Activity In U937 Cells Research Articles

P70S6 kinase is a target of the novel proteasome inhibitor 3,3′-diamino-4′-methoxyflavone during apoptosis in human myeloid tumor cells

Acute myeloid leukemia (AML) is a deadly disease characterized by the clonal expansion and accumulation of hematopoietic stem cells arrested at various stages of development. Clinical research efforts are currently focusing on targeted therapies that induce apoptosis in AML cells. Herein, the effects and mechanisms of the novel flavone 3,3′-diamino-4′-methoxyflavone (DD1) on AML cell dysfunction were investigated in AML cells (monoblast U937, myelomonocyte OCI-AML3, promyelocyte NB4, myeloblast HL-60) and blood samples from patients with AML. The administration of DD1 inhibited proliferation and induced death of AML cell lines and reduced the clonogenic activity of AML, but not normal, blood cells. The flavone's apoptotic action in U937 cells was associated with recruitment of mitochondria, Bax activation, Bad dephosphorylation (at Ser136), activation of caspases -8, -9, and -3 and cleavage of the caspase substrate PARP-1. DD1 induced a marked decrease in (i) Thr389-phosphorylation and (ii) protein levels of the caspase-3 substrate P70 ribosomal S6 kinase (P70S6K, known for its ability to phosphorylate Bad). Caspase-dependent apoptosis and P70S6K degradation were simultaneously prevented by the caspase inhibitors. Importantly, DD1 was shown to directly inhibit the proteasome's chymotrypsin-like activity in U937 cells. Apoptotic activity of the proteasome inhibitor bortezomib was also related to Bax activation and P70S6K downregulation. Accordingly, DD1 failed to induce P70S6K cleavage, Bax stimulation and apoptosis in K562 cells resistant to bortezomib. These results indicate that DD1 has the potential to eradicate AML cells and support a critical role for Bax and P70S6K in DD1-mediated proteasome inhibition and apoptosis of leukemia cells.

Defect of Adaptation to Hypoxia in Patients With COPD Due to Reduction of Histone Deacetylase 7

Background:Hypoxia inducible factor (HIF)-1 plays an important role in cellular adaptation to hypoxia by activating oxygen-regulated genes such as vascular endothelial growth factor (VEGF) and erythropoietin. Sputum VEGF levels are reported to be decreased in COPD, despite hypoxia. Here we show that patients with COPD fail to induce HIF-1α and VEGF under hypoxic condition because of a reduction in histone deacetylase (HDAC) 7.Methods:Peripheral blood mononuclear cells (PBMCs) were obtained from patients with moderate to severe COPD (n = 21), smokers without COPD (n = 12), and nonsmokers (n = 15). PBMCs were exposed to hypoxia (1% oxygen, 5% CO2, and 94% N2) for 24 h, and HIF-1α and HDAC7 protein expression in nuclear extracts were determined by sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS-PAGE)/Western blotting.Results:HIF-1α was significantly induced by hypoxia in each group when compared with the normoxic condition (12-fold induction in nonsmokers, 24-fold induction in smokers without COPD, fourfold induction in COPD), but induction of HIF-1α under hypoxia was significantly lower in patients with COPD than in nonsmokers and smokers without COPD (P < .05 and P < .01, respectively). VEGF messenger RNA detected by quantitative real-time polymerase chain reaction was correlated with HIF-1α protein in nuclei (r = 0.79, P < .05), and HDAC7 protein expression was correlated with HIF-1α protein in nuclei (r = 0.46, P < .05). HDAC7 knockdown inhibited hypoxia-induced HIF-1α activity in U937 cells, and HIF-1α nuclear translocation and HIF-1α binding to the VEGF promoter in A549 cells.Conclusions:HDAC7 reduction in COPD causes a defect of HIF-1α induction response to hypoxia with impaired VEGF gene expression. This poor cellular adaptation might play a role in the pathogenesis of COPD.

Isolation, purification, immunological and anti-tumor activities of polysaccharides from Gymnema sylvestre

Five water-soluble polysaccharides, including GSP11 (xylose:glucose=1:2.47), GSP22 (rhamnose:glucose:galactose=1.6:1:1.22), GSP33 (rhamnose:glucose:galactose=1:1.5:1.1), GSP44 (xylose:glucose:galactose=1:1.03:1.2) and GSP55 (rhamnose:xylose:glucose:galactose=1:1.21:1.81:2.58), were obtained from Gymnema sylvestre and tested for their immunological and anti-tumor activities. The molecular weights of the five water-soluble polysaccharides were 5.3×104, 5.4×105, 9.5×105, 1.0×106 and 1.9×106Da, respectively. Immunological activity tests revealed that both crude and deproteinated polysaccharides (GPS) possess good immunological activities, especially on blood serum hemolysin formation at 300mg/kg. MTT assays revealed that all samples possessed anti-tumor activities in a dose-dependent manner except for GSP22 in AGS cells. At a concentration of 640μg/mL, GSP11 and GSP33 showed good activities in U937 cells, with inhibitory rates of 78.6% and 83.8%, respectively, and GSP22 showed good activity in SGC cells with an inhibitory rate of 78.2%.

Different signaling mechanisms regulating IL-6 expression by LPS between gingival fibroblasts and mononuclear cells: seeking the common target

To reduce connective tissue IL-6 level stimulated by LPS, it is essential to control IL-6 expression in both mononuclear cells and fibroblasts. However, it is unclear whether the regulatory mechanisms for both cells are similar or not. In this study, we found that signaling pathways mediating LPS-stimulated IL-6 in mononuclear U937 cells and fibroblasts were different. Furthermore, our studies showed that while LPS activated AP-1 and NFκB in U937 cells, it only activated NFκB in fibroblasts. Analysis of nuclear AP-1 subunits showed that LPS stimulated c-Fos, Fra-1 and Jun D activities in U937 cells, but not fibroblasts. The lack of ERK involvement in LPS-stimulated IL-6 in fibroblasts was further supported by the observations that simvastatin, which is known to target ERK-AP-1, failed to inhibit LPS-stimulated IL-6 by fibroblasts. Finally, we showed that targeting NFκB pathway was highly effective in inhibition of LPS-stimulated IL-6 in coculture of U937 cells and fibroblasts.

Tipifarnib and tanespimycin show synergic proapoptotic activity in U937 cells

BackgroundFarnesyltransferase inhibitor tipifarnib (R115777) has been used for treatment of hematological malignancies; however, its observed anticancer effect was limited. This prompted us to search for inhibitors that would show synergic, proapoptotic effect when combined with R115777. We decided to study LY294002, which inhibits PI-3 kinase, and tanespimycin (17AAG), which inhibits Hsp90—a chaperone for a number of proteins, including Akt kinase.MethodsThe effect of drugs, used alone or in combination, was tested in U937 cells (human leukemic monocyte lymphoma), which are often used as a model for liquid tumor. The number of viable cells was evaluated with trypan blue staining, while apoptosis was assessed by presence of active caspase-3 and terminal dUTP nick-end labeling of DNA (TUNEL).ResultsAt concentrations in which R115777, LY294002 and 17AAG were only slowing down the proliferation rate, when used separately, the combination of R115777 + LY294002 and R115777 + 17AAG significantly reduced the number of cells and induced cellular apoptosis.ConclusionsOur results suggest that the combination of R115777 + 17AAG could be useful in treating some of the hematological malignancies.

3,5-Disubstituted-thiazolidine-2,4-dione analogs as anticancer agents: Design, synthesis and biological characterization

A series of 2,5-disubstituted-thiazolidine-2,4-dione analogs based on the newly identified lead 1, a potential anticancer agent via the inhibition of the Raf/MEK/extracellular signal regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling cascades, were synthesized and biologically characterized. A new lead structure, 15, was identified to have improved anti-proliferative activities in U937 cells, to induce apoptosis in U937, M12 and DU145 cancer cells, and to arrest U937 cells at the S-phase. Furthermore, Western blot analysis demonstrated a correlation of the anti-proliferative activity and blockade of the Raf/MEK/ERK and PI3K/Akt signaling pathways. Collectively, these results strongly encourage further optimization of 15 as a new lead with multi-target properties to develop more potent compounds as anticancer agents.

Synthesis, Structural Characterization, and Pro‐apoptotic Activity of 1‐Indanone Thiosemicarbazone Platinum(II) and Palladium(II) Complexes: Potential as Antileukemic Agents

In the search for alternative chemotherapeutic strategies against leukemia, various 1-indanone thiosemicarbazones, as well as eight novel platinum(II) and palladium(II) complexes, with the formula [MCl₂(HL)] and [M(HL)(L)]Cl, derived from two 1-indanone thiosemicarbazones were synthesized and tested for antiproliferative activity against the human leukemia U937 cell line. The crystal structure of [Pt(HL1)(L1)]Cl·2MeOH, where L1=1-indanone thiosemicarbazone, was solved by X-ray diffraction. Free thiosemicarbazone ligands showed no antiproliferative effect, but the corresponding platinum(II) and palladium(II) complexes inhibited cell proliferation and induced apoptosis. Platinum(II) complexes also displayed selective apoptotic activity in U937 cells but not in peripheral blood monocytes or the human hepatocellular carcinoma HepG2 cell line used to screen for potential hepatotoxicity. Present findings show that, in U937 cells, 1-indanone thiosemicarbazones coordinated to palladium(II) were more cytotoxic than those complexed with platinum(II), although the latter were found to be more selective for leukemic cells suggesting that they are promising compounds with potential therapeutic application against hematological malignancies.

Abstract 3262: Pharmaco-modulation of dihydroxy coumarins towards more selective antileukemic agents

Abstract Background: We described that 7,8-dihydroxy-4-methylcoumarin (DHMC) induces apoptosis in human leukemic U-937 cells, mediated by its pro-oxidant activity and activation of JNK. We also reported that DHMC shows selective toxicity for leukemic cells over normal peripheral blood monocytes. The aim of the present work is to establish the structural requirements for DHMC to display pro-apoptotic activity performing a structure activity relationship study of DHMC derivatives, cinnamic acid derivatives (as open-chain coumarin analogues) and a-pyrones (as representative of the d-lactone ring of the coumarin nucleus). Further, in an attempt to explain its selective toxicity, the mechanism of DHMC action through the JNK pathway will be characterized. Materials and Methods: DHMC analogues, cinnamic acid derivatives and a-pyrones were tested for their ability to induce biological effects in U-937 cells. Growth inhibition was determined through 3H-thymidine incorporation. Cellular viability was tested by trypan blue exclusion assays. Pro-apoptotic activity was evaluated by flow cytometry (annexin V), determination of caspase-3 activity and Western Blot analysis of caspase-3 and cleaved PARP levels. The oxidation potential and the capacity to generate free radicals were evaluated by cyclic voltammetry and electron paramagnetic resonance, respectively. To evaluate the role of cytoplasmic p21 (Cip1/WAF1) in the mechanism of DHMC selective action, we used U-937 cells transfected with a zinc-inducible p21 deletion mutant lacking the nuclear localization signal. Results: Only DHMC derivatives bearing two adjacent aromatic hydroxyl groups (catechol moiety) showed proliferation inhibition and pro-apoptotic activity in U-937 cells. a-pyrones had no effect on cellular viability, and cinnamic acid derivatives bearing the catechol moiety exhibited proliferation inhibition but no cytotoxic activity. Although ortho-dihydroxy coumarins and ortho-dihydroxy cinnamic acid derivatives showed a similar oxidation potential, only the former could generate stable free radicals in medium-like conditions, suggesting this capacity would be critical for their pro-apoptotic activity in U-937 cells. Finally, the pro-apoptotic effect of DHMC via JNK activation was abolished in U-937 cells expressing cytoplasmic p21, indicating this protein would be involved in the resistance that normal lymphocytes and monocytes, which normally express p21 in their cytoplasm, show for DHMC. Conclusions: DHMC-derived compounds carrying a catechol moiety can only induce apoptosis in U-937 cells if the integrity of the coumarinic nucleus is preserved. Selective toxicity of DHMC for leukemic cells would involve activation of JNK in the absence of cytoplasmic p21 expression. Collectively, our results point to ortho-dihydroxy coumarins as promising prototypes for the design of more selective antileukemic drugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3262. doi:10.1158/1538-7445.AM2011-3262

Novel Cinnamyl Hydroxyamides and 2‐Aminoanilides as Histone Deacetylase Inhibitors: Apoptotic Induction and Cytodifferentiation Activity

Four novel series of cinnamyl-containing histone deacetylase (HDAC) inhibitors 1-4 are described, containing hydroxamate (1 and 3) or 2-aminoanilide (2 and 4) derivatives. When screened against class I (maize HD1-B and human HDAC1) and class II (maize HD1-A and human HDAC4) HDACs, most hydroxamates and 2-aminoanilides displayed potent and selective inhibition toward class I enzymes. Immunoblotting analyses performed in U937 leukemia cells generally revealed high acetyl-H3 and low acetyl-α-tubulin levels. Exceptions are compounds 3 f-i, 3 m-o, and 4 k, which showed higher tubulin acetylation than SAHA. In U937 cells, cell-cycle blockade in either the G₂/M or G₁/S phase was observed with 1-4. Five hydroxamates (compounds 1 h-l) effected a two- to greater than threefold greater percent apoptosis than SAHA, and in the CD11c cytodifferentiation test some 2-aminoanilides belonging to both series 2 and 4 were more active than MS-275. The highest-scoring derivatives in terms of apoptosis (1 k, 1 l) or cytodifferentiation (2 c, 4 n) also showed antiproliferative activity in U937 cells, thus representing valuable tools for study in other cancer contexts.

Roles of Bcl-2 family members, PI3K and NF-κB pathways in Escherichia coli-induced apoptosis in human monocytic U937 cells

We previously showed that infection of human monocytic U937 cells with nonpathogenic Escherichia coli (E. coli) induced rapid apoptosis in a dose- and time-dependent manner. We also found that E. coli increase p38 mitogen-activated protein Kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK), and decrease extracellular-Regulated Kinase1/2 (ERK1/2) phosphorylation and increase caspase-3 and -9 activity in U937 cells. The current study determines if Bcl-2, Bax, the phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor kappa B (NF-κB) regulates E. coli–induced U937 cell apoptosis. Studying the underlying mechanisms we found that the E. coli-induced apoptosis in U937 cells was associated with a more prominent reduction in expression of Bcl-2, levels of P-Akt and NF-κB. Because levels of inhibition of apoptosis protein (cIAP), and X-chromosomelinked inhibitor of apoptosis protein (XIAP) are regulated by NF-κB, E. coli decreased the levels of these proteins in U937 cells through inhibition of NF-κB. Moreover, E. coli markedly elevated Bax expression and cytochrome c redistribution. LY294002, PDTC and Embelin, specific inhibitors of PI3K, NF-κB and XIAP, induced U937 cell apoptosis and the apoptosis is dependent on activity of caspase-3 and -9 in E. coli-treated U937 cells. Through using LY294002 and western blotting, we identified NF-κB was the downstream Akt target regulated by E. coli. Taken together, these results clearly indicate reduced activation of NF-κB via impaired PI3K/Akt activation could result in increased apoptosis of U937 cells infected by E. coli. Moreover, E. coli can induce apoptosis with an increased expression of Bax and a reduced expression of Bcl-2, which resulted in increased levels of cytochrome c release and increase caspase-3 and -9 in U937 cells.

Novel Histone Deacetilase (HDAC) Inhibitors: In Vitro Effects on Leukemic Cells

AbstractAbstract 4946Histone deacetylase inhibitors (HDAC-I) are a class of agents with the capacity to induce acetylation of histone and non-histone proteins. These molecules have been intensively investigated in a variety of malignancies because of their ability to inhibit proliferation, induce differentiation and apoptosis in tumor cells. However, clinical response to clinically available HDAC-I have been obtained only in a proportion of patients, prompting further studies aimed at identifying more active compounds and at defining the molecular mechanisms of response to this class of agents. Acetyl-L-carnitine (ALCAR) is a metabolic intermediate that facilitates the influx and efflux of acetyl groups across the mitochondrial inner membrane, thereby contributing to the regulation of energy production and metabolism. ALCAR activity as a modulator of cellular stress response has prompted its use to protect against chemotherapy-induced neurotoxicity. However, ALCAR effects on neoplastic cells are still not defined, especially in combination with chemotherapy. Here we investigated the effects of MS-275, a HDAC-I, on cell proliferation and apoptosis in cell line models of acute myeloid leukemia (AML) acute lymphoblastic leukemia (ALL), and multiple myeloma (MM), in comparison with vorinostat also known as SAHA (suberoyl anilid hydroxamic acid), the most widely used HDAC-I in clinical setting. HDAC-I were tested at doses ranging from 5 to 5000nM. In addition, the effects of simultaneous exposure to 10 mM of ALCAR and selected sub-toxic concentration of HDAC-I were analyzed. The cytotoxic effect of the treatment was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The drug concentration inducing 50 % cell killing (IC-50) was calculated from the dose-response curve. Cell cycle inhibition and induction of apoptosis were analyzed by flow cytometry using the Acridine- Orange (AO) technique. Results indicated that the tested compound MS-275 significantly inhibited cell growth, as assessed by MTT assay, when used at of 5000nM. Comparative analysis of the efficacy of the two different HDAC-I compounds indicated that MS-275 was the more effective agent and the only one with clear dose-dependent activity, while SAHA displayed a flat dose-response curve, which dropped only at the highest concentration. In particular, the myeloid cell line Molm-13 was strikingly sensitive to MS-275 (IC50: < 15 nM), U937 and HL60 myeloid cell lines, the lymphoid cell line Jurkat and the MM cell line ARH-77 showed intermediate sensitivity (IC50: < 1000 nM), while the lymphoid cell line CEM R was resistant (IC50 > 10 uM). SAHA showed no activity in U937 cells when used at concentrations ranging from 100 to 1000 nM, with a dramatic reduction of absorbance at 5000 nM (>80% reduction compared to the control). Nevertheless, the combination of 500 nM SAHA with 10mM ALCAR reveled a synergistic interaction, with a 46% reduction in absorbance. We then analyzed the effects on apoptosis induction, as determined by the percentage of cells with a sub-G1 DNA content. MS-275 dose-dependently induced apoptosis in HL-60 cells (4.2%, 17.1%, 60.8%, and 87.5% in the presence of 100, 500, 1000, 5000 nM of MS-275, respectively). Conversely, SAHA induced minimal apoptosis (< 10%) at concentration ranging from 100 to 1000 nM, although > 75% of cells became apoptotic after treatment with the compound at 5000 nM. In summary, our results show that the HDAC-I MS-275 is a potent inhibitor of leukemic cell growth, capable of inducing apoptosis particularly in cell lines derived from myeloid leukemia and MM. Preliminary studies exploring the combined use of ALCAR with the SAHA support a potential anti-neoplastic synergism in selected hematological malignancies. Disclosures:Petrucci: Celgene: Honoraria; Janssen Cilag: Honoraria. Pisano: Sigma-Tau: Employment. Tafuri: Sigma-Tau: Research Funding.

Intracellular Prooxidant Activity of Melatonin Induces a Survival Pathway Involving NF‐κB Activation

We have shown that melatonin exerts a prooxidant activity in U937 cells, a tumor human promonocytic cell line. (1) Here we show that melatonin induces a strong canonical activation of NF-kappaB, inducing IkappaBalpha degradation and the consequential nuclear translocation of p50/p65 subunits. The timing of NF-kappaB activation overlaps with the timing of reactive oxygen species (ROS) production due to melatonin. Overexpression of dominant-negative IkappaB, which prevents a possible NF-kappaB activation, transformed melatonin in a proapoptotic molecule. These data indicate for the first time that melatonin can trigger NF-kappaB activation and might suggest a possible role for ROS induced by melatonin. Results indicate a possible involvement in the survival pathway of melatonin-generated ROS as secondary messengers.

A protein deacetylase SIRT1 is a negative regulator of metalloproteinase‐9

Inappropriate elevation of matrix metalloproteinase-9 (MMP9) is reported to be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). The object of this study was to identify the molecular mechanism underlying this increase of MMP9 expression, and here we show that oxidative stress-dependent reduction of a protein deacetylase, SIRT1, known as a putative antiaging enzyme, causes elevation of MMP9 expression. A sirtuin inhibitor, splitomycin, and SIRT1 knockdown by RNA interference led an increase in MMP9 expression in human monocytic U937 cells and in primary sputum macrophages, which was detected by RT-PCR, Western blot, activity assay, and zymography. In fact, the SIRT1 level was significantly decreased in peripheral lungs of patients with COPD, and this increase was inversely correlated with MMP9 expression and MMP9 promoter activation detected by a chromatin immunoprecipitation assay. H(2)O(2) reduced SIRT1 expression and activity in U937 cells; furthermore, cigarette smoke exposure also caused reduction of SIRT1 expression in lung tissue of A/J mice, with concomitant elevation of MMP9. Intranasal treatment of a selective and novel SIRT1 small molecule activator, SRT2172, blocked the increase of MMP9 expression in the lung as well as pulmonary neutrophilia and the reduction in exercise tolerance. Thus, SIRT1 is a negative regulator of MMP9 expression, and SIRT1 activation is implicated as a novel therapeutic approach to treating chronic inflammatory diseases, in which MMP9 is abundant.

Induction of type 1 interferon receptor by zinc in U937 cells

This study aims to determine whether zinc enhances interferon (IFN)-α activity in U937 cells. Type 1 IFN2 receptor (IFNAR2) protein in U937 cells was measured by flow cytometry. After 24 h of exposure to zinc chloride or polaprezinc (a chelate of zinc and l-carnosine) at concentrations ranging from 50 to 200 μM, histograms showing anti-IFNAR2 antibody-positive cells shifted to a higher FITC intensity. Zinc chloride and polaprezinc increased IFNAR2 mRNA levels approximately 30% and 40%, respectively, compared to the control. l-Carnosine alone did not alter IFNAR2 mRNA or protein levels. Cellular levels of 2′–5′ oligoadenylate synthetases (OAS) were markedly increased by IFN-α, and the increase was significantly accelerated by polaprezinc. However, polaprezinc alone did not increase 2′–5′OAS levels. The finding suggests that zinc, especially polaprezinc, enhances the expression of INFAR2 in U937 cells, thereby inducing production of the anti-viral protein 2′–5′OAS.

Effect of Cadmium on Macrophage U937 Cell Proliferation and Activation

The effect of cadmium concentration on the proliferation and activation of U937 cells was studied. Cadmium at different concentrations was incubated with the cell line U937 in culture. Three days later, it was observed that cadmium inhibited the proliferation of the cells in a concentration-dependent manner which may be attributed to the formation of adducts with some growth factors. Later, U937 cells were also pre-treated with phorbol 12- myristate, 13-acetate to transform the cells from the monocyte-like morphology to the macrophage form. The macrophage U937 cells were then incubated with different concentrations of cadmium and the supernatants of the cell cultures were analyzed for the production of nitric oxide via the Griess reaction. Tumour necrosis factor alpha (TNF-α), and the interleukins 6 and 1 (IL 6 and IL 1) were also analyzed by enzyme linked immunosorbent assay. The results reveal that cadmium significantly enhanced the macrophages to produce nitric oxide and the cytokines when compared to lipopolysaccharide. This shows that cadmium significantly activated macrophages therefore the heavy metal has the capacity of initiating the inflammatory response. It is thus considered as an immunotoxicant. Keywords : cadmium, hepatotoxic, nephrotoxic, inflammatory, nitric oxide, lipopolysaccharide.

DMNQ-S17 inhibits constitutive NF-kappaB activation leading to induction of apoptosis through the activation of caspase-3 in human myeloid leukemia U937 cells

Through cytotoxicity screening with naphthoquinone derivatives, a novel compound 6-(1-oxoallkyl)-5,8-dimethoxy-1,4-naphthoquinone-S17 (DMNQ-S17) showed its potency against human myeloid leukemia U937 cells. Thus, to elucidate the apoptotic mechanism of DMNQ-S17, this study was performed in myeloid leukemia U937 cells by 2,3-bis [2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) assay, eletrophoretic mobility shift assay (EMSA), terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay, 4′,6-diamidino-2-phenylindole (DAPI) staining, and Western blotting. In the present study, DMNQ-S17 inhibited constitutive NF κB activation and its transcriptional activity in U937 cells. In addition, DMNQ-S17 induced apoptotic features such as apoptotic bodies, cell shrinkage and chromatin condensation in U937 cells. Consistently, flow cytometric analysis showed that DMNQ-S17 increased sub-G1 portion and TUNEL positive cells in a concentration-dependent manner. Furthermore, DMNQ-S17 effectively attenuated mitochondrial membrane potential, released cytochrome C, activated caspase-3 expression, and cleaved poly (ADP-ribose) polymerase (PARP). Reversely, caspase-3 and -9 inhibitors also blocked the DMNQ-S17 induced caspase-3 activation and PARP cleavage in U937 cells. Taken together, these findings suggest that DMNQ-S17 can be a potent anticancer candidate for myeloid leukemias by the suppression of NF-κB activation leading to the activation of caspase-3 in human myeloid leukemia U937 cells.

Lutein prevents the effect of high glucose levels on immune system cells in vivo and in vitro

Diabetic patients present an increased susceptibility to frequent and protracted infections. The recognition of an impaired immune system has implications for the diagnosis, treatment and outcome of infections. Nuclear Factor kappa B (NF-kappaB) is a redox sensitive transcription factor involved in immune response, cell proliferation and apoptosis that has been associated to the development of diabetic complications. Herein we study the effects of high glucose on oxidative stress markers (malondialdeyde and glutathione contents) and NF-kappaB activity in U937 cells (a human promonocytic cell line). Furtheremore effects of lutein treatment in lymphocytes from diabetic rats was studied. The results show that high glucose induces oxidative stress in immune system cells, both in vitro and in vivo, as well as an increase in their NF-kappaB activity. It is also showed that lutein, a natural antioxidant without hypoglycemiant properties, is able to prevent all the alterations observed. Thus, this study confirms the role of oxidative stress in the immune system impairment described in diabetes, and allows the proposal of antioxidants for the clinical management of the diabetes-associated susceptibility to infections.

Effects of haloperidol, clozapine and olanzapine on the survival of human neuronal and immune cells in vitro

Cytotoxic effects on neuronal as well as on immune cells have been reported for both typical and atypical antipsychotic drugs. We evaluated the effects of different concentrations of a typical (haloperidol) and two atypical (clozapine, olanzapine) antipsychotics on the survival of human neuronal (SH-SY5Y cells) and immune cells (U937 cells) by determining the metabolic activity after 24 h of incubation by the modified tetrazolium method. The dopaminergic neuroblastoma SH-SY5Y and the lymphoma U-937 cell line are well established models for in vitro investigations. To further elucidate possible mechanisms of action we also determined the ATP content in the cultured cells. After experimental treatment, significant effects were detected by Kruskal Wallis test for all treatment conditions. Post-hoc tests (Dunn's method) showed that haloperidol and clozapine at the two highest concentrations (25 and 50 microg/ml) caused a significant decrease of metabolic activity in both cell systems, which was also detectable after treatment with clozapine at a concentration of 12.5 microg/ml in U937 cells. In contrast, olanzapine induced a significant increase in metabolic activity of SH-SY5Y cells at all concentrations except for the concentration of 3.1 microg/ml, whereas the metabolic activity in U937 cells was increased at concentrations of 1.6 and 6.25 microg/ml. For the determination of ATP content, the LD(50) values of the metabolic activity were used, except for olanzapine for which no distinct LD(50) value was available. Significant changes were detected for all treatments and post-hoc tests revealed that haloperidol caused a significant decrease compared to the control condition in both cell systems. These findings suggest that antipsychotic substances of different classes exert differential metabolic effects in both neuronal and immune cell systems.

Effects of gambogic acid on the regulation of nucleoporin Nup88 in U937 cells

In order to investigate the anti-leukemia effects of gambogic acid (GA) and its relation to the regulation of nucleoporin Nup88 in U937 cells in vitro, the inhibitory effect of GA on the growth of U937 cells was examined by using MTT assay. Apoptosis was detected by Annexin-V FITC/PI double-labeled cytometry. Cell cycle regulation was studied by propidium iodide method. Both flow cytometry (FCM) and RT-PCR were employed to assess the expression of Nup88, and the localization of Nup88 was determined by confocal microscopy. The results indicated that GA had strong inhibitory effect on cell proliferation and apoptosis induction activity in U937 cells in vitro in a time-and dose-dependent manner. The 24-h IC(50) value was (1.019+/-0.134) mg/L. Moreover, GA induced arrest of U937 cells in G(0)/G(1) phase. Over-expression of Nup88 was found in U937 cells, whereas GA could significantly down-regulate both the protein and mRNA levels of Nup88. Nup88 was diffusely distributed between nucleus and cytoplasm and was located at the cytoplasmic side of nuclear rim, and occasionally in cytoplasm. It is suggested that GA exerts its anti-leukemia effects by regulating the expression and distribution of nucleoporin Nup88. It promises to be new agent for the treatment of acute leukemia.

Regulation of Ionizing Radiation-induced Apoptosis by Mitochondrial NADP+-dependent Isocitrate Dehydrogenase

Ionizing radiation induces the production of reactive oxygen species, which play an important causative role in apoptotic cell death. By supplying NADPH for antioxidant systems, we recently demonstrated that the control of mitochondrial redox balance and the cellular defense against oxidative damage are some of the primary functions of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm). In this study, we demonstrate that modulation of IDPm activity in U937 cells regulates ionizing radiation-induced apoptosis. When we examined the regulatory role of IDPm against ionizing radiation-induced apoptosis in U937 cells transfected with the cDNA for mouse IDPm in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPm expressed in target cells and their susceptibility to apoptosis. Upon exposure to 2 gray gamma-irradiation, there was a distinct difference between the IDPm transfectant cells in regard to the morphological evidence of apoptosis, DNA fragmentation, cellular redox status, oxidative damage to cells, mitochondrial function, and the modulation of apoptotic marker proteins. In addition, transfection of HeLa cells with an IDPm small interfering RNA decreased the activity of IDPm, enhancing the susceptibility of radiation-induced apoptosis. Taken together, these results indicate that IDPm may play an important role in regulating the apoptosis induced by ionizing radiation, and the effect of IDPm small interfering RNA on HeLa cells offers the possibility of developing a modifier of radiation therapy.