Differentiation Of HL-60 Cells Research Articles

LRRK2 is involved in the chemotaxis of neutrophils and differentiated HL-60 cells, and the inhibition of LRRK2 kinase activity increases fMLP-induced chemotactic activity

BackgroundNeutrophils depend heavily on glycolysis for energy production under normal conditions. In contrast, neutrophils require energy supplied by mitochondrial oxidative phosphorylation (OXPHOS) during chemotaxis. However, the mechanism by which the energy supply changes from glycolysis to OXPHOS remains unknown. Leucine-rich repeat kinase 2 (LRRK2) is partially present in the outer mitochondrial membrane fraction. Lrrk2-deficient cells show mitochondrial fragmentation and reduced OXPHOS activity. We have previously reported that mitofusin (MFN) 2 is involved in chemotaxis and OXPHOS activation upon chemoattractant N-formyl-Met-Leu-Phe (fMLP) stimulation in differentiated HL-60 (dHL-60) cells. It has been previously reported that LRRK2 binds to MFN2 and partially colocalizes with MFN2 at the mitochondrial membranes. This study investigated the involvement of LRRK2 in chemotaxis and MFN2 activation in neutrophils and dHL-60 cells.MethodsLrrk2 knockout neutrophils and Lrrk2 knockdown dHL-60 cells were used to examine the possible involvement of LRRK2 in chemotaxis. Lrrk2 knockdown dHL-60 cells were used a tetracycline-inducible small hairpin RNA (shRNA) system to minimize the effects of LRRK2 knockdown during cell culture. The relationship between LRRK2 and MFN2 was investigated by measuring the GTP-binding activity of MFN2 in Lrrk2 knockdown dHL-60 cells. The effects of LRRK2 kinase activity on chemotaxis were examined using the LRRK2 kinase inhibitor MLi-2.ResultsfMLP-induced chemotactic activity was reduced in Lrrk2 knockout neutrophils in vitro and in vivo. Lrrk2 knockdown in dHL-60 cells expressing Lrrk2 shRNA also reduced fMLP-induced chemotactic activity. Lrrk2 knockdown dHL-60 cells showed reduced OXPHOS activity and suppressed mitochondrial morphological change, similar to Mfn2 knockdown dHL-60 cells. The amount of LRRK2 in the mitochondrial fraction and the GTP-binding activity of MFN2 increased upon fMLP stimulation, and the MFN2 GTP-binding activity was suppressed in Lrrk2 knockdown dHL-60 cells. Furthermore, the kinase activity of LRRK2 and Ser935 phosphorylation of LRRK2 were reduced upon fMLP stimulation, and LRRK2 kinase inhibition by MLi-2 increased the migration to fMLP.ConclusionsLRRK2 is involved in neutrophil chemotaxis and the GTP-binding activity of MFN2 upon fMLP stimulation. On the other hand, the kinase activity of LRRK2 shows a negative regulatory effect on fMLP-induced chemotactic activity in dHL-60 cells.-J661noP-dBkFUp6aS7aWrVideo

Signaling dynamics distinguish high- and low-priority neutrophil chemoattractant receptors.

Human neutrophils respond to multiple chemoattractants to guide their migration from the vasculature to sites of infection and injury, where they clear pathogens and amplify inflammation. To properly focus their responses during this complex navigation, neutrophils prioritize pathogen- and injury-derived signals over long-range inflammatory signals, such as the leukotriene LTB4, secreted by host cells. Different chemoattractants can also drive qualitatively different modes of migration even though their receptors couple to the same Gαi family of G proteins. Here, we used live-cell imaging to demonstrate that the responses differed in their signaling dynamics. Low-priority chemoattractants caused transient responses, whereas responses to high-priority chemoattractants were sustained. We observed this difference in both primary neutrophils and differentiated HL-60 cells, for downstream signaling mediated by Ca2+, a major regulator of secretion, and Cdc42, a primary regulator of polarity and cell steering. The rapid attenuation of Cdc42 activation in response to LTB4 depended on the phosphorylation sites Thr308 and Ser310 in the carboxyl-terminal tail of its receptor LTB4R in a manner independent of endocytosis. Mutation of these residues to alanine impaired chemoattractant prioritization, although it did not affect chemoattractant-dependent differences in migration persistence. Our results indicate that distinct temporal regulation of shared signaling pathways distinguishes between receptors and contributes to chemoattractant prioritization.

Novel NADPH Oxidase-2 Inhibitors as Potential Anti-Inflammatory and Neuroprotective Agents.

A family of seven NADPH oxidase enzymes (Nox1-5, Duox1-2) has been implicated in a variety of diseases, including inflammatory lung diseases, neurodegenerative diseases, cardiovascular diseases, and cancer. Here, we report the results of our studies aimed at developing novel brain-permeable Nox2 inhibitors with potential application as neuroprotective agents. Using cell-based assays, we identified a novel Nox2 inhibitor, TG15-132, that prevents PMA-stimulated oxygen consumption and reactive oxygen species (superoxide radical anion and hydrogen peroxide) formation upon acute treatment in differentiated HL60 cells. Long-term treatment with TG15-132 attenuates the induction of genes encoding Nox2 subunits, several inflammatory cytokines, and iNOS in differentiated THP-1 cells. Moreover, TG15-132 shows a relatively long plasma half-life (5.6 h) and excellent brain permeability, with a brain-to-plasma ratio (>5-fold) in rodent models. Additionally, TG15-132 does not cause any toxic effects on vital organs or blood biomarkers of toxicity in mice upon chronic dosing for seven days. We propose that TG15-132 may be used as a Nox2 inhibitor and a potential neuroprotective agent, with possible further structural modifications to increase its potency.

Artemisia gmelinii Extract Attenuates Particulate Matter-Induced Neutrophilic Inflammation in a Mouse Model of Lung Injury.

Particulate matter (PM) induces and augments oxidative stress and inflammation, leading to respiratory diseases. Although Artemisia gmelinii Weber ex Stechm has antioxidant and anti-inflammatory effects, there are no reports on whether Artemisia gmelinii extract (AGE) regulates lung inflammation in a PM-induced model. Thus, we investigated the protective effects of AGE using a PM-induced mouse lung inflammation model. AGE significantly decreased the expression of inflammatory chemokines, neutrophil extracellular trap formation, and the total number of inflammatory cells in the bronchoalveolar lavage fluid (BALF). Furthermore, AGE attenuated lung inflammation through the suppression of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)/mitogen-activated protein kinase (MAPK) signaling pathway, while promoting the nuclear factor erythroid-2-related factor 2 (NRF2)/heme oxygenase-1 (HO-1) signaling pathway in lung tissues. Concordant with these observations, AGE suppressed inflammatory cytokines, chemokines, reactive oxygen species, NETosis, myeloperoxidase, and neutrophil elastase by decreasing the mRNA expression of High mobility group box 1, Runt-related transcription factor 1, and Kruppel-like factor 6 in differentiated HL-60 cells. In summary, our data demonstrated that AGE suppresses PM-induced neutrophil infiltration, lung damage, and pulmonary inflammation by suppressing NF-κB/MAPK signaling pathways and enhancing the NRF2/HO-1 signaling pathway. These findings suggest that AGE administration is an effective approach for preventing and treating PM-induced respiratory inflammation.

Unripe Rubus occidentalis, Ellagic Acid, and Urolithin A Attenuate Inflammatory Responses in IL-1β-Stimulated A549 Cells and PMA-Stimulated Differentiated HL-60 Cells.

Unripe Rubus occidentalis (uRO) contains various natural polyphenols with beneficial physiological activities and is particularly rich in ellagic acid (EA). EA has ameliorated type 2 inflammation and airway hyperresponsiveness in animal models of eosinophilic asthma. EA is metabolized by the gut microbiota to urolithin A (UA), which exhibits anti-inflammatory properties. However, it remains unclear whether uRO, EA, and UA reduce inflammatory responses and oxidative stress in respiratory epithelial cells and neutrophils. In this study, inflammation was induced in A549 (human lung epithelial cells) and dHL-60 cells (neutrophil-like cells differentiated from human promyelocytic leukemia HL-60 cells) and treated with various concentrations of water extract of uRO (uRO-w), EA, and UA. EA, uRO-w and UA suppressed the inflammatory cytokine and chemokine levels and reduced the expression of matrix metalloproteinase-9 in A549 cells stimulated with IL-1β. As a result of analyzing the mechanism by which these inflammatory molecules are expressed, it was found that EA, uRO-w, and UA regulated corticosteroid-sensitive mitogen activated protein kinase, nuclear factor κB, and corticosteroid-insensitive AKT. In addition, uRO-w, EA, and UA significantly reduced reactive oxygen species levels in phorbol 12-myristate 13-acetate-stimulated dHL-60 cells and inhibited neutrophil extracellular trap formation. Therefore, our results suggest that uRO-w, EA, and UA are potential therapeutic agents for preventing and treating inflammatory respiratory diseases.

Peptides Derived from Soybean β-Conglycinin Induce the Migration of Human Peripheral Polymorphonuclear Leukocytes.

Food-derived peptides have various biological activities. When food proteins are ingested orally, they are digested into peptides by endogenous digestive enzymes and absorbed by the immune cell-rich intestinal tract. However, little is known about the effects of food-derived peptides on the motility of human immune cells. In this study, we aimed to understand the effects of peptides derived from a soybean protein β-conglycinin on the motility of human peripheral polymorphonuclear leukocytes. We illustrated that MITL and MITLAIPVNKPGR, produced by digestion using in-vivo enzymes (trypsin and pancreatic elastase) of β-conglycinin, induces the migration of dibutyryl cAMP (Bt2 cAMP)-differentiated human promyelocytic leukemia 60 (HL-60) cells and human polymorphonuclear leukocytes in a dose- and time-dependent manner. This migration was more pronounced in Bt2 cAMP-differentiated HL-60 cells; mRNA expression of formyl peptide receptor (FPR) 1 increased significantly than in all-trans-retinoic acid (ATRA)-differentiated HL-60 cells. This migration was inhibited by tert-butoxycarbonyl (Boc)-MLP, an inhibitor of FPR, and by pretreatment with pertussis toxin (PTX). However, the effect was weak when treated with WRW4, a selective inhibitor of the FPR2. We then demonstrated that MITLAIPVNKPGR induced intracellular calcium responses in human polymorphonuclear leukocytes and Bt2 cAMP-HL60 cells. Furthermore, pre-treatment by fMLP desensitized the calcium response of MITLAIPVNKPGR in these cells. From the above, MITLAIPVNKPGR and MITL derived from soybean β-conglycinin induced polymorphonuclear leukocyte migration via the FPR1-dependent mechanism. We found chemotactic peptides to human polymorphonuclear leukocytes, which are the endogenous enzyme digests of soybean protein.

PKM2/STAT1-mediated PD-L1 upregulation on neutrophils during sepsis promotes neutrophil organ accumulation by serving an anti-apoptotic role

BackgroundDelayed neutrophil apoptosis during sepsis may impact neutrophil organ accumulation and tissue immune homeostasis. Elucidating the mechanisms underlying neutrophil apoptosis may help identify potential therapeutic targets. Glycolysis is critical to neutrophil activities during sepsis. However, the precise mechanisms through which glycolysis regulates neutrophil physiology remain under-explored, especially those involving the non-metabolic functions of glycolytic enzymes. In the present study, the impact of programmed death ligand-1 (PD-L1) on neutrophil apoptosis was explored. The regulatory effect of the glycolytic enzyme, pyruvate kinase M2 (PKM2), whose role in septic neutrophils remains unaddressed, on neutrophil PD-L1 expression was also explored.MethodsPeripheral blood neutrophils were isolated from patients with sepsis and healthy controls. PD-L1 and PKM2 levels were determined by flow cytometry and Western blotting, respectively. Dimethyl sulfoxide (DMSO)-differentiated HL-60 cells were stimulated with lipopolysaccharide (LPS) as an in vitro simulation of septic neutrophils. Cell apoptosis was assessed by annexin V/propidium iodide (annexin V/PI) staining, as well as determination of protein levels of cleaved caspase-3 and myeloid cell leukemia-1 (Mcl-1) by Western blotting. An in vivo model of sepsis was constructed by intraperitoneal injection of LPS (5 mg/kg) for 16 h. Pulmonary and hepatic neutrophil infiltration was assessed by flow cytometry or immunohistochemistry.ResultsPD-L1 level was elevated on neutrophils under septic conditions. Administration of neutralizing antibodies against PD-L1 partially reversed the inhibitory effect of LPS on neutrophil apoptosis. Neutrophil infiltration into the lung and liver was also reduced in PD-L1−/− mice 16 h after sepsis induction. PKM2 was upregulated in septic neutrophils and promoted neutrophil PD-L1 expression both in vitro and in vivo. In addition, PKM2 nuclear translocation was increased after LPS stimulation, which promoted PD-L1 expression by directly interacting with and activating signal transducer and activator of transcription 1 (STAT1). Inhibition of PKM2 activity or STAT1 activation also led to increased neutrophil apoptosis.ConclusionIn this study, a PKM2/STAT1-mediated upregulation of PD-L1 on neutrophils and the anti-apoptotic effect of upregulated PD-L1 on neutrophils during sepsis were identified, which may result in increased pulmonary and hepatic neutrophil accumulation. These findings suggest that PKM2 and PD-L1 could serve as potential therapeutic targets.

ATRA treatment slowed P-selectin-mediated rolling of flowing HL60 cells in a mechano-chemical-dependent manner.

All-trans retinoic acid (ATRA)-induced differentiation of acute promyelocytic leukemia (APL) toward granulocytes may trigger APL differentiation syndrome (DS), but there is less knowledge about the mechano-chemical regulation mechanism of APL DS under the mechano-microenvironment. We found that ATRA-induced changes in proliferation, morphology, and adhesive molecule expression levels were either dose or stimulus time dependent. An optimal ATRA stimulus condition for differentiating HL60 cells toward neutrophils consisted of 1 × 10-6 M dose and 120h of stimulus time. Under wall shear stresses, catch-slip bond transition governs P-selectin-mediated rolling for neutrophils and untreated or ATRA-treated (1 × 10-6 M, 120h) HL60 cells. The ATRA stimuli slowed down the rolling of HL60 cells on immobilized P-selectin no matter whether ICAM-1 was engaged. The β2 integrin near the PSGL-1/P-selectin axis would be activated within sub-seconds for each cell group mentioned above, thus contributing to slow rolling. A faster β2 integrin activation rate and the higher expression levels of PSGL-1 and LFA-1 were assigned to induce the over-enhancement of ATRA-treated HL60 adhesion in flow, causing APL DS development. These findings provided an insight into the mechanical-chemical regulation for APL DS development via ATRA treatment of leukemia and a novel therapeutic strategy for APL DS through targeting the relevant adhesion molecules.

Effects of Garlicin on Proliferation and Differentiation of HL-60 Cells

We have explored the effect of garlicin, a biologically active compound isolated from garlic, on the proliferation and differentiation of HL-60 leukemia cells. To this end, we measured the effect of garlicin on cell proliferation, cell morphology, cell cycle, and the expression cluster of differentiation molecules 11b and 15, B-cell lymphoma 2 protein, and BCL2-associated X proteins. All-trans retinoic acid and arsenic trioxide treatments served as positive controls for comparison. Results show that garlicin significantly inhibited the proliferation of HL-60 cells and blocked most cells in the G0/G1 phase, with an effect stronger than all-trans retinoic acid or arsenic trioxide treatments. The ability to induce cell maturation and differentiation was like that of all-trans retinoic acid, which may play a role by downregulating the expression of B-cell lymphoma 2 protein and upregulating the expression of BCL2-associated X protein. In conclusion, garlicin significantly inhibits the proliferation and induces differentiation of HL-60 cells, and thus it may become a new clinically viable differentiation-inducing agent.

MP34-15 THE ROLE OF FERROPTOSIS IN NEUTROPHILS-MEDIATED INNATE IMMUNE DEFENSE AGAINST URINARY TRACT INFECTION

You have accessJournal of UrologyCME1 Apr 2023MP34-15 THE ROLE OF FERROPTOSIS IN NEUTROPHILS-MEDIATED INNATE IMMUNE DEFENSE AGAINST URINARY TRACT INFECTION Yan Liu, Hongying Huang, Herbert Lepor, Xue-Ru Wu, and Ellen Shapiro Yan LiuYan Liu More articles by this author , Hongying HuangHongying Huang More articles by this author , Herbert LeporHerbert Lepor More articles by this author , Xue-Ru WuXue-Ru Wu More articles by this author , and Ellen ShapiroEllen Shapiro More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003268.15AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Ferroptosis is a recently discovered programmed cell death involved in disorders including infectious disease, autoimmune disease, and cancer. It is characterized by iron accumulation and reactive oxygen species (ROS)-induced lipid peroxidation. Neutrophils are the first line of innate defense against urinary tract infection (UTI), and their interaction with uropathogenic E.coli (UPEC) triggers generation of ROS and leads to cell death. However, whether UPECs induce ferroptosis in neutrophils remains elusive. This study aims to elucidate the role of ferroptosis in neutrophils-mediated host defense against UTI. METHODS: Human myeloblastic cells HL-60 were induced to terminally differentiated neutrophils by 1.5% DMSO for 72 hours. The differentiated cells were challenged by type 1 piliated UPEC strains (UTI89, NU14), isogenic FimH adhesin deletion strain (NU14-1), or non-fimbriated laboratory strain (HB101) at a multiplicity of infection of 40. ROS level was examined by H2DCFDA cellular ROS assay. Iron level was measured by Phen Green FL diacetate assay. Cell membrane integrity was checked by DiO cell labeling. Lipid peroxidation was detected by cell-based lipid peroxidation assay. The cells were exposed to UPEC virulent factors lipopolysaccharide (LPS) and flagellin. HL-60 cells pretreated with or without the ferroptosis inhibitor ferrostatin-1 were infected, and cell suspensions were lysed, diluted, and plated to assess viable UPECs. RESULTS: Type 1 piliated UPEC strains UTI89 and NU14 induced a rapid elevation in ROS and iron accumulation in differentiated HL-60 cells in a dose- and time-dependent manner and accompanied by lipid peroxidation with multiple perforations on the membrane, all of which are hallmarks of ferroptosis. On the contrary, pretreatment of HL-60 cells with ferroptosis inhibitor ferrostatin-1 reduced 20% of UPEC-induced ROS production, alleviated lipid peroxidation, and led to a 36% higher number of viable UPECs compared to the controls. Furthermore, FimH deletion isogenic strain NU14-1 and non-fimbriated laboratory strain HB101 did not elicit activation of ferroptosis in HL-60 cells, indicating FimH-mediated adherence is required for inducing ferroptosis. Lastly, UPEC virulent factors LPS or flagellin alone did not evoke ferroptosis. CONCLUSIONS: Our data demonstrate for the first time that UPECs induce ferroptosis in neutrophils, and the induction depends on the adhesion of UPECs to the cell surface. The results also suggest that enhancing ferroptosis holds the therapeutic potential for improved clearance of UPECs. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e464 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yan Liu More articles by this author Hongying Huang More articles by this author Herbert Lepor More articles by this author Xue-Ru Wu More articles by this author Ellen Shapiro More articles by this author Expand All Advertisement PDF downloadLoading ...

Retinoic Acid-Induced HL-60 Cell Differentiation Is Augmented by Copper Supplementation1–4

The objective of this study was to determine how copper influences the ability of HL-60 cells to differentiate into cells of the granulocytic lineage. We hypothesized that granulopoiesis requires copper because copper-deficient humans become neutropenic. Differentiation of HL-60 cells along the granulocytic lineage with retinoic acid was enhanced by copper. The results showed a greater number of cells were more differentiated when copper was added to the medium for 96 h. The respiratory burst activity of retinoic acid-induced cells was increased by copper supplementation, but intracellular superoxide anion generation was not affected. Supplementation with copper resulted in more cell-associated copper in both noninduced and induced cells; however, the induced cells accumulated three times more copper than the noninduced cells. Even though the amount of copper associated with retinoic acid-treated cells was greater than in untreated cells, the activity of a copper-requiring enzyme, copper/zinc superoxide dismutase, was significantly lower. Copper supplementation increased the activity of this enzyme in both retinoic acid-treated and untreated cells. Cytochrome c oxidase activity was not affected by retinoic acid treatment or by copper supplementation. Copper seems to play a specific role during the early stages of granulocyte differentiation.

Diallyl disulfide downregulating RhoGDI2 induces differentiation and inhibit invasion via the Rac1/Pak1/LIMK1 pathway in human leukemia HL-60 cells.

Leukemia is a type of disease in which hematopoietic stem cells proliferate clonally at the genetic level. We discovered previously by high-resolution mass spectrometry that diallyl disulfide (DADS), which is one of the effective ingredients of garlic, reduces the performance of RhoGDI2 from APL HL-60 cells. Although RhoGDI2 is oversubscribed in several cancer categories, the effect of RhoGDI2 in HL-60 cells has remained unexplained. We aimed to investigate the influence of RhoGDI2 on DADS-induced differentiation of HL-60 cells to elucidate the association among the effect of inhibition or over-expression of RhoGDI2 with HL-60 cell polarization, migration and invasion, which is important for establishing a novel generation of inducers to elicit leukemia cell polarization. Co-transfection with RhoGDI2-targeted miRNAs apparently decreases the malignant biological behavior of cells and upregulates cytopenias in DADS-treated HL-60 cell lines, which increases CD11b and decreases CD33 and mRNA levels of Rac1, PAK1 and LIMK1. Meanwhile, we generated HL-60 cell lines with high-expressing RhoGDI2. The proliferation, migration and invasion capacity of such cells were significantly increased by the treated with DADS, while the reduction capacity of the cells was decreased. There was a reduction in CD11b and an increase in CD33 production, as well as an increase in the mRNA levels of Rac1, PAK1 and LIMK1. It also confirmed that inhibition of RhoGDI2 attenuates the EMT cascade via the Rac1/Pak1/LIMK1 pathway, thereby inhibiting the malignant biological behavior of HL-60 cells. Thus, we considered that inhibition of RhoGDI2 expression might be a new therapeutic direction for the treatment of human promyelocytic leukemia. The anti-cancer property of DADS against HL-60 leukemia cells might be regulated by RhoGDI2 through the Rac1-Pak1-LIMK1 pathway, which provides new evidence for DADS as a clinical anti-cancer medicine.

Hydrogen Sulfide Downregulates Oncostatin M Expression via PI3K/Akt/NF-κB Signaling Processes in Neutrophil-like Differentiated HL-60 Cells

The cytokine oncostatin M (OSM) is regarded as a critical mediator in various inflammatory responses. While the gaseous signaling molecule hydrogen sulfide (H2S) plays a role in a variety of pathophysiological conditions, such as hypertension, inflammatory pain, osteoarthritis, ischemic stroke, oxidative stress, retinal degeneration, and inflammatory responses, the underlying mechanism of H2S action on OSM expression in neutrophils needs to be clarified. In this work, we studied how H2S reduces OSM expression in neutrophil-like differentiated (d)HL-60 cells. To evaluate the effects of H2S, sodium hydrosulfide (NaHS, a donor that produces H2S), ELISA, real-time PCR (qPCR), immunoblotting, and immunofluorescence staining were utilized. Although exposure to granulocyte-macrophage colony-stimulating factor (GM-CSF) resulted in upregulated levels of production and mRNA expression of OSM, these upregulated levels were reduced by pretreatment with NaHS in dHL-60 cells. Similarly, the same pretreatment lowered phosphorylated levels of phosphatidylinositol 3-kinase, Akt, and nuclear factor-kB that had been elevated by stimulation with GM-CSF. Overall, our results indicated that H2S could be a therapeutic agent for inflammatory disorders via suppression of OSM.

Novel Insights into the Role of Kras in Myeloid Differentiation: Engaging with Wnt/β-Catenin Signaling.

Cells of the HL-60 myeloid leukemia cell line can be differentiated into neutrophil-like cells by treatment with dimethyl sulfoxide (DMSO). The molecular mechanisms involved in this differentiation process, however, remain unclear. This review focuses on the differentiation of HL-60 cells. Although the Ras proteins, a group of small GTP-binding proteins, are ubiquitously expressed and highly homologous, each has specific molecular functions. Kras was shown to be essential for normal mouse development, whereas Hras and Nras are not. Kras knockout mice develop profound hematopoietic defects, indicating that Kras is required for hematopoiesis in adults. The Wnt/β-catenin signaling pathway plays a crucial role in regulating the homeostasis of hematopoietic cells. The protein β-catenin is a key player in the Wnt/β-catenin signaling pathway. A great deal of evidence shows that the Wnt/β-catenin signaling pathway is deregulated in malignant tumors, including hematological malignancies. Wild-type Kras acts as a tumor suppressor during DMSO-induced differentiation of HL-60 cells. Upon DMSO treatment, Kras translocates to the plasma membrane, and its activity is enhanced. Inhibition of Kras attenuates CD11b expression. DMSO also elevates levels of GSK3β phosphorylation, resulting in the release of unphosphorylated β-catenin from the β-catenin destruction complex and its accumulation in the cytoplasm. The accumulated β-catenin subsequently translocates into the nucleus. Inhibition of Kras attenuates Lef/Tcf-sensitive transcription activity. Thus, upon treatment of HL-60 cells with DMSO, wild-type Kras reacts with the Wnt/β-catenin pathway, thereby regulating the granulocytic differentiation of HL-60 cells. Wild-type Kras and the Wnt/β-catenin signaling pathway are activated sequentially, increasing the levels of expression of C/EBPα, C/EBPε, and granulocyte colony-stimulating factor (G-CSF) receptor.

Biofabrication of nanovesicles for brain diseases.

Biofabrication of nanovesicles for brain diseases.

Anti-leukemic activity of Cyperus rotundus L. on human acute myeloid leukemia HL-60 cells in vitro

Context: Acute myeloid leukemia (AML) is the most common form of acute leukemia. Currently, many people use medicinal herbs for the treatment of cancers. Nut grass (Cyperus rotundus L.) is a medicinal plant widely used in conventional medicine due to its role as an anti-cancer. Aims: To evaluate the effect of Cyperus rotundus tuber (CRT) ethanolic extract on cell proliferation, differentiation, cell cycle, and apoptotic of HL-60 cells. Methods: HL-60 cells line as a model for AML was cultured under the influence of CRT extract concentrations (35.4, 354, and 3540 µg/mL) for 48 h. Furthermore, the cell was subjected to carboxyfluorescein succinimidyl ester staining for proliferation, using a CD117 marker for differentiation. Annexin V-FITC and PI for cell cycle and apoptosis. The effect of this herb was studied by flow cytometry. The data were statistically analyzed with one-way ANOVA (p≤0.05) and the Tukey test using SPSS version 16 for Windows. Results: The results showed that the CRT inhibited proliferation activity, differentiation, cell cycle arrest, and apoptosis. Conclusions: The data clearly showed the potential anti-cancer activity of CRT on HL-60 cells and suggested that it could help develop promising therapeutic agents for AML treatments.

Oligoarginine-Conjugated Peptide Foldamers Inhibiting Vitamin D Receptor-Mediated Transcription.

The vitamin D receptor (VDR) is a nuclear receptor, which is involved in several physiological processes, including differentiation and bone homeostasis. The VDR is a promising target for the development of drugs against cancer and bone-related diseases. To date, several VDR antagonists, which bind to the ligand binding domain of the VDR and compete with the endogenous agonist 1α,25(OH)D3, have been reported. However, these ligands contain a secosteroidal skeleton, which is chemically unstable and complicated to synthesize. A few VDR antagonists with a nonsecosteroidal skeleton have been reported. Alternative inhibitors against VDR transactivation that act via different mechanisms are desirable. Here, we developed peptide-based VDR inhibitors capable of disrupting the VDR-coactivator interaction. It was reported that helical SRC2-3 peptides strongly bound to the VDR and competed with the coactivator in vitro. Therefore, we designed and synthesized a series of SRC2-3 derivatives by the introduction of nonproteinogenic amino acids, such as β-amino acids, and by side-chain stapling to stabilize helical structures and provide resistance against digestive enzymes. In addition, conjugation with a cell-penetrating peptide increased the cell membrane permeability and was a promising strategy for intracellular VDR inhibition. The nona-arginine-conjugated peptides 24 with side-chain stapling and 25 with cyclic β-amino acids showed strong intracellular VDR inhibitory activity, resulting in suppression of the target gene expression and inhibition of the cell differentiation of HL-60 cells. Herein, the peptide design, structure-activity relationship (SAR) study, and biological evaluation of the peptides are described.

Biofilm formation, its removal, and consequent effect on the osteoblast response to titanium surfaces: A model for re-osseointegration.

The aim of the study was to characterize pathogenic biofilm formation on titanium surfaces, the ability to remove the biofilm and the osteoblast response to infected and cleaned titanium surfaces as a model for re-osseointegration. Multispecies biofilm composed of Pseudomonas. gingivalis, F. nucleatum, S. sanguis, and A. naeslundii were grown on smooth, acid-etched, and acid-etched-aluminum-sprayed titanium surfaces. Bacterial viability was determined with live/dead staining. The biofilm was removed mechanically or together with adjunctive antibiotics. The osteoblast (Saos2) response to previously infected, treated and non-infected titanium surfaces were measured according to 4'-6-diamidino-2-phenylindole staining. Alkaline phosphatase levels and receptor activator of nuclear factor kappa-Β ligand/osteoprotegerin expression were measured with enzyme-linked immunosorbent assay and immunofluorescence staining, respectively The inflammatory environment was established by using differentiated HL-60 cells (neutrophils) pre-inoculated onto the biofilm clusters that were more prominent and less scattered on infected titanium surfaces before osteoblast attachment. Biofilm formed on all the tested surfaces, with an increased thickness on rough surfaces and no differences in bacterial viability. All the treatments reduced the amount of biofilm, but none led to bacteria-free surfaces. The treated surfaces showed reduced osteoblast attachment and reduced alkaline phosphatase activity compared with non-infected surfaces. Additionally, treated surfaces showed an osteoblast shift to a pro-osteoclastic-induction phenotype, compared with non-infected surfaces. The presence of experimental inflammation before osteoblast attachment reduced the levels of osteoblast attachment compared with that of the non-inflamed control. Biofilm removal from titanium surfaces is incomplete when hand instruments are used alone or in combination with antibiotics. The treated surfaces showed impaired osteoblast attachment and function, particularly in the presence of inflammation, which may prevent or decrease the ability for re-osseointegration.

Constitutively active GPR43 is crucial for proper leukocyte differentiation.

The G protein-coupled receptors, GPR43 (free fatty acid receptor 2, FFA2) and GPR41 (free fatty acid receptor 3, FFA3), are activated by short-chain fatty acids produced under various conditions, including microbial fermentation of carbohydrates. Previous studies have implicated this receptor energy homeostasis and immune responses as well as in cell growth arrest and apoptosis. Here, we observed the expression of both receptors in human blood cells and a remarkable enhancement in leukemia cell lines (HL-60, U937, and THP-1 cells) during differentiation. A reporter assay revealed that GPR43 is coupled with Gαi and Gα12/13 and is constitutively active without any stimuli. Specific blockers of GPR43, GLPG0974 and CATPB function as inverse agonists because treatment with these compounds significantly reduces constitutive activity. In HL-60 cells, enhanced expression of GPR43 led to growth arrest through Gα12/13 . In addition, the blockage of GPR43 activity in these cells significantly impaired their adherent properties due to the reduction of adhesion molecules. We further revealed that enhanced GPR43 activity induces F-actin formation. However, the activity of GPR43 did not contribute to butyrate-induced apoptosis in differentiated HL-60 cells because of the ineffectiveness of the inverse agonist on cell death. Collectively, these results suggest that GPR43, which possesses constitutive activity, is crucial for growth arrest, followed by the proper differentiation of leukocytes.

O-GlcNAc-Mediated Regulation of Galectin Expression and Secretion in Human Promyelocytic HL-60 Cells Undergoing Neutrophilic Differentiation.

In this study, we have tested the hypothesis that the expression and secretion of galectins are driven through mechanisms globally impacted by homeostatic regulation involving the post-translational modification of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc). We showed that neutrophilic differentiation of HL-60 cells induced by all-trans retinoic acid (ATRA) and 6-diazo-5-oxo-L-norleucine (DON) was associated with a significant drop of cellular O-GlcNAc levels in serum-contained and serum-free cell culture media. Galectin gene and protein expression profiles in HL-60 cells were specifically modified by ATRA and by inhibitors of O-GlcNAc cycle enzymes, however overall trends for each drug were similar between cells growing in the presence or absence of serum except for LGALS9 and LGALS12. The secretion of four galectins (-1, -3, -9, and -10) by HL-60 cells in a serum-free medium was stimulated by O-GlcNAc-reducing ATRA and DON while O-GlcNAc-elevating thiamet G (O-GlcNAcase inhibitor) failed to change the basal levels of extracellular galectins. Taken together, these results demonstrate that O-GlcNAc homeostasis is essential not only for regulation of galectin expression in cells but also for the secretion of multiple members of this protein family, which can be an important novel aspect of unconventional secretion mechanisms.