Acute Monocytic Leukemia Research Articles

Small interfering RNA-mediated LPXN silencing suppresses proliferation and enhances drug sensitivity of human acute monocytic leukemia SHI-1 cells in vitro

To investigate the effect of silencing LPXN expression by RNA interference on the proliferation and drug sensitivity of human acute monocytic leukemia SHI-1 cells in vitro. Small interfering RNA (siRNA) sequences targeting LPXN were designed and transiently transfected in SHI-1 cells via Lipofectamine 2000, and the most efficient siRNA sequence for LPXN silencing was identified using Western blotting. The protein expression levels of LPXN, p-JNK, p-P38 MAPK and p-ERK were in the cells transfected with the selected siRNA were detected using Western blotting, and the cell proliferation changes were assessed using CCK-8 reagent. LPXN silencing by siRNA transfection resulted in significant proliferation suppression in SHI-1 cells with an inhibition rate of(27.04±2.05) % (P < 0.05). Western blotting showed that treatment of the siRNA-transfected SHI-1 cells with 0-25 μmol/L curcumin or with 0-2.0 μmol/L Ara-C further increased the cell inhibition rate and obviously enhanced the expressions of p-P38 MAPK and p-JNK without significantly affecting p-ERK expression. Down-regulation of LPXN expression by siRNA transfection can suppress the proliferation and increase the drug sensitivity of SHI-1 cells probably by activating JNK and P38 MAPK.

Panax notoginseng Saponins Regulate Macrophage Polarization under Hyperglycemic Condition via NF-κB Signaling Pathway.

Panax notoginseng saponins (PNS), the principal constituents derived from Panax notoginseng, have been extensively used for treating cardiocerebral vascular diseases in China and other Asian countries. The main effects of PNS were anti-inflammatory properties, inhibition of platelet aggregation, improvement of blood flow and insulin resistance, and so on. This study was carried out to explore the effects of PNS on macrophage polarization under hyperglycemic conditions. Human acute monocyte leukemia cell line THP-1 cells were induced into macrophages with Phorbol ester (PMA). Macrophages were then divided into five groups as follows: control (5.5mMol/l glucose), hyperglycemia group (15mMol/l glucose), hyperglycemia plus low-dose PNS (20ug/ml), hyperglycemia plus moderate-dose PNS (40ug/ml), and hyperglycemia plus high-dose PNS (60ug/ml). After 48-hour cell culture, the percentages of M1- and M2-polarized macrophages were measured by flow cytometry analysis. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was used to evaluate the Ym1 and arginase 1 expression in macrophages. Protein expression of arginase 1, NF-κB p50, p65, and inhibitor of κB (IκB) alpha phosphorylation in macrophages was identified with Western blotting. PNS, especially the high-dose PNS, remarkably increased M2 phenotype ratio in macrophages cultured with hyperglycemia, and the mRNA expression of Ym1 and arginase 1 in macrophages was also upregulated. Meanwhile, PNS remarkably increased the protein expression of arginase 1 and decreased IκB-alpha phosphorylation and subunits of NF-κB p50 and p65 from macrophages in culture medium with hyperglycemia. Taken together, our work demonstrated that PNS promote macrophages to transform M2 phenotype under hyperglycemic conditions through downregulating NF-κB signaling pathway.

Abstract 5198: Androgen receptor signaling affects macrophage differentiation in the human prostate cancer microenvironment

Abstract Increasing evidence from epidemiologic and pathologic studies suggests a role of macrophages in the initiation and progression of prostate cancer (PCa). During PCa development, macrophages (CD68+) are recruited into the human prostate cancer microenvironment, including inflammation-associated M1 and cancer-promoting M2 macrophages. Like other prostate cancer-associated stromal cells, macrophages express the androgen receptor (AR); however, its functionality is not known. CD68+ cells generated in vitro from peripheral white blood cells and PMA activated and IFN and LPS polarized human acute monocytic leukemia THP-1 cells also expressed AR. In these cells AR is an 80KD splice variant, lacking parts of exon 1 and exon 2. AR translocated to the nucleus upon testosterone stimulation, which was counteracted by the AR signaling inhibitor RD162. In vitro generated M1 macrophages expressed the M2 markers CD163 and CD206 upon testosterone stimulation, which was reversed by RD162. AR-ChIP sequencing suggested AR regulated Macrophage Triggering Receptor 1 (TREM1) signaling. Expression of key cytokines (e.g., CCL2, CXCL8, CCL7, IL10, IL1B) involved in TREM1 signaling was upregulated upon testosterone stimulation. Actions of these cytokines is generally associated with a protumor phenotype of macrophages. Conditioned medium from macrophages stimulated with testosterone for 8 hrs resulted in enhanced proliferation and migration of human prostate LNCap and CWRR1 cells. In conclusion, AR signaling might affect differentiation of prostate cancer-associated macrophages into cancer-promoting M2, which affects prostate cancer cell proliferation and migration. Maintaining macrophages in M1 might be a novel mechanism of action of androgen receptor inhibitors. Citation Format: Bianca Cioni, Ekatarina Nevedomskaya, Suzan Stelloo, Monique Melis, Johan van Burgsteden, Jeroen de Jong, Henk van der Poel, Jan Paul de Boer, Wilbert Zwart, Andries M. Bergman. Androgen receptor signaling affects macrophage differentiation in the human prostate cancer microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5198.

Regulation and biological role of the peptide/histidine transporter SLC15A3 in Toll-like receptor-mediated inflammatory responses in macrophage

The peptide/histidine transporter SLC15A3 is responsible for transporting histidine, certain dipeptide and peptidomimetics from inside the lysosome to cytosol. Previous studies have indicated that SLC15A3 transcripts are mainly expressed in the lymphatic system, however, its regulation and biological role in innate immune responses and inflammatory diseases are as yet unknown. In this study, mouse peritoneal macrophages (PMs), mouse bone marrow-derived macrophages (BMDMs), the human acute monocytic leukemia cell line THP-1 and the human lung epithelial carcinoma cell line A549 were used to investigate the regulation and biological role of SLC15A3 in TLR-mediated inflammatory responses. Our results showed that SLC15A3 was upregulated by TLR2, TLR4, TLR7 and TLR9 ligands in macrophages at both the mRNA and protein levels via activation of NF-κB (nuclear factor-kappa-B), MAPK (mitogen-activated protein kinase) and IRF3 (interferon regulatory factor 3). Furthermore, knockdown or overexpression of SLC15A3 influenced the TLR4-triggered expression of proinflammatory cytokines. A reporter gene assay showed that the SLC15A3 promotor contained potential NF-κB binding sites, which were reasonable for regulating SLC15A3 by TLR-activation through NF-κB signaling. Additionally, SLC15A3 expression was increased and positively related to inflammation in mice with bacterial peritonitis. The collective findings suggest that SLC15A3 is regulated by various TLRs, and that it plays an important role in regulating TLR4-mediated inflammatory responses.

Antibacterial, Antioxidant, and Antiproliferative Activities of Corymbia citriodora and the Essential Oils of Eight Eucalyptus Species.

Background: Essential oils (EOs) have shown antimicrobial, antioxidant, and antiproliferative activity, which may, alone or in combination with other substances, potentially be used for the development of new drugs. However, their chemical variability, depending on the species, varieties, or geographical origin (among other factors) determines different bioactivities that need to be evaluated. Methods: The antioxidant activity of Corymbia citriodora and eight Eucalyptus species EOs was determined using two different methods: the scavenging ability of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS+•) and peroxyl free radicals or oxygen radical absorbance capacity (ORAC). Antibacterial activity was evaluated using the microorganisms Streptococcus pneumoniae (strains D39 and TIGR4), and Haemophilus influenza (strain DSM 9999). The essential oils’ minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) was assessed using a microdilution method. The antiproliferative activity was determined using the THP-1 cell line (human acute monocytic leukaemia) with methylthiazolyldiphenyl-tetrazolium bromide assay (MTT). Results: Corymbia citriodora and Eucalyptus viminalis EOs showed the highest ABTS and peroxyl free radical scavenging capacity. Eucalyptus globulus EO showed a high potential to treat Streptococcus pneumoniae infections. Haemophilus influenzae was the respiratory pathogen that showed the highest resistance to all EOs, including tea tree EO. After 96 h of incubation, at 25 μg/mL, Eucalyptus radiata and Eucalyptus viminalis EOs showed highest cytotoxic activity against the THP-1 cell line. Conclusions: Despite their specific bioactivities, no single EO showed simultaneously good antioxidant, antimicrobial, and antiproliferative activity.

Biogenic Synthesis of Novel Functionalized Selenium Nanoparticles by Lactobacillus casei ATCC 393 and Its Protective Effects on Intestinal Barrier Dysfunction Caused by Enterotoxigenic Escherichia coli K88.

Selenium (Se) is an essential element for human and animal health. Biogenic selenium nanoparticles (SeNPs) by microorganism possess unique physical and chemical properties and biological activities compared with inorganic Se and organic Se. The study was conducted to investigate the mainly biological activities of SeNPs by Lactobacillus casei ATCC 393 (L. casei 393). The results showed that L. casei 393 transformed sodium selenite to red SeNPs with the size of 50–80 nm, and accumulated them intracellularly. L. casei 393-SeNPs promoted the growth and proliferation of porcine intestinal epithelial cells (IPEC-J2), human colonic epithelial cells (NCM460), and human acute monocytic leukemia cell (THP-1)-derived macrophagocyte. L. casei 393-SeNPs significantly inhibited the growth of human liver tumor cell line-HepG2, and alleviated diquat-induced IPEC-J2 oxidative damage. Moreover, in vivo and in vitro experimental results showed that administration with L. casei 393-SeNPs protected against Enterotoxigenic Escherichia coli K88 (ETEC K88)-caused intestinal barrier dysfunction. ETEC K88 infection-associated oxidative stress (glutathione peroxidase activity, total superoxide dismutase activity, total antioxidant capacity, and malondialdehyde) was ameliorated in L. casei 393-SeNPs-treated mice. These findings suggest that L. casei 393-SeNPs with no cytotoxicity play a key role in maintaining intestinal epithelial integrity and intestinal microflora balance in response to oxidative stress and infection.

Roles of Dectin-1 in phagocytosis of Candida albicans by macrophage-like cells derived from a human acute monocytic leukemia cell line THP-1

Objective To investigate the roles of Dectin-1 in phagocytosis of Candida albicans (C. albicans) by macrophage-like cells derived from a human acute monocytic leukemia cell line THP-1. Methods THP-1 macrophage-like cells served as the target cells, and were transfected with small interfering RNA (siRNA) targeting Dectin-1 to down-regulate the expression of Dectin-1 receptor (siRNA-Dectin-1 group) . THP-1 macrophage-like cells transfected with nonsense siRNA (siRNA-NC) served as a negative control group. After transfection, the THP-1 macrophage-like cells in the above 2 groups were co-cultured with heat-killed C. albicans separately. And then, fluorescence microscopy was performed to count THP-1 macrophage-like cells phagocytosing C. albicans, and flow cytometry was used to determine the mean fluorescence intensity (MFI) of dihydrorhodamine (DHR) -123 fluorescent cells. Statistical analysis was done by one-way analysis of variance (ANOVA) and t test with the SPSS19.0 software. Results After transfection with siRNA-Dectin-1, the mRNA and protein expression of Dectin-1 significantly decreased in THP-1 macrophage-like cells (t= 26.163, P < 0.001) . After 1-, 2-, 4-hour co-culture of THP-1 macrophage-like cells with C. albicans, fluorescence microscopy showed that the phagocytosis rates of C. albicans by THP-1 macrophage-like cells were significantly lower in the siRNA-Dectin-1 group than in the negative control group (17.5% vs. 22.1%, 18.6% vs. 24.3%, 39.2% vs. 59.1%, respectively, all P < 0.05) , so were the percentage of THP-1 macrophage-like cells phagocytosing more than 3 C. albicans cells (2.2% vs. 4.7%, 2.5% vs. 5.4%, 5.1% vs. 8.3%, respectively, all P < 0.05) . After 30-minute, 1-, 2- and 4-hour co-culture of THP-1 macrophage-like cells with DHR-123-labelled C. albicans, flow cytometry showed that the MFI of C. albicans-phagocytosing cells was significantly lower in the siRNA-Dectin-1 group than in the negative control group (36.8 vs. 45.7, 54.3 vs. 62.4, 72.1 vs. 84.9, 93.6 vs. 116.7, respectively, all P < 0.05) . Conclusion Dectin-1 receptor plays an important role in the phagocytosis of C. albicans by macrophages. Key words: Candida albicans; Macrophages; Phagocytosis; Leukemia, monocytic, acute; Dectin-1

Haemophagocytic lymphohistiocytosis occurred during induction chemotherapy in an acute monocytic leukemia patient with FLT3-ITD and DNMT3A mutations

BackgroundHaemophagocytic lymphohistiocytosis (HLH) is considered to be a large challenge for clinicians due to the variable overlaps of symptoms with other severe diseases and a high rate of mortality. Prompt diagnosis and treatment are crucial to avoid a fatal outcome. However, very limited reports have focused on HLH during chemotherapy (Ch-HLH) due to a low incidence and insufficient knowledge.Case presentationA 22-year-old male was diagnosed with acute monocytic leukemia with FLT3-ITD and DNMT3A mutations and pulmonary infection. He received IA regimen (Idarubicin, 8 mg/m2/d for 3 days and cytarabine, 100 mg/m2/d for 7 days) chemotherapy, anti-infection drugs and blood components transfusions. During the stage of bone marrow suppression, he presented with a fever, cytopenia (WBC, 0.43 × 109/L; Hb, 73 g/L and PLT, 1 × 109/L), refractory coagulation dysfunction (APTT, 104.0 s; PT, 30.5 s and Fbg, 0.87 g/L), splenomegaly (3 cm below the costal margin), hyperferritinemia (SF > 3000 μg/L), increased soluble interleukin-II receptors (sIL-2R > 7500 u/mL) and haemophagocytosis in the bone marrow and was diagnosed with HLH. After he was treated with methylprednisolone at 500 mg/d for 3 days, 120 mg/d for 3 days and 80 mg/d for 3 days, followed by a gradually reduced dose combined with powerful anti-infection drugs, his symptoms subsided and his abnormal parameters recovered to normal levels.ConclusionPatients with HLH in acute leukemia have a high rate of mortality. This case report provides helpful clinical experiences relative to the recognition and treatment of Ch-HLH for clinicians.

Inhibition of pericellular plasminogen activation by apolipoprotein(a): Roles of urokinase plasminogen activator receptor and integrins αMβ2 and αVβ3

Background and aimsLipoprotein(a) (Lp(a)) is a causal risk factor for cardiovascular disorders including coronary heart disease and calcific aortic valve stenosis. Apolipoprotein(a) (apo(a)), the unique glycoprotein component of Lp(a), contains sequences homologous to plasminogen. Plasminogen activation is markedly accelerated in the presence of cell surface receptors and can be inhibited in this context by apo(a). MethodsWe evaluated the role of potential receptors in regulating plasminogen activation and the ability of apo(a) to mediate inhibition of plasminogen activation on vascular and monocytic/macrophage cells through knockdown (siRNA or blocking antibodies) or overexpression of various candidate receptors. Binding assays were conducted to determine apo(a) and plasminogen receptor interactions. ResultsThe urokinase-type plasminogen activator receptor (uPAR) modulates plasminogen activation as well as plasminogen and apo(a) binding on human umbilical vein endothelial cells (HUVECs), human acute monocytic leukemia (THP-1) cells, and THP-1 macrophages as determined through uPAR knockdown and overexpression. Apo(a) variants lacking either the kringle V or the strong lysine binding site in kringle IV type 10 are not able to bind to uPAR to the same extent as wild-type apo(a). Plasminogen activation is also modulated, albeit to a lower extent, through the Mac-1 (αMβ2) integrin on HUVECs and THP-1 monocytes. Integrin αVβ3 can regulate plasminogen activation on THP-1 monocytes and to a lesser extent on HUVECs. ConclusionsThese results indicate cell type-specific roles for uPAR, αMβ2, and αVβ3 in promoting plasminogen activation and mediate the inhibitory effects of apo(a) in this process.

Clonal neutrophil infiltrates in concurrent Sweet's syndrome and acute myeloid leukemia: A case report and literature review

Sweet's syndrome (SS), also known as acute febrile neutrophilic dermatosis is often associated with a hematological malignancy, especially acute myeloid leukemia (AML) and myeloid dysplasia syndrome. Histopathologically, SS is characterized by diffuse infiltrates in the upper dermis, predominantly consisting of mature neutrophils. The origin of neutrophils invading the skin remains unknown. Herein, we report a patient with concurrent acute monoblastic leukemia and SS who initially presented with discrete erythematous papules and nodules on the neck. Single nucleotide polymorphism (SNP) array and next generation sequencing (NGS) revealed a concordant fms-related tyrosine kinase-3 (FLT-3) gene mutation in the bone marrow and skin lesion, indicating that the neutrophilic infiltrates were clonally related to the underlying myeloid neoplasm. This is the first case report of concurrent SS and AML, in which SNP array and NGS analysis were applied to confirm the clonality of the neutrophilic infiltrates.

Histiocytic sarcoma of the neck: A case report.

Histiocytic sarcoma (HS) is a term used to describe malignant hyperplasia of cells exhibiting morphological and immunophenotypical characteristics similar to those of mature cells, with expression of one or more tissue cell markers, excluding acute monocytic leukemia and primitive monocytic sarcoma. We herein describe a case of histiocytic sarcoma of the neck supported by histopathological and immunohistological evidence. A 53-year-old female patient of Chinese descent presented with a rapidly enlarging right neck mass. Imaging studies revealed multiple right cervical lymphadenectases with right jugular vein involvement. The tumor was composed of diffusely distributed large non-cohesive tumor cells, round or oval and focally spindle-shaped. The tumor cells were immunopositive for macrophage-associated antigen CD68 and lysosomes, mostly consistent with a diagnosis of HS. HS is very prone to systemic metastasis; therefore, early diagnosis and timely treatment are crucial.

Expression of interleukin-6 in cystic lesions of patients with acne vulgaris and in vitro effect of Propionibacterium acnes on the production of interleukin-6 by human THP-1 monocytes

Objective To determine the expression of interleukin-6 (IL-6) in cystic lesions of patients with acne vulgaris, and to evaluate the in vitro effect of Propionibacterium acnes (P. acnes) on the production of IL-6 and activation of p38 mitogen-activated protein kinase (p38MAPK) in the human acute monocytic leukemia cell line THP-1. Methods Real-time fluorescence-based quantitative PCR was performed to determine the mRNA expression of IL-6 in cystic lesions of 6 patients with acne vulgaris, as well as in skin tissues of 6 healthy persons. Some cultured THP-1 cells were divided into 5 groups to betreated with 2 × 106 CFU/ml, 2 × 107 CFU/ml and 2 × 108 CFU/ml heat-killed P. acnes suspensions (P. acnes groups) , 100 μg/L lipopolysaccharide (LPS group) and RPMI 1640 medium (control group) respectively. After 1-, 3- and 6-hour treatment, real-time fluorescence-based quantitative PCR was conducted to determine the mRNA expression of IL-6 in the above groups. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the level of IL-6 in the culture supernatant of cells in the 2 × 108-CFU/ml P. acnes group, LPS group and control group at 24 hours after the treatment. Western blot analysis was conducted to determine the protein expression of p38MAPK and phosphorylated p38MAPK in the 2 × 108-CFU/ml P. acnes group after 15-, 30- and 60-minute treatment, as well as in the LPS group after 30-minute treatment and in the control group. Some other THP-1 cells were divided into 3 groups: 2 × 108-CFU/ml P. acnes group treated with 2 × 108 CFU/ml P. acnes suspensions, SB203580 (an inhibitor of p38MAPK) group treated with 20 μmol/L SB203580 for 30 minutes followed by the treatment with 2 × 108 CFU/ml P. acnes suspensions, and control group treated with RPMI 1640 medium alone. After 6-hour treatment, the mRNA expression of IL-6 in the above 3 groups was measured by real-time fluorescence-based quantitative PCR. Results The mRNA expression of IL-6 was significantly higher in the cystic lesions of acne vulgaris than in the normal skin tissues (3.680 ± 0.790 vs. 1.155 ± 0.250, t= 3.047, P < 0.05) . Two-way analysis of variance showed that there were significant difference in the mRNA expression of IL-6 among the 2 × 106-CFU/ml, 2 × 107-CFU/ml and 2 × 108-CFU/ml P. acnes groups, LPS group and control group (F= 532.3, P < 0.001, v= 4) , and the mRNA expression of IL-6 significantly differed among different time points (F= 526.6, P < 0.001, v= 2) . There were also significant differences in the IL-6 level in the culture supernatant of cells among the 2 × 108-CFU/ml P. acnes group ([1 618.22 ± 32.23]ng/L) , LPS group ([3 212.06 ± 353.00]ng/L) and control group ([147.10 ± 0.53]ng/L; v= 2, F= 102.35, P < 0.01) . After 15-, 30- and 60-minute treatment with 2 × 108 CFU/ml P. acnes suspensions, the protein expression of phosphorylated p38MAPK obviously increased. The mRNA expression of IL-6 in THP-1 cells was significantly lower in the SB203580 group than in the 2 × 108-CFU/ml P. acnes group (t= 15.91, P= 0.004) . Conclusions The mRNA expression of IL-6 evidently increases in the cystic lesions of patients with acne vulgaris. P. acnes can activate the signaling molecule p38MAPK in THP-1 cells, and promote the production of IL-6 by THP-1 cells. Key words: Acne vulgaris; Propionibacterium acnes; Interleukin-6; Leukemia, monocytic, acute; p38 Mitogen-activated protein kinases

Akt, mTOR and NF-κB pathway activation in Treponema pallidum stimulates M1 macrophages

The polarization of macrophages and the molecular mechanism involved during the early process of syphilis infection remain unknown. This study was conducted to explore the influence of Treponema pallidum (T. pallidum) treatment on macrophage polarization and the Akt-mTOR-NFκB signaling pathway mechanism involved in this process. M0 macrophages derived from the phorbol-12-myristate-13-acetate-induced human acute monocytic leukemia cell line THP-1 were cultured with T. pallidum. T. pallidum induced inflammatory cytokine (IL-1β and TNF-α) expression in a dose- and time-dependent manner. However IL-10 cytokine expression decreased at the mRNA and protein levels. Additionally, the expression of the M1 surface marker iNOS was up-regulated with incubation time, and the expression of the M2 surface marker CD206 was low (vs. PBS treated macrophages, P < 0.001) and did not fluctuate over 12 h. Further studies revealed that Akt-mTOR-NFκB pathway proteins, including p-Akt, p-mTOR, p-S6, p-p65, and p-IκBα, were significantly higher in the T. pallidum-treated macrophages than in the PBS-treated macrophages (P < 0.05). In addition, inflammatory cytokine expression was suppressed in T. pallidum-induced M1 macrophages pretreated with LY294002 (an Akt-specific inhibitor) or PDTC (an NF-κB inhibitor), while inflammatory cytokine levels increased in T. pallidum-induced M1 macrophages pretreated with rapamycin (an mTOR inhibitor). These findings revealed that T. pallidum promotes the macrophage transition to pro-inflammatory M1 macrophages in vitro. The present study also provides evidence that Akt, mTOR and NF-κB pathway activation in T. pallidum stimulates M1 macrophages. This study provides novel insights into the innate immune response to T. pallidum infection.

Imidazole Alkaloids and Their Zinc Complexes from the Calcareous Marine Sponge Leucetta chagosensis.

Five new imidazole derivatives (1-5), together with eight related known alkaloids, were isolated from a calcareous marine sponge, Leucetta chagosensis, collected from the South China Sea. Their structures were fully characterized by spectroscopic methods. Structurally, 1 possesses an unusual skeleton featuring imidazole and oxazolone rings linked via a nitrogen atom, whereas 2 bears an intriguing guanylurea-substituted imidazole ring. Compounds 4 and 5 were identified as zinc complexes; they represent the metal complex analogues of naamidine J (6) and pyronaamidine (7), respectively. Among the isolated compounds, 2 and 5 showed significant inhibitory activities toward the LPS-induced production of IL-6 in the human acute monocytic leukemia cell line THP-1, and 7 displayed cytotoxicity against MCF-7, PC9, A549, and breast cancer stem cells (MCF-7-Oct4-GFP) with IC50 values of 5.2, 5.6, 7.8, and 10 μM, respectively.

Inhibition of STAT3 phosphorylation by curcumin partly reduces adhesion molecule expression in vascular endothelial cell

OBJECTIVESWe investigated whether curcumin, an ingredient of turmeric, can suppress the expression of intercellular adhesion molecule 1 (ICAM‐1) and vascular cell adhesion molecule 1 (VCAM‐1) in human umbilical vein endothelial cells (HUVEC) stimulated with lipopolysaccharide (LPS), and the underlying mechanisms.MATERIALS AND METHODOLOGYThe levels of adhesion molecules, activities of nuclear factor kappaB (NF‐kB) and signal transducer and activator of transcription 3 (STAT3) were determined using Western blot analysis. The effect of curcumin on monocyte binding to endothelial cells was determined using THP‐1 monocyte (human acute monocytic leukemia cell line) adhesion assay.RESULTSCurcumin prevented the LPS‐mediated increase in ICAM‐1 and VCAM‐1 expression in HUVEC in a dose‐dependent manner. Curcumin did not affect LPS induced degradation of IkappaBα (IkBα). On the other hand, curcumin reduced the LPS‐induced STAT3 phosphorylation. Curcumin significantly reduced LPS‐mediated THP‐1 monocyte adhesion to HUVEC. Furthermore, in C57BL/6 mice, curcumin prevented the LPS induced increase in aortic ICAM‐1 and VCAM‐1 expression.CONCLUSIONSCurcumin prevented LPS‐induced ICAM–1 and VCAM–1 expression on vascular endothelial cells associated with inhibition of STAT3 phosphorylation.Support or Funding InformationThis work was supported by the research grants of the Chungbuk National University in 2014.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Acute monocytic leukemia diagnosed by flow cytometry includes acute myeloid leukemias with weakly or faintly positive non-specific esterase staining.

A diagnosis of acute monocytic leukemia (AML-M5) based on α-naphthyl butyrate esterase (α-NB) staining has some problems, because AML-M5 leukemic cells often show weak or faint positivity on α-NB staining. In these situations, some cases of AML-M5 tend to be misdiagnosed as AML-M0. Therefore, we evaluated the significance of weak or faint α-NB staining in AML-M5 diagnosed by flow cytometry (FCM). Nineteen AML cases in which leukemic cells were negative for naphthol AS-D chloroacetate esterase staining were studied. For FCM, we defined leukemic cells as having a monocytic nature when more than 10% of the leukemic cells were positive for at least one of the following antigens: CD4, CD11c, CD14, and CD64. The monocytic nature determined by FCM was consistent with positive or weak positivity on α-NB staining. Five of 6 cases in which leukemic cells exhibited faint positivity for α-NB staining could be diagnosed as AML-M5 by FCM, while negative α-NB staining was consistent with a diagnosis of AML-M0. These results suggest that AML-M5 should be taken into consideration even when leukemic cells are faintly positive for α-NB staining.

Screening and identification of RNA interference sequence for human acute monocytic leukemia related antigen 22

Objective To screen the optimal RNA interference sequence of acute monocytic leukemia associated antigen 22 (MLAA-22) gene in order to study gene function of it. Methods MLAA-22 coding sequence (CDS) was cloned by reverse transcription polymerase chain reaction (RT-PCR) and the CDS was inserted in to pEGFP-N1-3FLAG vector to construct eukaryotic expression vector of MLAA-22. At the same time, four RNA interference sequences were designed and cloned to the vector. Expression vector and RNA interference vector were co-transfected into 293T cells, and the optimal RNA interference sequence was screened by fluorescence and Western blot analyses. Results MLAA-22 eukaryotic expression vector pEGFP-N1-3FLAG and four RNA interference vectors were successfully constructed. After co-transfected 293T cells, KD2 was selected as the optimal interference sequence of MLAA-22. Conclusion KD2 is an optimal interference sequence for targeting MLAA-22 antigen gene. Key words: Leukemia, monocytic, acute; RNA interference; Acute monocytic leukemia associated antigen 22

Intramacrophage Mycobacterium tuberculosis efflux pump gene regulation after rifampicin and verapamil exposure.

Since resistance of Mycobacterium tuberculosis (Mtb) partially derives from efflux pumps (EPs) in the plasma membrane, the current study evaluates EPs in Mtb exposed to rifampicin in the presence of the EP inhibitor verapamil, within a macrophage environment. Human acute monocytic leukaemia cell line THP-1 was infected with Mtb H37Rv and exposed to rifampicin and verapamil alone and in combination for 24 and 72 h. After RNA extraction, quantitative PCR was carried out for 11 EP genes using SYBR green PCR master mix in the StepOne™ Real-Time PCR System. After 24 h of exposure to rifampicin, Mtb H37Rv showed that 10 EP genes were up-regulated when compared with the control. The rifampicin/verapamil combination induced down-regulation of 54.5% (6/11) of the EP genes. At 72 h, rifampicin exposure induced up-regulation of 10 EP genes and rifampicin/verapamil induced down-regulation of 8 EP genes, which suggests effective EP-inhibitory activity of verapamil against Mtb H37Rv in an intramacrophage environment. The current study demonstrated that rifampicin/verapamil caused down-regulation of several EP genes in Mtb inside the macrophage environment. In vivo trials may show that rifampicin/verapamil therapy could be of value in enhancing anti-TB treatment.

High glucose induces dysfunction of human umbilical vein endothelial cells by upregulating miR-137 in gestational diabetes mellitus

Recent studies have revealed considerable dysfunction of vascular endothelial cells (VECs) and abnormal expression of microRNA (miR)-137 in women with gestational diabetes mellitus (GDM), and the aim of this study was to clarify the underlying mechanism and possible role of microRNA (miR)-137 in dysfunction of VECs during GDM. We found increased levels of miR-137 in the plasma of GDM women and high-glucose (HG)-exposed HUVECs. Upregulating miR-137 in HUVECs elevated the chemokine (C-C motif) ligand 2 (CCL2) secretion and enhanced the chemotaxis and adhesion of U937 and THP-1 (two human acute monocytic leukemia cell lines) cells to HUVECs in a co-culture system. Moreover, HG stimulation and/or overexpression of miR-137 inhibited the viability, upregulated the expression levels of vascular cell adhesion molecule-1 (VCAM-1), intercellular cell adhesion molecule-1 (ICAM-1), E-selectin, and inflammatory cytokine interleukin (IL)-6, and downregulated the production of IL-8, vascular endothelial growth factor (VEGF), and angiogenesis of HUVECs in vitro. These results imply that up-regulated miR-137 by HG can restrict the viability and angiogenesis, promote the activation and inflammatory cytokine secretion of VECs, and stimulate the monocyte chemotaxis and adhesion to VECs. Ultimately, we have concluded that miR-137 is crucial to HG-induced VEC dysfunction and may be involved in pathology of GDM.

Plasticity of Human THP-1 Cell Phagocytic Activity during Macrophagic Differentiation.

Studies of the role of macrophages in phagocytosis are of great theoretical and practical importance for understanding how these cells are involved in the organism's defense response and in the development of various pathologies. Here we investigated phagocytic plasticity of THP-1 (acute monocytic human leukemia) cells at different stages (days 1, 3, and 7) of phorbol ester (PMA)-induced macrophage differentiation. Analysis of cytokine profiles showed that PMA at a concentration of 100nM induced development of the proinflammatory macrophage population. The functional activity of macrophages was assessed on days 3 and 7 of differentiation using unlabeled latex beads and latex beads conjugated with ligands (gelatin, mannan, and IgG Fc fragment) that bind to the corresponding specific receptors. The general phagocytic activity increased significantly (1.5-2.0-fold) in the course of differentiation; phagocytosis occurred mostly through the Fc receptors, as shown previously for M1 macrophages. On day 7, the levels of phagocytosis of gelatin- and Fc-covered beads were high; however, the intensity of ingestion of mannan-conjugated beads via mannose receptors increased 2.5-3.0-fold as well, which indicated formation of cells with an alternative phenotype similar to that of M2 macrophages. Thus, the type and the plasticity of phagocytic activity at certain stages of macrophage differentiation can be associated with the formation of functionally mature morphological phenotype. This allows macrophages to exhibit their phagocytic potential in response to specific ligands. These data are of fundamental importance and can be used to develop therapeutic methods for correcting the M1/M2 macrophage ratio in an organism.