Monocytic Leukemia THP-1 Cells Research Articles

Tetranuclear Polypyridylruthenium(II) Complexes as Selective Nucleic Acid Stains for Flow Cytometric Analysis of Monocytic and Epithelial Lung Carcinoma Large Extracellular Vesicles.

Selective staining of extracellular vesicles (EVs) is a major challenge for diagnostic and therapeutic applications. Herein, the EV labeling properties of a new class of tetranuclear polypyridylruthenium(II) complexes, Rubb7-TNL and Rubb7-TL, as phosphorescent stains are described. These new stains have many advantages over standard stains to detect and characterize EVs, including: high specificity for EV staining versus cell staining; high phosphorescence yields; photostability; and a lack of leaching from EVs until incorporation with target cells. As an example of their utility, large EVs released from control (basal) or lipopolysaccharide (LPS)-stimulated THP-1 monocytic leukemia cells were studied as a model of immune system EVs released during bacterial infection. Key findings from EV staining combined with flow cytometry were as follows: (i) LPS-stimulated THP-1 cells generated significantly larger and more numerous large EVs, as compared with those from unstimulated cells; (ii) EVs retained native EV physical properties after staining; and (iii) the new stains selectively differentiated intact large EVs from artificial liposomes, which are models of cell membrane fragments or other lipid-containing debris, as well as distinguished two distinct subpopulations of monocytic EVs within the same experiment, as a result of biochemical differences between unstimulated and LPS-stimulated monocytes. Comparatively, the staining patterns of A549 epithelial lung carcinoma-derived EVs closely resembled those of THP-1 cell line-derived EVs, which highlighted similarities in their selective staining despite their distinct cellular origins. This is consistent with the hypothesis that these new phosphorescent stains target RNA within the EVs.

Mast-cell expressed membrane protein-1 is expressed in classical monocytes and alveolar macrophages in idiopathic pulmonary fibrosis and regulates cell chemotaxis, adhesion, and migration in a TGFβ-dependent manner.

Mast-cell expressed membrane protein-1 (MCEMP1) is higher in patients with idiopathic pulmonary fibrosis (IPF) with an increased risk of death. Here we aimed to establish the mechanistic role of MCEMP1 in pulmonary fibrosis. We identified increased MCEMP1 expression in classical monocytes and alveolar macrophages in IPF compared with controls. MCEMP1 is upregulated by transforming growth factor beta (TGFβ) at the mRNA and protein levels in monocytic leukemia THP-1 cells. TGFβ-mediated MCEMP1 upregulation results from the cooperation of SMAD3 and SP1 via concomitant binding to SMAD3/SP1 cis-regulatory elements within the MCEMP1 promoter. We also found that MCEMP1 regulates TGFβ-mediated monocyte chemotaxis, adhesion, and migration. Our results suggest that MCEMP1 may regulate the migration and transition of monocytes to monocyte-derived alveolar macrophages during pulmonary fibrosis development and progression.NEW & NOTEWORTHY MCEMP1 is highly expressed in circulating classical monocytes and alveolar macrophages in IPF, is regulated by TGFβ, and participates in the chemotaxis, adhesion, and migration of circulating monocytes by modulating the effect of TGFβ in RHO activity.

SARS-CoV-2 E protein-induced THP-1 pyroptosis is reversed by Ruscogenin.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an emerging pathogenic coronavirus, has been reported to cause excessive inflammation and dysfunction in multiple cells and organs, but the underlying mechanisms remain largely unknown. Here we showed exogenous addition of SARS-CoV-2 envelop protein (E protein) potently induced cell death in cultured cell lines, including THP-1 monocytic leukemia cells, endothelial cells, and bronchial epithelial cells, in a time- and concentration-dependent manner. SARS-CoV-2 E protein caused pyroptosis-like cell death in THP-1 and led to GSDMD cleavage. In addition, SARS-CoV-2 E protein upregulated the expression of multiple pro-inflammatory cytokines that may be attributed to activation of NF-κB, JNK and p38 signal pathways. Notably, we identified a natural compound, Ruscogenin, effectively reversed E protein-induced THP-1 death via inhibition of NLRP3 activation and GSDMD cleavage. In conclusion, these findings suggested that Ruscogenin may have beneficial effects on preventing SARS-CoV-2 E protein-induced cell death and might be a promising treatment for the complications of COVID-19.

Abstract 1877: Measurement of cytosolic DNA sensing cGAS-STING pathway functional activity using in vitro phenotypic assay models

Abstract Innate immunity plays a vital role in detecting cytoplasmic nucleic acids resulting from viral infection or the presence of tumor cells. Cytosolic DNA is sensed by the cyclic-GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, and in the case of tumors, activation of this pathway has potential to both positively and negatively modulate cancer development. Activation of the STING pathway is a key prerequisite for type I IFN production that is needed for either endogenous or treatment-induced cancer immune responses, and reduced cGAS-STING expression is associated with poorer survival of patients with lung adenocarcinoma, invasive breast ductal carcinoma, and gastric cancer. Intrinsic activation of the cGAS-STING pathway in the tumor microenvironment (TME) promotes cancer cell-intrinsic senescence and recruitment of immune cells, resulting in antitumor effects. For cancers that are difficult to treat and characterized by immune tolerance, combination therapies of STING agonists with immune checkpoint inhibitors represents a new paradigm in disease management. To date, only two early clinical trials of the STING agonist ADU-S100 in combination with checkpoint inhibitors are underway (NCT02675439 and NCT03172936). To progress clinical trials more rapidly and with better safety outcomes for patients with advanced treatment-refractory metastatic solid tumors or lymphomas, being able to reliably screen a variety of STING agonists in different types of in vitro cancer models is of paramount importance. We have previously reported a novel functional assay that is capable of demonstrating the potency level of different STING agonists, based on the IFNβ response in THP-1 monocytic leukemia cells. We further investigated the role of newly developed STING agonists and clinical compounds in phenotypic in vitro cancer cell and TME models to inform on oncotherapeutic development. STING-expressing cancer cell lines, chosen based on microarray mRNA expression, were treated with agonists and assayed for cGAS-STING activation status using a fully automated platform for high-content imaging. In vitro analysis demonstrated high phospho-STING activation at 4 hours. In the BioMAP® Oncology Colorectal TME model, STING activation increased IL-6 release and the effect on other primary immune and tissue remodeling biomarkers will be discussed. Preclinical studies indicate that STING agonists, used as adjuvants in combination with other agents or radiation therapy, suppress tumor progression, reduce cellular toxicity, and eliminate metastases in breast and pancreatic cancer models. Phenotypic assays that provide human, translational data early in discovery are a valuable tool to accelerate progress in this area. Citation Format: Satheesh K. Sainathan, Justin H. Lipner, Jennifer I. Drake, Brogan A. Epkins, Brianna M. Roux, Alastair J. King. Measurement of cytosolic DNA sensing cGAS-STING pathway functional activity using in vitro phenotypic assay models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1877.

Sweet Bee Venom Triggers Multiple Cell Death Pathways or Spurs Acute Cell Rupture According to Its Concentration in THP-1 Monocytic Leukemia Cells.

Sweet bee venom (sBV) contains various pharmacologically active components of bee venom (BV), but it is modified via the removal of the harmful substances found in BV. Thus, sBV has been used for pain relief in Oriental medicine but has only recently been applied for the treatment of various diseases. In this study, we examined the pharmacological effects and immunomodulatory functions of sBV in THP-1 monocytic leukemia cells. Growth inhibition and cell death were observed according to the concentration of sBV. However, the rapid collapse of cell cycle distribution was shown at 20 μg/mL sBV treatment, indicating that sBV led to cell death or acute cell rupture according to concentration. sBV administration activated Caspase-9, PARP1, RIPK1, and RIPK3, suggesting that the pharmacological actions of sBV were associated with induction of apoptosis and necroptosis. On the other hand, sBV or LPS administration increased cytokine expression, including IL-1β, and showed synergistic cell death in combinatory treatment conditions. Moreover, combinatory administration of sBV and LPS induced severe damage or death during egg development. This result implies that sBV exhibits both pharmacological and toxic effects depending on its concentration. Therefore, sBV might be a promising therapeutic approach, but optimal concentration should be considered before treatment.

Andrographolide, isolated from Andrographis paniculata, induces apoptosis in monocytic leukemia and multiple myeloma cells via augmentation of reactive oxygen species production

Background: Andrographolide (Andro) is a diterpenoid component of the plant Andrographis paniculata that is known for its anti-tumor activity against a variety of cancer cells. Methods: We studied the effects of Andro on the viability of the human leukemia monocytic cell line THP-1 and the human multiple myeloma cell line H929. Andro was compared with cytosine arabinoside (Ara-C) and vincristine (VCR), which are well-established therapeutics against hematopoietic tumors. The importance of reactive oxygen species (ROS) production for the toxicity of each agent was investigated by using an inhibitor of ROS production, N-acetyl-L-cysteine (NAC). Results: Andro reduced the viability of THP-1 and H929 in a dose-dependent manner. H929 viability was highly susceptible to Andro, although only slightly susceptible to Ara-C. The agents Andro, Ara-C, and VCR each induced apoptosis, as shown by cellular shrinkage, DNA fragmentation, and increases in annexin V-binding, caspase-3/7 activity, ROS production, and mitochondrial membrane depolarization. Whereas Ara-C and VCR increased the percentages of cells in the G0/G1 and G2/M phases, respectively, Andro showed little or no detectable effect on cell cycle progression. The apoptotic activities of Andro were largely suppressed by NAC, an inhibitor of ROS production, whereas NAC hardly affected the apoptotic activities of Ara-C and VCR. Conclusions: Andro induces ROS-dependent apoptosis in monocytic leukemia THP-1 and multiple myeloma H929 cells, underlining its potential as a therapeutic agent for treating hematopoietic tumors. The high toxicity for (thus forming: The high toxicity for H929 cells, by a mechanism that is different from that of Ara-C and VCR, is encouraging for further studies on the use of Andro against multiple myeloma.) H929 cells, by a mechanism that is different from that of Ara-C and VCR, is encouraging for further studies on the use of Andro against multiple myeloma.

Andrographolide, isolated from Andrographis paniculata, induces apoptosis in monocytic leukemia and multiple myeloma cells via augmentation of reactive oxygen species production

Background: Andrographolide (Andro) is a diterpenoid component of the plant Andrographis paniculata that is known for its anti-tumor activity against a variety of cancer cells. Methods: We studied the effects of Andro on the viability of the human leukemia monocytic cell line THP-1 and the human multiple myeloma cell line H929. Andro was compared with cytosine arabinoside (Ara-C) and vincristine (VCR), which are well-established therapeutics against hematopoietic tumors. Results: Andro reduced the viability of THP-1 and H929 in a dose-dependent manner. H929 viability was highly susceptible to Andro, although only slightly susceptible to Ara-C. The agents Andro, Ara-C, and VCR each induced apoptosis, as shown by cellular shrinkage, DNA fragmentation, and increases in annexin V-binding, caspase-3/7 activity, reactive oxygen species (ROS) production, and mitochondrial membrane depolarization. The apoptotic activities of Andro were largely suppressed by N-acetyl-L-cysteine (NAC), an inhibitor of ROS production, whereas NAC hardly affected the apoptotic activities of Ara-C and VCR. Furthermore, whereas Ara-C and VCR increased the percentages of cells in the G0/G1 and G2/M phases, respectively, Andro showed little or no detectable effect on cell cycle progression. Conclusions: Andro induces ROS-dependent apoptosis in monocytic leukemia THP-1 and multiple myeloma H929 cells, underlining its potential as a therapeutic agent for treating hematopoietic tumors. Notably, the high sensitivity of H929 cells is encouraging for further studies on the use of Andro against multiple myeloma.

Apolipoprotein M promotes the anti-inflammatory effect of high-density lipoprotein by binding to scavenger receptor BI.

BackgroundInflammation participates pivotally in the pathogenesis of atherosclerosis. Apolipoprotein M (apoM) is a high-density lipoprotein (HDL)-associated plasma protein that affects HDL metabolism and shows various anti-inflammatory functions in atherosclerosis. In this study, we aim to determine whether apoM is expressed in peripheral blood mononuclear cells (PBMCs) and promoted the anti-inflammatory effect of HDL by combing with scavenger receptor BI (SR-BI).MethodsThe expression of apoM in PBMCs is detected by a confocal fluorescence microscope and flow cytometry. The interactions between apoM and SR-BI are detected with co-immunoprecipitation. The multiplexed Luminex xMAP assay detects the inflammatory factors induced by apoM+ HDL and apoM– HDL in inflammatory cell model.ResultsApoM is expressed on CD14+ monocytes, CD3+ T cells, and CD19+ B cells, CD16+ and CD56+ NK cells. CD14+ monocytes have the highest ratio of apoM+ cells. ApoM+ HDL, apoM– HDL, and recombinant apoM protein could be co-precipitated with SR-BI on the surface of human THP-1 monocytic leukemia cells. In vitro, apoM+ HDL induces significantly less expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β than apoM– HDL.ConclusionsApoM was expressed on all PBMCs. ApoM interacted with SR-BI on THP-1. ApoM+ HDL has a more significant anti-inflammatory effect than apoM– HDL.

Antileukemic activity of sulfoxide nutraceutical allicin against THP-1 cells is associated with premature phosphatidylserine exposure in human erythrocytes.

BackgroundAllicin (ACN), a sulfoxide in freshly crushed garlic, is known for its diverse bioactive properties. Among the most notable effects of ACN is its antitumor activity against a wide array of cancer types. Thus, ACN may be a promising anticancer therapeutic. Nevertheless, chemotherapy-induced anemia is a major obstacle in cancer management with a prevalence of up to 70%. Although the pathophysiology behind it remains elusive, a number of medications known to cause anemia in patients have been shown to induce premature programmed cell death in red blood cells (RBCs) known as eryptosis. This study, thus, investigates the anticancer potential of ACN against THP-1 monocytic leukemia cells, its toxic effects on human RBCs, and delineate the underlying biochemical mechanisms.MethodsCytotoxicity was detected using the MTT assay, while hemoglobin leakage was used as a surrogate for hemolysis which was photometrically measured. Major eryptotic events were examined using flow cytometry with fluorescent probes. Phosphatidylserine (PS) exposure was detected by Annexin-V-FITC, cytosolic calcium with Fluo4/AM, and reactive oxygen species with H2DCFDA.ResultsOur results show that ACN induces hemolysis in a dose-dependent fashion, which is significantly abrogated in absence of extracellular calcium. Moreover, ACN stimulates PS exposure, intracellular calcium overload, and oxidative stress. Using small-molecule inhibitors, we demonstrate that the pro-eryptotic activity of ACN is ameliorated in presence of zVAD(OMe)-FMK, SB203580, and D4476.ConclusionACN possesses both hemolytic and eryptotic properties mediated through elevated intracellular calcium levels, oxidative stress, caspase, p38 MAPK, and CK1α.

P4501Identification and characterization of endothelial microRNAs that modulate leukocyte adhesion through novel mechanistic pathways

Abstract Introduction Inflammation is essential for the protective response of the immune system. However, hyperactivated inflammation and dysregulated resolution strongly associates with the pathophysiology of atherosclerosis and ischemia-induced injury after myocardial infarction. Therefore, attenuation of inflammatory response has emerged as a promising approach to reduce cardiovascular disease burden. A limiting step of inflammation is the local recruitment of leukocytes to the lesion, a process regulated by intense cross-talk between immune and endothelial cells. A better understanding of the modulatory mechanisms of adhesion is paramount for the development of better therapies. Purpose Identify endothelial miRNAs that impact leukocyte adhesion and characterize the underlying pathways that regulate this process. Methods A functional high-throughput screening (HTS) of human miRNA libraries (mimics and inhibitors) measured miRNA impact on monocyte (THP-1) adhesion to an endothelial monolayer (HAEC). Individually miRNAs were transfected in HAEC and fluorescently-labeled monocyte attachment was recorded by a robotic automated microscopy platform. Computational analysis lead to identification of potential targets and relevant pathways associated to the action of candidate miRNAs. Further validation of promising targets was performed by qPCR and western blotting. Additional endothelial phenotypic properties such as cytoskeleton morphology or endothelial barrier function were analyzed in the presence of specific miRNAs. Results Functional HTS and secondary screening resulted in 38 microRNAs that reduced and 2 that increased monocyte adhesion. Bioinformatic target prediction and pathway analysis narrowed the set of miRNA candidates used for characterization studies. These miRNAs significantly modulated cell adhesion of both monocytic-leukemia THP-1 cells and freshly isolated human CD14+ monocytes, but effect on CD14+ was weaker compared to THP-1. Several miRNAs induced severe changes on endothelial cell morphology, likely due to cytoskeleton rearrangement. We identified and validated several miRNA targets belonging to the Ras GTPase family of actin remodeling modulators (RalA, RAP1A). Additionally, a few miRNAs targeted Ephrin signaling molecules (EFNs, EPHs) which mediate multiple cell functions including cell-cell contacts. We also explored miRNA effects on endothelial barrier function and measured monocyte adhesion under physiological and disturbed flow conditions. Conclusions We identified a set of miRNAs able to modulate monocyte cell adhesion to endothelial cells under inflammatory conditions. Potential mechanistic pathways of miRNA modulation of adhesion included Ephrin signaling pathway and Ras GTPase family. A better understanding of the role of specific microRNAs regulating the immune-endothelial cell interaction may lead to novel therapeutic strategies in atherosclerosis and myocardial infarction. Acknowledgement/Funding DZHK (German Centre for Cardiovascular Research), partner site Berlin, Germany; Berlin Institute of Health (BIH)

Abstract 6: Computationally assisted target screening of STING agonist for immunologic therapy

Abstract Stimulator of interferon genes (STING) is a receptor protein involved in the propagation of innate immune sensing of cytosolic DNA through the production of IFN-β. Mechanistic studies have shown IFN-β production within the tumor microenvironment can result in activation of tumor antigen-specific CD8+ T-cell immunity that can lead to tumor regression [1, 2]. STING activation by STING agonists should result in innate T-cell mediated anti-tumor immunity in the tumor microenvironment and have significant potential as a cancer therapeutic. Conversely, inhibition of STING would lead to a decreased production of IFN-β which could have implications in the treatment of autoimmune disease such as lupus erythematosus. MD equilibrated crystal structures for human HAQ, REF, and WT alleles were clustered to find optimal conformations for diverse chemical library screening. Novel consensus docking protocols utilizing rigid receptor, induced fit, and quantum polarized ligand docking were applied for quantifying and refining proposed binding mechanisms of STING isoforms. Models for STING agonists and antagonists were developed. From directed virtual screening, a novel low-molecular-weight organic molecule (GF3-002) that is not based on a cyclic dinucleotide was found as a potential STING activator and is currently under investigation. GF3-002 and analogs were synthesized and structures were confirmed with LCMS and proton NMR. Both the HAQ and WT alleles, representing 78.3% of the human population were tested against compounds and controls. 2,3-cGAMP and DMSO were used as positive and negative controls, respectively. Microscale thermophoresis and SPR confirmed binding of GF3-002 to WT STING CTD with a kD of 3.2 ± 1.7 μM. IFN-dependent luciferase expression was measured by luminescence in THP-1 monocytic leukemia cells and found an EC50 of 29 ± 1.6 μM, compared to the native ligand 2,3-cGAMP EC50 of 42 ± 4.1 μM. Finally, qPCR was used quantify production of IFN-β via treatment of dendritic cells with GF3-002. 1. Sali, Characterization of a Novel Human-Specific STING Agonist that Elicits Antiviral Activity Against Emerging Alphaviruses. 2017. 2. Corrales, L., et al., Direct activation of STING in the tumor microenvironment leads to potent and systemic tumor regression and immunity. Cell reports, 2015. 11(7): p. 1018-1030. Citation Format: Grace A. Binder, Christine S. Gambino, Anna Kharitonova, Rainer S. Metcalf, Kenyon G. Daniel, Wayne C. Guida. Computationally assisted target screening of STING agonist for immunologic therapy [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 6.

Extract From Plectranthus amboinicus Inhibit Maturation and Release of Interleukin 1β Through Inhibition of NF-κB Nuclear Translocation and NLRP3 Inflammasome Activation.

Uncontrolled inflammation may produce massive inflammatory cytokines, in which interleukin 1β (IL-1β) plays a key role, resulting in tissue damage and serious disorders. The activation of NLRP3 inflammasome is one of the major mechanisms in maturation and release of IL-1β. Plectranthus amboinicus is a perennial herb. Several pharmacological activities of natural components and crude extracts from P. amboinicus have been reported including anti-inflammation; however, the underlying mechanism is not clear. Phorbol-12-myristate 13-acetate-differentiated THP-1 monocytic leukemia cells were used as a reliable model in this study to examine the effect on inflammasome signaling pathway by PA-F4, an extract from Plectranthus amboinicus. PA-F4 inhibited ATP-induced release of caspase-1, IL-1β, and IL-18 from lipopolysaccharides (LPS)-primed cells. PA-F4 induced a concentration-dependent inhibition of both ASC dimerization and oligomerization in cells under LPS priming plus ATP stimulation. Co-immunoprecipitation of NLRP3 and ASC demonstrated that PA-F4 significantly blunted the interaction between NLRP3 and ASC. Furthermore, PA-F4 completely abolished ATP-induced K+ efflux reaction in LPS-primed cells. Taken together, PA-F4 displayed an inhibitory activity on NLRP3 inflammasome activation. Moreover, PA-F4 also inhibited LPS-induced p65 NF-κB activation, suggesting an inhibitory activity on LPS priming step. Further identification showed that rosmarinic acid, cirsimaritin, salvigenin, and carvacrol, four constituents in PA-F4, inhibited LPS-induced IL-6 release. In contrast, rosmarinic acid, cirsimaritin and carvacrol but not salvigenin inhibited ATP-induced caspase-1 release from LPS-primed cells. In conclusion, PA-F4 displayed an inhibitory activity on activation of NLRP3 inflammasome. PA-F4 inhibited LPS priming step through block of p65 NF-κB activation. It also inhibited ATP-induced signaling pathways in LPS-primed cells including the inhibition of both ASC dimerization and oligomerization, K+ efflux reaction, and the release reaction of caspase-1, IL-1β, and IL-18. Rosmarinic acid, cirsimaritin, salvigenin, and carvacrol could partly explain PA-F4-mediated inhibitory activity on blocking the activation of NLRP3 inflammasome.

Inhibitory Effects of Menadione on Helicobacter pylori Growth and Helicobacter pylori-Induced Inflammation via NF-κB Inhibition.

H. pylori is classified as a group I carcinogen by WHO because of its involvement in gastric cancer development. Several reports have suggested anti-bacterial effects of menadione, although the effect of menadione on major virulence factors of H. pylori and H. pylori-induced inflammation is yet to be elucidated. In this study, therefore, we demonstrated that menadione has anti-H. pylori and anti-inflammatory effects. Menadione inhibited growth of H. pylori reference strains and clinical isolates. Menadione reduced expression of vacA in H. pylori, and translocation of VacA protein into AGS (gastric adenocarcinoma cell) was also decreased by menadione treatment. This result was concordant with decreased apoptosis in AGS cells infected with H. pylori. Moreover, cytotoxin-associated protein A (CagA) translocation into H. pylori-infected AGS cells was also decreased by menadione. Menadione inhibited expression of several type IV secretion system (T4SS) components, including virB2, virB7, virB8, and virB10, that are responsible for translocation of CagA into host cells. In particular, menadione inhibited nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) activation and thereby reduced expression of the proinflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF-α in AGS as well as in THP-1 (monocytic leukemia cell) cell lines. Collectively, these results suggest the anti-bacterial and anti-inflammatory effects of menadione against H. pylori.

Traditional chinese medicine ingredients Rosa damascena and Poria cocos promote phagocytosis and a dendritic cell phenotype in THP-1 cells

Background: Rosa damascena and Poria cocos are ingredients commonly used in Traditional Chinese Medicine. R. damascena is used to promote blood circulation as well as liver and stomach function, while P. cocos is used to eliminate dampness and enhance spleen function. Objective: The objective of the study is to investigate possible mechanisms by which R. damascena and P. cocos may promote immune function. Materials and Methods: Phagocytosis and dendritic cell (DC) surface marker expression assays were used to evaluate the effect of R. damascena and P. cocos extracts on human THP-1 monocytic leukemia cell biology. Results: R. damascena and P. cocos extracts both enhanced phagocytosis of latex beads by THP-1 cells, and when combined, phagocytosis was enhanced to a level greater than what might be expected by adding the individual phagocytosis responses together. In addition, both extracts enhanced maturation of THP-1 cells into a DC phenotype as measured by increased surface expression of the costimulatory molecules CD14, CD40, CD80, and CD86. Conclusion: These results suggest that Rosa damascena and P. cocos may promote monocyte phagocytosis and then stimulate differentiation of the cells into DCs thereby bridging innate and adaptive immune responses. Abbreviations used: AKT: Protein kinase B; AP-1: Activator protein-1; Bcl2: B cell lymphoma-2; CD: Cluster of differentiation; COX-2: Cyclooxygenase-2; DC: Dendritic cell; EGFR: Epidermal growth factor receptor; FOXO1: Forkhead box protein-1; GM-CSF: Granulocyte/macrophage colony stimulating factor; HLA: Human leukocyte antigen; HPLC: High-performance liquid chromatography; IL-1β: Interleukin-1β; IL-4: Interleukin-4; M1: Classically activated macrophage; M2: Alternatively activated macrophage; MAPK: Mitogen-activated protein kinase; NFκB: Nuclear factor kappa-light-chain-enhancer of activated B cells; NLRP3: NLR Family Pyrin Domain Containing 3; ϕ: Phagocytic index; p53: Tumor protein p53; PARP: Poly (ADP-ribose) polymerase; PMA: Phorbol 12-myristate 13-acetate; PRR: Pattern recognition receptor; STAT: Signal transducer and activator of transcription SD: Standard deviation; Syk: Spleen tyrosine kinase; TCM: Traditional Chinese Medicine.

SINCERITIES: inferring gene regulatory networks from time-stamped single cell transcriptional expression profiles.

MotivationSingle cell transcriptional profiling opens up a new avenue in studying the functional role of cell-to-cell variability in physiological processes. The analysis of single cell expression profiles creates new challenges due to the distributive nature of the data and the stochastic dynamics of gene transcription process. The reconstruction of gene regulatory networks (GRNs) using single cell transcriptional profiles is particularly challenging, especially when directed gene-gene relationships are desired.ResultsWe developed SINCERITIES (SINgle CEll Regularized Inference using TIme-stamped Expression profileS) for the inference of GRNs from single cell transcriptional profiles. We focused on time-stamped cross-sectional expression data, commonly generated from transcriptional profiling of single cells collected at multiple time points after cell stimulation. SINCERITIES recovers directed regulatory relationships among genes by employing regularized linear regression (ridge regression), using temporal changes in the distributions of gene expressions. Meanwhile, the modes of the gene regulations (activation and repression) come from partial correlation analyses between pairs of genes. We demonstrated the efficacy of SINCERITIES in inferring GRNs using in silico time-stamped single cell expression data and single cell transcriptional profiles of THP-1 monocytic human leukemia cells. The case studies showed that SINCERITIES could provide accurate GRN predictions, significantly better than other GRN inference algorithms such as TSNI, GENIE3 and JUMP3. Moreover, SINCERITIES has a low computational complexity and is amenable to problems of extremely large dimensionality. Finally, an application of SINCERITIES to single cell expression data of T2EC chicken erythrocytes pointed to BATF as a candidate novel regulator of erythroid development.Availability and implementationMATLAB and R version of SINCERITIES are freely available from the following websites: http://www.cabsel.ethz.ch/tools/sincerities.html and https://github.com/CABSEL/SINCERITIES. The single cell THP-1 and T2EC transcriptional profiles are available from the original publications (Kouno et al., 2013; Richard et al., 2016). The in silico single cell data are available on SINCERITIES websites.Supplementary information Supplementary data are available at Bioinformatics online.

A Flavone Constituent from Myoporum bontioides Induces M-Phase Cell Cycle Arrest of MCF-7 Breast Cancer Cells.

Myoporum bontioides is a traditional medicinal plant in Asia with various biological activities, including anti-inflammatory and anti-bacterial characteristics. To identify the bioactive constituents from M. bontioides, a newly-identified flavone, 3,4′-dimethoxy-3′,5,7-trihydroxyflavone (compound 1), along with eight known compounds, were investigated in human MCF-7 breast cancer, SCC4 oral cancer, and THP-1 monocytic leukemia cells. Among these compounds, compound 1 exhibited the strongest antiproliferative activity with half-maximal inhibitory concentration (IC50) values ranging from 3.3 μM (MCF-7) to 8.6 μM (SCC4). Flow cytometric analysis indicated that compound 1 induced G2/M cell cycle arrest in MCF-7 cells. Mechanistic evidence suggests that the G2/M arrest could be attributable to compound 1’s modulatory effects on the phosphorylation and expression of numerous key signaling effectors, including cell division cycle 2 (CDC2), CDC25C, and p53. Notably, compound 1 downregulated the expression of histone deacetylase 2 (HDAC2) and HDAC4, leading to increased histone H3 acetylation and p21 upregulation. Together, these findings suggest the translational potential of compound 1 as a breast cancer treatment.

1α-Dihydroxyvitamin D3 and Retinoic Acid Increase Nuclear Vitamin D Receptor Expression in Monocytic THP-1 Cells.

1α,25-Dihydroxyvitamin D3 [1,25(OH)2D3] and retinoic acid, such as all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9cRA), are known to induce differentiation of myeloid leukemia cells. Combined treatment effectively enhances the differentiation effect, particularly in monocytic leukemia cells. The underlying mechanism of this combined effect remains unknown. THP-1 monocytic leukemia cells were treated with 1,25(OH)2D3 in combination with 9cRA, ATRA or selective synthetic ligand for retinoic acid receptor (RAR) or retinoid X receptor (RXR), and the nuclear expression and function of vitamin D receptor (VDR) were examined. Combined treatment with 1,25(OH)2D3 and RAR ligand, not RXR ligand, effectively increased nuclear VDR expression and induced expression of the VDR target gene cathelicidin antimicrobial peptide (CAMP) in a gene-selective manner. Combination of 1,25(OH)2D3 plus RAR ligand is effective in induction of nuclear VDR expression and of target gene.

Synthesis and Cytotoxicity of bis(pyrazol-1-yl)-Alkane Derivatives with Polymethylene Linkers and Related Mono- and Dipyrazolium Salts

Reactions of α,ω-dibromoalkanes with pyrazole and 3,5-dimethylpyrazole in superbasic medium of dimethyl sulfoxide – potassium hydroxide were used for the synthesis of bidentate ligands: bis(pyrazol-1-yl)alkanes and bis(3,5-dimethylpyrazol-1-yl)alkanes linked by alkyl chains containing between four and twelve methylene groups. Oxidative iodination of the obtained compounds with I2–HIO3–H2SO4 system in acetic acid provided diiodo derivatives: bis(4-iodopyrazol-1-yl)alkanes and bis(4-iodo-3,5-dimethylpyrazol-1-yl)alkanes. The obtained diiodo derivatives of pyrazole were further converted to mono- and dipyrazolium salts by alkylation of nitrogen atom at position 2 of the pyrazole rings with iodomethane, as well as with methyl triflate. These products are of interest as precursors to mesoionic N-heterocyclic carbene complexes. The cytotoxicity of the obtained compounds was studied against THP-1 monocytic leukemia cells.

IL-32θ inhibits monocytic differentiation of leukemia cells by attenuating expression of transcription factor PU.1.

PU.1 is a key transcription factor regulating the myeloid differentiation. PU.1-induced monocytic differentiation into macrophage is also important for blood cancer development. Therefore, we chose THP-1 monocytic leukemia cells to investigate the function of a recently discovered IL-32θ. Genetic analyses identified differences in the sequences of IL-32θ and IL-32β. Using previously established cell lines that stably express IL-32θ and IL-32β and cell lines transiently expressing IL-32θ, we observed that expression of IL-32θ inhibited phorbol 12-myristate 13-acetate (PMA)-induced monocytic differentiation in both THP-1 and HL-60 cells. IL-32θ also suppressed expression of the macrophage cell surface markers, CD11b, CD18, and CD36. Interestingly, expression of IL-32β or IL-32θ had no effect on the expression levels of cell cycle related factors. As a result, we concluded that these isoforms did not contribute to PMA-induced cell cycle arrest. IL-32θ was found to modulate expression of PU.1, a transcription factor necessary for myeloid lineage commitment. Transient expression of PU.1 in THP-1/IL-32θ cells rescued the observed differentiation defect. Additionally, transient expression of both CCAAT-enhancer-binding protein α (C/EBPα) and PU.1 in THP-1/IL-32θ cells exhibited synergistic effects in rescuing the differentiation defect. These observations indicate that intracellular IL-32θ inhibits the differentiation of monocytes into macrophages by attenuating PU.1 expression.

Abstract 483: EGR1 Mediates Suppression of PKD1 Expression, an Action Antagonized by Pioglitazone in Differentiated Angiotensin II-Treated THP-1 Cells

Aims: Perixosome proliferator-activated receptor γ (PPARγ) agonists have been shown to inhibit Angiotensin II-induced experimental abdominal aortic aneurysms. Since increased macrophage infiltration in the vascular wall is a hallmark for initiation of aneurysmal pathology, we set out to explore the effects of the PPARγ agonist pioglitazone on Angiotensin II-treated, phorbol-ester-differentiated monocytes, with the scope to identify molecular changes that may be responsible for the infiltrating phenotype of macrophages. Approach and Results: Using microarray-based expression profiling of phorbol-ester-stimulated THP-1 monocytic leukemia cells, we found that Angiotensin II (Ang II) or Ang II and pioglitazone were able to cause a number of aneurysm-related gene changes. Among those, polycystic kidney disease 1 (PKD1) was significantly up-regulated. Mutations that lead to changes in PKD1 expression are the main cause of autosomal dominant polycystic kidney disease, which is linked to increased incidence of aneurysms. Online-software analysis of the PKD1 proximal promoter revealed a putative Early Growth Response 1 (EGR1) binding site, which we confirmed by chromatin immuneprecipitation (ChIP) and quantitative real time (RT) PCR. Further analyses of publicly available ChIP-sequencing data (ENCODE project) revealed that this putative binding site in the PKD1 proximal promoter overlapped with a conserved EGR1-binding peak present in 5 other cell lines. Quantitative RT-PCR showed that EGR1 suppressed PKD1, while Ang II significantly up-regulated PKD1, an effect counteracted by pioglitazone. Conversely, in THP-1 cells that were lenti-virally transduced with EGR1 short-hairpin RNA, reduced EGR1 levels led to significant up-regulation of PKD1, especially after treatment with pioglitazone. Conclusion: PKD1 is an EGR1-target gene that is suppressed during monocytic differentiation at the time of EGR1 expression. Ang II treatment up-regulates the expression of PKD1 and pioglitazone mediates the opposite effect by displacement of EGR1 from their shared binding site in the PKD1 proximal promoter through a mechanism that requires further investigation.