Human Lymphoma Cell Line U937 Research Articles

In-Vitro Screening of Cytotoxicity and Potential Anti-Cancer Activity of Mangrove Crab Soup on Human Lymphoma Cell Line U937

<i>In-Vitro</i> Screening of Cytotoxicity and Potential Anti-Cancer Activity of Mangrove Crab Soup on Human Lymphoma Cell Line U937

INVERSE RELATIONSHIP BETWEEN THE ANTIOXIDANT ACTIVITY OF STRUCTURALLY RELATED SYNTHETIC MONOPHENOLS AND THEIR TOXICITY IN TUMOR CELLS

The aim of the study was to study investigate the relationship between the antioxidant activity of new synthetic structurally related water-soluble monophenols and their effect on the tumor cell viability in vitro. Material and methods. Five original hydrophilic sulfur- and selenium-containing monophenols with varying length of the hydrocarbon chain of the para -alkylthiosulfonate substituent, the amount of tert -butyl ortho -substituents and the «S-S» fragment structure are synthesized: sodium 3-(3'- tert -butyl-4'-hydroxyphenyl)ethyl thiosulfonate (TS-12), sodium 3-(3'- tert -butyl-4'-hydroxyphenyl)propyl sulfonate (S-13), sodium 3-(3'- tert -butyl-4'-hydroxyphenyl)propyl seleniumsulfonate (SeS-13), sodium 3-(3'- tert -butyl-4'-hydroxyphenyl)propyl thiosulfonate (TS-13), sodium 3-(3',5'-di- tert -butyl-4’-hydroxyphenyl)propyl thiosulfonate (TS-17). The antioxidant activity of the compounds was determined in a cell-free model test system by the ability to inhibit the luminol-dependent chemiluminescence of free radical-generating azo compound AAPH. Antitumor cytotoxicity was evaluated by their effect on the viability of human histiocytic lymphoma cell line U937 (monocyte/macrophage-like cells) and human breast adenocarcinoma cell line MCF-7 in the MTT test. Results and discussion. All tested phenolic compounds exerted antioxidant activity in the cell-free model system (inhibition of azo initiator radical AAPH • ) and cytotoxicity against U937 and MCF-7 cells, the effect depended on the dose and structure of the molecule. There was a direct relationship between the structure of monophenols and their ability to inhibit the viability of tumor cells of different lines, regardless of the origin of the latter, myeloid (U937) or epithelial (MCF-7), and growth type (respectively anchorage-independent and attached), as well as the concentration range of compounds (respectively from 100 to 500 μM and from 2 to 150 μM). At the same time, the relationship between the antioxidant activity of monophenols and their cytotoxicity was inverse (when SeS-13 was excluded from analysis), which may be related both to the ability of the tumor to self-defense against reactivate oxygen metabolites and to the indirect pro-oxidant effect of phenolic compounds.

Linezolid-induced Apoptosis through Mitochondrial Damage and Role of Superoxide Dismutase-1 in Human Monocytic Cell Line U937

Cytopenia is a major adverse event associated with linezolid therapy. The objective of this study was to examine whether the cytotoxicity of linezolid to eukaryotic cells was associated with mitochondrial dysfunction and apoptosis-like cell death in human leukemic monocyte lymphoma cell line U937. Apoptosis-like cell death was clearly observed when cells were incubated with linezolid, depending on the duration and linezolid concentration. Mitochondrial membrane potential of cells treated with linezolid collapsed in a short period of time, but the number of mitochondria did not decrease. Cytotoxicity of linezolid was relieved by the knockdown of superoxide dismutase-1 in U937 cells. On the other hand, no autophagy was observed in cells treated with linezolid. These results suggest that mitochondrial damages would be linked to the induction of apoptosis in U937 cells treated with linezolid and that its mechanism does not involve autophagy.

Antibacterial and antitumor activity of Bogorol B-JX isolated from Brevibacillus laterosporus JX-5.

Antimicrobial peptides are promising anti-infective agent candidates because they have a broad antimicrobial spectrum and bioactivity and are unlikely to elicit antibiotic resistance. The bogorols represent a new cationic antibiotic peptide and possess great therapeutic potential because of their bioactivity and precise mode of action. Here, we report that Bogorol B-JX (BBJX), a peptide previously isolated from Brevibacillus laterosporus JX-5 by us, has significant antibacterial and antitumor activities in vitro. BBJX was found to inhibit methicillin-resistant Staphylococcus aureus (MRSA) at 2.5µg/mL with distinct mechanisms of action from those against Bacillus bombyseptieus and Escherichia coli. It penetrates MRSA membrane with little visible destruction and binds to genomic DNA. BBJX could inhibit the proliferation of human histiocytic lymphoma cell line U-937 and ConA-activated spleen cells at 5µg/mL, but was not cytotoxic to the Jurkat cells, resting spleen cells or differentiated macrophage-like U-937 immunocytes. Moreover, BBJX caused apoptosis of U-937 cells by opening the mitochondrial permeability transition pore and stimulating the production of reactive oxygen species. Taken together, these studies provided basis for future medical application of the bogorols.

Phorbol-12-myristate-13-acetate-mediated stabilization of leukemia inhibitory factor (lif) mRNA: involvement of Nucleolin and PCBP1.

Leukemia inhibitory factor (LIF) is a potent pleiotropic cytokine involved in diverse biological activities, thereby requiring precise spatial and temporal control of its expression. The present study reveals that enhanced expression of LIF in response to PMA (phorbol-12-myristate-13-acetate) in human histiocytic lymphoma cell line U937 largely happens through stabilization of its mRNA. Functional characterization of the long 3'-untranslated region of human lif mRNA revealed several conserved sequences with conventional cis-acting elements. A 216 nucleotide containing proximal cis-element with two AUUUA pentamers and four poly-rC sequences demonstrated significant mRNA destabilizing potential, which, on treatment with PMA, showed stabilizing activity. Affinity chromatography followed by western blot and RNA co-immunoprecipitation of PMA-treated U937 extract identified Nucleolin and PCBP1 as two protein trans-factors interacting with lif mRNA, specifically to the proximal non-conventional AU-rich region. PMA induced nucleo-cytoplasmic translocation of both Nucleolin and PCBP1. RNA-dependent in vivo co-association of both these proteins with lif mRNA was demonstrated by decreased co-precipitation in the presence of RNase. Ectopic overexpression of Nucleolin showed stabilization of both intrinsic lif mRNA and gfp reporter, whereas knockdown of Nucleolin and PCBP1 demonstrated a significant decrease in both lif mRNA and protein levels. Collectively, this report establishes the stabilization of lif mRNA by PMA, mediated by the interactions of two RNA-binding proteins, Nucleolin and PCBP1 with a proximal cis-element.

Targeted label-free quantification of interleukin-8 in PMA-activated U937 cell secretome by nanoLC-ESI-MS/MS-sSRM.

Monocytes are a part of the innate immune system. Their differentiation into macrophages changes their cellular proteome and secretome. Particularly secretome components such as cytokines are crucial for immune response and inflammation in many diseases. Differentiation of human lymphoma cell line U937 can be triggered by phorbol 12-myristate 13-acetate (PMA). Screening of the cytokine release in U937 upon PMA stimulation by cytometric bead array almost exclusively showed interleukin-8 (IL-8). Next, a label-free nanoLC-ESI-MS/MS-sSRM method for quantification of IL-8 in the cell secretome was established and applied to monitor the time kinetics of PMA treatment in different concentrations. Targeted secretome analysis was achieved by scheduled SRM-MS using one proteotypic peptide as precursor ion and four mass transitions. Label-free quantification was performed by external calibration using IL-8 standard. Validation results indicated that the method was suited for the quantification of IL-8 in the secretome. The maximal IL-8 release of 62.4 ng/mL was observed after incubating cells treated by 50 ng/mL PMA for 48 h. The method can now be used for quantification of IL-8 release from different cells under various conditions. Furthermore, it can be easily expanded to other secreted proteins detected by untargeted screening methods.

Extracellular heat shock proteins protect U937 cells from H2O2-induced apoptotic cell death.

The cytoprotective role of heat shock proteins (HSPs) has been demonstrated in various cell types however, only few studies have investigated the role of extracellular exposure to HSPs in the survival of human lymphoma cell line U937. In the present study, we investigated the effect of extracellular exposure to four HSPs (HSP90, HSP70, HSP60, and HSP47) on apoptotic cell death induced by either oxidative stress (hydrogen peroxide) or endoplasmic reticulum stress-mediated intracellular calcium overload. It was found that extracellular exposure to HSPs reduced the cytotoxicity induced by hydrogen peroxide, but not that evoked by thapsigargin (a specific inhibitor of cytosolic calcium reuptake which is able to induce endoplasmic reticulum stress with subsequent intracellular calcium overload). Similarly, it was observed that exogenous HSPs were able to suppress the caspase-3 activation induced by hydrogen peroxide. These findings indicate that extracellular HSPs increase the resistance of human lymphoma cell line U937 to apoptotic cell death induced by hydrogen peroxide and diminish oxidative stress-mediated injures.

Role of diallyl disulfide-mediated cleavage of c-Myc and Sp-1 in the regulation of telomerase activity in human lymphoma cell line U937

ObjectiveGarlic (Allium sativum) has been considered a wonder herb for years with a reputation of disease prevention. Telomerase, a ribonucleoprotein enzyme responsible for telomere integrity, is strongly up-regulated in different types of cancers. The aim of this study was to reveal the role of diallyl disulfide (DADS), an organosulfur component of garlic, on telomerase activity in human lymphoma with an emphasis on key transcription factors c-Myc and Sp-1. MethodsHuman lymphoma cell line U937 was used as model cell line. Telomerase activity was measured by telomerase repeat amplification protocol assay, levels of related proteins and mRNAs were measured by Western blot and reverse transcriptase polymerase chain reaction, respectively. Moreover, in vitro binding assay was performed using radiolabeled double-stranded DNA having specific sequences to detect involvement of transcription factors in DADS-dependent modulation of telomerase activity. ResultsThe present study demonstrated DADS-mediated decrease in telomerase activity in U937 cells with concomitant transcriptional down-regulation of human telomerase reverse transcriptase (hTERT) that is caused by reduced binding of c-Myc and Sp-1 to their respective binding sites on hTERT promoter. Lowering of DNA-binding activity of c-Myc and Sp-1 due to DADS treatment is caused by the deactivation of these transcription factors due to cleavage. Additionally, Mad1—the repressor protein of hTERT expression—is also overexpressed in DADS-treated U937 cells. ConclusionsThese findings strongly suggest that DADS down-regulate telomerase activity through c-Myc-, Sp-1-, and Mad1-dependent transcriptional down-regulation of hTERT.

Oxidative damage of U937 human leukemic cells caused by hydroxyl radical results in singlet oxygen formation.

The exposure of human cells to oxidative stress leads to the oxidation of biomolecules such as lipids, proteins and nuclei acids. In this study, the oxidation of lipids, proteins and DNA was studied after the addition of hydrogen peroxide and Fenton reagent to cell suspension containing human leukemic monocyte lymphoma cell line U937. EPR spin-trapping data showed that the addition of hydrogen peroxide to the cell suspension formed hydroxyl radical via Fenton reaction mediated by endogenous metals. The malondialdehyde HPLC analysis showed no lipid peroxidation after the addition of hydrogen peroxide, whereas the Fenton reagent caused significant lipid peroxidation. The formation of protein carbonyls monitored by dot blot immunoassay and the DNA fragmentation measured by comet assay occurred after the addition of both hydrogen peroxide and Fenton reagent. Oxidative damage of biomolecules leads to the formation of singlet oxygen as conformed by EPR spin-trapping spectroscopy and the green fluorescence of singlet oxygen sensor green detected by confocal laser scanning microscopy. It is proposed here that singlet oxygen is formed by the decomposition of high-energy intermediates such as dioxetane or tetroxide formed by oxidative damage of biomolecules.

ARHGEF3 controls HDACi-induced differentiation via RhoA-dependent pathways in acute myeloid leukemias

Altered expression and activity of histone deacetylases (HDACs) have been correlated with tumorigenesis. Inhibitors of HDACs (HDACi) induce acetylation of histone and non-histone proteins affecting gene expression, cell cycle progression, cell migration, terminal differentiation and cell death. Here, we analyzed the regulation of ARHGEF3, a RhoA-specific guanine nucleotide exchange factor, by the HDACi MS275 (entinostat). MS275 is a well-known benzamide-based HDACi, which induces differentiation of the monoblastic-like human histiocytic lymphoma cell line U937 to monocytes/macrophages. Incubation of U937 cells with MS275 resulted in an up regulation of ARHGEF3, followed by a significant enhancement of the marker of macrophage differentiation CD68. ARHGEF3 protein is primarily nuclear, but MS275 treatment rapidly induced its translocation into the cytoplasm. ARHGEF3 cytoplasmic localization is associated with activation of the RhoA/Rho-associated Kinase (ROCK) pathway. In addition to cytoskeletal rearrangements orchestrated by RhoA, we showed that ARHGEF3/RhoA-dependent signals involve activation of SAPK/JNK and then Elk1 transcription factor. Importantly, MS275-induced CD68 expression was blocked by exposure of U937 cells to exoenzyme C3 transferase and Y27632, inhibitors of Rho and ROCK respectively. Moreover, ARHGEF3 silencing prevented RhoA activation leading to a reduction in SAPK/JNK phosphorylation, Elk1 activation and CD68 expression, suggesting a crucial role for ARHGEF3 in myeloid differentiation. Taken together, our results demonstrate that ARHGEF3 modulates acute myeloid leukemia differentiation through activation of RhoA and pathways directly controlled by small GTPase family proteins. The finding that GEF protein modulation by HDAC inhibition impacts on cell differentiation may be important for understanding the antitumor mechanism(s) by which HDACi treatment stimulates differentiation in cancer.

Nanoceria protects from alterations in oxidative metabolism and calcium overloads induced by TNFα and cycloheximide in U937 cells: pharmacological potential of nanoparticles

The present study is aimed to determine the protective effect of a novel nanoparticle with antioxidant properties, nanoceria, on reactive oxygen species (ROS) production, and calcium signaling evoked by the tumor necrosis factor-alpha (TNFα) in combination with cycloheximide (CHX) on apoptosis in the human histiocytic lymphoma cell line U937. Our results show that treatment of U937 cells with 10ng/mL TNFα in combination with 1μg/mL CHX led to several Ca(2+) alterations. These stimulatory effects on calcium signals were followed by intracellular ROS production and mitochondria membrane depolarization, as well as a time-dependent increase in caspase-8 and -9 activities. Our results show that the pretreatment with well known antioxidants such as trolox and N-acetyl cysteine (NAC) partially reduced the apoptotic effects due to the administration of TNFα plus cycloheximide. Furthermore, nanoceria had a stronger protective effect than trolox or NAC. Our findings also suggest that TNFα plus cycloheximide-induced apoptosis is dependent on alterations in cytosolic concentration of calcium [Ca(2+)]c and ROS generation in human histiocytic U937 cells.

Hemin Protects U937 Cells from Oxidative Stress via Glutathione Synthesis

Although heme oxygenase-1 and glutathione play important roles in the antioxidant defense system, the sharing and/or cross-talking of the HO-1 and GSH system remain poorly understood. The object of this study is to determine whether the glutathione system is involved in the antioxidant function of hemin. Hydrogen peroxide decreased the viability of the human leukemic monocyte lymphoma cell line U937. When these cells were pretreated with hemin before the addition of hydrogen peroxide, cell death was prevented. An inhibitor of heme oxygenase-1 or glutathione biosynthesis significantly abolished this protective effect of hemin. These results suggest that both heme oxygenase-1 and glutathione are involved in the protective effects of hemin against U937 cell death, which was induced by hydrogen peroxide. Hemin induced an increase in glutathione levels following the upregulation of the gene expression and protein levels of glutamate-cysteine ligase catalytic subunit. The inhibitor of heme oxigenase-1 inhibited the upregulation of glutamate-cysteine ligase catalytic subunit expression. These results suggest that hemin induces glutathione synthesis through heme oxygenase-1 activation to protect cells from hydrogen peroxide-induced oxidative stress.

Benzaldehyde thiosemicarbazone complexes of platinum: Syntheses, structures and cytotoxic properties

The reaction of 4-R-benzaldehyde thiosemicarbazones (denoted in general as H2L-R, where H2 stands for the two dissociable protons and R (R=OCH3, CH3, H, Cl and NO2) for the substituent on the phenyl ring) with [Pt(PPh3)2Cl2] in the presence of NEt3 afforded a family of organometallic complexes of platinum(II) of the type [Pt(PPh3)(L-R)]. Reaction of the same group of ligands with K2[PtCl4] in the presence of NEt3 afforded complexes of the type [Pt(HL-R)2]. The crystal structure of [Pt(PPh3)(L-CH3)] and [Pt(HL-CH3)2] have been determined. In the [Pt(PPh3)(L-R)] complexes, the benzaldehyde thiosemicarbazones are coordinated to platinum as dianionic tridentate C,N,S-donors. In the [Pt(HL-R)2] complexes, the benzaldehyde thiosemicarbazones are coordinated to the metal center as bidentate N,S-donors, forming five-membered chelate rings, and with reference to the structure of the uncoordinated thiosemicarbazone ligand, this coordination mode is associated with a change in stereochemistry around the CN bond. All the [Pt(PPh3)(L-R)] and [Pt(HL-R)2] complexes display intense absorptions in the visible and ultraviolet regions. The cytotoxic effects of these complexes, examined on the human leukemia cell line HL-60 and human lymphoma cell line U-937, have shown that all the [Pt(PPh3)(L-R)] and [Pt(HL-R)2] complexes are cytotoxic in nature and their IC50 values indicate their potential use as antitumor agents.

Antitumor activity of antimicrobial peptides against U937 histiocytic cell line.

We investigated cytotoxic activity of antimicrobial peptides of different origin (both naturally occurring and synthetic), structure and known mechanisms of action against human histiocytic lymphoma cell line U937. The strongest cytotoxic activity against U937 cell line was shown by Pexiganan MSI-78, followed by Citropin 1.1, Protegrin 1 and a synthetic lipopeptide, N-α-palmitoyl-L-lysyl-L-lysine amide (Pal-Lys-Lys-NH₂). The cytotoxic activity of the peptides was more dependent on the time of incubation than concentration. Only for the lipopeptide, whose mode of action was restricted to disruption of electric potential of the cell membrane, the correlation between cytotoxicity and concentration was almost linear. The high cytotoxicity of Pexiganan MSI-78, Protegrin 1 and the lipopeptide could be basically explained by their membranolytic activity leading to necrosis. However, in the case of Citropin 1.1, the cell membrane integrity was disrupted only slightly and independently of the peptide concentration. Therefore, some other mechanism of action might be responsible for its strong dose-dependent cytotoxic activity, e.g., membranolytic activity leading to apoptosis. Furthermore, TNF-α production due to LPS (lipopolysaccharide) stimulation was suppressed by the presence of Citropin 1.1, Pexiganan MSI-78 or Protegrin 1, but not by Buforin 2 or the lipopeptide. Our experiments have shown that cytotoxic activity is not limited to some specific molecular structure of a peptide, but rather to the length of the peptide chain as it is likely to affect the efficiency of the tumor cell membrane disruption and interaction with LPS.

Gallic Acid Inhibits Cell Viability and Induces Apoptosis in Human Monocytic Cell Line U937

In the present study, we investigated the effects of gallic acid (GA) (3,4,5-trihydroxybenzoic acid), a polyhydroxyphenolic compound, isolated from Rhus chinensis, on the human monocytic lymphoma cell line U937. In vitro experiments showed that treating U937 cells with various amounts of GA inhibited cell viability and induced apoptosis in a dose-dependent manner. In order to understand the mechanism by which GA induces apoptosis, we examined the gene expression of p53, nuclear factor κB (NF-κB), and inhibitor of NF-κB (I-κB) after treating the cells with GA and found that expression levels of the genes for p53 and NF-κB increased and that for I-κB decreased. The results obtained from western blotting with U937 cells showed up-regulation of NF-κB protein and down-regulation of proliferating cell nuclear antigen and I-κB protein. These results demonstrate that GA efficiently induces apoptosis in U937 cells and that GA is a potential chemotherapeutic agent against lymphoma.

Cytotoxicity and its mechanism of zinc oxide nanoparticles on human leukemic monocyte lymphoma cell line U937

To investigate the cytotoxicity and its mechanism of ZnO nanoparticles on human leukemic monocyte lymphoma cell line U937. Four different size ZnO (10, 30, 60, 500 nm) were carefully characterized. The survival rate and viability were measured by trypan blue assay and MTT assay for each size ZnO particles at different concentrations (12, 120, 240, 600, 1200 µmol/L). The zinc probe, Fluozin-3, was used to detect the intracellular free zinc. Transmission electron microscopy was adopted to observe the cellular ultrastructure and the uptake of ZnO. All four kinds of ZnO were rod shape, with a purity of > 99.9 wt%, and they were classified as zincite phase crystal and their surface areas were in accordance with the sizes. The viability (ZnO-n10: (97 ± 19)%, (91 ± 4)%, (24 ± 4)%, (15 ± 2)%; ZnO-n30: (111 ± 4)%, (81 ± 3)%, (24 ± 2)%, (27 ± 8)%; ZnO-n60: (105 ± 11)%, (73 ± 20)%, (43 ± 11)%, (28 ± 14)%; ZnO-µm: (88 ± 16)%, (62 ± 7)%, (22 ± 4)%, (13 ± 5)%) of cells exposed to ZnO decreased with the increasing of the concentration of ZnO from 12 to 600 µmol/L (r values were 0.965, 0.979, 0.998, 0.992, and the t values were 19.8, 25.3, 76.3, 40.9, respectively, P < 0.05). The liability (ZnO-n10: (98 ± 1)%, (67 ± 2)%, (59 ± 7)%, (13 ± 13)%, (5 ± 4)%; ZnO-n30: (98 ± 1)%, (97 ± 2)%, (50 ± 3)%, (20 ± 14)%, (7 ± 2)%; ZnO-n60: (97 ± 2)%, (88 ± 5)%, (48 ± 10)%, (12 ± 5)%, (4 ± 1)%; ZnO-µm: (96 ± 1)%, (76 ± 3)%, (58 ± 3)%, (19 ± 5)%, (20 ± 10)%) of cells exposed to ZnO decreased with the increasing of the concentration of ZnO from 12 to 600 µmol/L (r valued at 0.982, 0.956, 0.972, 0.980, and the t valued at 19.3, 12.1, 15.6, 18.5, respectively, P < 0.05). The increase of the zinc concentration showed by the zinc fluorescence probe was 121 ± 11, which was similar to the fluorescence of cells treated with ZnAc(2) (132 ± 14, F = 0.6, P > 0.05) at the Zn-equivalent concentration. There was no statistic difference for the percents of high zinc content cells in total cells exposed to ZnO-n30 (87.6 ± 2.6)% and these exposed to ZnAc(2) (86.9 ± 3.2)% (F = 1.5, P > 0.05). ZnO nanoparticles are highly cytotoxic to U937 cells and the solubilization of ZnO is the main toxicological mechanism.

Abstract 2261: Antitumor and anti-inflammatory effect of a novel nutrient mixture on human lymphoma cell line U-937

Abstract Introduction: Phytochemicals and dietary antioxidants are known to decrease the risk of inflammation and prevent cancer development. We have developed strategies to inhibit cancer progression and its spread by natural products. A nutrient mixture (NM) containing ascorbic acid, lysine, proline and green tea extract has exhibited anticancer activity in vitro and in vivo in a number of cancer cell lines. Objective: We investigated the effect of NM on human lymphoma cell line U-937 in vitro by measuring: cell proliferation, MMP expression, invasion, apoptosis, and Cox-2 and Cox-1 protein expression. Materials and Methods: Human lymphoma cell line U-937 (ATCC) was cultured in RPMI medium supplemented with fetal bovine serum and antibiotics. U-937 cells were seeded on 24-well tissue culture plates with 40nM phorbol 12-myristate 13-acetate (PMA). After 24 hrs, the cells were treated with NM at 0, 50, 100, 250, 500 and 1000 mcg/ml, in triplicate at each dose. Cell proliferation was evaluated by MTT assay, MMP expression by gelatinase zymography, invasion through Matrigel, apoptosis by using live green caspase detection kit (Molecular Probe), and COX-2 and COX-1 expression by Western blot. Results: NM was not toxic to U-937 cells at a concentration of 250 mcg/ml and exhibited an antiproliferative effect at 500 mcg/ml concentration. Zymography demonstrated only one band corresponding to MMP-9, which was inhibited by NM in a dose-dependent manner. Matrigel invasion was significantly reduced (by 95%) at 250 mcg/ml NM and completely blocked at 500 mcg/ml NM. NM induced slight apoptosis at 100 mcg/ml and moderate at 500 and 1000 mcg/ml concentration. NM inhibited Cox-2 expression in a dose-dependent fashion and had no effect on Cox-1 expression. Conclusions: Our results suggest that NM is an excellent candidate for therapeutic use in the treatment of hematological malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2261.

Mechanism of cell death by 5‐aminolevulinic acid‐based photodynamic action and its enhancement by ferrochelatase inhibitors in human histiocytic lymphoma cell line U937

Photodynamic therapy (PDT) for tumors is based on the tumor-selective accumulation of a photosensitizer, protoporphyrin IX (PpIX), followed by irradiation with visible light. However, the molecular mechanism of cell death caused by PDT has not been fully elucidated. The 5-aminolevulinic acid (ALA)-based photodynamic action (PDA) was dependent on the accumulation of PpIX, the level of which decreased rapidly by eliminating ALA from the incubation medium in human histiocytic lymphoma U937 cells. PDA induced apoptosis characterized by lipid peroxidation, increase in Bak and Bax/Bcl-xL, decrease in Bid, membrane depolarization, cytochrome c release, caspase-3 activation, phosphatidylserine (PS) externalization. PDT-induced cell death seemed to occur predominantly via apoptosis through distribution of PpIX in mitochondria. These cell death events were enhanced by ferrochelatase inhibitors. These results indicated that ALA-based-PDA induced apoptotic cell death through a mitochondrial pathway and that ferrochelatase inhibitors might enhanced the effect of PDT for tumors even at low concentrations of ALA.

ApoG2 inhibits antiapoptotic Bcl-2 family proteins and induces mitochondria-dependent apoptosis in human lymphoma U937 cells

Lymphoma is one of the most common types of hematological malignancies and proteins from the Bcl-2 family are highly expressed in human lymphomas. Apogossypolone (ApoG2), the most potent gossypol derivative, has been classified as a novel small-molecule inhibitor of antiapoptotic Bcl-2 family proteins. Here, we assessed the in-vitro cytotoxicity of ApoG2 on human U937 lymphoma cells, and explored the underlying intracellular molecular mechanisms of ApoG2. Using the WST-8 assay, we found that ApoG2 inhibited growth of U937 cells in a dose-dependent and time-dependent manner, and the IC50 values were 30.08, 14.81, and 9.26 mumol/l for 24, 48, and 72 h treatments, respectively. ApoG2 also induced apoptosis in U937 cells, as noted through changes in morphological characteristics, including cellular internucleosomal DNA fragmentation and the appearance of a sub-G1 apoptotic peak. Treatment with ApoG2 downregulated Bcl-xL and Mcl-1 protein expression and blocked the binding of Bcl-2 with Bax protein. Furthermore, ApoG2 led to an abundant release of cytochrome c from mitochondria and a five-fold increase in the activity of caspase-3 and caspase-9. Taken together, our results suggest that ApoG2 could effectively suppress the growth of human lymphoma cell line U937 through the inhibition of the antiapoptotic Bcl-2 family proteins and the induction of mitochondria-dependent apoptotic cell death.

Requirement of aggregation propensity of Alzheimer amyloid peptides for neuronal cell surface binding

BackgroundAggregation of the amyloid peptides, Aβ40 and Aβ42, is known to be involved in the pathology of Alzheimer's disease (AD). Here we investigate the relationship between peptide aggregation and cell surface binding of three forms of Aβ (Aβ40, Aβ42, and an Aβ mutant).ResultsUsing confocal microscopy and flow cytometry with fluorescently labelled Aβ, we demonstrate a correlation between the aggregation propensity of the Alzheimer amyloid peptides and their neuronal cell surface association. We find that the highly aggregation prone Aβ42 associates with the surface of neuronal cells within one hour, while the less aggregation prone Aβ40 associates over 24 hours. We show that a double mutation in Aβ42 that reduces its aggregation propensity also reduces its association with the cell surface. Furthermore, we find that a cell line that is resistant to Aβ cytotoxicity, the non-neuronal human lymphoma cell line U937, does not bind either Aβ40 or Aβ42.ConclusionTaken together, our findings reveal that amyloid peptide aggregation propensity is an essential determinant of neuronal cell surface association. We anticipate that our approach, involving Aβ imaging in live cells, will be highly useful for evaluating the efficacy of therapeutic drugs that prevent toxic Aβ association with neuronal cells.