/PKA pathway. ET challenge of macrophages induced a differential expression of genes that participate in multiple macrophage effector functions such as cytokine production, cell adhesion, and the inflammatory response. Furthermore, ET influenced the expression of components of the ERK1/2, as well as the NF-αB signaling pathways. We also showed that ET treatments inhibit the phosphorylation of the ERK1/2 protein. ET also attenuated NF-αB subunit p65 phosphorylation and transcriptional activity of NF-αB via the cAMP/PKA pathway in macrophages. Since the observed modulatory effects were characteristic only of the bacterial exotoxin ET, we propose this may be a mechanism used by B. anthracis to manipulate macrophages and establish systemic infection.

Transient high salt intake causes epigenetic changes and leads to persistent inflammatory activation to produce “salt memory”

Transient high salt intake causes a sustained increase in blood pressure (BP) even after returning to a normal-salt diet, a phenomenon known as “salt memory.” However, the molecular mechanisms of this phenomenon remain to be elucidated. Dahl salt-sensitive (SS) rats were fed a high-salt (8% NaCl) or high-salt diet and treated with drugs for 8 to 16 weeks and then returned to a normal-salt diet for 3 months. This study investigated the molecular mechanisms of salt memory and its mediation of SS hypertension and renal damage. We show that transient high salt intake caused persistent elevation of BP and exacerbation of kidney damage in Dahl SS rats even after returning to a normal-salt diet. Both epigenetic changes and inflammatory activation also persisted after resumption of a normal diet. Arterial BP, renal injury and the inflammatory response returned to normal levels in rats administered mycophenolate mofetil (MMF) during the 8-week period of high salt intake, resulting in the disappearance of salt memory. However, the vasodilator hydralazine did not ameliorate kidney damage or inflammatory activation, although it decreased BP to control levels. Transient high salt intake increased histone 3 lysine 4 monomethylation (H3K4me1) levels at the nuclear factor κB (NF-κB) subunit p65 promoter in SS rats, promoting p65 gene transcription and NF-κB activation and further leading to a series of inflammatory responses. Our findings demonstrate that transient high salt-induced epigenetic changes and persistent inflammatory activation play important roles in salt memory and its mediation of SS hypertension and renal damage.

A palmitate-rich metastatic niche enables metastasis growth via p65 acetylation resulting in pro-metastatic NF-κB signaling.

Metabolic rewiring is often considered an adaptive pressure limiting metastasis formation; however, some nutrients available at distant organs may inherently promote metastatic growth. We find that the lung and liver are lipid-rich environments. Moreover, we observe that pre-metastatic niche formation increases palmitate availability only in the lung, whereas a high-fat diet increases it in both organs. In line with this, targeting palmitate processing inhibits breast cancer-derived lung metastasis formation. Mechanistically, breast cancer cells use palmitate to synthesize acetyl-CoA in a carnitine palmitoyltransferase 1a-dependent manner. Concomitantly, lysine acetyltransferase 2a expression is promoted by palmitate, linking the available acetyl-CoA to the acetylation of the nuclear factor-kappaB subunit p65. Deletion of lysine acetyltransferase 2a or carnitine palmitoyltransferase 1a reduces metastasis formation in lean and high-fat diet mice, and lung and liver metastases from patients with breast cancer show coexpression of both proteins. In conclusion, palmitate-rich environments foster metastases growth by increasing p65 acetylation, resulting in a pro-metastatic nuclear factor-kappaB signaling.

DOP34 Human MD2 deficiency - an inborn error of immunity predisposing to early onset Inflammatory Bowel Disease

Abstract Background Pattern recognition receptors (PRRs) serve as a hub of immune responses to microbes in the gut and play a critical role in maintaining intestinal homeostasis. Toll-like receptor 4 (TLR4) represents an important PRR that can recognize the gram-negative bacterial cell wall component lipopolysaccharide (LPS). Upon binding to LPS, TLR4 forms a multimeric complex with the indispensable co-receptor myeloid differentiation protein 2 (MD2) facilitating selection of TLR4 as cargo for endocytosis and TLR4-mediated activation of downstream signalling. Despite the critical role of TLR4 in innate immunity, no patients with TLR4 or MD2 deficiency have yet been reported. Methods To investigate potential genetic causes for two related patients presenting with very early onset Inflammatory Bowel Disease (VEO-IBD) and/or pneumonia, we have performed whole exome sequencing (WES). To assess the functional consequences of the identified LY96 (encoding for MD2) variant, we generated a CRISPR/Cas9-mediated knockout (KO) of MD2 or knockin (KI) of the patient mutation in induced pluripotent stem cells (iPSCs) and studied TLR4-mediated signalling, cytokine responses, and bacterial handling in iPSC-derived macrophages. Results Genetic analysis identified a 3 bp homozygous in-frame deletion in the LY96 gene (NM_015364.5, c.347_349delCAA; p.Thr116del) in our index patients following an autosomal recessive inheritance pattern. Immunoblotting revealed an altered protein expression of overexpressed Flag-tagged mutant MD2 protein due to impaired N-linked glycosylation. Correspondingly, iPSC-derived MD2-deficient macrophages showed an impaired LPS-induced TLR4 endocytosis. As a functional consequence, we could detect reduced NF-κB and MAPK signalling (phosphorylation of NF-κB p65 subunit, ERK1/2, and p38 MAPK) and dysregulated inflammasome activation (IL-1b production and secretion) upon challenge with LPS. Gentamycin protection assays and live cell imaging suggested that MD2-deficient macrophages exhibit an impaired phagocytosis of microbial pathogens. In addition, macrophages with mutant MD2 showed decreased cytokine expression (e.g., IFNB, IL6, IL10, and TNF) in response to LPS or gram-negative bacteria (E. coli and Salmonella typhimurium) but not gram-positive bacteria (Listeria monocytogenes). Conclusion Human MD2 deficiency is an inborn error of immunity that may predispose to VEO-IBD associated with altered TLR4-mediated signalling, cytokine responses, and bacterial handling. The first description of patients with MD2 deficiency provides critical insights on human TLR4/MD2 biology and warrants caution on therapeutics strategies targeting TLR4 signalling.

Diabetes and Its Cardiovascular Complications: Potential Role of the Acetyltransferase p300.

Diabetes has been shown to accelerate vascular senescence, which is associated with chronic inflammation and oxidative stress, both implicated in the development of endothelial dysfunction. This condition represents the initial alteration linking diabetes to related cardiovascular (CV) complications. Recently, it has been hypothesised that the acetyltransferase, p300, may contribute to establishing an early vascular senescent phenotype, playing a relevant role in diabetes-associated inflammation and oxidative stress, which drive endothelial dysfunction. Specifically, p300 can modulate vascular inflammation through epigenetic mechanisms and transcription factors acetylation. Indeed, it regulates the inflammatory pathway by interacting with nuclear factor kappa-light-chain-enhancer of activated B cells p65 subunit (NF-κB p65) or by inducing its acetylation, suggesting a crucial role of p300 as a bridge between NF-κB p65 and the transcriptional machinery. Additionally, p300-mediated epigenetic modifications could be upstream of the activation of inflammatory cytokines, and they may induce oxidative stress by affecting the production of reactive oxygen species (ROS). Because several in vitro and in vivo studies shed light on the potential use of acetyltransferase inhibitors, a better understanding of the mechanisms underlying the role of p300 in diabetic vascular dysfunction could help in finding new strategies for the clinical management of CV diseases related to diabetes.

SARS-CoV-2 Nsp2 Contributes to Inflammation by Activating NF-κB.

COVID-19 is associated with robust inflammation and partially impaired antiviral responses. The modulation of inflammatory gene expression by SARS-CoV-2 is not completely understood. In this study, we characterized the inflammatory and antiviral responses mounted during SARS-CoV-2 infection. K18-hACE2 mice were infected with a Wuhan-like strain of SARS-CoV-2, and the transcriptional and translational expression interferons (IFNs), cytokines, and chemokines were analyzed in mouse lung homogenates. Our results show that the infection of mice with SARS-CoV-2 induces the expression of several pro-inflammatory CC and CXC chemokines activated through NF-κB but weakly IL1β and IL18 whose expression are more characteristic of inflammasome formation. We also observed the downregulation of several inflammasome effectors. The modulation of innate response, following expressions of non-structural protein 2 (Nsp2) and SARS-CoV-2 infection, was assessed by measuring IFNβ expression and NF-κB modulation in human pulmonary cells. A robust activation of the NF-κB p65 subunit was induced following the infection of human cells with the corresponding NF-κB-driven inflammatory signature. We identified that Nsp2 expression induced the activation of the IFNβ promoter through its NF-κB regulatory domain as well as activation of p65 subunit phosphorylation. The present studies suggest that SARS-CoV-2 skews the antiviral response in favor of an NF-κB-driven inflammatory response, a hallmark of acute COVID-19 and for which Nsp2 should be considered an important contributor.

Caudatin attenuates inflammatory reaction by suppressing JNK/AP-1/NF-κB/caspase-1 pathwaysin activatedHMC-1 cells.

One of the interfering factors in Coronavirus disease 2019 (COVID-19) is the cytokine storm, which contributes to hyperinflammation. Mast cells cause COVID-19 hyperinflammation by increasing inflammatory cytokine levels. We investigated whether caudatin, an active compound of Cynanchum auriculatum, could suppress inflammatory response signaling in human mast cell line, HMC-1 cells. Caudatin suppressed activation of c-Jun N-terminal kinase (JNK) and activator protein-1 (AP-1) in HMC-1 cells. Caudatin suppressed nuclear translocation of catalytic subunit (p65) of nuclear factor (NF)-κB by blocking IκBα phosphorylation and degradation. Caudatin also reduced levels of activated-caspase-1 protein and activation of caspase-1. Non-toxic caudatin doses inhibited the mRNA expression and protein synthesis of pro-inflammatory cytokines. A significant finding was that caudatin inhibited JNK/AP-1/NF-κB/caspase-1 signaling molecules, reducing the secretion of inflammatory cytokines. Consequently, we propose that caudatin might be used as a material in health functional foods to alleviate mast cell-mediated inflammatory conditions like COVID-19.

The Involvement of Canonical NFκB Pathway in Megakaryocyte Differentiation Induction by Nanocurcumin.

Background: Megakaryopoiesis is characterized by progressive polyploidization and the expression of megakaryocytic markers. Numerous transcription factors and physiological signaling pathways regulate this phenomenon. Megakaryocyte differentiation induction in the K562 cell line and hematopoietic stem cells via nanocurcumin drug has been identified in our previous study. K562 cells are typical Chronic Myelogenous Leukemia (CML) cells that are resistant to apoptosis and express the bcr-abl fusion gene. These cells have the potential to differentiate into erythrocytes and megakaryocytes. Curcumin is well known as a component with strong potential to alter NFκB activity in various cells. NFκB pathway regulates various genes such as apoptotic and immune response genes. The current study attempted to evaluate the possible role of nanocurcumin in NFκB pathway regulation during the megakaryopoiesis process in the K562 cell line. Materials and Methods: Megakaryocyte markers expression and phenotype alteration of nanocurcumin-treated K562 cells have been detected by flow cytometry and microscopy imaging. The nuclear level of the RelA (p65) subunit of NFκB was determined by western blot test in K562 cells during megakaryopoiesis induction via nanocurcumin treatment at different times. The expression of NFκB target genes including c-MYC, BAX, and NQO1 was also analyzed in nanocurcumin-treated K562 cells by quantitative RT-PCR assay at different times. Results: The study has shown that nanocurcumin causes an increase in NFκB activity transiently during megakaryocyte differentiation, followed by a change in the expression of c-MYC, BAX, and NQO1 target genes. Conclusion: The NFκB pathway can be considered a new pathway for inducing megakaryocyte differentiation by nanocurcumin in vitro and in vivo megakaryopoiesis experiments.

Two target gene activation pathways for orphan ERR nuclear receptors.

Estrogen-related receptors (ERRα/β/γ) are orphan nuclear receptors that function in energy-demanding physiological processes, as well as in development and stem cell maintenance, but mechanisms underlying target gene activation by ERRs are largely unknown. Here, reconstituted biochemical assays that manifest ERR-dependent transcription have revealed two complementary mechanisms. On DNA templates, ERRs activate transcription with just the normal complement of general initiation factors through an interaction of the ERR DNA-binding domain with the p52 subunit of initiation factor TFIIH. On chromatin templates, activation by ERRs is dependent on AF2 domain interactions with the cell-specific coactivator PGC-1α, which in turn recruits the ubiquitous p300 and MED1/Mediator coactivators. This role of PGC-1α may also be fulfilled by other AF2-interacting coactivators like NCOA3, which is shown to recruit Mediator selectively to ERRβ and ERRγ. Importantly, combined genetic and RNA-seq analyses establish that both the TFIIH and the AF2 interaction-dependent pathways are essential for ERRβ/γ-selective gene expression and pluripotency maintenance in embryonic stem cells in which NCOA3 is a critical coactivator.

Molecular characterization and immune functions of lipasin in Nile tilapia (Oreochromis niloticus): Involvement in the regulation of tumor necrosis factor-α secretion

Lipasin, the product of the angiopoietin-like 8 (angptl8) gene, is known as a critical regulator of plasma lipid metabolism. However, its immune function in vertebrates is currently poorly understood. By 5'/3′-rapid amplification of cDNA ends (RACE), we established the structural identity of Nile tilapia (Oreochromis niloticus) angptl8. The transcripts of tilapia angptl8 were widely expressed in various tissues, with the highest levels in the liver. Following lipopolysaccharide in vivo challenges, time-dependent angptl8 gene expression was observed in the head kidney and liver. On the basis of the sequence obtained, we produced recombinant lipasin that inhibited lipoprotein lipase activity. Treatment of head kidney leukocytes with lipasin stimulated tumor necrosis factor-α (TNF-α) secretion and gene expression. In addition, lipasin-induced TNF-α secretion could be prevented by inhibiting the nuclear factor-kappa B (NF-κB) signaling pathway. Furthermore, lipasin enhanced the phosphorylation and degradation of IκBα and promoted translocation of the p65 subunit of NF-κB to the nucleus. Collectively, the current findings suggested that lipasin was involved in the immune response of Nile tilapia and stimulated TNF-α secretion by activating the NF-κB pathway in tilapia head kidney leukocytes.

Targeting a transcription factor NF-κB by green tea catechins using in silico and in vitro studies in pancreatic cancer.

Pancreatic cancer remains a lethal disease and a major public health problem globally. Nuclear factor-kappa B (NF-κB) has been identified as a therapeutic target in several cancers and plays an important role in inflammatory responses. Many phytochemicals, including catechins, have been reported in the scientific literature with efficient anticancer potential and minimal side effects. This study aims to gain insights into the inhibitory mechanism of catechin derivatives epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG) using in silico and in vitro studies especially considering NF-κB targeting. We explored the binding pose, interacting residues and molecular interactions for catechin derivatives with NF-κB. Docking analysis showed that the catechin derivatives acted as covalent inhibitors with the p65 subunit of NF-κB and interacted with other residues through non-bonding interactions and hydrogen bonds. Further, we validated the effect of EGCG on NF-κB activity in pancreatic cancer cell lines MIAPaCa-2 and SU 86.86. Our in vitro data showed EGCG effectively reduced cell growth and proliferation, induced apoptosis, and inhibited NF-κB activity in the studied cell lines. In addition, EGCG repressed the expression of NF-κB target genes including MMP9, MMP2, cMyc, and BCL-2. Thus, targeting NF-κB with EGCG could be a potential therapeutic alternative for pancreatic cancer treatment.

Hyperforin Elicits Cytostatic/Cytotoxic Activity in Human Melanoma Cell Lines, Inhibiting Pro-Survival NF-κB, STAT3, AP1 Transcription Factors and the Expression of Functional Proteins Involved in Mitochondrial and Cytosolic Metabolism.

Hyperforin (HPF), the main component responsible for the antidepressant action of Hypericum perforatum, displays additional beneficial properties including anti-inflammatory, antimicrobic, and antitumor activities. Among its antitumor effects, HPF activity on melanoma is poorly documented. Melanoma, especially BRAF-mutated melanoma, is still a high-mortality tumor type and the currently available therapies do not provide solutions. We investigated HPF's antimelanoma effectiveness in A375, FO1 and SK-Mel-28 human BRAF-mutated cell lines. Cell viability assays documented that all melanoma cells were affected by low HPF concentrations (EC50% 2-4 µM) in a time-dependent manner. A Br-deoxy-uridine incorporation assay attested a significant reduction of cell proliferation accompanied by decreased expression of cyclin D1 and A2, CDK4 and of the Rb protein phosphorylation, as assessed by immunoblots. In addition, the expression of P21/waf1 and the activated form of P53 were increased in A375 and SK-Mel-28 cells. Furthermore, HPF exerts cytotoxic effects. Apoptosis is induced 24 h after HPF administration, documented by an increase of cleaved-PARP1 and a decrease of both Bcl2 and Bcl-xL expression levels. Autophagy is induced, attested by an augmented LC3B expression and augmentation of the activated form of AMPK. Moreover, HPF lowers GPX4 enzyme expression, suggesting ferroptosis induction. HPF has been reported to activate the TRPC6 Ca++ channel and/or Ca++ and Zn++ release from mitochondria stores, increasing cytosolic Ca++ and Zn++ concentrations. Our data highlighted that HPF affects many cell-signaling pathways, including signaling induced by Ca++, such as FRA1, pcJun and pCREB, the expression or activity of which are increased shortly after treatment. However, the blockage of the TRPC6 Ca++ channel or the use of Ca++ and Zn++ chelators do not hinder HPF cytostatic/cytotoxic activity, suggesting that damages induced in melanoma cells may pass through other pathways. Remarkably, 24 h after HPF treatment, the expression of activated forms of the transcription factors NF-κB P65 subunit and STAT3 are significantly lowered. Several cytosolic (PGM2, LDHA and pPKM2) and mitochondrial (UQCRC1, COX4 and ATP5B) enzymes are downregulated by HPF treatment, suggesting a generalized reduction of vital functions in melanoma cells. In line with these results is the recognized ability of HPF to affect mitochondrial membrane potential by acting as a protonophore. Finally, HPF can hinder both melanoma cell migration and colony formation in soft agar. In conclusion, we provide evidence of the pleiotropic antitumor effects induced by HPF in melanoma cells.

Glucocorticoids Impair the 7α-Hydroxycholesterol-Enhanced Innate Immune Response.

Glucocorticoids suppress the vascular inflammation that occurs under hypercholesterolemia, as demonstrated in an animal model fed a high-cholesterol diet. However, the molecular mechanisms underlying these beneficial effects remain poorly understood. Because cholesterol is oxidized to form cholesterol oxides (oxysterols) that are capable of inducing inflammation, we investigated whether glucocorticoids affect the immune responses evoked by 7α-hydroxycholesterol (7αOHChol). The treatment of human THP-1 monocytic cells with dexamethasone (Dex) and prednisolone (Pdn) downregulated the expression of pattern recognition receptors (PRRs), such as TLR6 and CD14, and diminished 7αOHChol-enhanced response to FSL-1, a TLR2/6 ligand, and lipopolysaccharide, which interacts with CD14 to initiate immune responses, as determined by the reduced secretion of IL-23 and CCL2, respectively. Glucocorticoids weakened the 7αOHChol-induced production of CCL2 and CCR5 ligands, which was accompanied by decreased migration of monocytic cells and CCR5-expressing Jurkat T cells. Treatment with Dex or Pdn also reduced the phosphorylation of the Akt-1 Src, ERK1/2, and p65 subunits. These results indicate that both Dex and Pdn impair the expression of PRRs and their downstream products, chemokine production, and phosphorylation of signaling molecules. Collectively, glucocorticoids suppress the innate immune response and activation of monocytic cells to an inflammatory phenotype enhanced or induced by 7αOHChol, which may contribute to the anti-inflammatory effects in hypercholesterolemic conditions.

Insampaedok-San Extract Exerts an Immune-Enhancing Effect through NF-κB p65 Pathway Activation.

Insampaedok-san (IS) has traditionally been prescribed as a medication for cold-related symptoms in Northeast Asia, including Korea and China. In this study, we focused on elucidating the molecular mechanism underlying the immunomodulatory activity of IS water extract (ISE) in macrophages. ISE significantly enhanced the levels of nitric oxide (NO) and prostaglandin E2 (PGE2) by increasing the expression of inducible NO synthase and cyclooxygenase-2 (COX-2) in a dose-dependent manner. ISE, which consists of many herbs, contains a large number of active compounds whose pharmacological targets and mechanisms are complicated. Therefore, network pharmacology analysis was used to predict the potential key components, targets, and mechanisms of ISE as immunomodulators. Subsequently, the network pharmacology results were validated experimentally. Seven key components were identified through HPLC-QTOF-MS. As predicted by the network pharmacology analysis, ISE increased the mRNA expression of Tnf and Il6. Furthermore, ISE increased the phosphorylation, nuclear translocation, and transcriptional activity of the p65 subunit of the nuclear factor-κB (NF-κB) signaling pathway. In contrast, rapamycin, an NF-κB inhibitor, suppressed the ISE-induced mRNA expression of Tnf and Il6. In conclusion, ISE is an immune activator that can elevate the production of NO, PGE2, and proinflammatory cytokines mediated by NF-κB signaling.

Pirfenidone Attenuates the EMT Process and the Secretion of VEGF in TGF-β2-Induced ARPE-19 Cells via Inhibiting the Activation of the NF-κB/Snail Signaling Pathway.

Pirfenidone (PFD), an antifibrotic drug, has various beneficial functions such as antioxidant, antifibrotic, and anti-inflammatory effects. This study aimed to explore the molecular mechanisms underlying how PFD modulates retinal pigment epithelial (RPE) cells involved in neovascularization and subretinal fibrosis. ARPE-19 cell lines were treated with transforming growth factor-beta 2 (TGF-β2) alone or in combination with PFD. RPE cell viability, as a consequence of PFD use, was determined by the CCK-8 assay. Cell migration was assessed by the wound closure assay and quantified by the Image J software. Protein expression of the following markers was measured by the western blot analysis: an epithelial cell marker and E-cadherin; mesenchymal cell markers, fibronectin, matrix metalloprotein-9 (MMP-9), and alpha-smooth muscle actin (α-SMA); a fibrotic marker and connective tissue growth factor (CTGF); an angiogenesis marker and vascular endothelial growth factor (VEGF); NF-κB/Snail. The mRNA levels of fibronectin and α-SMA were determined by quantitative real-time PCR. VEGF was quantitatively measured by the enzyme-linked immunosorbent assay. The cell viability assay revealed that PFD had no significant cytotoxic effect on RPE cells at concentrations of less than 1 mg/mL. The cell scratch assay showed that TGF-β2 stimulation significantly improved the migration of RPE cells and that PFD attenuated this effect. PFD significantly inhibited the TGF-β2-induced protein expression of E-cadherin and increased the TGF-β2-induced protein expression of fibronectin, MMP-9, α-SMA, CTGF, and VEGF in ARPE-19 cells. The mRNA expression of fibronectin and α-SMA was inhibited by PFD in TGF-β2-inducedARPE-19 cells. Additionally, the increased intracellular and supernatant expression of VEGF protein was suppressed by PFD. Mechanistically, RPE cells treated with PFD + TGF-β2 exhibited a decrease in phosphorylation of the NF-κB P65 subunit and activation of Snail, compared with the RPE cells treated with TGF-β2 alone. PFD ameliorated TGF-β2-induced neovascularization and fibrosis by suppressing the NF-κB/Snail signaling pathway. Therefore, PFD may be a potential drug in the treatment of age-related macular degeneration.