Western Immunoblotting Research Articles

Radiotherapy Increases aMMP-8-Levels and Neutrophil/Lymphocyte Ratio Rapidly in Head and Neck Cancer Patients: A Pilot Study.

Radiotherapy for head and neck carcinoma (HNC) has both curative and palliative purposes. This study investigated mouthrinse aMMP-8 levels, molecular forms of MMP-8, blood neutrophil counts and neurophil/lymphocyte ratios before and 3 weeks after HNC radiotherapy started. Thirteen HNC patients undergoing radiotherapy were included. Mouthrinse samples (before and 3 weeks after HNC radiotherapy had started) were assayed quantitatively by aMMP-8 point-of-care-kit (PerioSafe®/ORALyzer®) and by western immunoblot. Total neutrophil counts and neutrophil/lymphocyte ratios were evaluated in the hemogram results. Three weeks after HNC radiotherapy started, significant increases in aMMP-8 levels and neutrophil/lymphocyte ratios were observed. No significant difference was found in total neutrophil counts. Elevations of the activated and fragmented MMP-8 levels after HNC radiotherapy application were observed on western immunoblot analysis. The increase in the aMMP-8 levels and neutrophil/lymphocyte ratios indicate inflammation both locally and systemically suggesting increased risk for periodontitis due to the HNC radiotherapy.

Stress-Induced Changes in Trophic Factor Expression in the Rodent Urinary Bladder: Possible Links With Angiogenesis.

Substantive evidence supports a role of chronic stress in the development, maintenance, and even enhancement of functional bladder disorders such as interstitial cystitis/bladder pain syndrome (IC/BPS). Increased urinary frequency and bladder hyperalgesia have been reported in rodents exposed to a chronic stress paradigm. Here, we utilized a water avoidance stress (WAS) model in rodents to investigate the effect of chronic stress on vascular perfusion and angiogenesis. Female Wistar-Kyoto rats were exposed to WAS for 10 consecutive days. Bladder neck tissues were analyzed by western immunoblot for vascular endothelial growth factor (VEGF) and nerve growth factor precursor (proNGF). Vascular perfusion was assessed by fluorescent microangiography followed by Hypoxyprobe testing to identify regions of tissue hypoxia. The expression of VEGF and proNGF in the bladder neck mucosa was significantly higher in the WAS rats than in the controls. There was a trend toward increased vascular perfusion, but without a statistically significant difference from the control group. The WAS rats displayed a 1.6-fold increase in perfusion. Additionally, a greater abundance of vessels was observed in the WAS rats, most notably in the microvasculature. These findings show that chronic psychological stress induces factors that can lead to increased microvasculature formation, especially around the bladder neck, the region that contains most nociceptive bladder afferents. These findings may indicate a link between angiogenesis and other inflammatory factors that contribute to structural changes and pain in IC/BPS.

Sulforaphane attenuates cancer cell-induced atrophy of C2C12 myotubes

Cancer cachexia is common in many cancers and the loss of skeletal muscle mass compromises the response to therapies and quality of life. A contributing mechanism is oxidative stress and compounds able to attenuate it may be protective. Sulforaphane (SFN), a natural antioxidant in cruciferous vegetables, activates nuclear factor erythroid 2-related factor 2 (Nrf2) signaling to decrease oxidative stress. Although SFN has potential as a cancer therapeutic, whether it can attenuate muscle wasting in the absence or presence of chemotherapy is unknown. In healthy C2C12 myotubes, SFN administration for 48 h induced hypertrophy through increased myoblast fusion via Nrf2 and ERK signaling. To determine whether SFN could attenuate wasting induced by cancer cells, myotubes were cocultured with or without Colon-26 (C-26) cancer cells for 48 h and treated with 5-fluorouracil (5-FU, 5 µM) or vehicle (DMSO). SFN (10 µM) or DMSO was added for the final 24 h. Coculture with cancer cells in the absence and presence of 5-FU reduced myotube width by ∼30% (P < 0.001) and ∼20% (P < 0.01), respectively, which was attenuated by SFN (P < 0.05). Exposure to C-26 conditioned media reduced myotube width by 15% (P < 0.001), which was attenuated by SFN. Western immunoblotting and qRT-PCR confirmed activation of Nrf2 signaling and antioxidant genes. Coadministration of Nrf2 inhibitors (ML-385) or MEK inhibitors (PD184352) revealed that SFN's attenuation of atrophy was blocked by ERK inhibition. These data support the chemoprotective and antioxidative function of SFN in myotubes, highlighting its therapeutic potential for cancer-related muscle wasting.

Quercetin alleviates subarachnoid hemorrhage-induced early brain injury via inhibiting ferroptosis in the rat model.

Early brain injury (EBI) refers to a series of pathophysiological brain lesions that occur within 72 hr after subarachnoid hemorrhage (SAH), which is an extremely crucial factor in the poor prognosis of patients. In EBI, ferroptosis has been proven to cause neuronal death. Quercetin (QCT) is effective in deactivating reactive oxygen species (ROS), inhibiting lipid peroxidation, and even chelating iron, but its role in SAH remains unclear. In this study, the mortality rate, severity grade of SAH, brain water content (BWC), blood-brain barrier permeability, and neurological function of the rats were detected. Moreover, mitochondrial morphology in cortical neurons were observed and their sizes were subsequently quantified. The levels of lipid peroxidation on glutathione and malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) were determined, whereas the protein expressions of glutathione peroxidase 4 (GPX4), SLC7A11 (xCT), transferrin receptor 1 (TfR1), and ferroportin-1 (FPN1) were analyzed by western immunoblotting. The neurodegeneration involved in EBI was investigated by fluoro-Jade C staining, while iron staining was utilized to measure iron content. Our results showed that inhibition of ferroptosis by QCT could suppress EBI and improve neurological function in SAH rats. QCT increased the expression levels of GPX4, xCT, and FPN1, while downregulated TfR1, and exerted protective effects on neurons as well as alleviated iron accumulation and lipid peroxidation in the cortex of SAH rats. In conclusion, our study revealed that QCT might alleviate the EBI by inhibiting ferroptosis in SAH rats.

Preparation and qualification of soluble amyloid beta oligomers for use in bioanalytic assays supporting Alzheimer’s disease therapeutics

AbstractBackgroundSoluble amyloid beta oligomers (sAβOs) accumulate early in Alzheimer’s disease (AD) and trigger AD‐related neuropathologies and impairments in memory. sAβO structures contributing to the neurotoxic effects remain ill‐defined due to their low concentration, instability, and heterogeneity, impeding the effective design and use of sAβO reference standards in bioanalytic assays for use in clinical trials. At present, no assays for sAβOs are available with proven robustness and clinical performance, due to absence of well‐characterized critical raw materials (antibodies, reference materials for preparation of sAβO calibrators) and quality control specimens.MethodWe have used amyloid‐derived diffusible ligands (ADDLs), a synthetic model preparation commonly used in the literature (Stine et al. 2003), as a sAβO standard integrated into different assays designs developed on the Mesoscale Discovery (MSD) technology. As proof‐of‐concept, we have utilized these ADDL assays to study the specificity and selectivity of antibodies targeted against sAβOs.ResultOptimization of ADDL coating, stabilization of coated materials, and long‐term plate stability indicated that ADDL‐coated plates provided a suitable platform to assess the performance of sAβO‐targeting antibodies.Prior to coating, ADDL preparations were tested for reproducible SDS‐stable size distribution and immunoreactivity using SDS‐PAGE with silver stain and Western immunoblotting. The native size distribution (SEC) and globular conformation (AFM) of ADDLs and their ability to bind neuronal synapses in culture (ICC) and subsequently elicit AD‐related pathologies has been well reported in the literature. ADDLs were coated onto 96‐well MSD plates and the ADDL‐coated plates were stored at 4 °C for long‐term use; plate stability was confirmed up to one year. Biotinylated and non‐biotinylated sAβO‐targeting antibodies with variable selectivity towards monomeric or oligomeric Aβ species were used for comparative binding analysis. Additional sAβO preparations, including low concentration ADDL preparations and commercially available sAβOs were used to further understand the selectivity and specificity of the panel of sAβO‐targeting antibodies.ConclusionQualified sAβO preparations (ADDLs) can be used as tools for coating plates to determine specificity and selectivity of sAβO‐targeting antibodies. Other expected uses are: (i) calibrator in immunoassays; or (ii) to screen for the presence of sAβO auto‐antibodies in biological fluids.

Patient-derived three-dimensional cortical neurospheres to model Parkinson's disease.

There are currently no preventive or disease-modifying therapies for Parkinson's Disease (PD). Failures in clinical trials necessitate a re-evaluation of existing pre-clinical models in order to adopt systems that better recapitulate underlying disease mechanisms and better predict clinical outcomes. In recent years, models utilizing patient-derived induced pluripotent stem cells (iPSC) have emerged as attractive models to recapitulate disease-relevant neuropathology in vitro without exogenous overexpression of disease-related pathologic proteins. Here, we utilized iPSC derived from patients with early-onset PD and dementia phenotypes that harbored either a point mutation (A53T) or multiplication at the α-synuclein/SNCA gene locus. We generated a three-dimensional (3D) cortical neurosphere culture model to better mimic the tissue microenvironment of the brain. We extensively characterized the differentiation process using quantitative PCR, Western immunoblotting and immunofluorescence staining. Differentiated and aged neurospheres revealed alterations in fatty acid profiles and elevated total and pathogenic phospho-α-synuclein levels in both A53T and the triplication lines compared to their isogenic control lines. Furthermore, treatment of the neurospheres with a small molecule inhibitor of stearoyl CoA desaturase (SCD) attenuated the protein accumulation and aberrant fatty acid profile phenotypes. Our findings suggest that the 3D cortical neurosphere model is a useful tool to interrogate targets for PD and amenable to test small molecule therapeutics.

Euphol from Tapinanthus sp. Induces Apoptosis and Affects Signaling Proteins in Glioblastoma and Prostate Cancer Cells

Plants play an important role in cancer therapy. They are source of natural molecules which can induce apoptosis in cancer cells by affecting molecular mechanisms implicated in cancer progression. The MAP Kinase/ERK1/2 and PI3K/AKT signaling pathways are two classical signaling pathways implicated in cancer progression and constitute therapeutic targets against cancer. This study aimed to evaluate the effect of euphol on MAP Kinase/ERK1/2 and PI3K/AKT signaling pathways in glioblastoma and prostate cancer cells. Euphol is a tetracyclique triterpene alcohol isolated from Tapinanthus sp. which is a hemi parasitic plant belonging to Loranthaceae family. Plant powder was extracted by maceration and euphol was isolated and described using respectively column chromatography separation on silica gel and spectroscopic data. Cytotoxic effect of euphol was evaluated using XTT assay and its effect on MAP Kinase/ERK1/2 and PI3K/AKT protein expression was investigated by Western immunoblot analysis. Apotosis was analyzed by evaluating caspase-3/7 activity. Our investigations demonstrated that this compound has an important cytotoxic effect on C6 and U87 MG glioblastoma (GBM) cells and PC-3 prostate cancer cells. Furthermore, euphol-induced apoptosis revealed by elevated caspase 3/7 activity, was correlated with a significant inhibition of MAP kinase/Erk 1/2 and PI3K/Akt signaling pathway in glioblastoma U87 MG cells. The reverse effect was observed in C6 glioblastoma cells, where apoptosis was correlated with a long-lasting activation of Erk 1/2. In PC-3 cells, euphol had no or limited effect on Erk 1/2 and Akt activity. These results indicate that euphol induces cell death in glioblastoma and prostate cancer cells and regulates significantly Erk1/2 and Akt activity in glioblastoma cells.

Human coagulation factor X and CD5 antigen-like are potential new members of the zonulin family proteins

Zonulin is a physiologic epithelial and endothelial permeability modulator. Zonulin increases antigen trafficking from the gut lumen into the bloodstream and in between body compartments, a mechanism linked to many chronic inflammatory diseases. Upon its initial discovery, it was noted that zonulin was not a single protein, but rather a family of structurally and functionally related proteins referred to as the zonulin family proteins (ZFPs). ZFPs are members of the mannose associated serine proteases (MASP) family and are the result of high mutation rates leading to many zonulin polymorphisms. Pre-haptoglobin 2, the precursor of haptoglobin 2, was identified as the first eukaryotic member of the ZFPs, and properdin, a key positive regulator of the alternative pathway, as a second member. In this study, we report two additional proteins that are likely ZFPs. Human coagulation factor X (FX) and CD5 antigen-like (CD5L). Both FX and CD5L recombinant proteins were detected by anti-zonulin antibody in Western immunoblot analysis, and both proteins decreased epithelial barrier competency of Caco-2 cell monolayers as established by the Trans Epithelial Electrical Resistance (TEER) assay. These results indicate that FX and CD5L have structural and functional similarities with previously identified ZFPs and, therefore, can be considered new members of this family of proteins.

C/EBPα-p30 Confers AML Cell Susceptibility to the Terminal Unfolded Protein Response and Resistance to Venetoclax By Activating DDIT3 Transcription

Introduction: CCAAT-enhancer-binding protein α (C/EBPα) is one of the most essential lineage-specific transcription factors involved in myeloid differentiation and includes two isoforms of protein: full-length C/EBPα-p42 and truncated C/EBPα-p30. Acute myeloid leukemia (AML) with CEBPA biallelic (CEBPAbi) mutation or single mutation in the bZIP region is associated with a favorable prognosis, but the mechanisms are still unclear. The unfolded protein response (UPR) is a conserved adaptive signal transduction pathway triggered by endoplasmic reticulum (ER) stress. UPR first strives to restore protein homeostasis and maintain cell survival through a pro-survival arm called adaptive UPR. However, in the setting of irremediable ER stress, the UPR transforms into pro-apoptotic terminal UPR signaling that promotes cell apoptosis. DNA damage-inducible transcript 3 (DDIT3) is a key transcription factor responsible for terminal UPR under severe ER stress. Here, we hypothesize that C/EBPα two isoforms regulate DDIT3 transcription, resulting in easier elimination of AML cells with CEBPA mutations and dysregulation by terminal UPR. Methods: Human AML cell lines and mouse 32Dcl3 were transduced with recombinant lentiviruses to knockdown CEBPA expression or over-express of C/EBPα two isoforms. RT-qPCR was used to detected the mRNA levels and western immunoblotting was used to determine the protein levels. The cell apoptosis rates were determined as the percentage of Annexin V-positive cells by flow cytometry. We performed CFU assays to confirm the declined granulocytic differentiation of 32Dcl3 cells with Cebpa knockdown. To identify potential C/EBPα-binding DNA sequences in the genome, we performed ChIP-seq. Luciferase reporter analysis was used to confirm the transcriptional regulation ability of C/EBPα two isoforms and independent ChIP-qPCR of candidate sequences were performed to further verify the transcription factor binding sites of C/EBPα. Finally, we generated a xenograft model with NCG mice to determine the potential application value of our findings. Results: Firstly, we found a negative correlation between CEBPA expression and DDIT3 levels in AML cell lines and primary de novo AML patients. After knockdown of CEBPA in THP-1 cells, which has high endogenous C/EBPα expression, DDIT3 expression was upregulated, and an increased apoptotic rate of AML cells was induced by ER stress. Cebpa knockdown also induced easier elimination of mouse myeloid progenitor cells 32Dcl3 due to upregulation of Ddit3, so as to avoid leukemogenesis since their differentiation was blocked. After the overexpression of C/EBPα-p42 and C/EBPα-p30 in NB4 cells, which have relatively low levels of endogenous C/EBPα isoforms, we found that C/EBPα-p30 activated DDIT3 expression to increase the sensitivity of AML cells to tunicamycin. However, C/EBPα-p42 had the opposite effect to C/EBPα-p30. Through luciferase reporter analysis, we confirmed that C/EBPα-p30 and C/EBPα-p42 directly regulate the transcription of DDIT3 in a contrasting manner, indicating that the two isoforms maintain protein homeostasis corporately. ChIP-seq and ChIP-qPCR revealed that C/EBPα-p30 has a special binding site in DDIT3 gene promoter with stronger affinity than C/EBPα-p42 (hg38, Chr12:57520654-57521026). Given that the BCL2 family proteins are major targets of DDIT3, we further observed that a low p42/p30 ratio was related to reduced BCL2 levels-a major target of DDIT3, increased MCL1 levels, and resistance of AML cells to venetocalx. Finally, in vitro and in vivo we found that tunicamycin and sorafenib could synergistically reverse venetoclax resistance in AML cells by activating the terminal UPR and inhibiting MCL1. Conclusions: Collectively, our results identified a critical regulation of C/EBPα two isoforms on the transcription of DDIT3 in AML cells as well as normal myeloid progenitor cells and confirmed the importance of this regulation in leukemia surveillance and elimination of AML cells. Moreover, we demonstrated that patients with AML having a low C/EBPα p42/p30 ratio and CEBPAbi mutation may not benefit from monotherapy with BCL2 inhibitors. This can be resolved by a combination of ER stress inducers or MCL1 inhibitors, including tunicamycin and sorafenib, which may represent a novel therapeutic strategy for AML patients with CEBPA mutations and dysregulation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 10138: Exercise Inhibits Doxorubicin-Induced Cardiotoxicity by Preventing Cardiomyocyte Cellular Senescence

Introduction: Doxorubicin (Dox) induces cardiomyocyte cellular senescence thru proinflammatory mechanisms and by damaging DNA, resulting in Dox-induced cardiotoxicity. Inhibiting cardiomyocyte cellular senescence inhibits Dox-induced cardiac damage. We showed that exercise inhibits both acute and late Dox-induced cardiotoxicity using our juvenile cardiotoxicity mouse model. Here we used p16-3MR transgenic mice to track the induction of senescent cardiomyocytes to determine whether exercise prevents Dox-induced cardiotoxicity by inhibiting senescence. Hypothesis: Cellular senescence plays a key role in Dox-induced cardiotoxicity in the heart following therapy. Exercise prevents Dox-induced cardiotoxicity by inhibiting myocardial cell senescence. Methods: To confirm induction of senescence by Dox in vitro , mouse cardiomyocytes were isolated and treated with 250 nmol/L Dox for 24 h for 24h cells, washed and then analyzed 7 days later for senescent markers. Mice were treated with PBS, Dox (2.5mg/kg 2x/wk for 2 wks), or Dox + exercise (walking 45 min/day at 12 meter/min). Cardiac function was evaluated by echocardiography 24 h after therapy to confirm Dox-induced cardiotoxicity. Heart tissue was collected 14 days after therapy. Tissue sections were analyzed by immunohistochemical and Western immunoblot. Results: Dox-induced senescence was confirmed by a significant increase in β-galactosidase in cardiomyocytes from the in vitro study. Decreased ejection fraction, fractional shortening and F-actin distribution was seen in Dox but not Dox + exercise-treated mice confirming Dox-induced cardiotoxicity and prevention of heart damage by exercise. There was an increase in p16 INK4a expression (signifying senescent cells) in cardiac tissue from Dox-treated but not Dox + exercise-treated mice compared with PBS controls. Conclusion: Taken together our data demonstrated that exercise inhibited Dox-induced cardiotoxicity by inhibiting the induction of cardiomyocyte senescence.

HIV Increases the Risk of Cigarette Smoke-Induced Emphysema Through MMP-9.

HIV is associated with an increased risk for emphysema. Matrix metalloproteinase 9 (MMP-9) is a lung tissue remodeling enzyme associated with emphysema. We previously found MMP-9 activity increases with increases in oxidative stress and that HIV increases alveolar oxidative stress. We hypothesized that HIV proteins would increase the risk of cigarette smoke-induced emphysema due to MMP-9. HIV-1 transgenic rats and wild-type littermates were exposed to cigarette smoke or sham for 8 weeks. Lung compliance and histology were assessed. Bronchoalveolar lavage (BAL), primary alveolar macrophages (AM), and serum samples were obtained. A rat alveolar macrophage cell line was exposed to the HIV protein Tat, and MMP-9 levels were assessed by Western immunoblotting. MMP-9 protein expression and activity were assessed in AM from the HIV rat model by ELISA and cytoimmunofluoresence, respectively. Serum from human subjects with and without HIV and tobacco dependence was assessed for MMP-9 levels. MMP-9 expression was significantly increased in rat alveolar macrophages after Tat exposure. HIV-1 transgenic rats developed emphysema while wild-type littermates did not. MMP-9 expression was also increased in the serum, BAL, and AM of HIV-1 transgenic rats after exposure to cigarette smoke compared with wild-type rats. In parallel, serum samples from HIV+ smokers had higher levels of MMP-9 than subjects without HIV and those who did not smoke. The combination of HIV and cigarette smoke increases MMP-9 expression in experimental rat HIV models and human subjects. HIV and cigarette smoke both induce alveolar oxidative stress and thereby increase MMP-9 activity.

Reversion of methionine addiction of osteosarcoma cells to methionine independence results in loss of malignancy, modulation of the epithelial-mesenchymal phenotype and alteration of histone-H3 lysine-methylation.

Methionine addiction, a fundamental and general hallmark of cancer, known as the Hoffman Effect, is due to altered use of methionine for increased and aberrant transmethylation reactions. However, the linkage of methionine addiction and malignancy of cancer cells is incompletely understood. An isogenic pair of methionine-addicted parental osteosarcoma cells and their rare methionine-independent revertant cells enabled us to compare them for malignancy, their epithelial-mesenchymal phenotype, and pattern of histone-H3 lysine-methylation. Methionine-independent revertant 143B osteosarcoma cells (143B-R) were selected from methionine-addicted parental cells (143B-P) by their chronic growth in low-methionine culture medium for 4 passages, which was depleted of methionine by recombinant methioninase (rMETase). Cell-migration capacity was compared with a wound-healing assay and invasion capability was compared with a transwell assay in 143B-P and 143B-R cells in vitro. Tumor growth and metastatic potential were compared after orthotopic cell-injection into the tibia bone of nude mice in vivo. Epithelial-mesenchymal phenotypic expression and the status of H3 lysine-methylation were determined with western immunoblotting. 143B-P cells had an IC50 of 0.20 U/ml and 143B-R cells had an IC50 of 0.68 U/ml for treatment with rMETase, demonstrating that 143B-R cells had regained the ability to grow in low methionine conditions. 143B-R cells had reduced cell migration and invasion capability in vitro, formed much smaller tumors than 143B-P cells and lost metastatic potential in vivo, indicating loss of malignancy in 143B-R cells. 143B-R cells showed gain of the epithelial marker, ZO-1 and loss of mesenchymal markers, vimentin, Snail, and Slug and, an increase of histone H3K9me3 and H3K27me3 methylation and a decrease of H3K4me3, H3K36me3, and H3K79me3 methylation, along with their loss of malignancy. These results suggest that shifting the balance in histone methylases might be a way to decrease the malignant potential of cells. The present results demonstrate the rationale to target methionine addiction for improved sarcoma therapy.

OR07-5 PARP1 Contributes to the Glucocorticoid Receptor Transcriptional Response And Influences the Skeletal Muscle Phenotype

Abstract Background NAD+-dependent PARP1 (Poly-(ADP-ribosyl)polymerase 1) generates the post-translational modification ADP-Ribosylation (ADPR). Given PARP1-ADPR determines cellular NAD+ availability, which in turn impacts a range of hormonally governed actions, we sought to identify the molecular roles of PARP1 in relation to glucocorticoids in the endocrine sensitive tissue skeletal muscle. Importance: Activity of the glucocorticoid receptor (GR) is dictated by molecular partners whose actions skew the frequency with which GR produces meaningful transcriptional outcomes. As a result, our molecular understanding of this steroid hormone receptor remains incomplete. Hypothesis PARP1 through ADPR actions impacts skeletal muscle phenotype and co-regulates glucocorticoid-mediated transcriptional responses. Experimental Design: Murine muscle myoblasts (C2C12) or primary murine muscle cultures were treated with PARP inhibitors (PJ34(10uM) or BYK204165(1uM)) or transfected with silencing RNA targeting PARP1. Cells were treated with ± dexamethasone (1uM). Major Results Analysis of differentiating muscle revealed PARP1 and ADPR are dynamic at days 0-5 (measured by Western immunoblot. p&amp;lt;0.001; n=6). Chemical inhibition of PARP1 significantly decreased ADPR (Western immunoblot. p&amp;lt;0.001; n=9) but did not prevent myotube formation. To identify if initial ADPR dynamics held consequence for fully differentiated myotubes, cells were treated with a single dose of the PARP1 inhibitor BYK204165 at induction of differentiation. Myotube lysates were collected 5 days later and were subjected to unlabelled SWATH-MS. This measured the abundance of 180 proteins as significantly different in PARP inhibitor-treated cells (n=7). Analyses of these proteins revealed over-representation of pathways governing muscle development (FDR 8.99-08), contraction (FDR 9.47-08, myofiber assembly (FDR 8.06-05), and energy metabolism (FDR 2.93-06). Indicating PARP1-ADPR regulates myogenesis and plays a role in the striated muscle contractile response. ChIP assays for PARP1 binding in cells showed a (30-fold enrichment ±3.2SEM) at GR response elements and detectable ADPR was reduced when exposed to dexamethasone (Western immunoblot n=5). These data support ADPR as responsive to glucocorticoid exposure and PARP1 influences GR recruitment to the genome. RNAseq of siPARP1 (n=4) versus scrambled myoblasts (n=5) identified differential expression of 165 genes. Gene ontology revealed over-representation in siPARP1 cells of those pathways regulating muscle differentiation, including actin-binding, cytoskeletal structure, and NAD+ binding genes. SiRNA of PARP1 had no influence over the ability of glucocorticoids to activate archetype target genes (DUSP1, MURF1, ATROGIN1 &amp; GILZ), but differentially impacted the expression of 434 other genes. Conclusions PARP1 mediated ADPR is critical to the 'early phase' events of muscle cell differentiation. PARP1 influences short-term transcriptional behaviors of the glucocorticoid receptor; in turn, glucocorticoids themselves impact PARP1-genome interaction events. These findings are important for the understanding of general GR mechanisms and have relevance for the widespread functional decline observed during tissue glucocorticoid excess. Presentation: Saturday, June 11, 2022 12:30 p.m. - 12:45 p.m.

Anti-Ribosomal-P Antibody Association with Neuropsychiatric Lupus in Sudanese Patients Attending Rheumatology Clinic in Omdurman Military Hospital.

To assess and establish the relationship between neuropsychiatric systemic lupus erythematosus (NPSLE) involvement and serological biomarkers like antiribosomal-P antibodies. This is an analytical cross-sectional hospital-based study conducted on patients attending Omdurman Military Hospital from July 2019 to December 2019. A total of 90 patients were enrolled, 30 of whom had NPSLE compared with 60 SLE patients without NPSLE. SLE diagnosis was established based on the revised SLICC criteria (presence of at least 4 criteria) for SLE classification, with neuropsychiatric manifestations defined based on the ACR nomenclature. The immunological examination results have been performed by (ELISA immune-enzymatic method, immunofluorescence, and Western immunoblotting test). SPSS v 21.0 software was utilised for data analysis. NPSLE patients exhibited +ve ANA in 96.7% vs 75% in non-NPSLE (P-value = 0.008), antiribosomal-P antibodies (46.7% vs 20%; P-value = 0.0001), anti-nucleosome antibodies (26.7% vs 5%; P-value = 0.005), and anti-histones antibodies (40% vs 20%; P-value = 0.04). ANA antibodies were significantly associated with neurological manifestations as ANA antibodies were common in epilepsy (n = 9; 91%) and stroke (n = 8; 27.6%) (P-value < 0.001). Neuropsychiatric manifestation of systemic lupus erythematosus exhibits variable clinical manifestations. Neuropsychiatric manifestations of SLE are strongly associated with the anti-ribosomal P antibody presence and can be employed as a powerful diagnostic tool.

Peptide Lv augments intermediate-conductance calcium-dependent potassium channels (KCa3.1) in endothelial cells to promote angiogenesis.

Peptide Lv is a small endogenous secretory peptide that is expressed in various tissues and conserved across different species. Patients with diabetic retinopathy, an ocular disease with pathological angiogenesis, have upregulated peptide Lv in their retinas. The pro-angiogenic activity of peptide Lv is in part through promoting vascular endothelial cell (EC) proliferation, migration, and sprouting, but its molecular mechanism is not completely understood. This study aimed to decipher how peptide Lv promotes EC-dependent angiogenesis by using patch-clamp electrophysiological recordings, Western immunoblotting, quantitative PCR, and cell proliferation assays in cultured ECs. Endothelial cells treated with peptide Lv became significantly hyperpolarized, an essential step for EC activation. Treatment with peptide Lv augmented the expression and current densities of the intermediate-conductance calcium-dependent potassium (KCa3.1) channels that contribute to EC hyperpolarization but did not augment other potassium channels. Blocking KCa3.1 attenuated peptide Lv-elicited EC proliferation. These results indicate that peptide Lv-stimulated increases of functional KCa3.1 in ECs contributes to EC activation and EC-dependent angiogenesis.

Inhibition of Ribonucleotide Reductase Subunit 2 (RRM2) Induces Radiosensitization in Gastroenteropancreatic Neuroendocrine Tumors

<h3>Purpose/Objective(s)</h3> Novel radiotherapy, such as Peptide Radionuclide Receptor Therapy, is becoming a milestone treatment for advanced Gastroenteropancreatic neuroendocrine tumors (GEP-NETs). Ribonucleotide Reductase (RnR) subunit M2 (RRM2) activates RnR, the rate-limiting enzyme of DNA synthesis and repair. The purpose of this study was to determine the therapeutic rationale of RnR-targeted therapy and to evaluate 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP) as a radiosensitizer in treating GEP-NETs <i>in vitro</i> and <i>in vivo.</i> <h3>Materials/Methods</h3> (1) RRM2 inhibition was achieved by siRNA knockdown or a RRM2 specific inhibitor, 3-AP, in three GEP-NET cell lines (QGP-1, BON and NT-3) and patient-derived spheroids. Radiation (XRT) was delivered via an X-ray irradiator. Immunohistochemistry confirmed RRM2 expression in cell lines and spheroids, and western immunoblotting was used to examine phosphorylation of DNA repair pathways (DNA-PK, ATM, ATR), cell cycle arrest markers (Rb, Chk1, Chk2) and apoptotic protein (cleaved PARP). Proliferation assays and cell cycle analyses were used to evaluate cytostatic cellular response. Colorimetric survival and DNA fragmentation assays were used to investigate radiosensitization of RRM2 inhibition. (2) For <i>in vivo</i> studies<i>,</i> we utilized an athymic nude mouse GEP-NET xenograft model to assess tumor growth using a combination of 3-AP and XRT. <h3>Results</h3> (1) RRM2 receptor expression, predominantly in the cytoplasm, was noted in all GEP-NET cell lines and spheroids. Knockdown of RRM2 significantly inhibited cellular proliferation at 48 and 72h. Similarly, 3-AP inhibited cellular proliferation with an absolute IC-50 of 1240nM and 1297nM for QGP-1 and BON cells, respectively. Induction of ATM and DNA-PK phosphorylation, but not ATR, was detected with RRM2 inhibition. Importantly, siRNA RRM2 knockdown and 3-AP increased expression of cell cycle arrest markers (pRb, pChk1, and pChk2), and 3-AP (2500nM) arrested cells before entering the G2/M phase, significantly reducing the percentage of G2/M cells from 8.4% to 0.5% at 72h. Combination of RRM2 inhibition with XRT increased apoptosis as noted by increased expression of cleaved PARP. RRM2 inhibition by 3-AP potentiated XRT by reducing 14d cellular survival and inducing DNA fragmentation at 96h post-XRT. (2) We observed a significant decrease in tumor size in mice treated with XRT (2Gy) alone compared to control (146 mm² vs. 280 mm², respectively); combination treatment (2Gy XRT, followed by immediate 3-AP [10mg/kg] i.p. injection) demonstrated a further 48% decrease in tumor size compared to XRT alone. <h3>Conclusion</h3> Our studies demonstrate that inhibition of RRM2 potentiates XRT and results in a significant increase in apoptosis <i>in vitro</i> and a decrease in tumor size <i>in vivo</i>, suggesting enhanced cytotoxicity and radiosensitization. Selective RRM2 inhibition by 3-AP provides a potent therapeutic strategy as a radiosensitizer and offers a novel approach in treating advanced GEP-NETs.

Anti-Inflammatory and Chondro-Protective Effects of Acidic Polysaccharide from Enteromorpha Prolifera in Experimental Models of Osteoarthritis In-Vitro and In-Vivo.

Osteoarthritis (OA) progression has been shown to increase the expression of inflammatory cytokines in joints, leading to the destruction of cartilage matrix. Interleukin (IL)-1β is a potent inflammatory cytokine associated with osteoarthritic synovial fluid. The protective effects of polysaccharides from Enteromorpha prolifera against acute hepatic injury was reported. In this study, we examined the effects of Enteromorpha polysaccharide extracts (EPEs) in the treatment of OA. The effects of the EPEs were assessed using an IL-1β-stimulated SW1353 and SW982 cells. The expression levels of specific mRNA and proteins were evaluated using semi-quantitative reverse transcription polymerase chain reaction (sqRT-PCR) and western immunoblotting. An OA animal study involving C57BL/6J mice was also conducted to assess the effects on tactile sensitivity and anterior cruciate ligament transection (ACLT). Acidic polysaccharide extract (APE) was shown to significantly reduce cytokine and chemokine mRNA levels in IL-1β-stimulated SW1353 and SW982 cells and attenuate the expression of proinflammatory cytokines and p38/AP-1 in SW1353 cells. APE was also shown to minimize the effect of osteolytic lesions in the knee joints of ACLT-induced osteoarthritic mice. APE is a potent inhibitor of joint degeneration associated with OA.

CLL-162 PTX3 is Constitutively Active in CLL Cells

The pentraxin-related PTX3, commonly produced by myeloid and endothelial cells, is a humoral pattern-recognition protein of the innate immune system. Because CLL patients' PTX3 plasma levels are high and most circulating cells in patients with chronic lymphocytic leukemia (CLL) are CLL cells, we reasoned that CLL cells produce PTX3. Western immunoblotting revealed that low-density cells from 7 out of 7 CLL patients produce high levels of PTX3, flow cytometry analysis revealed that the PTX3-producing cells are B lymphocytes co-expressing CD19 and CD5, and confocal microscopy demonstrated that PTX3 co-localized with STAT3. Because signal transducer and activator of transcription (STAT)-3 is constitutively activated in CLL cells and we identified putative STAT3 binding sites within the PTX3 gene promoter, we postulated that phosphorylated STAT3 triggers transcriptional activation of PTX3. Immunoprecipitation analysis of CLL cells' chromatin fragments showed that STAT3 antibodies precipitated PTX3 DNA. STAT3 knockdown induced a marked reduction in PTX3 expression, indicating a STAT3-induced transcriptional activation of the PTX3 gene in CLL cells. Using an electromobility shift assay that we established and a dual-reporter luciferase assay, we confirmed that STAT3 binds the PTX3 gene promoter. Downregulation of PTX3 induced apoptotic disassembly of CLL cells, suggesting that inhibition of PTX3 might benefit patients with CLL.

The Role of 5' Adenosine Monophosphate-Activated Protein Kinase in the Chemo-Sensitivity and Metabolic Behaviour of Breast Cancer Cells Exposed to Hypoxia and Hyperglycaemia

Background: 5' adenosine monophosphate-activated protein kinase (AMPK) is a key enzyme for maintaining energy homeostasis in the cell and is associated with many downstream targets of metabolic processes such as mTORC1, p53 and fatty acid synthase (FASN) and insulin-like growth factor binding protein-2 (IGFBP-2).&#x0D; Aim: To investigate the interactions between AMPK, FASN and IGFBP-2 and how the activity of AMPK affects the metabolism and response of breast cancer cells to chemotherapy with changes in oxygenation and under different glucose concentrations.&#x0D; Methods: MCF-7 breast cancer cells were exposed to different glucose levels (5mM and 25mM) in the presence or absence of doxorubicin under normoxic and hypoxic conditions with and without AMPK silenced using siRNA. Changes in protein abundance were monitored using Western Immunoblotting. Cell death was measured by the Muse® Cell Analyser using a count and viability assay. Hypoxia was chemically induced using cobalt chloride or with low levels of oxygen (2%). Lactate and citrate levels were measured using commercially available kits.&#x0D; Results: In normoxic conditions, AMPK activity was higher in normal levels of glucose (5mM) compared with high levels of glucose (25mM). Under hypoxic conditions, AMPK phosphorylation remained high in 5mM glucose with levels in 25 mM glucose being equivalent. Upregulation of AMPK in normoxic and hypoxic conditions was associated with a reduction in FASN and IGFBP-2, which resulted in a better response to chemotherapy. Moreover, the cells increased the production of lactate and reduced production of citrate under normoxic conditions in 25mM glucose compared to 5mM glucose. Silencing AMPK under normoxic conditions or inducing hypoxia promoted a more glycogenic phenotype. However, silencing AMPK under hypoxic conditions reduced levels of lactate comparable to normoxic levels. The citrate profile was unaffected by silencing AMPK or altering levels of oxygen.&#x0D; Conclusions: AMPK plays an important role in regulating metabolic signalling and this alters the sensitivity of breast cancer cells to chemotherapy.

15-Deoxy-Delta-12,14-prostaglandin J2 modulates pro-labour and pro-inflammatory responses in human myocytes, vaginal and amnion epithelial cells.

BackgroundPrematurity is the leading cause of childhood death under the age of five. The aetiology of preterm birth is multifactorial; however, inflammation and infection are the most common causal factors, supporting a potential role for immunomodulation as a therapeutic strategy. 15-Deoxy-Delta-12,14-prostaglandin J2 (15dPGJ2) is an anti-inflammatory prostaglandin and has been shown to delay lipopolysaccharide (LPS) induced preterm labour in mice and improve pup survival. This study explores the immunomodulatory effect of 15dPGJ2 on the transcription factors NF-κB and AP-1, pro-inflammatory cytokines, and contraction associated proteins in human cultured myocytes, vaginal epithelial cell line (VECs) and primary amnion epithelial cells (AECs).MethodsCells were pre-incubated with 32µM of 15dPGJ2 and stimulated with 1ng/mL of IL-1β as an in vitro model of inflammation. Western immunoblotting was used to detect phosphorylated p-65 and phosphorylated c-Jun as markers of NF-κB and AP-1 activation, respectively. mRNA expression of the pro-inflammatory cytokines IL-6, IL-8, and TNF-α was examined, and protein expression of COX-2 and PGE2 were detected by western immunoblotting and ELISA respectively. Myometrial contractility was examined ex-vivo using a myograph.Results15dPGJ2 inhibited IL-1β-induced activation of NF-κB and AP-1, and expression of IL-6, IL-8, TNF-α, COX-2 and PGE2 in myocytes, with no effect on myometrial contractility or cell viability. Despite inhibiting IL-1β-induced activation of NF-κB, expression of IL-6, TNF-α, and COX-2, 15dPGJ2 led to activation of AP-1, increased production of PGE2 and increased cell death in VECs and AECs.ConclusionWe conclude that 15dPGJ2 has differential effects on inflammatory modulation depending on cell type and is therefore unlikely to be a useful therapeutic agent for the prevention of preterm birth.