Expression and secretion of galectin-12 in the context of neutrophilic differentiation of human promyeloblastic HL-60 cells.

Galectin-12 is a tissue-specific galectin that has been largely defined by its role in the regulation of adipocyte differentiation and lipogenesis. This study aimed to evaluate the role of galectin-12 in the differentiation and polarization of neutrophils within a model of acute myeloid leukemia HL-60 cells. All-trans retinoic acid and dimethyl sulfoxide were used to induce differentiation of HL-60 cells which led to the generation of two phenotypes of neutrophil-like cells with opposite changes in galectin-12 gene (LGALS12) expression and different functional responses to N-formyl- l-methionyl- l-leucyl- l-phenylalanine. These phenotypes showed significant differences of differentially expressed genes on a global scale based on bioinformatics analysis of available Gene Expression Omnibus (GEO) data sets. We also demonstrated that HL-60 cells could secrete and accumulate galectin-12 in cell culture medium under normal growth conditions. This secretion was found to be entirely inhibited upon neutrophilic differentiation and was accompanied by an increase in intracellular lipid droplet content and significant enrichment of 22 lipid gene ontology terms related to lipid metabolism in differentiated cells. These findings suggest that galectin-12 could serve as a marker of neutrophilic plasticity or polarization into different phenotypes and that galectin-12 secretion may be influenced by lipid droplet biogenesis.

Acetylation of FOXO1 activates Bim expression involved in CVB3 induced cardiomyocyte apoptosis.

Viral myocarditis (VMC) is the major reason for sudden cardiac death among both children and young adults. Of these, coxsackievirus B3 (CVB3) is the most common causative agent of myocarditis. Recently, the role of signaling pathways in the pathogenesis of VMC has been evaluated in several studies, which has provided a new perspective on identifying potential therapeutic targets for this hitherto incurable disease. In the present study, in vivo and in vitro experiments showed that CVB3 infection leads to increased Bim expression and triggers apoptosis. In addition, by knocking down Bim using RNAi, we further confirmed the biological function of Bim in apoptosis induced by CVB3 infection. We additionally found that Bim and forkhead box O1 class (FOXO1) inhibition significantly increased the viability of CVB3-infected cells while blocking viral replication and viral release. Moreover, CVB3-induced Bim expression was directly dependent on FOXO1 acetylation, which is catalyzed by the co-regulation of CBP and SirTs. Furthermore, the acetylation of FOXO1 was an important step in Bim activation and apoptosis induced by CVB3 infection. The findings of this study suggest that CVB3 infection induces apoptosis through the FOXO1 acetylation-Bim pathway, thus providing new insights for developing potential therapeutic targets for enteroviral myocarditis.

Granulocyte pro-myeloperoxidase is redundantly processed by proprotein convertase furin and PC7 in HL-60 cells.

Neutrophil myeloperoxidase/H2O2/chloride system is a key mechanism to control pathogen infection. This enzyme, myeloperoxidase, plays a pivotal role in the arsenal of azurophilic granules that are released through degranulation upon neutrophil activation, which trigger local hypochlorous acid production. Myeloperoxidase gene encodes a protein precursor named promyeloperoxidase that arbors a propeptide that gets cleaved later during secretory routing in post-endoplasmic reticulum compartments. Although evidence suggested that this processing event was performed by one or different enzymes from the proprotein convertases family, the identity of this enzyme was never investigated. In this work, the naturally producing myeloperoxidase promyelocytic cell line HL-60 was used to investigate promyeloperoxidase cleavage during granulocytic differentiation in response to proprotein convertase inhibitors decanoyl-RVKR-chloromethylketone and hexa-d-arginine. Stable PC knockdown of endogenously expressed proprotein convertases, furin and PC7, was achieved using lentiviral delivery of shRNAs. None of the knockdown cell line could reproduce the effect of the pan-proprotein convertases inhibitor decanoyl-RVKR-chloromethylketone that accumulated intracellular promyeloperoxidase stores in HL-60 cells, therefore illustrating that both furin and PC7 redundantly process this proprotein.

Molecular and biochemical evaluation of oxidative effects of cord blood CD34+ MPs on hematopoietic cells.

A graft source for allogeneic hematopoietic stem cell transplantation is umbilical cord blood, which contains umbilical cord blood mononuclear cells (MNCs and mesenchymal stem cells, both an excellent source of extracellular microparticles (MPs). MPs act as cell communication mediators, which are implicated in reactive oxygen species formation or detoxification depending on their origin. Oxidative stress plays a crucial role in both the development of cancer and its treatment by triggering apoptotic mechanisms, in which CD34+ cells are implicated. The aim of this work is to investigate the oxidative stress status and the apoptosis of HL-60 and mononuclear cells isolated from umbilical cord blood (UCB) following a 24- and 48-hour exposure to CD34 + microparticles (CD34 + MPs). The activity of superoxide dismutase, glutathione reductase, and glutathione S-transferase, as well as lipid peroxidation in the cells, were employed as oxidative stress markers. A 24- and 48-hour exposure of leukemic and mononuclear cells to CD34 + -MPs resulted in a statistically significant increase in the antioxidant activity and lipid peroxidation in both cells types. Moreover, CD34 + MPs affect the expression of BCL2 and FAS and related proteins and downregulate the hematopoietic differentiation program in both HL-60 and mononuclear cells. Our results indicate that MPs through activation of antioxidant enzymes in both homozygous and nonhomozygous cells might serve as a means for graft optimization and enhancement.

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.

Genetic and functional modulation by agonist MRS5698 and allosteric enhancer LUF6000 at the native A3 adenosine receptor in HL-60 cells.

The A3 adenosine receptor (AR) is an important inflammatory and immunological target. However, the underlying mechanisms are not fully understood. Here, we report the gene regulation in HL-60 cells treated acutely with highly selective A3AR agonist MRS5698, positive allosteric modulator (PAM) LUF6000, or both. Both pro- and anti-inflammatory genes, such as IL-1a, IL-1ß, and NFκBIZ, are significantly upregulated. During our observations, LUF6000 alone produced a lesser effect, while the MRS5698 + LUF6000 group demonstrated generally greater effects than MRS5698 alone, consistent with allosteric enhancement. The number of genes up- and down-regulated are similar. Pathway analysis highlighted the critical involvement of signaling molecules, including IL-6 and IL-17. Important upstream regulators include IL-1a, IL-1ß, TNF-α, NF-κB, etc. PPAR, which modulates eicosanoid metabolism, was highly downregulated by the A3AR agonist. Considering previous pharmacological results and mathematical modeling, LUF6000's small enhancement of genetic upregulation suggested that MRS5698 is a nearly full agonist, which we demonstrated in both cAMP and calcium assays. The smaller effect of LUF6000 on MRS5698 in comparison to its effect on Cl-IB-MECA was shown in both HL-60 cells endogenously expressing the human (h) A3AR and in recombinant hA3AR-expressing CHO cells, consistent with its HL-60 cell genetic regulation patterns. In summary, by using both selective agonists and PAM, we identified genes that are closely relevant to immunity and inflammation to be regulated by A3AR in differentiated HL-60 cells, a cell model of neutrophil function. In addition, we demonstrated the previously uncharacterized allosteric signaling-enhancing effect of LUF6000 in cells endogenously expressing the hA3AR.

Sialyl LewisX glycomimetics bearing an extended anionic chain targeting E- and P- selectin binding sites.

Neutrophil binding to vascular P- and E-selectin is the rate-limiting step in the recruitment of immune cells to sites of inflammation. Many diseases, including sickle cell anemia, post-myocardial infarction reperfusion injury, and acute respiratory distress syndrome are characterized by dysregulated inflammation. We have recently reported sialyl Lewisx analogues as potent antagonists of P- and E-selectin and demonstrated their in vivo immunosuppressive activity. A key component of these molecules is a tartrate diester that serves as an acyclic tether to orient the fucoside and the galactoside moiety in the required gauche conformation for optimal binding. The next stage of our study involved attaching an extended carbon chain onto one of the esters. This chain could be utilized to tether other pharmacophores, lipids, and contrast agents in the context of enhancing pharmacological applications through the sialyl Lewisx / receptor-mediated mechanism. Herein, we report our preliminary studies to generate a small library of tartrate based sialyl Lewisx analogues bearing extended carbon chains. Anionic charged chemical entities are attached to take advantage of proximal charged amino acids in the carbohydrate recognition domain of the selectin receptors. Starting with a common azido intermediate, synthesized using copper-catalyzed Huisgen 1,3-dipolar cycloadditions, these molecules demonstrate E- and P-selectin binding properties.

Novel guanidine derivatives targeting leukemia as selective Src/Abl dual inhibitors: Design, synthesis and anti-proliferative activity.

A new series of benzene-sulfonamide derivatives 3a-i was designed and synthesized via the reaction of N-(pyrimidin-2-yl)cyanamides 1a-i with sulfamethazine sodium salt 2 as dual Src/Abl inhibitors. Spectral data IR, 1H-, 13C- NMR and elemental analyses were used to confirm the structures of all the newly synthesized compounds 3a-i and 4a-i. Crucially, we screened all the synthesized compounds 3a-i against NCI 60 cancer cell lines. Among all, compound 3b was the most potent, with IC50 of 0.018 µM for normoxia, and 0.001 µM for hypoxia, compared to staurosporine against HL-60 leukemia cell line. To verify the selectivity of this derivative, it was assessed against a panel of tyrosine kinase EGFR, VEGFR-2, B-raf, ERK, CK1, p38-MAPK, Src and Abl enzymes. Results revealed that compound 3b can effectively and selectively inhibit Src/Abl with IC500.25 µM and Abl inhibitory activity with IC500.08 µM, respectively, and was found to be more potent on these enzymes than other kinases that showed the following results: EGFR IC500.31 µM, VEGFR-2 IC500.68 µM, B-raf IC500.33 µM, ERK IC501.41 µM, CK1 IC500.29 µM and p38-MAPK IC500.38 µM. Moreover, cell cycle analysis and apoptosis performed to compound 3b against HL-60 suggesting its antiproliferative activity through Src/Abl inhibition. Finally, molecular docking studies and physicochemical properties prediction for compounds 3b, 3c, and 3 h were carried out to investigate their biological activities and clarify their bioavailability.

Electronic Health Record and Semantic Issues Using Fast Healthcare Interoperability Resources: Systematic Mapping Review.

BACKGROUND: The increasing use of electronic health records and the Internet of Things has led to interoperability issues at different levels (structural and semantic). Standards are important not only for successfully exchanging data but also for appropriately interpreting them (semantic interoperability). Thus, to facilitate the semantic interoperability of data exchanged in health care, considerable resources have been deployed to improve the quality of shared clinical data by structuring and mapping them to the Fast Healthcare Interoperability Resources (FHIR) standard. OBJECTIVE: The aims of this study are 2-fold: to inventory the studies on FHIR semantic interoperability resources and terminologies and to identify and classify the approaches and contributions proposed in these studies. METHODS: A systematic mapping review (SMR) was conducted using 10 electronic databases as sources of information for inventory and review studies published during 2012 to 2022 on the development and improvement of semantic interoperability using the FHIR standard. RESULTS: A total of 70 FHIR studies were selected and analyzed to identify FHIR resource types and terminologies from a semantic perspective. The proposed semantic approaches were classified into 6 categories, namely mapping (31/126, 24.6%), terminology services (18/126, 14.3%), resource description framework or web ontology language-based proposals (24/126, 19%), annotation proposals (18/126, 14.3%), machine learning (ML) and natural language processing (NLP) proposals (20/126, 15.9%), and ontology-based proposals (15/126, 11.9%). From 2012 to 2022, there has been continued research in 6 categories of approaches as well as in new and emerging annotations and ML and NLP proposals. This SMR also classifies the contributions of the selected studies into 5 categories: framework or architecture proposals, model proposals, technique proposals, comparison services, and tool proposals. The most frequent type of contribution is the proposal of a framework or architecture to enable semantic interoperability. CONCLUSIONS: This SMR provides a classification of the different solutions proposed to address semantic interoperability using FHIR at different levels: collecting, extracting and annotating data, modeling electronic health record data from legacy systems, and applying transformation and mapping to FHIR models and terminologies. The use of ML and NLP for unstructured data is promising and has been applied to specific use case scenarios. In addition, terminology services are needed to accelerate their use and adoption; furthermore, techniques and tools to automate annotation and ontology comparison should help reduce human interaction.

Systems Biology for Drug Target Discovery in Acute Myeloid Leukemia.

Combining new therapeutics with all-trans-retinoic acid (ATRA) could improve the efficiency of acute myeloid leukemia (AML) treatment. Modeling the process of ATRA-induced differentiation based on the transcriptomic profile of leukemic cells resulted in the identification of key targets that can be used to increase the therapeutic effect of ATRA. The genome-scale transcriptome analysis revealed the early molecular response to the ATRA treatment of HL-60 cells. In this study, we performed the transcriptomic profiling of HL-60, NB4, and K562 cells exposed to ATRA for 3-72 h. After treatment with ATRA for 3, 12, 24, and 72 h, we found 222, 391, 359, and 1032 differentially expressed genes (DEGs) in HL-60 cells, as well as 641, 1037, 1011, and 1499 DEGs in NB4 cells. We also found 538 and 119 DEGs in K562 cells treated with ATRA for 24 h and 72 h, respectively. Based on experimental transcriptomic data, we performed hierarchical modeling and determined cyclin-dependent kinase 6 (CDK6), tumor necrosis factor alpha (TNF-alpha), and transcriptional repressor CUX1 as the key regulators of the molecular response to the ATRA treatment in HL-60, NB4, and K562 cell lines, respectively. Mapping the data of TMT-based mass-spectrometric profiling on the modeling schemes, we determined CDK6 expression at the proteome level and its down-regulation at the transcriptome and proteome levels in cells treated with ATRA for 72 h. The combination of therapy with a CDK6 inhibitor (palbociclib) and ATRA (tretinoin) could be an alternative approach for the treatment of acute myeloid leukemia (AML).

Networking Aspects of the Electronic Health Records: Hypertext Transfer Protocol Version 2 (HTTP/2) vs HTTP/3.

The rapid development of the digital healthcare and the electronic health records (EHR) requires smooth networking infrastructure to access data using Hypertext Transfer Protocol (HTTP)-based applications. The new HTTP/3 standard should provide performance and security improvements over HTTP/2. The goal of our work was to test the performance of HTTP/2 and HTTP/3 in the context of the EHRs. We used 45,000 test FHIR Patient resources downloaded and uploaded using 20, 50, 100 and 200 resources per Bundle, which resulted in 2251, 901, 451 and 226 HTTP GET and POST requests respectively. The first test downloading 20 resources per Bundle showed that HTTP/3 outperformed HTTP/2 in the local (mean request time 16.57 ms ± 7.2 standard deviation [SD]) and in the remote network (71.45 ms ± 43.5 SD) which is almost 3 times faster. In the 50 and 100 resources per Bundle test the HTTP/3 protocol demonstrated again more than two times gain in downloading performance for remote requests with mean request time 91.13 ms ± 34.54 SD and 88.09 ms ± 21.66 SD respectively. Furthermore, HTTP/3 outperformed HTTP/2 in the constructed clinical dataset remote transfer. In the upload tests HTTP/3 showed only a slight gain in performance merely in the remote network. The HTTP/3 protocol is a relatively new development and a major improvement for the worldwide web. This new technology is still missing in the digital health and EHRs. Its use could offer a major performance gain in situations where data is gathered from multiple remote locations.

Measuring the Coverage of the HL7® FHIR® Standard in Supporting Data Acquisition for 3 Public Health Registries.

With the increasing need for timely submission of data to state and national public health registries, current manual approaches to data acquisition and submission are insufficient. In clinical practice, federal regulations are now mandating the use of data messaging standards, i.e., the Health Level Seven (HL7®) Fast Healthcare Interoperability Resources (FHIR®) standard, to facilitate the electronic exchange of clinical (patient) data. In both research and public health practice, we can also leverage FHIR® ‒ and the infrastructure already in place for supporting exchange of clinical practice data ‒ to enable seamless exchange between the electronic medical record and public health registries. That said, in order to understand the current utility of FHIR® for supporting the public health use case, we must first measure the extent to which the standard resources map to the required registry data elements. Thus, using a systematic mapping approach, we evaluated the level of completeness of the FHIR® standard to support data collection for three public health registries (Trauma, Stroke, and National Surgical Quality Improvement Program). On average, approximately 80% of data elements were available in FHIR® (71%, 77%, and 92%, respectively; inter-annotator agreement rates: 82%, 78%, and 72%, respectively). This tells us that there is the potential for significant automation to support EHR-to-Registry data exchange, which will reduce the amount of manual, error-prone processes and ensure higher data quality. Further, identification of the remaining 20% of data elements that are "not mapped" will enable us to improve the standard and develop profiles that will better fit the registry data model.

Suppressive effect of resveratrol, catechin and their conformationally constrained analogs on neutrophil extracellular trap formation by HL-60-derived neutrophils.

Neutrophil extracellular trap (NET) formation is a unique self-defense mechanism of neutrophils; however, it is also involved in many diseases, including atherosclerosis. Resveratrol and catechin are antioxidants with anti-atherosclerotic properties. Here, we examined the effects of resveratrol, catechin, and other related compounds on NET formation. HL-60-derived neutrophils were pretreated with resveratrol and other compounds before stimulation with phorbol-myristate acetate (PMA). DNA and myeloperoxidase released from neutrophils were determined. Resveratrol suppressed the DNA release from neutrophils in a dose-dependent manner. NET formation was enhanced by 1-palmitoyl-2-oxovaleroyl phosphatidylcholine (POVPC), a truncated form of oxidized phospholipid, and resveratrol suppressed NET formation induced by POVPC and PMA. Furthermore, we designed several analogs of resveratrol or catechin whose conformation was restricted by the inhibition of the free rotation of aromatic rings. The conformationally constrained analogs were more effective at inhibiting NET formation; however, their inhibitory function decreased when compound was a large, hydrophobic analog. The most potent compounds, planar catechin and resveratrol, suppressed myeloperoxidase release from activated neutrophils. In addition, these compounds suppressed DNA release from neutrophils stimulated with calcium ionophore. These results suggest that resveratrol, catechin and their analogs exert anti-NET effects, and that constraining the geometry of these compounds enhanced their inhibitory effects.

[The future of hematopoietic stem cell transplantation using DX].

The advancement of information and communication technology (ICT) is bringing significant changes to hematopoietic stem cell transplantation. Digital transformation (DX) simplifies the collection and analysis of medical data, enabling provision of medical services beyond geographical constraints through telemedicine. Convenient access to electronic medical records and vital data from wearable devices could facilitate personalized medicine, for example, by predicting of disease onset. Online consultations are effective in improving the efficiency of posttransplant follow-ups, donor recruitment, and donor screening in rural areas. Moreover, patient-reported outcomes are effective in improving treatment outcomes and patient management. The effective utilization of ICT necessitates the enhancement of information technology (IT) literacy among healthcare professionals and patients, as well as development of IT proficiency among medical personnel. DX in hematopoietic stem cell transplantation contributes to the improvement of treatment outcomes, quality of medical care, and patient convenience while introducing new possibilities for the future of healthcare.

First-in-human phase I dose-escalation and dose-expansion trial of the selective MEK inhibitor HL-085 in patients with advanced melanoma harboring NRAS mutations.

BACKGROUND: HL-085 is a selective, orally administered MEK1/2 inhibitor. We aimed to evaluate the safety and efficacy of HL-085 in patients with advanced melanoma harboring NRAS mutations. METHODS: This was a multicenter phase 1 study. HL-085 was administered twice daily in a standard 3 + 3 dose-escalation design (10 dose cohorts; 0.5-18 mg twice daily), followed by dose expansion at the recommended phase II dose (RP2D). The primary endpoints included tolerability, dose-limiting toxicity (DLT), maximum tolerated dose (MTD) and RP2D. RESULTS: Between September 13, 2017, and January 18, 2021, 42 patients were enrolled (dose escalation phase: n = 30; dose expansion phase: n = 12). No DLT was reported during dose escalation and MTD was not reached with HL-085 doses up to 18 mg twice daily. The RP2D was 12 mg twice daily. The most common all-grade drug-related adverse events (AEs) across all dose levels were rash (61.9%), increased creatine phosphokinase (CK, 59.5%), face edema (50.0%), increased aspartate aminotransferase (47.6%), peripheral edema (40.5%), diarrhea (33.3%), alanine aminotransferase (33.3%), and paronychia (19.0%), most of which were grade 1 and 2. Most frequency of grade ≥ 3 AEs were CK (14.2%), asthenia (7.1%), peripheral edema (4.8%), and acneiform dermatitis (4.8%). In the cohort of 12 mg twice daily dose (15 patients), confirmed objective response rate was 26.7%; disease control rate was 86.7%; median duration of response was 2.9 months; median progression-free survival was 3.6 months. CONCLUSIONS: The HL-085 showed acceptable tolerability and substantial clinical activity in patients with advanced melanoma harboring NRAS mutations. TRIAL REGISTRATION: Trial registration ClinicalTrials.gov number: NCT03973151.

Alterations in sphingolipid composition and mitochondrial bioenergetics represent synergistic therapeutic vulnerabilities linked to multidrug resistance in leukemia.

Modifications in sphingolipid (SL) metabolism and mitochondrial bioenergetics are key factors implicated in cancer cell response to chemotherapy, including chemotherapy resistance. In the present work, we utilized acute myeloid leukemia (AML) cell lines, selected to be refractory to various chemotherapeutics, to explore the interplay between SL metabolism and mitochondrial biology supportive of multidrug resistance (MDR). In agreement with previous findings in cytarabine or daunorubicin resistant AML cells, relative to chemosensitive wildtype controls, HL-60 cells refractory to vincristine (HL60/VCR) presented with alterations in SL enzyme expression and lipidome composition. Such changes were typified by upregulated expression of various ceramide detoxifying enzymes, as well as corresponding shifts in ceramide, glucosylceramide, and sphingomyelin (SM) molecular species. With respect to mitochondria, despite consistent increases in both basal respiration and maximal respiratory capacity, direct interrogation of the oxidative phosphorylation (OXPHOS) system revealed intrinsic deficiencies in HL60/VCR, as well as across multiple MDR model systems. Based on the apparent requirement for augmented SL and mitochondrial flux to support the MDR phenotype, we explored a combinatorial therapeutic paradigm designed to target each pathway. Remarkably, despite minimal cytotoxicity in peripheral blood mononuclear cells (PBMC), co-targeting SL metabolism, and respiratory complex I (CI) induced synergistic cytotoxicity consistently across multiple MDR leukemia models. Together, these data underscore the intimate connection between cellular sphingolipids and mitochondrial metabolism and suggest that pharmacological intervention across both pathways may represent a novel treatment strategy against MDR.

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.

BACKGROUND: Neutrophils 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. METHODS: Lrrk2 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. RESULTS: fMLP-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. CONCLUSIONS: LRRK2 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. Video Abstract.

Synthesis of raloxifene-like quinoxaline derivatives by intramolecular electrophilic cyclization with disulfides.

The intramolecular electrophilic cyclization of alkynes with disulfides to form thieno[2,3-b]quinoxaline structures and to introduce thioether substituents afforded quinoxaline derivatives (7a-7d, 8a-8d). Among obtained eight derivatives, the raloxifene analogues (7c, 8b) showed specifically high cytotoxicity against breast cancer cells (SK-BR-3), and raloxifene analogues (8a) showed the highest cytotoxicity against human leukemia cells (HL-60). None of the raloxifene analogues (7a-7d, 8a-8d) showed cytotoxicity against human lung fibroblasts (WI-38), which are normal cells.

Cytotoxicity and induction of G1 phase cell cycle arrest in human acute promyelocytic leukemia HL60 cells by indole alkaloids isolated from Dialium corbisieri seeds.

The phytochemical investigation of Dialium corbisieri seeds led to the isolation of five monoterpenoid indole alkaloids along with a phytoserotonin, 1-6 and among the known compounds, the spectroscopic data of (5S)-methoxy-akuammiline (1) was reported for the first time. The structures were elucidated based on nuclear magnetic resonance spectroscopic techniques such as ultraviolet, infrared, high-resolution electrospray ionization time-of-flight mass spectrometry, and electron-capture dissociation spectrum calculations. The isolated compounds were evaluated for their cytotoxicity and cell progression in the human acute promyelocytic leukemia HL60 cell line.

Novel benzo chromene derivatives: design, synthesis, molecular docking, cell cycle arrest, and apoptosis induction in human acute myeloid leukemia HL-60 cells.

A series of benzo[h]chromenes, benzo[f]chromenes, and benzo[h]chromeno[2,3-d]pyrimidines were prepared. All the newly synthesised compounds were selected by National Cancer Institute for single-dose testing against 60 cell lines. Benzo[h]chromenes 5a and 6a showed promising anti-cancer activity and selected for the five-dose testing. Compounds 5a and 6a suppressed cell growth in HL-60 by the induction of cell cycle arrest, which was confirmed using flow cytometry and Annexin V-FITC/PI assays showed at the G1/S phase by regulating the expression of CDK-2/CyclinD1, triggering cell apoptosis by activating both the extrinsic (Fas/Caspase 8) and intrinsic (Bcl-2/Caspase 3) apoptosis pathways, which were determined by the western blot. Benzo[h]chromenes 5a and 6a decreased the protein expression levels of Bcl-2, CDK-2, and CyclinD1 and increased the expression of caspase 3, caspase 8, and Fas. In silico molecular analysis of compounds 5a and 6a in CDK-2 and Bcl-2 was performed.