NF-κB Assay Research Articles

The influence of hyperglycemia on the safety of ultrasound in retinal pigment epithelial cells.

Ultrasound (US) assisted drug delivery is receiving interest in treating posterior eye diseases, such as diabetic retinopathy due to its ability to maximize drug penetration into difficult to reach tissues. Despite its promise, the technique has only been investigated using healthy cell and tissue models, with no evidence to date about its safety in active disease. As a result, the aim of this study was to evaluate the safety of US administration in vitro in retinal pigment epithelial cells under normal and high glucose conditions. US protocols within the presently accepted safety threshold were applied and their influence on cell membrane and tight junction integrity as well as intracellular inflammation was evaluated using lactate dehydrogenase (LDH), zona occludens-1 (ZO-1), fluorescein isothiocyanate (FITC)-dextran dye leak and nuclear factor-kappaB (NF-κB) assays, respectively. Under high glucose conditions, US application increased LDH release and resulted in loss of ZO-1 labeling at 2 h; however, normal levels were restored within 24 h. US within its safety parameters did not induce any FITC-dextran dye leak or NF-κB nuclear translocation in normal or high glucose conditions. In conclusion, our results suggest that while high glucose conditions increase cell susceptibility to US-mediated stress, basal conditions can be restored within 24 h without long-lasting cell damage.

Functional Multigenomic Screening of Human-Associated Bacteria for NF-κB-Inducing Bioactive Effectors.

The effect of the microbiota on its human host is driven, at least in part, by small-molecule and protein effectors it produces. Here, we report on the use of functional multigenomic screening to identify microbiota-encoded effectors. In this study, genomic DNA from 116 human-associated bacteria was cloned en masse, and the resulting multigenomic library was screened using a nuclear factor-κB reporter (NF-κB) assay. Functional multigenomics builds on the concept of functional metagenomics but takes advantage of increasing advances in cultivating and sequencing human-associated bacteria. Effector genes found to confer NF-κB-inducing activity to Escherichia coli encode proteins in four general categories: cell wall hydrolases, membrane transporters, lipopolysaccharide biosynthetic enzymes, and proteins of unknown function. The compact nature of multigenomic libraries, which results from the ability to normalize input DNA ratios, should simplify screening of libraries using diverse heterologous hosts and reporter assays, increasing the rate of discovery of novel effector genes.IMPORTANCE Human-associated bacteria are thought to encode bioactive small molecules and proteins that play an intimate role in human health and disease. Here, we report on the creation and functional screening of a multigenomic library constructed using genomic DNA from 116 bacteria found at diverse sites across the human body. Individual clones were screened for genes capable of conferring NF-κB-inducing activity to Escherichia coli NF-κB is a useful reporter for a range of cellular processes related to immunity, pathogenesis, and inflammation. Compared to the screening of metagenomic libraries, the ability to normalize input DNA ratios when constructing a multigenomic library should facilitate the more efficient examination of commensal bacteria for diverse bioactivities. Multigenomic screening takes advantage of the growing available resources in culturing and sequencing the human microbiota and generates starting points for more in-depth studies on the mechanisms by which commensal bacteria interact with their human host.

Cytotoxic constituents from Penicillium concentricum, an endophytic fungus from Trichocolea tomentella.

In our continuing effort to identify bioactive secondary metabolites from natural sources, the antiproliferative activity of 23 compounds, previously isolated from Penicillium concentricum, was assessed using the sulforhodamine B assay. The cytotoxic effect was determined against HeLa cervical, HT-29 colon, MDA-MB-321 breast, PC-3, and DU-145 prostate cancer cell lines. Compounds were also tested in the mitochondrial transmembrane potential (MTP) and nuclear factor kappa B (NF-κB) target-based assays. The results showed that 2-bromogentisyl alcohol (2) and 3-hydroxy-benzenemethanol (8) exhibited the highest cytotoxic activity against different cancer cell lines. Epoxydon (14) showed selectivity against DU-145 prostate cancer cells [inhibitory concentration 50 (IC50)=1.2 μmol/l]. Compounds 2, 8, 14, 18, 21 also induced damage of MTP (IC50=0.1, 0.2, 7.0, 9.6, and 1.8 μmol/l, respectively). In the NF-κB assay, only compound 8 exhibited potent inhibition (IC50=0.3 μmol/l). Compounds 2 and 14 showed cytotoxic activity and induction of damage in mitochondrial membrane potential while compound 8 inhibited NF-κB and MTP damage. Additionally, compound 14 with selectivity against DU-145 prostate cancer cells induced cell cycle arrested in G2/M phase. Thus, compounds 2, 8, and 14 could be useful leads in the development of new anticancer agents from natural sources.

Green synthesis of hematite (α-Fe2O3) nanoparticles using Rhus punjabensis extract and their biomedical prospect in pathogenic diseases and cancer

Hematite nanoparticles (NPs) are chemically and thermodynamically more stable among iron oxide NPs therefore these are considered highly suitable for biomedical applications. This work explores Rhus punjabensis extract potential as both reducing and capping agent for phytomediated synthesis of NPs from ferric chloride precursor. The structural, morphological and compositional investigations were carried out with XRD, SEM, TEM, EDS, and FFT. The synthesized NPs were hematite (Fe2O3) with rhombohedral crystal structure of an approximate size of 41.5 ± 5 nm. TEM, FTIR, and TG/DTA indicated phase-pure Fe2O3 formation with controlled size through use of capping agent. Biological activities depict that synthesized NPs had free radical scavenging, antioxidative potential and reducing power activities. These activities are due to phenolic and flavonoid like properties by attachment of different functional groups on the surface of NPs. The NPs exhibited significant Antileishmanial, antibacterial and protein kinase inhibition activities as compared with the corresponding plant extract. NPs depicted cytotoxic effect against HL-60 leukemic and DU-145 prostate cancer cell lines with ED50 values of 11.9 and 12.79 μg/ml, respectively. The inhibitory effect observed in the NF-κB assay suggested apoptotic potential of hematite NPs to cancerous cells. The green chemistry route reported in this paper for Fe2O3 NP synthesis can be safely and efficiently explored for treatment of cancer and as theranostic agent.

Wound healing activity of terpinolene and α-phellandrene by attenuating inflammation and oxidative stress in vitro

This study was undertaken to investigate the in vitro wound healing effects and the anti-inflammatory and antioxidant activities of terpinolene and α-phellandrene. The in vitro stimulatory effects on the proliferation and migration of fibroblasts were assessed using the scratch assay. The anti-inflammatory activity was evaluated using cell-based assays by investigating their influence on nitric oxide (NO), superoxide anion (O2•-), tumour necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6) production and using the TNF-α-induced nuclear factor kappa (NF-κB) assay. Antioxidant activity was determined by the ABTS cation radical scavenging capacity, ferric reducing/antioxidant potential (FRAP), and NO free radical scavenging assays. Terpinolene and α-phellandrene significantly increased the migration and proliferation of fibroblasts and suppressed the pro-inflammatory cytokines IL-6 and TNF-α in a dose-dependent manner. Terpinolene and α-phellandrene at a concentration of 100 μM significantly inhibited NO production (41.3 and 63.8%, respectively) in a macrophage cell-culture-based assay, and resulted in reductions in O2•- production of 82.1 ± 3.5% and 70.6 ± 4.3%, respectively. Moreover, these monoterpenes were verified to suppress NF-κB activity. In summary, terpinolene and α-phellandrene may contribute to broadening clinical options in the treatment of wounds by attenuating inflammation and oxidative stress in vitro.

PS02.049: HIGH GPX1 EXPRESSION PROMOTES ESOPHAGEAL SQUAMOUS CELL CARCINOMA INVASION, MIGRATION, PROLIFERATION AND CISPLATIN-RESISTANCE BUT CAN BE REDUCED BY VITAMIN D

Abstract Background Esophageal cancer is one of the most common cancers worldwide. Despite recent progress in the devel- opment of novel therapies, esophageal carcinoma remains an aggressive cancer associated with a poor prognosis. The glutathione peroxidase 1 (GPX1) gene located on chromosome 3p21.3 is associated with the cancer of several organs. According to available information, GPX1, a gene downstream of NF-κB, is considered to exert adverse ef- fects on tumour progression and enhance malignancy in some cancers but has not been reported in esophageal cancer. It is also reported that vitamin D (Vit. D), a widely used drug in the clinical setting, could suppress GPX1 expression through the NF-κB pathway. Thus, it is speculated that Vit. D could reduce malignancy in esophageal cancer by altering the NF-κB pathway. Methods Selection GPX1 high expression EC109 and EC9706 cell line K150, and K180 low expression cell lines transfected with siRNA and vector. Using RT-PCR to detect cell transfection efficiency GPX1’s, Western blot detect the expression of uPA, MMP-2, NF-κB, and CCK-8 assay inhibition or overexpression on GPX1 on ESCC cell cisplatin resistance and proliferation. Transwell assay inhibition or over-expression of GPX1 on invasive ability of ESCC cells. Results By CCK-8 and transwell law found within the cells expressing low GPX1 ESCC can inhibit cell proliferation, cisplatin resistance, invasion and migration, high GPX1 when the opposite phenomenon can be observed. Western blot GPX1 down when found, PTEN, Bax activity rises, PDK-1, AKT, Bcl-2, uPA, MMP-2 expression decreased, vimentin, E-cadherin, ß-catenin, snail is no significant change. After Vit.D treatment, CCK-8 assay showed that cell proliferation and drug resistance was significantly inhibited; Transwell showed cell invasion and migration also decreased. Cells was detected by Western blot after transfection, cells, PTEN, Bax increased activity, p65, PDK-1, AKT, Bcl-2, uPA, MMP-2 expression decreased, vimentin, E-cadherin, ß-catenin, snail is no significant change. Conclusion Treatment with vitamin D can downregulate GPX1, thus decreasing tumour cell capacity for invasion, migration, proliferation and cisplatin resistance via the NF-κB pathway. Vitamin D, already commonly used in clinical therapy, could play a greater role in the treatment of esophageal cancer patients. Disclosure All authors have declared no conflicts of interest.

What is driving the NF-κB response in environmental water extracts?

In vitro bioassays are increasingly applied for water quality monitoring, with assays indicative of adaptive stress responses commonly included in test batteries. The NF-κB assay is responsive to surface water and wastewater extracts, but the causative compounds are unknown and micropollutants typically found in water do not activate the NF-κB assay. The current study aimed to investigate if co-extracted organic matter and/or endotoxins could cause the NF-κB response in surface water extracts. The effect of model bacterial lipopolysaccharides (LPS) and dissolved organic carbon (DOC) was evaluated in the NF-κB assay both before and after solid-phase extraction (SPE), with 7% effect recovery for LPS and between 7 and 52% effect recovery for DOC observed. The NF-κB response, endotoxin activity, micropollutant concentration and total organic carbon concentration was measured in four surface water extracts. All water extracts showed a response in the NF-κB assay, but the detected micropollutants could not explain the effect. Comparison of predicted bioanalytical equivalent concentrations based on micropollutant, DOC and endotoxin concentrations in surface water with experimental bioanalytical equivalent concentrations suggest that co-extracted endotoxins are the most important drivers of the observed effect, with DOC only having a minor contribution. While in vitro bioassays typically detect mixtures of organic micropollutants, the current study shows that the NF-κB assay can integrate the effects of co-extracted endotoxins. Given that endotoxins can pose a risk for human health, the NF-κB assay is a valuable inclusion in bioanalytical test batteries used for water quality monitoring.

In Vitro Dissolution, Cellular Membrane Permeability, and Anti-Inflammatory Response of Resveratrol-Encapsulated Mesoporous Silica Nanoparticles.

Sizing drugs down to the submicron and nanometer scale using nanoparticles has been extensively used in pharmaceutical industries to overcome the poor aqueous solubility of potential therapeutic agents. Here, we report the encapsulation and release of resveratrol, a promising anti-inflammatory and anticancer nutraceutical, from the mesopores of MCM-48-type silica nanospheres of various particle sizes, i.e., 90, 150, and 300 nm. Furthermore, the influence of the carrier pore size on drug solubility was also evaluated (3.5 vs 7 nm). From our results, it is observed that the saturated solubility could depend not only on the pore size but also on the particle size of the nanocarriers. Moreover, with our resveratrol-mesoporous silica nanoparticles formulation, we have observed that the permeability of resveratrol encapsulated in MCM-48 nanoparticles (90 nm) can be enhanced compared to a resveratrol suspension when tested through the human colon carcinoma cell monolayer (Caco-2). Using an in vitro NF-κB assay, we showed that resveratrol encapsulation did not alter its bioactivity and, at lower concentration, i.e., 5 μg mL-1, resveratrol encapsulation provided higher anti-inflammatory activity compared to both resveratrol suspension and solution. All combined, the reported results clearly highlight the potential of small size mesoporous silica nanoparticles as next generation nanocarriers for hydrophobic drugs and nutraceuticals.

Highly sensitive electrochemical nuclear factor kappa B aptasensor based on target-induced dual-signal ratiometric and polymerase-assisted protein recycling amplification strategy

In this work, an amplified electrochemical ratiometric aptasensor for nuclear factor kappa B (NF-κB) assay based on target binding-triggered ratiometric signal readout and polymerase-assisted protein recycling amplification strategy is described. To demonstrate the effect of “signal-off” and “signal-on” change for the dual-signal electrochemical ratiometric readout, the thiol-hairpin DNA (SH-HD) hybridizes with methylene blue (MB)-modified protection DNA (MB-PD) to form capture probes, which is rationally introduced for the construction of the assay platform. On the interface, the probes can specifically bind to target NF-κB and expose a toehold region which subsequently hybridizes with the ferrocene (Fc)-modified DNA strand to take the Fc group to the electrode surface, accompanied by displacing MB-PD to release the MB group from the electrode surface, leading to the both “signal-on” of Fc (IFc) and “signal-off” of MB (IMB). In order to improve the sensitivity of the electrochemical aptasensor, phi29-assisted target protein recycling amplification strategy was designed to achieve an amplified ratiometric signal. With the above advantages, the prepared aptasensor exhibits a wide linear range of 0.1pgmL−1 to 15ngmL−1 with a low detection limit of 0.03pgmL−1. This strategy provides a simple and ingenious approach to construct ratiometric electrochemical aptasensor and shows promising potential applications in multiple disease marker detection by changing the recognition probe.

Bioactive Constituents from an Endophytic Fungus, Penicillium polonicum NFW9, Associated with Taxus fauna.

Endophytic fungi are being recognized as vital and untapped sources of a variety of structurally novel and unique bioactive secondary metabolites in the field of natural products drug discovery. Herein, this study reports the isolation and characterization of secondary metabolites from an endophytic fungus Penicillium polonicum (NFW9) associated with Taxus fuana. The extracts of the endophytic fungus cultured on potato dextrose agar were purified using several chromatographic techniques. Biological evaluation was performed based on their abilities to inhibit tumor necrosis factor-alpha (TNF-α)-induced nuclear factor-kappa B (NF-κB) and cytotoxicity assays. Bioactivity-directed fractionation of the ethyl acetate extract of a fermentation culture of an endophytic fungus, Penicillium polonicum led to the isolation of a dimeric anthraquinone, (R)- 1,1',3,3',5,5'-hexahydroxy-7,7'-dimethyl[2,2'-bianthracene]-9,9',10,10'-tetraone (1), a steroidal furanoid (-)-wortmannolone (2), along with three other compounds (3-4). Moreover, this is the first report on the isolation of compound 1 from an endophytic fungus. All purified metabolites were characterized by NMR and MS data analyses. The stereo structure of compound 1 was determined by the measurement of specific optical rotation and CD spectrum. The relative stereochemistry of 2 was confirmed by single-crystal X-ray diffraction. Compounds 2-3 showed inhibitory activities in the TNF-α-induced NF-κB assay with IC50 values in the range of 0.47-2.11 µM. Compounds 1, 4 and 5 showed moderate inhibition against NF-κB and cancer cell lines. The endophytic fungus Penicillium polonicum of Taxus fuana is capable of producing biologically active natural compounds. Our results provide a scientific rationale for further chemical investigations into endophyte-producing natural products, drug discovery and development.

Potential anti-inflammatory, antioxidant and antimicrobial activities of Sambucus australis

Context: Sambucus australis Cham. & Schltdl. (Adoxaceae) is used in Brazilian folk medicine to treat inflammatory disorders.Objective: To evaluate the in vitro anti-inflammatory, antioxidant and antimicrobial properties of S. australis.Materials and methods: The anti-inflammatory activity of ethanol extracts of the leaf and bark of S. australis (1–100 μg/mL) were studied in lipopolysaccharide/interferon γ stimulated murine macrophages RAW 264.7 cells (24 h incubation) by investigating the release of nitric oxide (NO) and tumour necrosis factor-alpha (TNF-α) and in the TNF-α-induced nuclear factor kappa (NF-κB) assay. Minimum inhibitory concentration (MIC) was determined by the microdilution test (24 h incubation). Antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP) and the NO scavenging assays. Chemical composition was assessed by LC-MS/MS.Results: Antioxidant activities in the DPPH (IC50 43.5 and 66.2 μg/mL), FRAP (IC50 312.6 and 568.3 μg/mL) and NO radical scavenging assays (IC50 285.0 and 972.6 μg/mL) were observed in the leaf and bark ethanol extracts, respectively. Solely the leaf extract showed significant inhibition of NO and TNF-α production in RAW264.7 cells at concentrations of 2 and 100 μg/mL, respectively, and suppression of TNF-α inhibition of NF-κB by 12.8 and 20.4% at concentrations of 50 and 100 μg/mL, respectively. The extract also exhibited antibacterial activity against Salmonella typhimurium (MIC 250 μg/mL) and Klebsiella pneumoniae (MIC 250 μg/mL). LC-MS/MS revealed the presence of chlorogenic acid and rutin as major compounds.Discussion and conclusion: The results indicate that the ethanol leaf extract of S. australis exhibit prominent anti-inflammatory effects.

Anti-inflammatory and Quinone Reductase Inducing Compounds from Fermented Noni (Morinda citrifolia) Juice Exudates.

A new fatty acid ester disaccharide, 2-O-(β-d-glucopyranosyl)-1-O-(2E,4Z,7Z)-deca-2,4,7-trienoyl-β-d-glucopyranose (1), a new ascorbic acid derivative, 2-caffeoyl-3-ketohexulofuranosonic acid γ-lactone (2), and a new iridoid glycoside, 10-dimethoxyfermiloside (3), were isolated along with 13 known compounds (4-16) from fermented noni fruit juice (Morinda citrifolia). The structures of the new compounds, together with 4 and 5, were determined by 1D and 2D NMR experiments, as well as comparison with published values. Compounds 2 and 7 showed moderate inhibitory activities in a TNF-α-induced NF-κB assay, and compounds 4 and 6 exhibited considerable quinone reductase-1 (QR1) inducing effects.

Hedera nepalensis K. Koch: A Novel Source of Natural Cancer Chemopreventive and Anticancerous Compounds.

Traditional medicinal plants are often used for both the prevention and the treatment of local diseases. Taking into consideration the medicinal importance of Hedera nepalensis within local Pakistani traditions, the present study was undertaken to analyze the in vitro cancer chemopreventive and cytotoxic properties of the plant. The in vitro cancer chemopreventive testing was performed using nitrite assay, NFκB assay, aromatase assay, and quinone reductase 1 (QR1) assay. The cytotoxic potential was evaluated on three cancer-cell lines: MCF-7, MDA-MB-231, and HeLa using sulforhodamine B (SRB) assay. The results of cancer chemopreventive assays show that n-hexane and ethyl acetate fractions of tested plant have promising cancer chemopreventive potential. Lupeol isolated from n-hexane as well as ethyl acetate fraction showed lowest IC50 (0.20 ± 1.9 μM) in NFκB assay. Crude extract and its fractions inhibited the growth of three cancer cell lines by more than 60%, IC50 value of lupeol varied from 2.32 to 10.2 μM. HPLC-DAD-based quantification of lupeol in different plant tissues demonstrated that leaves of H. nepalensis are a rich source of lupeol (0.196 mg/100 mg dry weight). Our data have shown that H. nepalensis harbors cancer chemopreventive and cytotoxic agents.

Epigenetic changes and nuclear factor-κB activation, but not microRNA-224, downregulate Raf-1 kinase inhibitor protein in triple-negative breast cancer SUM 159 cells.

Raf-1 kinase inhibitor protein (RKIP) is a tumor suppressor and metastasis inhibitor, which enhances drug-induced apoptosis of cancer cells. Downregulation of RKIP may be significant in the biology of highly aggressive and drug-resistant tumors, for example triple-negative breast cancers (TNBCs). Potential causes for the low levels of RKIP expressed by SUM 159 TNBC cells were investigated in the present study. Bisulphite modification, methylation specific-polymerase chain reaction (PCR) and a TransAM NF-κB assay were performed and the results suggested that various mechanisms, including methylation of the gene promoter, histone deacetylation and nuclear factor-κB (NF-κB) activation, but not targeting by microRNA-224 (miR/miRNA-224), as determined by transfection of pre-miR-224 miRNA precursor or anti-miR-224 miRNA inhibitor, may downregulate RKIP in these cells. Furthermore, reverse transcription-quantitative PCR, western blotting, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium cell growth assay and flow cytometry revealed that in SUM 159 cells, the demethylating agent 5-aza-2'-deoxycytidine (5-AZA), the histone deacetylase inhibitor trichostatin A (TSA) and the NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) enhanced RKIP expression and resulted in significant cell growth inhibition and induction of apoptosis. 5-AZA and TSA mainly produced additive antitumor effects, while the combination of DHMEQ and TSA exhibited significant synergy in cell growth inhibition and induction of apoptosis assays. Increasing evidence that aberrant activation of NF-κB signaling is a frequent characteristic of TNBC highlights the fact that this transcription factor may be a useful target for treatment of such tumors. In addition to DHMEQ, proteasome inhibitors may also represent valuable therapeutic resources in this context. Notably, proteasome inhibitors, in addition to the inhibition of NF-κB activation, may also restore RKIP levels by inhibiting proteasome degradation of the ubiquitinated protein. The current results contribute to the understanding of the molecular mechanisms of RKIP downregulation in TNBC and suggest possible novel therapeutic approaches for the treatment of these types of cancer.

Effects of capsicodendrin in MCF-7 hormone-dependent breast cancer cells

The transcription factor NF-κB plays a significant role in cancer cell survival signaling. Targeting the NF-κB pathway promotes pro-apoptotic down-stream effect and sensitizes malignant cells to existing chemotherapeutic drugs. The cytotoxic effects of compounds isolated from three Cinnamosma species (C. fragrans, C. macrocarpa and C. madagascariensis) were evaluated in multiple cancer cells as well as in a NF-κB-p65 assay. The results obtained showed that capsicodendrin displayed a significant cytotoxic activity in the MCF-7 cell line (IC50= 7.5µM). Further, capsicodendrin also displayed NF-κB inhibitory activity (IC50= 8.6µM) when tested in the NF-κB assay. Subsequently, the apoptotic effect of capsicodendrin was examined using fluorescence-activated cell sorter. In non- treated MCF-7 cells, the results indicated that 46% of cells were detected in sub G1-phase. Treatment with capsicodendrin at a concentration level of 10µM increased the cell population in sub G1-phase to 81% of the total cell population, suggesting that apoptosis had been induced. To further evaluate the mechanism of action, the effect on the mitochondria membrane potential was evaluated using the fluorescent dye JC-1 in treated and untreated cells.

Serum Mir-29a Is Up-Regulated In Acute Graft Versus Host Disease (aGVHD) After Allogeneic Hematopoietic Stem Cell Transplantation (allo HSCT) and Activates Dendritic Cells (DCs)

Background aGVHD is one of the most frequent and lethal complications after allo HSCT, underscoring the need to develop novel therapies. To achieve this goal, aGVHD mechanisms needs to be further elucidated. Recently it was reported that miRNAs are modulating aGVHD. In addition miRNAs are also present in the human serum and regulate immune responses. Here, we hypothesize that serum miRNAs expression is deregulated in aGVHD and could play a role in aGVHD pathogenesis. Methods To identify miRNAs associated with aGVHD we performed serum miRNA expression analysis using deep-sequencing from allo HSCT recipients samples at the time of clinical suspicion of aGVHD. Peripheral blood (PB) samples were collected weekly until day 100+ and at the time of clinical diagnosis of aGVHD from allo HSCT patients enrolled into OSU11002. After serum separation, total RNA was extracted using Trizol. Libraries were constructed using the small RNA profiling kit and sequenced on the Solid analyzer. A mouse model of aGVHD (B6 mice donor splenocytes and BM cells transplanted to lethally irradiated F1 recipients) was used to assess serum miRNA expression in animals with aGVHD. Results In this study we included 10 patients with aGVHD (bowel n=2; skin (n=5) and both skin and bowel aGVHD (n=3). Median age was 51.9, conditioning regimens were mainly non-myeloablative (n=9), with unrelated donors (n=9). PB samples from allo HSCT patients with no aGVHD and matched for age, disease, conditioning regimen, donor and timing of sample collection were obtained and used as controls. Sequence alignment was performed using miRBase. Normalization as reads per million was followed by quantiles. We compared miRNA expression between all patients with aGVHD (n=10) and controls (n=7) using class comparison (BRB). We found 7 miRNAs up-regulated (miR-146a, miR-323-b, miR-34c, miR-363, miR-4245, miR-29a, miR-181a* ) and 3 miRNAs down-regulated (miR-3168, miR-662, miR-550a) (Fold change (FC) >2, p<0.01). Since miR-146 and miR-29a were both involved in immune regulation we further validated these miRNAs by RT-PCR in the B6-F1 model of murine aGVHD. We found up-regulation of miR-146 FC 2, p<0.01 and miR-29a FC 4.9 p<0.01) in mice with aGVHD (n=6) with respect to controls (n=4). Next, we focus on miR-29a since our group found that this miR binds as ligands to TLR8. We hypothesized that serum miR-29a could bind to TLR8 of APCs activating NFkB and enhancing alloreactive responses during aGVHD. First, we examined whether extracellular miR-29a could activate dendritic cells (DCs). B6 splenocytes were stimulated with Dotap formulations (mimicking exosomes) of miR-29a. Negative controls included Dotap alone or Dotap-miR16 formulation. We found that CD69 expression measured by FACS is significantly elevated in CD11c+ DCs (34%), and CD8+ T cells (56%) populations treated with miR-29a compared to controls (p<0.01). CD86, a co-stimulatory molecule on DCs, was also significantly up-regulated after miR-29a stimulation (33%, p<0.01). To investigate whether T cells could be activated by the miR alone, independently of APCs, we isolated untouched resting T cells from mouse splenocytes suspension using the Pan-T cell isolation kit and stimulated them with Dotap-miR-29a, Dotap-miR-16 or Dotap alone. CD69 was not up-regulated under these culture conditions indicating that the activation of T cells was dependent on APCs activation. To further confirm that miR-29a could activate DCs, we isolated DCs from B6 mice using the pan DC isolation kit and repeated the above experiment. We found that miR-29a stimulation of DCs but not controls induced the up-regulation of both CD69 and CD86 (20%). Furthermore, miR-29a Dotap treatment of isolated DCs stimulated the release of TNFα in the supernatant (114.2±14.3 pg/ml vs. controls 26.98±2.09 pg/ml, p<0.01). We also performed coimmunoprecipitation assays for TLR8 in HEK-293 cells expressing GFP-TLR8 and treated with Dotap-miR-16, Dotap-miR-29a, or Dotap alone and determined miRNA levels by qRT-PCR. Only miR-29a expression was highly enriched (>50-fold). This binding leads to the activation of NFkB as measured by a NF-κB assay in TLR8–HEK-293 cells treated only with Dotap-miR-29a. Validation of these results using murine and human DCs are undergoing. Summary Altogether, our results indicate that serum miR-29a is up-regulated during aGVHD and activates DCs, likely by direct binding to TLR8 and inducing NFkB activation Disclosures: No relevant conflicts of interest to declare.

(+)-Altholactone exhibits broad spectrum immune modulating activity by inhibiting the activation of pro-inflammatory cytokines in RAW 264.7 cell lines

An evaluation of Indonesian plants to identify compounds with immune modulating activity revealed that the methanolic extract of an Alphonsea javanica Scheff specimen possessed selective anti-inflammatory activity in a nuclear factor-kappa B (NF-κB) luciferase and MTT assay using transfected macrophage immune (Raw264.7) cells. A high-throughput LC/MS-ELSD based library approach of the extract in combination with the NF-κB and MTT assays revealed the styryl lactone (+)-altholactone (2) was responsible for the activity. Compound 2, its acetylated derivate (+)-3-O-acetylaltholactone (3), and the major compound of this class, (+)-goniothalmin (1), were further evaluated to determine their anti-inflammatory potential in the NF-κB assay. Concentration–response studies of 1–3 indicated that only 2 possessed NF-κB based anti-inflammatory activity. Compound 2 reduced the LPS-induced NO production, phosphorylation of IκBα, and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) using Western blot analysis. Further studies using qPCR indicated 2 reduced the expression of eight pro-inflammatory cytokines/enzymes (0.8–5.0μM) which included: COX-2, iNOS, IP-10, IL-1β, MCP-1, GCS-F, IL-6 and IFN-β. These results indicated that 2 displays broad spectrum immune modulating activity by functioning as an anti-inflammatory agent against LPS-induced NF-κB signaling. Conversely the selective cytotoxicity and in vivo anti-tumor and anti-inflammatory activity previously reported for 1 do not appear to arise from a mechanism that is linked to the NF-κB immune mediated pathway.

Triptolide-mediated cell death in neuroblastoma occurs by both apoptosis and autophagy pathways and results in inhibition of nuclear factor–kappa B activity

BackgroundNeuroblastoma is an aggressive pediatric malignancy with significant chemotherapeutic resistance. We assessed triptolide as a potential therapy. MethodsSH-SY5Y and IMR-32 neuroblastoma cell lines were treated with triptolide. Viability, intracellular calcium, caspase activation, protein, and mRNA levels were measured. Autophagy was evaluated with confocal microscopy. Nuclear factor–kappa B (NF-κB) activation was measured using a dual luciferase assay. ResultsTriptolide treatment resulted in death in both cell lines within 72 hours, with sustained increases in intracellular calcium. IMR-32 cells underwent cell death by apoptosis. Conversely, light chain 3II (LC3II) protein levels were elevated in SH-SY5Y cells, which is consistent with autophagy. Confocal microscopy confirmed increased LC3 puncta in SH-SY5Y cells compared with control cells. Heat shock pathway protein and mRNA levels decreased with treatment. NF-κB assays demonstrated inhibition of tumor necrosis factor (TNF)-α–induced activity with triptolide. ConclusionsTriptolide treatment induces cell death in neuroblastoma by different mechanisms with multiple pathways targeted. Triptolide may serve a potential chemotherapeutic role in advanced cases of neuroblastoma.

Novel low molecular weight lignins as potential anti-emphysema agents: In vitro triple inhibitory activity against elastase, oxidation and inflammation

No molecule has been found to be effective against emphysema to date primarily because of its complex pathogenesis that involves elastolysis, oxidation and inflammation. We here describe novel unsulfated or sulfated low molecular weight lignins (LMWLs) chemo-enzymatically prepared from 4-hydroxycinnamic acids monomers, as the first potent triple-action inhibitors of neutrophil elastase, oxidation and inflammation. The inhibitory potencies of three different cinnamic acid-based LMWLs were determined in vitro using chromogenic substrate hydrolysis assays, radical scavenging and lung cellular oxidative biomarker reduced glutathione (rGSH) assays, and lung cellular inflammatory biomarker NFκB and IL-8 assays, respectively. Each LWML uniquely displayed triple-action inhibition, among which CDSO3, a sulfated caffeic acid-based LMWL, was most potent. The half-maximal anti-human neutrophil elastase (HNE) potency of CDSO3 was 0.43 μM. This high potency arose from lignin-like oligomerization, which was further potentiated by 6.6-fold due to sulfation. Mechanistically, this elastase inhibition was of mixed-type, time-dependent and more selective to positively charged elastases. The half-maximal anti-oxidative potency of CDSO3 was 3.52 μM, 4.8-fold potentiated from that of the monomer, caffeic acid (CA). In contrast, the half-maximal inhibitory potency to TNFα-induced inflammation was 5–10 μM, despite no activity with the monomer. More intriguingly, this anti-inflammatory activity was essentially identical with different stimuli, okadaic acid and hydrogen peroxide (H2O2), which implied that CDSO3 acts directly on inflammatory cascades within the cells. Overall, oligomerization and sulfation produced or significantly potentiated the activity, in comparison to the monomer. Thus, sulfated and unsulfated LMWLs are novel non-peptidic 2.8–4.1 kDa macromolecules that exhibit for the first time potent triple inhibitory activity against elastase, oxidation and inflammation, the three major pathogenic mechanisms known to cause emphysema.

Cytotoxic epipolythiodioxopiperazine alkaloids from filamentous fungi of the Bionectriaceae

Bioactivity-directed fractionation of the organic extracts of two filamentous fungi of the Bionectriaceae, strains MSX 64546 and MSX 59553 from the Mycosynthetix library, led to the isolation of a new dimeric epipolythiodioxopiperazine alkaloid, verticillin H (1), along with six related analogs, Sch 52900 (2), verticillin A (3), gliocladicillin C (4), Sch 52901 (5), 11'-deoxyverticillin A (6) and gliocladicillin A (7). The structures of compounds 1-7 were determined by extensive NMR and HRMS analyses, as well as by comparisons to the literature. All compounds (1-7) were evaluated for cytotoxicity against a panel of human cancer cell lines, displaying IC(50) values ranging from 1.2 μM to 10 nM. Compounds 1-5 were examined for activity in the NF-κB assay, where compounds 2 and 3 revealed activity in the sub-micromolar range. Additionally, compounds 1, 3 and 4 were tested for EGFR inhibition using an enzymatic assay, while compound 3 was examined against an overexpressing EGFR(+ve) cancer cell line.