Thymidine Incorporation Assay Research Articles

Abstract 15406: Loss of Estrogen Receptor α Inhibits Angiogenic Function in Right Ventricular Endothelial Cells (RVECs)

Introduction: Right ventricular failure (RVF) is the leading cause of death in patients with pulmonary hypertension (PH). While the pathophysiology of RVF is complex and multifactorial, insufficient angiogenesis and impaired RV endothelial cell (RVEC) function have been identified as key contributors. 17β-estradiol (E2), the most abundant female sex steroid, exerts RV-protective effects including stimulation of angiogenesis. However, the mechanism underlying E2’s pro-angiogenic function are unknown. Hypothesis: Estrogen Receptor (ER) α is necessary for RV angiogenesis. Methods: To identify the role of ERα in RVEC function and RV angiogenesis, ERα loss-of-function mutant (ERα Mut ) rats were generated by CRISPR-Cas9. In vivo studies were performed in both male and female wild-type (WT) and ERα Mut rats. RV capillary density was assessed by immunofluorescence. RVECs were isolated from male or female WT and ERα Mut rats. Angiogenic function was evaluated by matrigel tube formation assay at 10h. RVEC proliferation (at 24h) was measured by Incucyte image-based analysis and Thymidine incorporation assay. RVEC migration (at 24h) was examined by transwell migration assay. Angiogenesis mediators in RVECs were quantified via Western Blot. P<0.05 was considered significant. Results: Female WT rats exhibited increased RV capillary density compared to males (p<0.001). However, loss of functioning ERα decreased RV capillary density (p<0.05). Loss of ERα impaired proliferation (24h) capability in female, but not male, RVECs (p<0.001). On the other hand, male, but not female, ERα Mut RVECs exhibited reduced cell migration in response to VEGF-A (p<0.01). Loss of ERα reduced RVEC tube formation in both sexes (p<0.05). At a molecular level, male ERα Mut RVECs expressed less VEGF-A, angiopoietin-2, and Id1 than WT (p<0.01) . Conclusions: ERα is necessary for RVEC angiogenic function. However, ERα effects on angiogenesis are sexually dimorphic. Male ERα Mut RVECs exhibit reduced cell migration potential and less angiogenesis mediators, whereas female ERα Mut RVECs exhibit decreased proliferation potential in vitro and reduced female RV capillary density in vivo . Targeting ERα may allow for sex-specific therapies aimed at enhancing RV adaptation in PH.

Sphingosine-1-phosphate induces airway smooth muscle hyperresponsiveness and proliferation

The emerging role of sphingosine-1-phosphate (S1P) in regulating smooth muscle functions has led to the exploration of the possibility that this sphingolipid could represent a potential therapeutic target in asthma and other lung diseases. Several studies in animal surrogates have suggested a role for S1P-mediated signalling in the regulation of airway smooth muscle contraction, airway hyperresponsiveness and airway remodelling but evidence from human studies is lacking. We have compared the responsiveness of the airways to S1P in healthy and asthmatic individuals in vivo, in isolated human airways ex vivo and in murine airways dissected from healthy and house dust mite (HDM) sensitised animals. Airway responsiveness was measured by spirometry during inhalation challenges and by wire myography in airways isolated from human and mouse lungs. Thymidine incorporation and calcium mobilisation assays were used to study human airway smooth muscle cell responses. S1P did not induce contraction of airways isolated from healthy and HDM exposed mice, nor in human airways. Similarly, there was no airway constriction observed in healthy and asthmatic subjects in response to increasing concentrations of inhaled S1P. However, 30 minutes exposure to S1P induced a significant, concentration-dependent enhancement of airway reactivity to methacholine and to histamine in murine and human airways respectively. HDM-sensitised mice demonstrated a significant increase in methacholine responsiveness which was not further enhanced by S1P treatment. S1P also concentration-dependently enhanced proliferation of human airway smooth muscle cells, an effect mediated through the S1P type 2 receptor, as shown by selective antagonism and S1PR2 siRNA knockdown. Our data suggest that S1P released locally into the airways may be involved in the regulation of airway smooth muscle hyperresponsiveness and hyperplasia, defining a novel target for future therapies.

OP38 Modulation of the septin cytoskeleton ameliorates intestinal fibrogenesis

Abstract Background Fibrosis-induced intestinal strictures leading to obstruction are a common and serious complication of Crohn’s disease (CD) with currently no available specific anti-fibrotic therapies. Cytoskeletal dynamics in mesenchymal cells are associated with organ fibrogenesis. Septin filaments are a novel cytoskeletal element, but the functional role of septins in regulating intestinal inflammation and fibrosis remain unknown. We examined the involvement of the septin cytoskeleton in modulating fibrogenic signaling in intestinal fibrogenesis in vitro and in vivo. Methods Expression of different septins in human intestinal resection tissues, primary human intestinal myofibroblasts (HIMF) and immortalized colonic myofibroblast (CCD 18co cells) were examined by real-time PCR and immunoblotting. Functional inhibition of the septin cytoskeleton was achieved using siRNA for septin 7 and the cell-permeable small molecule forchlorfenuron (FCF). Migration and proliferation of HIMF were examined by wound healing and thymidine incorporation assays, respectively. Levels of secreted cytokines were measured using cytometric bead array. Roles of septins in the development intestinal fibrosis in vivo was studied using a chronic DSS colitis model in mice with administration of FCF or vehicle. Results All septin genes were expressed in intestinal tissues with a predominant expression of septin 7. Pro-fibrotic transforming growth factor-β1 (TGFβ1) triggered HIMF activation manifested by the increased production of ECM proteins such as fibronectin and collagen 1 and enhanced expression of the myofibroblast markers α-smooth muscle actin, L-caldesmon and tropomyosin. Septin 7 knockdown resulted in loss of other septin proteins thereby disrupting the entire septin cytoskeleton. Either pharmacological, or genetic disruption of the septin cytoskeleton reversed the effect of TGFβ1 by attenuating production of ECM proteins and reducing expression of myofibroblast markers. Interestingly, these effects were only found on protein level and were not due to changes in gene expression, suggesting inhibition of their expression at a post-transcriptional level. Moreover, pharmacological or genetic disruptions of the septin cytoskeleton resulted in reduced migration, and attenuated cytokine secretion by activated myofibroblasts. FCF administration did not affect intestinal inflammation, but decreased the fibrosis score during chronic DSS colitis in mice. Conclusion Our data highlights the septin cytoskeleton as a novel regulator of intestinal myofibroblast activation and function. This for the first time implicates the cytoskeleton as an active participant in intestinal fibrosis and as a potential novel target for the development of anti-fibrotic therapies in IBD patients.

Basigin is necessary for normal decidualization of human uterine stromal cells.

Does basigin (BSG) regulate human endometrial stromal cell (HESC) decidualization in vitro? BSG regulates HESCs proliferation and decidualization. Studies have shown that in the human endometrium, BSG expression is menstrual-cycle dependent and its expression was significantly lower in uterine endometrium during the luteal phase of women experiencing multiple implantation failures after IVF than in women with normal fertility. We utilized a telomerase-immortalized HESCs in an in vitro cell culture model system to investigate whether BSG regulates decidualization of stromal cells. Further, we used microarray analysis to identify changes in the gene expression profile of HESCs treated with BSG small interfering RNA (siRNA). All experiments were repeated at least three times. The effect of BSG knockdown (using siRNA) on HESC proliferation was determined by counting cell number and by tritiated thymidine incorporation assays. The effect of BSG on decidualization of HESCs was determined by RT-qPCR for the decidualization markers insulin-like growth factor-binding protein 1 (IGFBP1) and prolactin (PRL). Immunoblotting was used to determine the effect of BSG siRNA on the expression of MMP-2,3. Microarray analysis was used to identify BSG-regulated genes in HESCs at Day 6 of decidualization. Functional and pathway enrichment analyses were then carried out on the differentially expressed genes (DEGs). The STRING online database was used to analyze protein-protein interaction (PPI) between DEG-encoded proteins, and CytoScape software was used to visualize the interaction. MCODE and CytoHubba were used to construct functional modules and screen hub genes separately. Several BSG-regulated genes identified in the microarray analysis were confirmed by qPCR. Knockdown of BSG expression in cultured stromal cells by siRNA significantly (P < 0.05) inhibited HESC proliferation, disrupted cell decidualization and down-regulated MMP-2 and MMP-3 expression. Microarray analysis identified 721 genes that were down-regulated, and 484 genes up-regulated with P < 0.05 in BSG siRNA treated HESCs. GO term enrichment analysis showed that the DEGs were significantly enriched in cell communication, signaling transduction and regulation, response to stimulus, cell adhesion, anatomical structure morphogenesis, extracellular matrix organization, as well as other functional pathways. KEGG pathway analysis identified upregulated gene enriched in pathways such as the MAPK signaling pathway, colorectal cancer, melanoma and axon guidance. In contrast, downregulated genes were mainly enriched in pathways including ECM-receptor interaction, PI3K-Akt signaling pathway, pathways in cancer, antigen processing, type I diabetes mellitus and focal adhesion. The top 10 hub nodes were identified using 12 methods analyses. The hub genes that showed up in two methods were screened out. Among these genes, upregulated genes included EGFR, HSP90AA1, CCND1, PXN, PRKACB, MGAT4A, EVA1A, LGALS1, STC2, HSPA4; downregulated genes included WNT4/5, FOXO1, CDK1, PIK3R1, IGF1, JAK2, LAMB1, ITGAV, HGF, MXRA8, TMEM132A, UBE2C, QSOX1, ERBB2, GNB4, HSP90B1, LAMB2, LAMC1 and ITGA1. Hub genes and module genes involved in the top three modules of PPI analysis were analyzed through the string database. Analysis showed that hub and module genes were related mainly to the WNT signaling pathway, PI3K-AKT signaling pathway and pathways in cancer. The microarray data set generated in this study has been published online at databank.illinois.edu. Most of the findings were obtained using an in vitro cell culture system that may not necessarily reflect in vivo functions. Our results demonstrate that BSG plays a vital role in decidualization and that downregulation of BSG in the uterine endometrium may be associated with infertility in women. The identified hub genes and pathways increase our understanding of the genetic etiology and molecular mechanisms underlying the regulation of decidualization by BSG. This work was supported by the NIH U54 HD40093 (R.A.N.). The authors have no competing interests to declare.

Effects of Nivolumab and Ipilimumab on the suppression of cisplatin resistant small cell lung cancer cells.

Small cell lung cancer (SCLC) accounts for nearly 10-15% of all lung cancer cases. Although many chemotherapy drugs, such as cisplatin and etoposide, were approved as primary therapy for SCLC patients, the prognosis is poor. In this study, we aimed to explore novel therapeutic strategy against SCLC. Two SCLC cell lines, LTEP-P and LTEP-P/DDP1.0, were treated with cisplatin, in the absence or presence of Nivolumab + Ipilimumab combination, and the cell viability was measured. Tumor size and mouse survival rate were examined upon different drug treatments. Protein levels of PD-1 and CTLA4 were detected in normal and SCLC cells by Western blot. Cellular cytotoxicity induced by T lymphocytes was measured by thymidine incorporation assay. Tumor infiltrated T cell populations from LTEP-P and LTEP/DDP1.0 tumor-bearing mice were analyzed by flow cytometry. LTEP-P cells, but not LTEP/DDP1.0 cells, exhibited decreased cell viability upon cisplatin, Nivolumab and Ipilimumab combinational treatment. T lymphocytes significantly inhibited the growth of LTEP-P cells in the presence of nivolumab and ipilimumab. The combinational therapy improved survival rate and inhibited tumor growth in LTEP-P tumor-bearing mice, but showed no effect on LTEP/DDP1.0 tumor-bearing mice. Nivolumab and Ipilimumab synergized with cisplatin in increasing CD8 + and CD4 + T cell population, while decreasing Treg population in LTEP-P tumor-bearing mice. The combinational therapy by cisplatin, Nivolumab and Ipilimumab could be an effective strategy against LTEP-P cells, accompanied with increased cytotoxic T cell populations, but has no significant effect against DDP-resistant lung adenocarcinoma cells.

Tick-Borne Encephalitis Specific Lymphocyte Response after Allogeneic Hematopoietic Stem Cell Transplantation Predicts Humoral Immunity after Vaccination.

The aim of this prospective study was to assess lymphocyte proliferative and cytokine response prior to and following tick-borne encephalitis (TBE) immunization among patients after allogeneic hematopoietic stem cell transplantation (HSCT). Seventeen adult patients 11–13 months after HSCT and eight unvaccinated healthy adults received up to three TBE vaccinations. Following in vitro stimulation with TBE-antigen, lymphocyte proliferation and cytokine secretion (IL-2, IL-10, IL-13, TNF-alpha, IFN-gamma, GM-CSF) were analyzed by thymidine incorporation assay and the Luminex system. Ten patients (59%) showed significant baseline TBE-specific lymphocyte proliferation (stimulation index (SI) > 3) prior to vaccination, but none of the unvaccinated controls (p = 0.002). All patients with a TBE-specific antibody response after two vaccinations (at least 2-fold increase of neutralization test titers) exhibited a strong TBE-specific lymphocyte proliferative response at baseline (SI > 10). Patients with sibling donors had a significantly stronger baseline TBE-specific lymphocyte proliferative and IL-13 cytokine response than patients with unrelated donors (p < 0.05). In conclusion, a relevant proportion of patients showed TBE-specific lymphocyte proliferative and cytokine responses prior to vaccination after HSCT, which predicted the humoral response to the vaccine. Patients with vaccinated sibling donors were more likely to elicit a cellular immune response than patients with unrelated donors of unknown vaccination status.

Targeting Bromodomain and External Domain Epigenetic Reader Protein As Effective Strategy for Double-Hit and Triple-Hit B-Cell Lymphoma

Approximately 12% of diffuse large B cell lymphoma (DLBCL) are double-hit lymphomas (DHL) with translocations in both MYC (MYC/8q24) and BCL2 (14;18) (q32; q21) and/or BCL6 (3q27) and prognosis is generally poor. Triple hit lymphomas (THL) are a subset of DHL exhibiting simultaneous gene translocations of MYC, BCL2 and BCL6, and demonstrate aggressive behavior, worse than that of DHL. Currently, there are no guidelines for the treatment of this group of patients, and disease remains challenging to treat. Bromodomain and external domain (BET) proteins are known as “epigenetic readers” and are enriched at the regions regulating transcription of oncogenes and many BET inhibitors (BETi) are now being clinically evaluated in several cancers.The overall goal of this study was to investigate the effect of BET inhibition through pharmacological inhibitors on single hit, DHL and THL lymphomas. We have performed extensive cytogenetic analysis on 14 human B cell lymphoma cell lines to identify MYC, BCL2 and BCL6 translocations. We identified DOHH2, OCILy1, OCILy10 as DHL, while only VAL represents THL. Raji was identified as single MYC translocation (SHL) and U2932 as WT MYC. Proliferation rate for each cell line was assessed by thymidine incorporation assays in the presence or absence of BET inhibition through pharmacological inhibitors JQ1, IBET762 or OTX015 in WT, SHL, DHL and THL B cells. BET inhibition through each BET inhibitors used individually led to significant (p=0.001) inhibition of cell proliferation in DHL and THL cells or WT- and SH- MYC expressing cells. As expected, BET inhibition led to almost complete inhibition of MYC mRNA and MYC protein in multiple lymphoma cell lines regardless of cytogenetic features. Overexpression of MYC partially rescued the effects of BET inhibition in these cells. Chromatin immunoprecipitation assay was performed to further examine the MYC regulatory mechanisms by BET inhibition. JQ1 treatment reduced BRD4 occupancy on MYC promoter but had no effect on BRD4 occupancy at the promoter region of BCL6. Overall these findings suggest that BET inhibition may suppress proliferation of DHL and THL cells via disruption of transcriptional network led by oncogenic MYC. Interestingly, the effect of BET inhibition on cell survival was modest in all MYC expressing cell lines regardless of their cytogenetic characteristics. Histone acetylation marks are major binding sites for BET proteins. We sought to assess the effect of combining BET inhibitors with histone deacetylase inhibitor vorinostat on proliferation and survival in WT, DHL and THL cells. Combination of sub-lethal doses of IBET and vorinostat decreased proliferation of WT, DHL and THL cells to a greater extent as compared to either drug alone (p<0.0001). IBET/vorinostat combination had no greater effect than IBET or SAHA alone on cell survival or signaling proteins (MCl-1, BCL2 and BIM) in any of the MYC expressing cell lines. These results suggest that combination of BET and/or HDAC pathway can greatly influence cell proliferation but had very limited impact on survival of MYC expressing WT, DHL or THL cells in-vitro. We observed that BET inhibition attenuated BCL6 expression but had no effect on BCL2 expression in DHL and THL expressing B cells. Next we tested the combination of JQ1 and IBET with a BCL2 inhibitor ABT199 (ABT) and assessed survival in THL and DHL cells. ABT alone decreased cell survival in a dose dependent manner, while co-treatment of IBET or JQ1 with ABT significantly suppressed survival as compared to ABT alone (p<0.001). We also evaluated the immunomodulatory activity of the BET inhibitors on the immune checkpoint PD-L1 and CD47, an anti-phagocytic ligand expressed on tumor cells. CD47 surface expression was observed in both DHL and THL cells, however constitutive PD-L1 was only expressed on THL cells; Both CD47 and PD-L1 expression decreased with JQ1 and IBET treatment (p<0.03). These data suggest that BET inhibitors may play an immunomodulatory role in suppressing MYC in the DHL and THL lymphomas.This study shows for the first time the role of BET epigenetic reader protein in DHL and THL aggressive B-cell lymphoma. Using several pharmacological inhibitors, we provide comprehensive mechanistic studies of its action to provide a rationale for the use of BET inhibitor therapy alone or in combination with BCL2 or checkpoint inhibitors for patients with double-hit and triple-hit lymphoma. DisclosuresNo relevant conflicts of interest to declare.

Syntheses of mono-acylated luteolin derivatives, evaluation of their antiproliferative and radical scavenging activities and implications on their oral bioavailability

Luteolin is a flavonoid found in a wide range of plant materials, including commonly eaten fruits and vegetables. It displays a wide range of biological activities but is known to have poor bioavailability. In this study, ten different mono-acyl (nine 5-O-acyl and one 7-O-acyl) derivatives of luteolin were synthesised for the purpose of improving bioactivity and bioavailability, and therefore enhance their therapeutic potential. The antiproliferative activity of these derivatives was assessed against the HCT116 colon cancer and MDA-MB-231 breast cancer cell lines using a 3[H] thymidine incorporation assay. The radical scavenging activity of these derivatives against 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical using Trolox as a standard, was also assessed. Some of these derivatives were found to have improved antiproliferative activity with comparable radical scavenging activity compared to luteolin. Increased lipophilicity has been shown to increase the bioavailability of flavonoids implying these analogues will also have increased bioavailability.

Immune response to dermatomyositis-specific autoantigen, transcriptional intermediary factor 1γ can result in experimental myositis

ObjectivesTo investigate whether autoimmunity to transcriptional intermediary factor 1 (TIF1)γ, a ubiquitous nuclear autoantigen for myositis-specific autoantibodies detected in patients with dermatomyositis (DM) is pathogenetic for inflammatory myopathy.MethodsWild-type, β2-microglobulin-null, perforin-null,...

Mushrooms as potential therapeutic agents in the treatment of cancer: Evaluation of anti-glioma effects of Coprinus comatus, Cantharellus cibarius, Lycoperdon perlatum and Lactarius deliciosus extracts

Cancer incidence rates are on the increase worldwide. The most common brain cancer in adults is glioblastoma. Currently available treatment modalities are limited and natural products such as mushrooms could enhance them. Apart from nutritional value, mushrooms are an excellent source of bioactive compounds and therefore could be used to treat various disorders. The aim of the study was to assess the anti-glioma potential of selected mushrooms on U87MG, LN-18 glioblastoma and SVGp12 normal human astroglial cell lines. The materials were Cantharellus cibarius, Coprinus comatus, Lycoperdon perlatum and Lactarius delicious. Aqueous, 70 % ethanol or 95 % ethanol extracts from mushrooms were used for analysis including assessment of antioxidant activity by DPPH assay, cell viability by MTT assay, DNA biosynthesis by thymidine incorporation assay, activity of metalloproteinase by gelatin zymography and cell cycle assay by flow cytometry. Mushroom extracts influenced the viability and DNA biosynthesis of cancer cells. Activity of ethanol mushroom extracts was stronger than that of aqueous extracts. Anti-glioma mechanism consisted in inhibition of cancer cell proliferation and induction of apoptosis associated with arrest of cells in subG1 or G2/M phase of cell cycle, and inhibition of metalloproteinases activity. Among investigated mushrooms, L. deliciosus and C. comatus showed the greatest anti-glioma potential.

Identification and characterization of antigen-specific CD4+ T cells targeting renally expressed antigens in human lupus nephritis with two independent methods

In the search for anti-renal autoreactivity in human lupus nephritis, we stimulated blood-derived CD4+ T cells from patients with systemic lupus erythematosus with various kidney lysates. Although only minor responses were detectable, these experiments led to the development of a search algorithm that combined autoantibody association with human lupus nephritis and target gene expression in inflamed kidneys. Applying this algorithm, five potential T cell antigens were identified. Blood-derived CD4+ T cells were then stimulated with these antigens. The cells were magnetically enriched prior to measurement with flow cytometry to facilitate the detection of very rare autoantigen-specific cells. The detected responses were dominated by IFN-γ-producing CD4+ T cells. Additionally, IL-10-producing CD4+ T cells were found. In a next step, T cell reactivity to each single antigen was independently evaluated with T cell libraries and [3H]-thymidine incorporation assays. Here, Vimentin and Annexin A2 were identified as the main T cell targets. Finally, Vimentin reactive T cells were also found in the urine of three patients with active disease. Overall, our experiments show that antigen-specific CD4+ T cells targeting renally expressed antigens arise in human lupus nephritis and correlate with disease activity and are mainly of the Th1 subset.

Chemopreventive Action of Garcinia Mangostana Linn. on Hepatic Carcinoma by Modulating Ornithine Decarboxylase Activity

Ornithine decarboxylase and alpha -L- fucosidase over-expression is associated with advanced hepatocellular carcinoma (HCC) development. The objective of this study was to elucidate the action of Garcinia Mangostana fruit extract (GME) on these overexpressed enzymes and apoptotic proteins in diethyl nitrosamine (DEN) induced hepatocellular carcinoma (HCC) rats. The cancer was induced using DEN to the experimental rats and treated with GME (400 mg/ kg) to find its anticancer property. The lysosomal enzymes such as alpha-L-fucosidase, beta- D-glucosidase, ornithine decarboxylase activity (ODC), apoptotic and antiapoptotic proteins such as Bcl-2, Bax and Bcl-xl and H3 thymidine incorporation assay were done to prove GME’s chemo preventive property. DEN induction caused significant increase in the activities of ornithine decarboxylase; lysosomal enzymes and increased cell proliferation with decreased apoptosis were observed. In contrast, the groups with GME treated rat’s elicited significant (P < 0.001) reduction in ornithine decarboxylase, lysosomal enzymes and decreased cell proliferation with increased apoptosis. GME has effective chemo preventive property and can serve as an anticarcinogenic therapeutic drug against hepatocellular carcinoma.

Sasa veitchii extract induces anticancer effects via inhibition of cyclin D1 expression in MCF-7 cells.

ABSTRACT Sasa veitchii and other Sasa species are traditional medicinal herbs belonging to a group of Japanese bamboos collectively called Kumazasa, and these species possess the potential for a wide variety of uses. The present study aimed to elucidate the anticancer mechanisms exerted by S. veitchii extract (SE) against a human breast cancer cell line, MCF-7 cells. Freeze-dried Sunchlon® was used as the SE, and cell proliferation activity was measured using the [3H]-thymidine incorporation assay. Induction of apoptosis was assessed via Annexin V and caspase-3 fluorescent staining, the induction of necrosis was measured via propidium iodide staining, and cell cycle-related protein expression was determined using western blotting. The IC50 value of the SE was 7.7 μg/mL in MCF-7 cells. Although the primary active ingredient in Sunchlon® is sodium copper chlorophyllin (0.25%), the present results indicated that ingredients other than SCC exert anti-cancer activities (the IC50 value of SCC was 715 μg/mL), and late apoptosis or necrosis was induced in an SE dose-dependent manner. The expression levels of cyclin D1 and Cdk6 were decreased after SE treatment, and there was no change in the Cdk1/2 expression levels. Additionally, the expression of the necrosis-related cell death indicators RIP1 and RIP3 was increased in response to high-dose SE treatments, and this was indicative of cells preparing for programmed cell death. SE induces cell death in MCF-7 cells via the inhibition of cyclin D1 expression at low concentrations, and this extract induces programmed necrosis (necroptosis) by potentiating RIP1/RIP3 expression.

Blockade of Pan-Fibroblast Growth Factor Receptors Mediates Bidirectional Effects in Lung Fibrosis.

FGFs (fibroblast growth factors) are major factors associated with the pathogenesis of pulmonary fibrosis. On the one hand, nintedanib, a tyrosine kinase inhibitor targeting several growth factor receptors, including the FGF receptor (FGFR), has been approved for the treatment of idiopathic pulmonary fibrosis. On the other hand, recent reports suggest that FGFs are required for epithelial recovery. In this study, we focused on FGF signaling to both fibroblasts and alveolar epithelial cells (AECs), and we examined the effect of a pan-FGFR blocker on experimental pulmonary fibrosis in mice. The effects of BGJ398, a pan-FGFR inhibitor, on the migration and proliferation of fibroblasts and AECs were assessed using Transwell migration or [3H]thymidine incorporation assays. The expression of FGFR was analyzed using IB or flow cytometry. We also investigated the effect of BGJ398 on pulmonary fibrosis induced by bleomycin in mice. Both lung fibroblasts and AECs expressed FGFRs. BGJ398 significantly inhibited the proliferation and migration of lung fibroblasts stimulated with FGF2. BGJ398 also reduced the proliferation of AECs in response to FGF2. Although the administration of BGJ398 ameliorated pulmonary fibrosis in bleomycin-treated mice, it increased mortality resulting from alveolar injury and inhibition of AEC regeneration. These data suggest that the total inhibition of FGFR signaling can suppress lung fibrosis by inhibiting fibroblast activities, although alveolar injury is simultaneously caused.

Effect of surface modified reduced graphene oxide nanoparticles on cerebellar granule neurons

Graphene has been the cornerstone material in various disciplines owing to its captivating properties. But graphene also exerts some toxic effects apart from its extraordinary characteristics. Various surface functionalizations on graphene are developed in order to overcome the toxicity. In this background, pluronics functionalization of reduced graphene oxide was done as an effort to modify its colloidal stability. Effect of pluronics functionalized reduced graphene oxide (rGO-P) on cerebellar granule neurons (CGNs) was examined to infer on its neurotoxic potential. Male Wister rats were exposed to rGO-P and were analyzed for acute toxicity and toxicokinetic studies. Immunotoxicity was performed on rGO-P exposed female rats on the 7th day of gestation by tritiated thymidine incorporation assay. CGNs were also isolated from rat newborns born to female rats dams exposed with rGO-P and examined for any subsequent toxic reactions. The findings suggest that CGNs isolated from rat newborns pups born to particle exposed female rats dams showed normal cell yield and morphology. Findings from toxicokinetic studies using confocal Raman mapping revealed that rGO-P has got cleared off from the blood within 21 days. Overall results validate rGO-P as safe for in vivo systems. Hence rGO-P can be fine-tuned to serve the purpose of neurobiological applications promisingly for neuroregeneration.

CD4+FOXP3+ T Cells in Rheumatoid Arthritis Bone Marrow Are Partially Impaired.

There is evolving evidence that dysregulation of immune homeostasis in the bone marrow (BM) adjacent to the inflamed joints is involved in the pathogenesis of. In this study, we are addressing the phenotype and function of regulatory T cells (Tregs) residing in the BM of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). BM and peripheral blood samples were obtained from RA and OA patients undergoing hip replacement surgery. The number and phenotype of Tregs were analyzed by flow cytometry and immunohistochemistry. The function of Tregs was investigated ex vivo, addressing their suppressive activity on effector T cells. [3H]-Thymidine incorporation assay and specific enzyme-linked immunosorbent assay were used for quantification of cell proliferation and pro-inflammatory (TNF, IFN-γ) cytokine release, respectively. Significantly lower numbers of CD4+FOXP3+ T cells were found in the BM of patients with RA compared to control patients with OA. High expression of CD127 (IL-7α receptor) and relatively low expression of CXCR4 (receptor for stromal cell-derived factor CXCL12) are characteristics of the CD4+FOXP3+ cells residing in the BM of RA patients. The BM-resident Tregs of RA patients demonstrated a limited suppressive activity on the investigated immune response. Our results indicate that the reduced number and impaired functional properties of CD4+FOXP3+ T cells present in the BM of RA patients may favor the inflammatory process, which is observed in RA BM.

Sclerotium rolfsii lectin induces opposite effects on normal PBMCs and leukemic Molt-4 cells by recognising TF antigen and its variants as receptors.

Sclerotium rolfsii lectin (SRL) exerts apoptotic effect against various cancer cells and an antitumor activity on mice with colon and breast cancer xenografts. The current study aimed to explore its exquisite carbohydrate specificity on human peripheral blood mononuclear cells (PBMCs) and leukemic T-cells. SRL, showed strong binding (>98%) to resting/activated PBMCs, leukemic Molt-4 and Jurkat cell lines. The glycans mediated binding to these cells was effectively blocked by mucin and fetuin, exhibiting 97% and 94% inhibition respectively. SRL showed mitogenic stimulation of PBMCs at 10μg/ml as determined by thymidine incorporation assay. In contrast, lectin induced a dose dependent growth inhibition of Molt-4 cells with 58% inhibition at 25μg/ml. Many common membrane receptors in activated PBMCs, Molt 4 and Jurkat cells were identified by lectin blotting. However, membrane receptors that are recognized by SRL in normal resting PBMCs were totally different and are high molecular weight glycoproteins. Treatment of membrane receptors with glycosidases prior to lectin probing, revealed that fucosylated Thomsen-Friedenreich(TF) antigen glycans are increasingly expressed on transformed Molt-4 leukemic cells compared to other cells. The findings highlight the opposite effects of SRL on transformed and normal hematopoietic cells by recognizing different glycan-receptors. SRL has promising potential for diagnostics and therapeutic applications in leukaemia.

Zn(ii)2,9-dimethyl-1,10-phenanthroline stimulates cultured bovine aortic endothelial cell proliferation.

Vascular endothelial cells cover the luminal surface of blood vessels in a monolayer. Proliferation of these cells is crucial for the repair of damaged endothelial monolayers. In the present study, we identified a zinc complex, Zn(ii)2,9-dimethyl-1,10-phenanthroline (Zn-12), that stimulates the proliferation of bovine aortic endothelial cells in a culture system. No such stimulatory activity was observed for the ligand alone or in combination with other metals; however, the ligand combined with iron weakly stimulated the proliferation, as evidenced by the [3H]thymidine incorporation assay. Inorganic zinc weakly but significantly stimulated proliferation, and intracellular accumulation of zinc was similar between inorganic zinc and Zn-12 treatment, suggesting that the mechanisms by which Zn-12 stimulates vascular endothelial cell proliferation contain processes that differ from those by which inorganic zinc stimulates proliferation. Although expression of endogenous fibroblast growth factor-2 (FGF-2) and its receptor FGFR-1 was unchanged by Zn-12, both siRNA-mediated knockdown of FGF-2 and FGFR inhibition partly but significantly suppressed the stimulation of vascular endothelial cell proliferation by Zn-12, indicating that the zinc complex activates the FGF-2 pathway to stimulate proliferation. Phosphorylation of ERK1/2 and MAPKs was induced by Zn-12, and PD98059, a MEK1 inhibitor, significantly suppressed the stimulatory effect of Zn-12 on vascular endothelial cell proliferation. Therefore, it is suggested that Zn-12 activates the FGF-2 pathway via activation of ERK1/2 signaling to stimulate vascular endothelial cell proliferation, although FGF-2-independent mechanisms are also involved in the stimulation. Zn-12 and related compounds may be promising molecular probes to analyze biological systems of vascular endothelial cells.

NLGP counterbalances the immunosuppressive effect of tumor-associated mesenchymal stem cells to restore effector T cell functions

BackgroundA dynamic interaction between tumor cells and its surrounding stroma promotes the initiation, progression, metastasis, and chemoresistance of solid tumors. Emerging evidences suggest that targeting the stromal events could improve the efficacies of current therapeutics. Within tumor microenvironment (TME), stromal progenitor cells, i.e., MSCs, interact and eventually modulate the biology and functions of cancer and immune cells. Our recent finding disclosed a novel mechanism stating that tumor-associated MSCs inhibit the T cell proliferation and effector functions by blocking cysteine transport to T cells by dendritic cells (DCs), which makes MSCs as a compelling candidate as a therapeutic target. Immunomodulation by nontoxic neem leaf glycoprotein (NLGP) on dysfunctional cancer immunity offers significant therapeutic benefits to murine tumor host; however, its modulation on MSCs and its impact on T cell functions need to be elucidated.MethodsBone marrow-derived primary MSCs or murine 10 T1/2 MSCs were tumor-conditioned (TC-MSCs) and co-cultured with B16 melanoma antigen-specific DCs and MACS purified CD4+ and CD8+ T cells. T cell proliferation of T cells was checked by Ki67-based flow-cytometric and thymidine-incorporation assays. Cytokine secretion was measured by ELISA. The expression of cystathionase in DCs was assessed by RT-PCR. The STAT3/pSTAT3 levels in DCs were assessed by western blot, and STAT3 function was confirmed using specific SiRNA. Solid B16 melanoma tumor growth was monitored following adoptive transfer of conditioned CD8+ T cells.ResultsNLGP possesses an ability to restore anti-tumor T cell functions by modulating TC-MSCs. Supplementation of NLGP in DC-T cell co-culture significantly restored the inhibition in T cell proliferation and IFNγ secretion almost towards normal in the presence of TC-MSCs. Adoptive transfer of NLGP-treated TC-MSC supernatant educated CD8+ T cells in solid B16 melanoma bearing mice resulted in better tumor growth restriction than TC-MSC conditioned CD8+ T cells. NLGP downregulates IL-10 secretion by TC-MSCs, and concomitantly, pSTAT3 expression was downregulated in DCs in the presence of NLGP-treated TC-MSC supernatant. As pSTAT3 negatively regulates cystathionase expression in DCs, NLGP indirectly helps to maintain an almost normal level of cystathionase gene expression in DCs making them able to export sufficient amount of cysteine required for optimum T cell proliferation and effector functions within TME.ConclusionsNLGP could be a prospective immunotherapeutic agent to control the functions and behavior of highly immunosuppressive TC-MSCs providing optimum CD8+ T cell functions to showcase an important new approach that might be effective in overall cancer treatment.

Abstract 1855: Re-purposing traditional Chinese anti-asthma formula ASHMI for triple negative breast cancer

Abstract Breast cancer is the most common cancer among women worldwide, contributing to 25.4% of newly diagnosed cancer cases in 2018. Triple negative breast cancer (TNBC) accounts for 10-20% of the breast cancer cases and represents an unmet clinical need because of its higher risk of recurrence and metastasis. Adjuvant therapies have often been employed to prevent secondary recurrence. Traditional Chinese medicine (TCM) formulations have long been used in prevention and cure of many types of diseases because of its anti-inflammatory property. Mainstream treatment modalities for TNBC involve surgery, radiation and chemotherapy (adjuvant or neoadjuvant). Unfortunately, chemotherapy in these patients often lead to resistance, which underscores the importance of alternative therapeutic approaches for this cancer. To this end, we wanted to evaluate anti-carcinogenic property of a TCM formulation ASHMITM developed primarily for asthma treatment. ASHMI™ is an extract of 3 traditional Chinese medicinal herbs-Ganoderma lucidum (Ling-Zhi), Sophora flavescens Ait (Ku-Shen) and Glycyrrhiza uralensis Fischer (Gan-Gao). For this study, triple-negative/basal B mammary carcinoma cell line MDA-MB157 was treated with two different concentrations of ASHMI and cellular proliferation was examined by [3H] thymidine incorporation assay. ASHMITM treated cells showed approximately 74% less [3H] thymidine incorporation indicating dampened proliferation. This observation was validated using western blot analysis with whole cell lysate, showing reduced expression of proliferation markers PCNA and NFκB. Interestingly, an increased expression of tumor suppressor protein cyclin-dependent kinase inhibitor 1 C (p57, Kip2) was observed after ASHMITM treatment. Next, we tested anti-proliferative property of the individual constituents and observed Ganoderma lucidum (GL) extract had a comparable efficacy as the whole formulation at a concentration of 100μg/ml. This observation was further confirmed by western blot and the same expression pattern was observed for PCNA, NFκB and p57. GL was further fractionated into 11 sub-components and each component was tested for the their anti-proliferative activity using similar assays. Fraction 2 with Ganoderenic acid D showed dose-dependent inhibition of cell proliferation and was further tested for its activity at lower concentrations. Ganoderenic acid D had similar efficiency as 100μg/ml of whole GL extract at a concentration as low as 2.5μg/ml suggesting this active compound might be primarily responsible for the anti-proliferative effect of GL. Our studies using an anti-inflammatory formulation of TCM have identified an active anti-carcinogenic compound, Ganoderenic acid D, that could be potentially used as an adjuvant in TNBC therapies. Citation Format: Sanjukta Chakraborty, Fei Mo, Martin Walsh, Changda Liu, Mingzhuo Cou, Rachana Maniyar, Ghada Ben Rahoma, Sarnath Singh, Tara Jarboe, Michelle Carnazza, Raj Tiwari, Xiu-min Li. Re-purposing traditional Chinese anti-asthma formula ASHMI for triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1855.