Human Histiocytic Lymphoma U937 Research Articles

O,N,S-tris-chelating ligand scaffolds flanked with cyclohexyl or adamantyl substituents anchored with diorganotin(IV) moieties: synthesis, structures and cytotoxicity

The O,N,S-tris-chelating ligand, H2NSCN-N-C(H)ArO− constitutes an attractive scaffold in coordination chemistry and we have expanded this scaffold by installing an alkyl group of different steric hindrance. Thus, the ligand scaffolds containing O,N,S (Ln) differ with respect to the chemical link between the coordinating O atoms, which is either a phenol or a naphthol moiety and carrying a cyclohexyl or a adamantyl groups (L1, phenol and cyclohexyl; L2, naphthol and cyclohexyl; L3, phenol and adamantyl; L4, naphthol and adamantyl). Six novel diorganotin(IV)-Ln compounds 1–6 were synthesized and structurally characterized. The solid-state structures were deduced from single crystal X-ray diffraction data, and revealed the Sn(IV) cations adopting square pyramid (in 2, 3 and 4) or trigonal bipyramid (in 1, 5 and 6) geometries with coordination spheres comprising κ-N,O,S (κ-N,S in 1) ligand chelators and two methyl or phenyl groups (plus a hydrosulfide in 1) co-ligands. Solution state characterizations were achieved by detailed 1H, 13C, 15N and 119Sn NMR analysis including selected 2D experiments. In vitro cytotoxic activities of the compounds were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay against human histiocytic lymphoma U-937 cells. The IC50 values revealed better activities of diphenyltin(IV) compounds 4–6 as compared to the respective pro-ligands and doxorubicin. More importantly, compounds 4–6 demonstrated maximum inhibition in U-937 cells (IC50 = 0.47–0.99 μM), with negligible effect on human peripheral blood mononuclear cells (PBMC). Compounds 4–6 effectively inhibited cell viability event, which was confirmed by detecting cytotoxicity, Live & Dead and Lactate dehydrogenase (LDH) release assays.

Antiproliferative activity of nickel(II), palladium(II) and zinc(II) thiosemicarbazone complexes

Complexes of nickel(II), palladium(II) and zinc(II) have been synthesized with four ONS thiosemicarbazone tridentate ligands. The ligands were obtained from the synthesis of 2-hydroxy-3-methoxybenzaldehyde or 2,3-dihydroxybenzaldehyde with thiosemicarbazide or 4-ethyl-3-thiosemicarbazide. The palladium and zinc complexes have formula [ML]2 (H2L, ligand), whereas in the case of nickel, two different types of complexes are formed: [NiL]2 or [Ni(HL)2] depending on the starting metal ion:ligand ratio (1:1 or 1:2, respectively). The nickel(II) complex [Ni(H2L)(HL)](CH3COO) was characterized also by single crystal X-ray diffraction.While the ligands did not exhibit antiproliferative activity on human histiocytic lymphoma U937 cells, the metal complexes showed GI50 values ranging from approximately 20 to 39 μM. The most promising metal complexes were also tested through Alkaline Comet Assay: while with the ligand DNA damages were not detected even at the highest ligand concentrations (100 µM), the complexes, and in particular the nickel dimers [NiL]2, exhibit genotoxic activity.

Silver Nanoparticles Functionalized With Antimicrobial Polypeptides: Benefits and Possible Pitfalls of a Novel Anti-infective Tool.

Silver nanoparticles (AgNPs) and antimicrobial peptides or proteins (AMPs/APs) are both considered as promising platforms for the development of novel therapeutic agents effective against the growing number of drug-resistant pathogens. The observed synergy of their antibacterial activity suggested the prospect of introducing antimicrobial peptides or small antimicrobial proteins into the gelatinized coating of AgNPs. Conjugates with protegrin-1, indolicidin, protamine, histones, and lysozyme were comparatively tested for their antibacterial properties and compared with unconjugated nanoparticles and antimicrobial polypeptides alone. Their toxic effects were similarly tested against both normal eukaryotic cells (human erythrocytes, peripheral blood mononuclear cells, neutrophils, and dermal fibroblasts) and tumor cells (human erythromyeloid leukemia K562 and human histiocytic lymphoma U937 cell lines). The AMPs/APs retained their ability to enhance the antibacterial activity of AgNPs against both Gram-positive and Gram-negative bacteria, including drug-resistant strains, when conjugated to the AgNP surface. The small, membranolytic protegrin-1 was the most efficient, suggesting that a short, rigid structure is not a limiting factor despite the constraints imposed by binding to the nanoparticle. Some of the conjugated AMPs/APs clearly affected the ability of nanoparticle to permeabilize the outer membrane of Escherichia coli, but none of the conjugated AgNPs acquired the capacity to permeabilize its cytoplasmic membrane, regardless of the membranolytic potency of the bound polypeptide. Low hemolytic activity was also found for all AgNP-AMP/AP conjugates, regardless of the hemolytic activity of the free polypeptides, making conjugation a promising strategy not only to enhance their antimicrobial potential but also to effectively reduce the toxicity of membranolytic AMPs. The observation that metabolic processes and O2 consumption in bacteria were efficiently inhibited by all forms of AgNPs is the most likely explanation for their rapid and bactericidal action. AMP-dependent properties in the activity pattern of various conjugates toward eukaryotic cells suggest that immunomodulatory, wound-healing, and other effects of the polypeptides are at least partially transferred to the nanoparticles, so that functionalization of AgNPs may have effects beyond just modulation of direct antibacterial activity. In addition, some conjugated nanoparticles are selectively toxic to tumor cells. However, caution is required as not all modulatory effects are necessarily beneficial to normal host cells.

Influence of ligand lipophilicity in Pt(II) complexes on their antiproliferative and apoptotic activities in tumour cell lines

One of the most widely used strategies for drug development is the coordination of bioactive ligands to transition metals, which could improve biological activity. Moreover, the incorporation of aromatic groups to ligands may allow an enhanced lipophilicity that can influence the cellular uptake and accumulation of the metallodrugs, thus increasing their activity. Herein, we have reported the synthesis and characterization of four Pt(II) complexes [PtCl2(L)], where L = 2-(1-pyrazolyl)-2-thiazoline (PzTn), 2-(1-pyrazolyl)-1,3-thiazine (PzTz), 2-(3,5-diphenyl-1-pyrazolyl)-2-thiazoline (DPhPzTn) or 2-(3,5-diphenyl-1-pyrazolyl)-1,3-thiazine (DPhPzTz). The study was aimed at analysing their potential anticarcinogenic ability in epithelial cervix carcinoma HeLa, human promyelocytic leukemia HL-60 and human histiocytic lymphoma U-937 tumour cell lines as well as checking whether the structural factors of the organic ligand may influence their biological activity. Our findings showed that PtDPhPzTn and PtDPhPzTz were far more effective in terms of cytotoxicity than their less lipophilic counterparts (PtPzTn and PtPzTz), especially in cells derived from solid cervical tumours, thereby suggesting that modulating the lipophilicity of the ligands can help improve the cytotoxic effect of the metal complexes.

Synthesis, Characterization and Antiproliferative Evaluation of Pt(II) and Pd(II) Complexes with a Thiazine-Pyridine Derivative Ligand.

Chemical, pharmacological, and clinical research on anticancer coordination complexes has led to noteworthy anticancer drugs such as cisplatin, carboplatin and oxaliplatin. Although these compounds are effective chemotherapeutic agents in the treatment of different tumors, they are associated with high toxicity and numerous side effects. Several studies have shown that the range of platinum complexes with antitumor activity is not limited to structural analogs of cisplatin. Therefore, the development of convenient anticancer drugs that can be effectively used for the treatment of human tumors has become the main goal of most research groups in this field. In this sense, active platinum complexes without NH groups, transplatinum complexes, multinuclear complexes, cationic complexes, and several classes of palladium(II) complexes have emerged. Herein, the synthesis and characterization of two Pt(II) or Pd(II) complexes with PyTz (2-(2-pyridyl)iminotetrahydro-1,3-thiazine), a thiazine derivative ligand, with the formula [MCl2(PyTz)]·C2H6O (M = Pt(II) or Pd(II)) were reported. The potential anticancer ability of both complexes was evaluated in epithelial cervix carcinoma HeLa, human ovary adenocarcinoma SK-OV-3, human histiocytic lymphoma U-937, and human promyelocytic leukemia HL-60 cell lines. Interestingly, the Pt(II) complex showed great cytotoxic potential against all tumor cell lines tested, whereas the Pd(II) complex displayed slight antitumor actions.

Pt(II) and Pd(II) complexes with a thiazoline derivative ligand: Synthesis, structural characterization, antiproliferative activity and evaluation of pro-apoptotic ability in tumor cell lines HT-29 and U-937

Eluding apoptosis represents the hallmark of tumoral cell behavior. Cisplatin (CisPt) is a very common chemotherapeutic agent to treat cancer by reestablishing apoptotic mechanisms of cell death. However, certain patients acquire resistance to CisPt as well as suffer nephrotoxicity, neurotoxicity, nausea and vomiting. The synthesis of new Pt(II) compounds represents an alternative to CisPt to avoid resistance and undesirable side effects. Pd(II) could be a Pt(II) surrogate given the similarity of coordination chemistry between them, thus widening the spectra of available anticancer drugs. Herein, we have synthesized and characterized two Pt(II) or Pd(II) complexes with TdTn (2-(3,4-dichlorophenyl)imino-N-(2-thiazolin-2-yl)thiazolidine), a thiazoline derivative ligand, with formula [PtCl2(TdTn)] and [PdCl2(TdTn)]. The potential anticancer ability was evaluated in human colon adenocarcinoma HT-29 and human histiocytic lymphoma U-937 cell lines. To that aim, U-937 and HT-29 cells were treated with TdTn, [PtCl2(TdTn)] and [PdCl2(TdTn)] for 24 h. The microscopy monitoring indicated that TdTn, [PtCl2(TdTn)] and [PdCl2(TdTn)] arrested the cell proliferation of U-937 and HT-29 cells with respect to control, in agreement with MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) analysis. Moreover, it is noteworthy that the ligand by its own showed antiproliferative effects in both cell lines. [PtCl2(TdTn)] and [PdCl2(TdTn)] caused caspase-3 activation in U-937 cells, simultaneously with caspase-9 activation due to complexes; however, in HT-29 caspase-3 activation occurred simultaneously with caspase-8 activation induced by the ligand TdTn. Only metal complexes were able to induce ROS (Reactive Oxygen Species) generation in U-937 cells, but not TdTn. In HT-29 cells neither the metal complexes, nor the ligand induced ROS generation.

Dynamics of the Functional Activity and Expression of Proteasome Subunits during Cellular Adaptation to Heat Shock

The ubiquitin-proteasome system (UPS) performs proteolysis of most intracellular proteins. The key components of the UPS are the proteasomes, multi-subunit protein complexes, playing an important role in cellular adaptation to various types of stress. We analyzed the dynamics of the proteasome activity, the content of proteasome subunits, and the expression levels of genes encoding catalytic subunits of proteasomes in the human histiocytic lymphoma U937 cell line immediately, 2, 4, 6, 9, 24, and 48 h after a heat shock (HS). The initial decrease (up to 62%) in the proteasome activity in cellular lysates was revealed, then 10 h after HS the activity began to recover. The amount of proteasomal α-subunits in the cells decreased 2 h after HS, and was restored to 24-48 h after HS. Fluctuations in the levels of mRNAs encoding proteasome catalytic subunits with the maximum expression 2 h after HS and a gradual decrease to 48 h after HS were observed. The average estimated number of mRNA copies per cell ranged from 10 for weakly to 150 for highly expressed proteasome genes. Thus, the recovery efficiency of UPS functionality after HS, which reflects the important role of proteasomes in maintaining cell homeostasis, was evaluated.

New Furanone Derivatives and Alkaloids from the Co-Culture of Marine-Derived Fungi Aspergillus sclerotiorum and Penicillium citrinum.

Six new compounds including two furanone derivatives sclerotiorumins A and B (1 and 2), one novel oxadiazin derivative sclerotiorumin C (3), one pyrrole derivative 1-(4-benzyl-1H-pyrrol-3-yl)ethanone (4), and two complexes of neoaspergillic acid aluminiumneohydroxyaspergillin (5) and ferrineohydroxyaspergillin (6) were isolated from the co-culture of marine-derived fungi Aspergillus sclerotiorum and Penicillium citrinum. Compound 3 was the first natural 1,2,4-oxadiazin-6-one. Compound 5 showed significant and selective cytotoxicity against human histiocytic lymphoma U937 cell line (IC50 =4.2μm) and strong toxicity towards brine shrimp (LC50 =6.1μm), and oppositely increased the growth and biofilm formation of Staphylococcus aureus.

Measurement of separase proteolytic activity in single living cells by a fluorogenic flow cytometry assay.

ESPL1/Separase, an endopeptidase, is required for centrosome duplication and separation of sister-chromatides in anaphase of mitosis. Overexpression and deregulated proteolytic activity of Separase as frequently observed in human cancers is associated with the occurrence of supernumerary centrosomes, chromosomal missegregation and aneuploidy. Recently, we have hypothesized that increased Separase proteolytic activity in a small subpopulation of tumor cells may serve as driver of tumor heterogeneity and clonal evolution in chronic myeloid leukemia (CML). Currently, there is no quantitative assay to measure Separase activity levels in single cells. Therefore, we have designed a flow cytometry-based assay that utilizes a Cy5- and rhodamine 110 (Rh110)-biconjugated Rad21 cleavage site peptide ([Cy5-D-R-E-I-M-R]2-Rh110) as smart probe and intracellular substrate for detection of Separase enzyme activity in living cells. As measured by Cy5 fluorescence the cellular uptake of the fluorogenic peptide was fast and reached saturation after 210 min of incubation in human histiocytic lymphoma U937 cells. Separase activity was recorded as the intensity of Rh110 fluorescence released after intracellular peptide cleavage providing a linear signal gain within a 90–180 min time slot. Compared to conventional cell extract-based methods the flow cytometric assay delivers equivalent results but is more reliable, bypasses the problem of vague loading controls and unspecific proteolysis associated with whole cell extracts. Especially suited for the investigaton of blood- and bone marrow-derived hematopoietic cells the flow cytometric Separase assay allows generation of Separase activity profiles that tell about the number of Separase positive cells within a sample i.e. cells that currently progress through mitosis and about the range of intercellular variation in Separase activity levels within a cell population. The assay was used to quantify Separase proteolytic activity in leukemic cell lines and peripheral blood samples from leukemia patients.

Cytotoxic polyketides from the deep-sea-derived fungus Engyodontium album DFFSCS021.

Eight new chromones, engyodontiumones A–H (1–8), and three new phenol derivatives (9–11) together with eight known polyketides (12–19) were isolated from the deep-sea-derived fungus Engyodontium album DFFSCS021. Their structures were identified by extensive spectroscopic analysis. Compounds 8 and 16 showed significant selective cytotoxicity against human histiocytic lymphoma U937 cell line with IC50 values of 4.9 and 8.8 μM, respectively. In addition, this is the first time to report that 8, 15 and 16 had mild antibacterial activity against Escherichia coli and Bacillus subtilis, and 15 showed potent antilarval activity against barnacle Balanus amphitrite larval settlement.

In vitro Response of Human Pathological Hematopoietic Cells to Cladribine

The influence of cladribine (2-chloro-2'-deoxyadenosine, CdA) on in vitro response of human acute lymphoblastic leukemia MOLT-4 cells, human histiocytic lymphoma U-937 cells, and human promyelocytic leukemia HL-60 cells, was determined using the MTT spectrophotometric and Beckman Coulter methods. Cell viability, cell volume and count were compared 24h and 48h after cladribine application at four concentrations--50 nM, 100 nM, 250 nM, and 500 nM. Different patterns of temporary changes in the viability, volume and count of pathological hematopoietic cells exposed to the action of CdA were found. The effects of CdA on MOLT-4, U-937, and HL-60 cells were dependent on the agent tested and its concentration, the time intervals after agent application, and the cell line used. The various in vitro cytotoxic activities of CdA against the three human pathological hematopoietic cell lines were shown.

Synthesis, structural characterization and leishmanicidal activity evaluation of ferrocenyl N-heterocyclic compounds

New ferrocenyl derivatives with the general formula FcC(O)L [Fc = (η5-C5H5)Fe(η5-C5H4)], where L is an aminoquinoline or hydroxyaminoquinoline, have been synthesized for evaluation of their leishmanicidal properties. The compounds were designed with ferrocene coupled to the quinolines by an amide or ester bridge. Ferrocenyl component is intended to act as quinoline carrier and ROS producer after in vivo oxidation to Fe(III), while decreasing normal cell cytotoxicity of coupled quinolines. The bridge was chosen based on its known ability to undergo hydrolysis by the protease/esterase rich media in phagolysosomes, the target of the intracellular form of leishmania parasites.The new compounds include N-(quinolin-3-yl)ferrocenamide (4), N-(quinolin-5-yl)ferrocenamide (5), N-(quinolin-6-yl)ferrocenamide (6), N-(2-methyl-quinolin-4-yl)ferrocenamide (7), N-(2-methylquinolin-6-yl)ferrocenamide (8), N-(6-methoxy-quinolin-8-yl)ferrocenamide (9), 2-amino-quinolin-8-yl ferrocenoate (10) and 2-amino-quinolin-4-yl ferrocenoate (11). They were characterized by NMR, cyclic voltammetry, mass spectrometry, UV/vis, FT-IR and elemental analysis, which confirmed all the proposed molecular structures. Compounds 7 and 8 were also structurally characterized by single crystal X-ray diffraction. In 7, the 4-amino-2-methylquinoline moiety is perpendicular to the substituted cyclopentadienyl ring (Cp), while in 8 the 6-amino-2-methylquinoline component is coplanar to the substituted Cp.The new compounds (4–11), same as four other previously published (1-ferrocenoyl-1H-(2-aminobenzimidazole) (1), 1-ferrocenoyl-1H-benzimidazole (2), 1-ferrocenoyl-1H-imidazole (3) and N-(pyridin-4-yl)ferrocenamide (12)), were evaluated in vitro in cultures of a Leishmania infantum strain, isolated from a human visceral leishmaniasis case, to establish their leishmanicidal activity. The toxicity against the human caucasian histiocytic lymphoma U-937 cell line was analyzed for the same set of compounds. All of them show activity against promastigote forms of L. infantum parasites at relatively high concentration (64–269 μM). Among the complexes that showed a better ratio between the toxic and the therapeutic dose, 3, 9 and 12 were selected for further studies in infected macrophages. Such compounds showed a very significant increase in their activity (17–23 times) giving very similar IC50 values (5.2–5.7 μM). All three compounds gave significantly better therapeutic indexes (88.5, 12; 56.4, 3; 16.6, 9) than the control miltefosine (6.1).22Abbreviations – MTD25, or Maximum Tolerated Dose, was defined as the drug concentration that decreases the number of viable macrophage cells by 25%. IC50, or inhibitory concentration, is the drug concentration that leads to a 50% inhibition of parasite growth, both in promastigote (IC50p) and amastigote (IC50a) phases.

Development of an Integrated Chip for Automatic Tracking and Positioning Manipulation for Single Cell Lysis

This study adopted a microelectromechanical fabrication process to design a chip integrated with electroosmotic flow and dielectrophoresis force for single cell lysis. Human histiocytic lymphoma U937 cells were driven rapidly by electroosmotic flow and precisely moved to a specific area for cell lysis. By varying the frequency of AC power, 15 V AC at 1 MHz of frequency configuration achieved 100% cell lysing at the specific area. The integrated chip could successfully manipulate single cells to a specific position and lysis. The overall successful rate of cell tracking, positioning, and cell lysis is 80%. The average speed of cell driving was 17.74 μm/s. This technique will be developed for DNA extraction in biomolecular detection. It can simplify pre-treatment procedures for biotechnological analysis of samples.

Protein kinase C-δ isoform mediates lysosome labilization in DNA damage-induced apoptosis

A lysosomal pathway, characterized by the partial rupture or labilization of lysosomal membranes (LLM) and cathepsin release into the cytosol, is evoked during the early events of 20-S-camptothecin lactone (CPT)-induced apoptosis in human cancer cells, including human histiocytic lymphoma U-937 cells. These lysosomal events begin rapidly and simultaneously with mitochondrial permeabilization and caspase activation within 3 h after drug treatment. Recently, in a comparative proteomics analysis performed on highly-enriched lysosomal extracts, we identified proteins whose translocation to lysosomes correlated with LLM induction after CPT treatment, including protein kinase C-δ (PKC-δ). In this study, we show that the PKC-δ translocation to lysosomes is required for LLM, as silencing its expression with RNA interference or suppressing its activity with the inhibitor, rottlerin, prevents CPT-induced LLM. PKC-δ translocation to lysosomes is associated with lysosomal acidic sphingomyelinase (ASM) phosphorylation and activation, which in turn leads to an increase in ceramide (CER) content in lysosomes. The accumulation of endogenous CER in lysosomes is a critical event for CPT-induced LLM as suppressing PKC-δ or ASM activity reduces both the CPT-mediated CER generation in lysosomes and CPT-induced LLM. These findings reveal a novel mechanism by which PKC-δ mediates ASM phosphorylation/activation and CER accumulation in lysosomes in CPT-induced LLM, rapidly activating the lysosomal pathway of apoptosis after CPT treatment.

Induction of DNA Breakage in U937 Cells by Oxazaphosphorines

Oxazaphosphorines are a class of DNA alkylating agents. The aim of the present study was to compare the possible influence of three new generation oxazaphosphorines, D-17272 (mafosfamide cyclohexylamine salt), D-18864 (4-hydro-peroxy-cyclophosphamide), and D-19575 (glufosfamide, beta-D-glucose-isophosphoramide mustard) on DNA damage induction in the human histiocytic lymphoma U937 cells. The flow cytometry APO-BRDU assay, based on the TUNEL method, was used for the in situ detection of DNA strand breaks. After exposure of U937 cells to the oxazaphosphorines, the patterns of temporary changes in the frequency of TUNEL positive U937 cells, expressing DNA breakage, were determined. The effects of the oxazaphosphorines on U937 cells were dependent on the agent tested and its dose, and the time intervals after the drug application. The different potential of D-17272, D-18864 and D-19575 to induce DNA strand breakage in the human histiocytic lymphoma U937 cells was shown.

Synthesis, structural characterization and antiproliferative and toxic bio-activities of copper(II) and nickel(II) citronellal N4-ethylmorpholine thiosemicarbazonates

This paper reports the syntheses and characterization of ethylmorpholine substituted citronellal thiosemicarbazone copper(II) and nickel(II) metal complexes. The compounds were characterized through elemental analyses and spectroscopic (IR, UV-Vis, NMR, MS) methods. The X-ray analysis of the two complexes shows that both Ni and Cu derivatives present a square planar coordination, where the coordinating homologous donor atoms bind in trans to each other. The compounds were tested for their biological activity after determination of their octanol-saline partition coefficients, followed by their radical scavenging properties. Eventually the complexes were tested for their proliferation inhibition on human histiocytic lymphoma U937 cell line. The GI(50) values resulted to be 2.3microM for the copper derivative and 12.3microM for the nickel derivative.

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.

Proteomic analysis of enriched lysosomes at early phase of camptothecin-induced apoptosis in human U-937 cells

A lysosomal pathway, characterized by partial rupture or labilization of lysosomal membranes and cathepsin activation, is evoked during camptothecin-induced apoptosis in human cancer cells, including human histiocytic lymphoma U-937 cells. These lysosomal events begin rapidly and simultaneously with mitochondrial permeabilization and caspase activation within 3 h after drug treatment. In this study, comparative and quantitative proteome analyses were performed to identify early changes in lysosomal protein expression/localization from U-937 cells undergoing apoptosis. In 2 independent experiments, among a total of more than 538 proteins putatively identified and quantitated by iTRAQ isobaric labeling and LC-ESI-MS/MS, 18 proteins were found to be upregulated and 9 downregulated in lysosomes purified from early apoptotic compared to control cells. Protein expression was validated by Western blotting on enriched lysosome fractions, and protein localization confirmed by fluorescence confocal microscopy of representative protein candidates, whose functions are associated with lysosomal membrane fluidity and dynamics. These include sterol-4-alpha-carboxylate 3-dehydrogenase (NSDHL), prosaposin (PSAP) and protein kinase C delta (PKC-delta). This comparative proteome analysis provides the basis for novel hypothesis and rationale functional experimentation, where the 3 validated candidate proteins are associated with lysosomal membrane fluidity and dynamics, particularly cholesterol, sphingolipid and glycosphingolipid metabolism.

Kalopanaxsaponin A induces apoptosis in human leukemia U937 cells through extracellular Ca 2+ influx and caspase-8 dependent pathways

In the present study, we investigated the effect of KPS-A on the apoptotic activity and the molecular mechanism of the action in human leukemia. Treatment with KPS-A significantly increased apoptotic DNA fragmentation in human histiocytic lymphoma U937 cells as shown by DAPI staining, flow cytometry, and agarose gel electrophoresis. In addition, stimulation of U937 cell with KPS-A induced a series of intracellular events: (1) the activations of caspase-8, caspase-9, and caspase-3; (2) the translocations of Bid and Bax proteins to mitochondria; (3) the loss of mitochondrial membrane potential; and (4) the increased release of cytochrome c to the cytosol. Pretreatment with a specific caspases-8, -9 or -3 inhibitor, neutralized the pro-apoptotic activity of KPS-A in U937 cells. We further demonstrated that KPS-A markedly induced an increase in intracellular Ca 2+ level, which was reversed by EGTA, a general calcium chelator, but not by TMB-8 and dantrolene, intracellular Ca 2+ release blockers. Moreover, KPS-A-induced DNA fragmentation and caspase activation were substantially reduced in the presence of EGTA. Taken together, these results suggest that KPS-A may play therapeutic role for leukemia via the potent apoptotic activity through Ca 2+/caspases-8/MPT/caspases-9/caspases-3 signaling pathway.

Preventive Effect of Trimidox on Oxidative Stress in U937 Cell Line

Trimidox (3,4,5-trihydroxybenzamidoxime) is one of the most potent ribonucleotide reductase inhibitors, revealing an antitumor effect in several experimental studies. We have examined the effect of trimidox on the induction of cytotoxicity and apoptosis via oxidative stress by typical free radical inducers, hydrogen peroxide (H(2)O(2)), tert-butylhydroperoxide (tBuOOH) or ultraviolet (UV) irradiation in a human diffuse histiocytic lymphoma U937 cell line. Trimidox showed strong radical scavenging activity by the DPPH reduction assay. The 50% rate inhibited the DPPH reduction concentration of trimidox, and its derivates didox, or gallic acid were 8.8 microM, 117.5 microM, or 41.8 microM, respectively. Induction of cytotoxicity by H(2)O(2) (500 microM) or tBuOOH (100 microM) was concentration-dependently attenuated by incubation with Trimidox (10-150 microM). Trimidox also prevented the effect of UV-induced apoptosis estimated by both nuclear morphological change and DNA fragmentation. This effect was due to inhibition of the production of reactive oxygen species. Moreover, the activity and mRNA expression of catalase, an antioxidant enzyme, was significantly increased by trimidox. These results indicate that trimidox has radical scavenging activity and prevents exogenous oxidative stress and increase in catalase; therefore, trimidox is suggested as an anticancer agent exhibiting potent antioxidant properties in this study.