Inhibition of tumor necrosis factor α–stimulated monocyte adhesion to human aortic endothelial cells by AMP-activated protein kinase

<b>Objective</b>— Proatherosclerotic adhesion of leukocytes to the endothelium is attenuated by NO. As AMP-activated protein kinase (AMPK) regulates endothelial NO synthesis, we investigated the modulation of adhesion to cultured human aortic endothelial cells (HAECs) by AMPK. <b>Methods and Results</b>— HAECs incubated with the AMPK activator, AICAR, or expressing constitutively active AMPK demonstrated reduced TNF α-stimulated adhesion of promonocytic U-937 cells. Rapid inhibition of TNF α-stimulated U-937 cell adhesion by AICAR was NO-dependent, associated with unaltered cell surface adhesion molecule expression, and reduced MCP-1 secretion by HAECs. In contrast, inhibition of TNF α-stimulated U-937 cell adhesion by prolonged AMPK activation was NO-independent and associated with reduced cell surface adhesion molecule expression. <b>Conclusions</b>— AMPK activation in HAECs inhibits TNF α-stimulated leukocyte adhesion by a rapid NO-dependent mechanism associated with reduced MCP-1 secretion and a late NO-independent mechanism whereby adhesion molecule expression, in particular E-selectin, is suppressed. We investigated the functional effects of AMPK activation in cultured human endothelial cells. Stimulation of AMPK inhibited TNF α-stimulated monocyte adhesion by two distinct mechanisms: a rapid NO-dependent mechanism associated with a reduction in chemokine release and a late NO-independent mechanism whereby adhesion molecule expression is suppressed

In vitro response of human pathological hematopoietic cells to fludarabine phosphate

The present study was undertaken to determine a possible influence of fludarabine (fludarabine phosphate, F-ara-AMP) on the cell viability and count. The experiments were performed in vitro on human acute lymphoblastic MOLT-4 cells, human acute myeloblastic ML-1 cells, and human histiocytic lymphoma U-937 cells. The research was conducted using the spectrophotometric and Beckman Coulter methods. The cell viability was analyzed using MTT assay. The cell count was detected using an electronic Z2 Coulter counter. Temporary changes in the cell viability and count were assessed at 24h and 48h after F-ara-AMP application. The in vitro activity of fludarabine phosphate against MOLT-4, ML-1, and U-937 cells was compared. F-ara-AMP applied at the four concentrations - 250 nM, 500 nM, 750 nM, and 1 μM - distinctly decreased the viability and count of the pathological hematopoietic cells. The effects of F-ara-AMP on MOLT-4, ML-1, and U-937 cells were dependent on the tested agent and its dose, the time intervals after the agent application, and the cell line used. ML-1 and U-937 cells appeared to be more resistant than MOLT-4 cells to the action of fludarabine phosphate. The in vitro response of the three human pathological hematopoietic cell lines to the F-ara-AMP action, was shown

Doxorubicin selectively induces apoptosis through the inhibition of a novel isoform of Bcl‑2 in acute myeloid leukaemia MOLM‑13 cells with reduced Beclin 1 expression

The overexpression of anti-apoptotic Bcl-2 in acute myeloid leukaemia (AML) may contribute to difficulties in eradicating these cells during chemotherapy. In the present study, doxorubicin (Dox) was evaluated for its potential to induce selective apoptotic cell death in AML MOLM-13 cells and to modulate autophagy through Bcl-2 and Beclin 1 protein expression. Annexin V/propidium iodide and 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) flow cytometric analyses were conducted to determine the effects of Dox on cell death and cell proliferation, respectively, following 48 h of co-incubation with AML MOLM-13 or U-937 monocytic cells. The protein expression levels of Bcl-2 and Beclin 1 in untreated and treated cells were quantified by western blot analysis. Dox reduced the viability of MOLM-13 cells partly by inhibiting cell division and inducing cell apoptosis. Dox demonstrated a level of selectivity in its cytotoxicity against MOLM-13 compared to U-937 cells (P<0.05). Dox induced a significant decrease in Beclin 1 protein levels in MOLM-13 cells without significantly affecting the protein levels in U-937 monocytes. A novel Bcl-2 15-20 kDa (p15-20-Bcl-2) isoform was found to be selectively expressed in AML MOLM-13 cells (but absent in the leukaemic cell lines tested, OCI-AML2, CML K562 and U-937). Dox induced a highly significant inhibition of p15-20-Bcl-2 at concentrations of 0.5, 0.75 and 1 µM (P<0.01). However, the usual 26 kDa Bcl-2 (p26-Bcl-2-α) isoform protein expression was not affected by the drug in either the MOLM-13 or U-937 cells. It was thus postulated that Dox exhibited some selectivity by targeting the p15-20-Bcl-2 isoform in MOLM-13 cells and activating Beclin 1 to induce cell death

Effect of Allium sativum (garlic) methanol extract on viability and apoptosis of human leukemic cell lines

Purpose: To investigate the effect of Allium sativum (garlic) methanol extract on viability and apoptosis of human leukemic cells.Methods: Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at concentrations of 3.125, 6.25, 12.5, 25, 50, 100, 200, 400 and 800 ug/mL of Allium sativum extract following 48-h treatment on U-937, Jurkat Clone E6-1 and K-562 cell lines. The mode of cell death was determined by Annexin V-FITC staining and analyzed by flow cytometry.Results: The results show that the half-maximal inhibitory concentration (IC50) of A. sativum on U-937, Jurkat Clone E6-1, K-562 cell lines was 105 ± 2.21, 489 ± 4.51 and 455 ± 3.13 μg/mL, respectively, compared with negative control, while apoptosis was 17.93 ± 0.95 % for U-937 cells (p ≤ 0.05), 38.37 ± 1.88 % for Jurkat Clone E6-1 cells (p ≤ 0.001) and 16.37 ± 1.10 % for K-562 cells. A majority of the cells were inhibited by the extract via apoptosis. Only U-937 cells (6.87 ± 0.65 %) showed significant necrosis compared to negative control (p ≤ 0.05).Conclusion: K-562 cells are the most resistant against garlic extract, in contrast to Jurkat Clone E6-1 cells. Garlic extract does not induce necrosis in Jurkat Clone E6-1 and K-562 cells.Keywords: Anti-leukemic, Garlic, Allium sativum, Annexin V-FITC staining,  Necrosis, Apoptosis, Flow cytometry, Jurkat Clone E6-1 cell

Regulation of 5-oxo-ETE synthesis in inflammatory cells

5-Oxo-ETE ist ein chemotaktischer Faktor für Granulozyten, der von der NADP+-abhängigen Dehydrogenase 5h-dh aus dem 5-Lipoxygenaseprodukt 5-HETE gebildet wird. Ziel dieser dreiteiligen Studie war es, die der 5-oxo-ETE-Produktion zugrunde liegenden Regulationsmechanismen aufzuklären. I. Einfluß von myeloider Zelldifferenzierung auf die Expression von 5h-dh in HL-60 und U-937 Zellen. Undifferenzierte HL-60 und U-937 Zellen produzieren vergleichbare Mengen von 5-oxo-ETE wie Monozyten oder Granulozyten. Differenzierung von U-937 Zellen mit PMA verdreifacht die Enzymaktivtät von 5h-dh, während die Behandlung von HL-60 Zellen mit dh-VitD3 diese verdoppelte. Der Einfluß von PMA auf 5h-dh wurde darüber hinaus in Mikrosomen von U-937 Zellen untersucht. Die Behandlung PMA verdreifachte Vmax, liess aber KM unbeeinflußt. II. Regulation der 5-oxo-ETE-Produktion durch oxidativen Stress und Glukose. Da der GSH-Redoxzyklus die Produktion von NADP+ zur Folge hat, stimulierten die Hydroperoxide H2O2 und tBOOH die Synthese von 5-oxo-ETE in U-937 Zellen. Aufgrund seiner Verarbeitung durch den Pentosephosphat Zyklus, der NAD+ in NADPH umwandelt, inhibierte Glucose diesen Effekt von H2O2. Die Synthese von 5-oxo-ETE wurde durch H2O2 auch in humanem Monozyten, Lymphocyten und Thrombozyten, aber nicht in Neutrophilen angeregt. Im Gegensatz zu Monozyten zeigten sich Thrombozyten und Lymphozyten allerdings glukose-resistent. T-BOOH ehöhte auch die Produktion von 5-oxo-ETE nach Zugabe von Ionophore und Arachidonsäure zu mononukleären Blutzellen. III. 5h-dh-Expression in human Strukturzellen. Zunächst rasterten wir mehrere sekundäre Epithelzelllinien und fanden 5h-dh in allen Zellen. Drei Indizien lassen vermuten, daß die epithele 5h-dh der myeloiden entspricht: (i) die enzymatische Aktivtät liegt vor allem in der mikrosomalen Fraktion vor, (ii) bei dem Kofaktor handelt es sich um NADP+ und nicht um NAD+, und (iii) 5S-HETE ist das bevorzugte Substrat. Weitere Studien zeigten, daß auch primäre humane Aorta-Endothelzellen 5h-dh expremieren. Vergleichbar zu Entzündungszellen wird die Produktion von 5-oxo-ETE auch in Endothel- und in Epithelzellen durch oxidativen Stress angeregt.5-Oxo-ETE is a highly potent granulocyte chemoattractant that is formed by the NADP+-dependent dehydrogenase 5h-dh by oxidation of the 5-lipoxygenase product 5-HETE. The objective of this study was to investigate underlying regulatory mechanisms of 5-oxo-ETE production in human cells. This matter was addressed from three directions. I. Expression of 5h-dh in HL-60 and U-937 cells and its activity changes during myeloid cell differentiation. Undifferentiated U-937 and HL-60 cells produce similar amounts of 5-oxo-ETE compared to monocytes or neutrophils. Differentiation of U-937 cells with PMA resulted in a 3-fold increase in 5-oxo-ETE production. Similarly, incubation of HL-60 cells with dh-VitD3 induced a 2-fold increase in 5-oxo-ETE production. The impact of PMA on 5h-dh was also investigated in the microsomal fraction of U-937 cells and compared to neutrophil microsomes. PMA treatment leads to a increase of Vmax but does not affect KM. II. Regulation of 5-oxo-ETE by oxidative stress and glucose levels. We found that H2O2 and t-butyl hydroperoxide strongly stimulate 5-oxo-ETE formation by U-937 cells through the GSH redox cycle by providing NADP+. Glucose inhibited the response to H2O2 through its metabolism by the pentose phosphate pathway, which converts NADP+ back to NADPH. 5-Oxo-ETE synthesis was also strongly stimulated by hydroperoxides in blood monocytes, lymphocytes, and platelets, but not neutrophils. Unlike monocytic cells, lymphocytes and platelets were resistant to the inhibitory effects of glucose. 5-Oxo-ETE synthesis following incubation of peripheral blood mononuclear cells with arachidonic acid and calcium ionophore was also strongly enhanced by t-BOOH. III. Expression of 5h-dh in human structural cells. We screened several secondary epithelial cell lines and detected 5h-dh in all cell lines. Epithelial 5h-dh and the inflammatory cell 5h-dh are identical: (i) the enzymatic activity is localized in microsomes, (ii) the cofactor is NADP+, and (iii) 5S-HETE is the preferred substrate. We also found that primary human aortic endothelial cells express 5h-dh. 5-oxo-ETE production by both endothelial and epithelial cells is regulated by oxidative stress in a manner similar to inflammatory cells

Expression of MICA, MICB and NKG2D in human leukemic myelomonocytic and cervical cancer cells

<p>Abstract</p> <p>Background</p> <p>Cancer cells are known to secrete the stress molecules MICA and MICB that activate cytotoxicity by lymphocytes and NK cells through their NKG2D receptor as a mechanism of immunological defense. This work was undertaken to evaluate if cancer cells can also express this receptor as a possible mechanisms of depletion of MIC molecules and thus interfere with their immune recognition.</p> <p>Methods</p> <p>Myelomonocytic leukemic (TPH-1 and U-937) and cervical cancer (CALO and INBL) cell lines were evaluated by Western Blot, ELISA, flow cytometry and immunocytochemistry to evaluate their capacity to express and secrete MICA and MICB and to be induced to proliferate by these molecules as well as to express their receptor NKG2D. Statistical analysis was performed by two-way ANOVA for time course analysis and Student's t-test for comparison between groups. Values were considered significantly different if p < 0.05.</p> <p>Results</p> <p>THP-1 and U-937 produce and secrete the stress MICA and MICB as shown by Western Blot of lysed cells and by ELISA of their conditioned media. By Western Blot and flow cytometry we found that these cells also express the receptor NKG2D. When THP-1 and U-937 were cultured with recombinant MICA and MICB they exhibited a dose dependent induction for their proliferation. CALO and INBL also produce MICA and MICB and were induced to proliferate by these stress molecules. By Western Blot, flow cytometry and immunocytochemistry we also found that these cells express NKG2D.</p> <p>Conclusions</p> <p>Our novel results that tumor cells can simultaneously secrete MIC molecules and express their receptor, and to be induced for proliferation by these stress molecules, and that tumor epithelial cells can also express the NKG2D receptor that was thought to be exclusive of NK and cytotoxic lymphocytes is discussed as a possible mechanism of immunological escape and of tumor growth induction.</p

Chlorinated Guaiane-Type Sesquiterpene Lactones as Cytotoxic Agents against Human Tumor Cells

This article belongs to the Section Bioactives and Nutraceuticals.Guaiane-type sesquiterpene lactones are naturally occurring compounds which have attracted attention due to their array of biological activities. In this study, chlorinated guaianolides 1–8, isolated from plants of the genus Centaurea, were evaluated against the human leukemia cell lines HL-60, U-937, a specific U-937 cell line that overexpresses the anti-apoptotic Bcl-2 protein and the human melanoma cell line SK-MEL-1. This established the relevant structure-growth inhibition relationships. Chlorohyssopifolins A (1), C (3) and D (4) and linichlorin A (6) were the most potent compounds in terms of inducing growth inhibition in the four cell lines. IC50 values were below 10 μM in all cases. Chlorohyssopifolins A (1) and D (4) and linichlorin A (6) were potent apoptotic inducers in human U-937 leukemia cells, as determined by fluorescent microscopy and flow cytometry, and their mechanism of action was associated with cytochrome c release, caspase activation and poly(ADP-ribose)polymerase cleavage. Overall this study shows that guaianolides induce cytotoxicity against human tumor cells and provides important insights into the cell death pathways that are involved.This research was funded by FEDER and AGENCIA CANARIA DE INVESTIGACIÓN, INNOVACIÓN Y SOCIEDAD DE LA INFORMACIÓN (PROID2017010095 FEDER/ACIISI) and in part by the Spanish Ministry of Science, Innovation and Universities and the European Regional Development Fund (PGC2018-094503-B-C21)

Immunomodulatory, antioxidant and anti-tumor capacity of acidic polysaccharides from Euglena gracilis.

Euglena sp. is a microalga producer of important molecules for the Biotechnology industry, since it is a producer of substances such as vitamins A, C and E, essential amino acids, polyunsaturated fatty acids, β-carotenes and paramilon (β-1,3-glucan). It has a modulating effect on the immune system, moderates blood glucose and the response to insulin, has anti-tumor activity and a cholesterol-lowering effect. In addition, its sulphated derivatives have anti-HIV activity. The present study was carried out with the objective of determining the immunomodulatory, antioxidant and anticancer activity of the acid polysaccharides extracted from Euglena gracilis. MTT colorimeter tests were carried out for the analysis of cytotoxicity on healthy cell lines murine macrophages (RAW 264.7) and human gingival fibroblasts (HGF-1) and for the anticancer activity cell lines were used colon cancer (HCT-116), breast cancer (MCF-7) and human leukemia (U-937) .The polysaccharide concentration at which cell survival was reduced by half (IC50) was estimated with these assays, showing that these polysaccharides have antitumor activity mainly on U-937 cells. (IC50 = 0.027 mg ml-1) against HCT-116 cells (IC50 = 0.036 mg ml-1) and MCF-7 (IC50 = 0.11 mg ml-1) An immunological test was performed to see the immunomodulatory capacity of the polysaccharides with which the production of proinflammatory cytokines IL-6 and TNF-α was determined by macrophages RAW 264.7 It was observed that these polysaccharides have a great stimulating capacity in the synthesis of these interleukins. Antioxidant capacity was (7.19 μmol TE g-1). In agreement with these results, it is suggested that E. gracilis polysaccharides could be considered for future studies as potential nutraceuticals that require their application when the activation of macrophages is needed.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech