-
Tuberculosis (Edinburgh, Scotland) Jul 2018Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) complex remains a deadly infectious disease worldwide. Mtb is an intracellular pathogen, and autophagy is an... (Comparative Study)
Comparative Study
Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) complex remains a deadly infectious disease worldwide. Mtb is an intracellular pathogen, and autophagy is an essential component of the immune response leading to TB clearance. Anti-TB treatment is based on classical isoniazid (INH) and rifampicin (RIF), but also new drugs, such as linezolid (LNZ) and bedaquiline (BDQ). However, little is known about these antibiotics' impact on Mtb intra-macrophagic behavior independent of their impact on host cells. We explored the effect of mycobacterial pre-treatment with these four antibiotics on the intra-macrophagic Mtb survival and trafficking, thanks to bacterial counts and microscopy confocal imaging. Our results showed that INH and BDQ impaired Mtb phagosome escape, RIF increased autolysosome formation, and LNZ and BDQ improved autophagy activation and efficacy. These data suggest that antibiotics favoring autophagy activation (LNZ and BDQ) may allowed better Mtb clearance by macrophages and could provide basis for future anti-TB strategies.
Topics: Antitubercular Agents; Autophagy; Diarylquinolines; Host-Pathogen Interactions; Humans; Isoniazid; Linezolid; Macrophages; Mycobacterium tuberculosis; Phagosomes; Rifampin; Tuberculosis; U937 Cells
PubMed: 30029917
DOI: 10.1016/j.tube.2018.05.014 -
Cytokine Jan 2019Interleukin (IL)-29 is known to modulate immune functions of monocytes or macrophages. In this study, we investigated the effect and its underlying mechanism of IL-29 on...
Interleukin (IL)-29 is known to modulate immune functions of monocytes or macrophages. In this study, we investigated the effect and its underlying mechanism of IL-29 on receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis using murine macrophage cell line RAW264.7 cells and bone-marrow-derived monocyte/macrophage precursor cells (BMMs), and human peripheral blood mononuclear cells (PBMCs). In response to human recombinant IL-29, cell viability and apoptosis were assessed by Cell Counting Kit-8 and flow cytometry; the osteoclast formation and activity by tartrate-resistant acid phosphatase (TRAP) staining and pit formation assay, respectively; the expression and activation of molecules that associated with osteoclastogenesis by real time-PCR, immunoblotting or immunofluorescent analysis. IL-28 receptor α (IL-28Rα), a specific receptor of IL-29 was expressed on RAW264.7 cells. Although IL-29 did not affect the viability and apoptosis of RAW264.7 cells, it inhibited multinucleated cells in the differentiation of osteoclastogenesis, the bone-resorbing activity of mature osteoclasts and osteoclastic specific genes expression including TRAP, cathepsin K (CTSK), nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), C-Fos and matrix metallopeptidase 9 (MMP-9). This inhibitory effect of IL-29 was confirmed on BMMs and PBMCs and mediated via IL-28Rα through the activation of Stat1 and 3 and the suppression of nuclear factor kappa B (NF-κB) and NFATc1 nuclear translocation in RAW264.7 cells. In conclusion, IL-29 inhibited osteoclastogenesis via activation of STAT signaling pathway, prevention of NF-κB activation and NFATc1 translocation, and suppression of downstream osteoclastogenic genes expression.
Topics: Animals; Cell Differentiation; Cell Line; Humans; Interferons; Interleukins; Leukocytes, Mononuclear; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; NF-kappa B; NFATC Transcription Factors; Osteoclasts; Osteogenesis; RANK Ligand; RAW 264.7 Cells; STAT Transcription Factors; Signal Transduction
PubMed: 30001863
DOI: 10.1016/j.cyto.2018.06.032 -
Archivum Immunologiae Et Therapiae... Oct 2018Mycobacterium tuberculosis (Mtb) survives and proliferates within the main cells of the innate immune system, macrophages. The goal of our study was to investigate the...
Mycobacterium tuberculosis (Mtb) survives and proliferates within the main cells of the innate immune system, macrophages. The goal of our study was to investigate the immunostimulatory effects of 13-cis retinoic acid (RA) and chicoric acid (CA) in human U937 macrophages against H37Ra Mtb infection by evaluating its potential role in the cell surface expression of HLA-DR, CD14 molecules as well as nitric oxide (NO) production and prevention of the Mtb growth within macrophages. In this study, we investigated the effects of 13-cis RA and CA on Mtb-infected macrophages using flowcytometry and Griess methods, respectively. Moreover, inhibitory effect of 13-cis RA and CA on Mtb growth within macrophages were assessed using colony-forming unit. 13-Cis RA and CA enhanced the cell surface expression of HLA-DR and CD14 molecules on U937 macrophages and prevented the growth of Mtb within macrophages. In addition, 13-cis RA and CA, have increased NO generation compared to untreated control macrophages, significantly (p < 0.001). Both drugs have a significant inhibitory effect on Mtb growth but CA at the highest concentration was more potent than 13-cis RA (p < 0.05). The results of our study showed that infected U937 macrophages treated with 13-cis RA and CA represented significant increases in NO production, CD14 and HLA-DR expression and also prevents intracellular survival of Mtb. Therefore, 13-cis RA and CA may have a significant therapeutic approach in the control of Mtb infection.
Topics: Caffeic Acids; Colony Count, Microbial; HLA-DR Antigens; Humans; Immunization; Isotretinoin; Lipopolysaccharide Receptors; Macrophages; Mycobacterium tuberculosis; Nitric Oxide; Succinates; Tuberculosis; U937 Cells; Up-Regulation
PubMed: 29704020
DOI: 10.1007/s00005-018-0511-0 -
European Review For Medical and... Feb 2018To detect the expression of long non-coding RNA-CRNDE in patients with acute myeloid leukemia and its effect on proliferation and apoptosis in acute myeloid leukemia...
OBJECTIVE
To detect the expression of long non-coding RNA-CRNDE in patients with acute myeloid leukemia and its effect on proliferation and apoptosis in acute myeloid leukemia cell line U937.
PATIENTS AND METHODS
81 cases of newly diagnosed acute myeloid leukemia (AML) were enrolled, and 35 non-malignant hematological patients were selected as controls. Quantitative RT-PCR (qRT-PCR) was performed to detect the expression of lncRNA-CRNDE in the bone marrow specimens of the subjects, and the difference between the two groups was also compared. The correlation between the expression of lncRNA-CRNDE and the sex, age, classification and total survival of clinical patients was analyzed according to the clinical data. U937 cells and monocytes isolated from normal people were cultured, and the expression of lncRNA-CRNDE in acute myeloid leukemia cell line U937 and normal monocytes was compared. SiRNA-CRNDE and pcDNA-CRNDE were transfected into U937 cells, and cell counting kit-8 (CCK-8) assay was performed to detect proliferation of U937 cells, Annexin V/PI flow cytometry was carried out to detect cell apoptosis. Cell cycle was measured by flow cytometry.
RESULTS
The expression of lncRNA-CRNDE in patients with AML and U937 cells was significantly higher than that in non-malignant hematological controls. Results of clinical data showed that the expression of lncRNA-CRNDE was associated with the classification and total survival of myeloid leukemia in clinical patients. After transfection of siRNA-CRNDE, the proliferation and cloning ability of U937 cells decreased, while the apoptosis increased (p < 0.01) and cells were arrested in G0-G1 phase. Meanwhile, after transfection of pcDNA-CRNDE, the proliferation ability of U937 cells increased significantly, which indicated that the expression of lncRNA-CRNDE might play an essential role in promoting the proliferation of U937 cells.
CONCLUSIONS
LncRNA-CRNDE is highly expressed in the bone marrow tissues of AML patients, and the expression level is negatively correlated with the total survival of those clinical patients. Meanwhile, the expression is higher in FAB type M4 and M5 than that in M1, M2 and M3. LncRNA-CRNDE promotes the proliferation and cell cycle of U937 cells, and inhibits cell apoptosis, which is expected to become a molecular marker for predicting and treating AML.
Topics: Adolescent; Adult; Biomarkers, Tumor; Cell Line, Tumor; Cell Proliferation; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Humans; Leukemia, Myeloid, Acute; Male; Middle Aged; RNA, Long Noncoding; Survival Rate; U937 Cells; Young Adult
PubMed: 29461608
DOI: 10.26355/eurrev_201802_14310 -
Scientific Reports Feb 2018Macrophages, apart from being the key effector cells of the innate immune system, also play critical roles during the development and progression of various complex...
Macrophages, apart from being the key effector cells of the innate immune system, also play critical roles during the development and progression of various complex diseases, including cancer. Tumor-associated macrophages, infiltrate tumors during different stages of cancer progression to regulate motility, invasion, and intravasation to metastatic sites. Macrophages can exist in different polarization states associated with unique function in tumors. Since tumor-associated macrophages constitute a very small proportion of tumor cells, analysis of gene expression pattern using normal extraction buffer-based methods remains a challenging task. Therefore, it is imperative to develop low-throughput strategies to investigate transcriptional regulations from a small number of immune cells. Here, we describe an efficient, sensitive, and cost-effective approach for gene expression analysis of a small number of fluorescence-activated sorted tumor-associated macrophages. Our analyses from the different number of stable, primary, and sorted macrophages suggest 5,000 cells is an optimal number for performing quantitative, real-time PCR analysis of multiple genes. Our studies could detect expression of macrophage-specific genes from cultured primary macrophages, and FACS-sorted macrophages from different biological tissues without introducing biases in comparative gene expression ratios. In conclusion, our kit-based method for quantitative gene expression analysis from a small number of cells found in biological tissues will provide an opportunity to study cell-specific, transcriptional changes.
Topics: Animals; Cell Count; Cell Separation; Female; Flow Cytometry; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Macrophages; Mice; Mice, Inbred C57BL; Neoplasm Proteins; Neoplasm Transplantation; Pancreatic Neoplasms; Real-Time Polymerase Chain Reaction; U937 Cells
PubMed: 29402936
DOI: 10.1038/s41598-018-20820-4 -
Zhonghua Xue Ye Xue Za Zhi = Zhonghua... Oct 2017To investigate the effect and mechanism of all-trans retinoic acid (ATRA) on leukemic cell line U937 cells with NPM1 mutation. Human acute myeloid leukemia cell line...
To investigate the effect and mechanism of all-trans retinoic acid (ATRA) on leukemic cell line U937 cells with NPM1 mutation. Human acute myeloid leukemia cell line U937 was explored, NPM1 mutated (A type) plasmids were transfected into U937 to form stable clones A1 and A2, which were identified by Western blot and Co-immunoprecipitation. The cell proliferation was measured by methylthiazolyl tetrazolium bromide (MTT) ; cell cycle and cell apoptosis were explored by flow cytometric; cell colony formation was measured by microscope count, the molecular pathways related to cell proliferation were measured by Western blot. ①The cell proliferations of mutant A1 and A2 were inhibited significantly by 52.6% and 35.8% (<0.05) , respectively under ATRA exposure. ②The percentages of G(0)/G(1) stage of mutant A1 and A2 increased by 20.1% and 35.8%, respectively under ATRA exposure. ③All the U937 leukemic cells were inhibited under ATRA exposure; the decreased percentages of vector, wild-type and mutant NPM1 cells were 32.7%, 57.9% and 90.9% respectively. ④p-ERK decreased obviously after ATRA exposure in NPM1 mutated leukemic cells. ⑤More mutant NPM1 cells inclined to apoptosis under the exposure of ATRA and cytotoxic drugs than cytotoxic drugs alone, meanwhile more cells apoptosis occurred when ATRA was administrated after cytotoxic drugs exposure. ATRA could inhibit cell proliferation and colony formation, blocked the cell cycle in the G(0)/G(1) stage accompanied by the significant reduction of p-ERK in U937 leukemic cells with NPM1 mutation. Besides, ATRA could synergize with drugs to suppress the leukemic cells survival more effectively when ATRA was administered after the cytotoxic drugs exposure in U937 leukemic cells with NPM1 mutation.
Topics: Cell Differentiation; Cell Proliferation; Humans; Mutation; Nuclear Proteins; Nucleophosmin; Tretinoin; U937 Cells
PubMed: 29166739
DOI: 10.3760/cma.j.issn.0253-2727.2017.10.008 -
Indian Journal of Experimental Biology Sep 2016Hydantoin derivatives, including phenytoin (5,5-diphenylhydantoin), have recently gained attention as they possess a variety of important biochemical and pharmacological...
Hydantoin derivatives, including phenytoin (5,5-diphenylhydantoin), have recently gained attention as they possess a variety of important biochemical and pharmacological properties. Nevertheless, available information on anticancer activity of hydantoin derivatives is still scarce. Here, we evaluated possible antileukemic potential of four phenytoin analogs, namely: methyl 2-(2,4-dioxo-5,5-diphenylimidazolidin-3-yl)propanoate (1), methyl 2-(1-(3-bromopropyl)-2,4-dioxo-5,5-diphenylimidazolidin-3-yl)propanoate (2), 1-(3-bromopropyl)-3-methyl-5,5-diphenylimidazolidine-2,4-dione (3) and 1-(3-bromobutyl)-3-methyl-5,5-diphenylimidazolidine-2,4-dione (4). The experiments were performed on human acute histiocytic lymphoma U937 cells and human promyelocytic leukemia HL-60 cells. The present study was conducted using spectrophotometric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay and the electronic Beckman-Coulter method. We observed temporary changes in the leukemia cell viability, volume and count. The effects of the four 5,5-diphenylhydantoin derivatives on U937 and HL-60 cells depended on the agent tested and its concentration, the time intervals after the compound application, and the leukemia cell line used. HL-60 cells were more sensitive than U937 cells to the action of the phenytoin analogs (1-4). The antileukemic activities of the three bromoalkyl diphenylhydantoin derivatives (2, 3, and 4) were stronger than that of the compound 1 [methyl 2-(2,4-dioxo-5,5-diphenylimidazolidin-3-yl) propanoate], with no bromoalkyl substituent. The structural modifications of 5,5-diphenylhydantoin are responsible for such varied antileukemic potential of its four derivatives.
Topics: Cell Survival; HL-60 Cells; Humans; Leukemia; Phenytoin; Structure-Activity Relationship; U937 Cells
PubMed: 28699720
DOI: No ID Found -
British Journal of Cancer Aug 2017Lurbinectedin is a novel anticancer agent currently undergoing late-stage (Phase II /III) clinical evaluation in platinum-resistant ovarian, BRCA1/2-mutated breast and...
BACKGROUND
Lurbinectedin is a novel anticancer agent currently undergoing late-stage (Phase II /III) clinical evaluation in platinum-resistant ovarian, BRCA1/2-mutated breast and small-cell lung cancer. Lurbinectedin is structurally related to trabectedin and it inhibits active transcription and the DNA repair machinery in tumour cells.
METHODS
In this study we investigated whether lurbinectedin has the ability to modulate the inflammatory microenvironment and the viability of myeloid cells in tumour-bearing mice.
RESULTS
Administration of lurbinectedin significantly and selectively decreased the number of circulating monocytes and, in tumour tissues, that of macrophages and vessels. Similar findings were observed when a lurbinectedin-resistant tumour variant was used, indicating a direct effect of lurbinectedin on the tumour microenviroment. In vitro, lurbinectedin induced caspase-8-dependent apoptosis of human purified monocytes, whereas at low doses it significantly inhibited the production of inflammatory/growth factors (CCL2, CXCL8 and VEGF) and dramatically impaired monocyte adhesion and migration ability. These findings were supported by the strong inhibition of genes of the Rho-GTPase family in lurbinectedin-treated monocytes.
CONCLUSIONS
The results illustrate that lurbinectedin affects at multiple levels the inflammatory microenvironment by acting on the viability and functional activity of mononuclear phagocytes. These peculiar effects, combined with its intrinsic activity against cancer cells, make lurbinectedin a compound of particular interest in oncology.
Topics: Animals; Antineoplastic Agents, Alkylating; Apoptosis; Carbolines; Caspase 8; Cell Adhesion; Cell Movement; Chemokine CCL2; Dioxoles; Down-Regulation; Female; Fibrosarcoma; Gene Expression; Gene Expression Profiling; HL-60 Cells; Heterocyclic Compounds, 4 or More Rings; Humans; Interleukin-8; Leukocyte Count; Macrophages; Mice; Mice, Inbred C57BL; Monocytes; Neovascularization, Pathologic; Ovarian Neoplasms; Tetrahydroisoquinolines; Trabectedin; Tumor Microenvironment; U937 Cells; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays; rho GTP-Binding Proteins
PubMed: 28683469
DOI: 10.1038/bjc.2017.205 -
Journal of Ultrasound in Medicine :... Dec 2017Low-intensity pulsed ultrasound (US) has been reported to promote periodontal tissue regeneration and reduce inflammation in soft tissues and in bone infectious...
OBJECTIVES
Low-intensity pulsed ultrasound (US) has been reported to promote periodontal tissue regeneration and reduce inflammation in soft tissues and in bone infectious diseases. Here we investigated the effect of low-intensity pulsed US on the expression of lipopolysaccharide (LPS)-induced inflammatory factors in U937 macrophage cells.
METHODS
U937 cells were stimulated with different concentrations of LPS and exposed to different intensities of low-intensity pulsed US. Cell viability and apoptosis of U937 cells were determined by cell-counting kit assays and flow cytometry. A real-time polymerase chain reaction and an enzyme-linked immunosorbent assay were used to test the expression of inflammatory factors. The expression levels of toll-like receptor 4, p65, p-IκBα, and IκBα were assessed by western blots.
RESULTS
Tumor necrosis factor α began to increase in U937 cells on induction with 1-μg/mL LPS. Low-intensity pulsed US at the intensity of 60 mW/cm was more effective in reducing interleukin 8 (IL-8) expression. Furthermore, LPS inhibited the viability and increased apoptosis of U937 cells, whereas low-intensity pulsed US significantly reversed these effects (P < .05). Low-intensity pulsed US reduced the protein expression of IL-6 and IL-8 at both gene and protein levels in U937 cells. The western blot and immunofluorescence showed that low-intensity pulsed US primarily suppressed the degradation and phosphorylation of IκBα and the translocation of p65 into the nuclei.
CONCLUSIONS
Low-intensity pulsed US alleviated the expression of inflammatory factors induced by LPS in U937 cells. This process was modulated by suppressing the toll-like receptor 4-nuclear factor κB signaling pathway. Therefore, low-intensity pulsed US might be a potential immunomodulatory therapy for the treatment of periodontitis.
Topics: Apoptosis; Cell Survival; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Humans; Inflammation; Lipopolysaccharides; Real-Time Polymerase Chain Reaction; U937 Cells; Ultrasonic Therapy; Ultrasonic Waves
PubMed: 28600899
DOI: 10.1002/jum.14239 -
Pediatric Blood & Cancer Dec 2017There is no consensus on the treatment for pediatric patients with acute myeloid leukemia and initial central nervous system (CNS) involvement.
BACKGROUND
There is no consensus on the treatment for pediatric patients with acute myeloid leukemia and initial central nervous system (CNS) involvement.
METHODS
To evaluate different CNS-directed treatment options (intrathecal [IT] therapy, CNS irradiation, hematopoietic stem cell transplantation [HSCT]), 261 patients (excluding acute promyelocytic leukemia) with initial CNS involvement treated in trials with similar intensive chemotherapy by four cooperative European study groups (1998-2013) were studied and compared with CNS-negative patients from the Berlin-Frankfurt-Münster group.
RESULTS
Patient characteristics in the different study groups were comparable. Young age, high white blood cell count, extramedullary involvement other than the CNS, monoblastic morphology, and inv(16) were associated with CNS involvement (each P < 0.0001). There were no major differences in outcome between the study groups. The cumulative incidence of relapse (CIR) regarding the CNS was higher in initially CNS-positive versus initially CNS-negative patients (all: 8 ± 2% vs. 3 ± 1%, P = 0.001; isolated: 4 ± 1% vs. 1 ± 0%, P = 0.03). However, global outcome of the CNS-positive cohort (overall survival, 64 ± 3%; event-free survival 48 ± 3%; and CIR 33% ± 3%) did not differ significantly from CNS-negative patients. Risk groups defined by cytogenetics were of likewise prognostic significance in CNS-positive and -negative patients. CNS treatment with cranial irradiation was not superior compared to IT therapy and systemic chemotherapy (± HSCT).
CONCLUSION
Although CNS relapses occurred more frequently in initially CNS-positive patients, their global outcome was similar as in CNS-negative patients. Intensified IT therapy was heterogeneous; however, at least eight applications, preferably with triple IT chemotherapy, seem to be appropriate to accompany dose-intensive systemic chemotherapy.
Topics: Adolescent; Central Nervous System Neoplasms; Child; Child, Preschool; Cranial Irradiation; Hematopoietic Stem Cell Transplantation; Humans; Infant; Infant, Newborn; Leukemia, Myeloid, Acute; Neoplasms, Second Primary; Proportional Hazards Models; Recurrence; Treatment Outcome
PubMed: 28598536
DOI: 10.1002/pbc.26664