Lymphoma U937 Cells Research Articles

Nicaraven induces programmed cell death by distinct mechanisms according to the expression levels of Bcl-2 and poly (ADP-ribose) glycohydrolase in cancer cells

The PARP-1 expression level and poly (ADP-ribosyl)ation activity in cancer markedly affect the therapeutic outcome. Nicaraven, a free radical scavenger has been found to inhibit PARP, but the effect on cancer cells is still unclear. In this study, we investigated the potential role and molecular mechanism of nicaraven on cancer cells. Using U937 lymphoma cells and HCT-8 colorectal cancer cells, we found that nicaraven moderately reduced the cell viability of both cells in a dose-dependent manner. Interestingly, nicaraven significantly induced apoptosis of U937 cells that are dominantly expressing Bcl-2 but induced PAR-dependent cell death (parthanatos) of HCT-8 cells that are highly expressing poly (ADP-ribose) glycohydrolase (PARG). Based on our data, nicaraven seems to induce programmed cell death through distinct mechanisms, according to the expression levels of Bcl-2 and PARG in cancer cells.

Synthesis, crystal structure, and in vitro evaluation of the anticancer activity of new Pt (Pd) complexes with 1-[(dimethylamino)methyl]-2-naphthol ligand.

The synthesis of new Pt(II) and Pd(II) complexes with 1-aminomethyl-2-naphtol ligands has been first performed. The adducts of [PtCl4]2- and [PdCl4]2- anions with the 1-aminomethyl-2-naphtol NH cation were synthesized. The structure for four Pt (Pd)-containing compounds was investigated using X-ray diffraction. The obtained compounds were examined for in vitro cytotoxic activity against Jurkat and K562 human leukemia cells, lymphoma U937cells, A2780 and the cisplatin-resistant A2780cis lines of human ovarian cancer, and normal fibroblasts. Study of induction of apoptosis and the effect of new palladium and platinum complexes on the cell cycle was carried out. The cells showed a higher sensitivity to Pt(II) compounds than to Pd(II) ones. All the synthesized metal complexes show much more antitumor activity compared with a platinum-containing cisplatin drug.

Silencing PEX26 as an unconventional mode to kill drug-resistant cancer cells and forestall drug resistance

ABSTRACT Promoting the macroautophagy/autophagy-mediated degradation of specific proteins and organelles can potentially be utilized to induce apoptosis in cancer cells or sensitize tumor cells to therapy. To examine this concept, we enriched for autophagosomes from histone deacetylase inhibitor (HDACi)-sensitive U937 lymphoma cells and isogenic HDACi-resistant cells. Mass spectrometry on autophagosome-enriched fractions revealed that HDACi-resistant cells undergo elevated pexophagy, or autophagy of the peroxisome, an organelle that supports tumor growth. To disturb peroxisome homeostasis, we enhanced pexophagy in HDACi-resistant cells via genetic silencing of peroxisome exportomer complex components (PEX1, PEX6, or PEX26). This consequently sensitized resistant cells to HDACi-mediated apoptosis, which was rescued by inhibiting ATM/ataxia-telangiectasia mutated (ATM serine/threonine kinase), a mediator of pexophagy. We subsequently engineered melanoma cells to stably repress PEX26 using CRISPR interference (CRISPRi). Melanoma cells with repressed PEX26 expression showed evidence of both increased pexophagy and peroxisomal matrix protein import defects versus single guide scrambled (sgSCR) controls. In vivo studies showed that sgPEX26 melanoma xenografts recurred less compared to sgSCR xenografts, following the development of resistance to mitogen-activated protein kinase (MAPK)-targeted therapy. Finally, prognostic analysis of publicly available datasets showed that low expression levels of PEX26, PEX6 and MTOR, were significantly associated with prolonged patient survival in lymphoma, lung cancer and melanoma cohorts. Our work highlighted that drugs designed to disrupt peroxisome homeostasis may serve as unconventional therapies to combat therapy resistance in cancer. Abbreviations: ABCD3/PMP70: ATP binding cassette subfamily D member 3; ACOX1: acyl-CoA oxidase 1; AP: autophagosome; COX: cytochrome c oxidase; CQ: chloroquine; CRISPRi: clustered regularly interspaced short palindromic repeats interference; DLBCL: diffuse large B-cell lymphoma; GO: gene ontology; dCas9: Cas9 endonuclease dead, or dead Cas9; HDACi: histone deacetylase inhibitors; IHC: Immunohistochemistry; LAMP2: lysosomal associated membrane protein 2; LCFAs: long-chain fatty acids; LFQ-MS: label-free quantitation mass spectrometry; LPC: lysophoshatidylcholine; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; PBD: peroxisome biogenesis disorders; PTS1: peroxisomal targeting signal 1; ROS: reactive oxygen species; sgRNA: single guide RNA; VLCFAs: very-long chain fatty acids; Vor: vorinostat; WO: wash-off.

Type I collagen or gelatin stimulates mouse peritoneal macrophages to aggregate and produce pro-inflammatory molecules through upregulated ROS levels

BackgroundExtracellular matrix (ECM) comprising the environments of multicellular society has a dynamic network structure. Collagen is one of the ubiquitous components of ECM. Collagen affects the inflammatory response by regulating the release of pro-inflammatory cytokines from cells. Gelatin, denatured collagen found temporally in tissues, is supposed to be pathophysiologically involved in tissue remodeling, inflammation caused by tissue damage. Previous reports indicate that, phorbol myristate (PMA)-stimulated human U937 (lymphoma cell line) cells that are often used as macrophage-like cells, show cell aggregations when cultured on type I collagen (col I) or gelatin-coated dishes, accompanying the changes of production and release of proinflammatory factors. However, it still remains to be examined whether collagen and gelatin affects normal macrophages as well. AimThis study aims to investigate the effect of col. I, the main component of collagenous protein and its denatured product, gelatin, on mouse peritoneal macrophages (MPMs). MethodsMTT assay, flow cytometric analysis of ROS, biochemical detection of antioxidant levels, ELISA assay, and western blot were used. ResultsMPMs formed multicellular aggregates on col. I - and gelatin-coated dishes with a concentration- and time-dependent manner. Further studies showed that the culture on col. I and gelatin up-regulated the protein expression and secretion of pro-inflammatory molecules such as IL-1β, TNFα and prostaglandin E2 (PGE2) in MPMs. The levels were higher in the cells on gelatin than those on col. I. ROS levels are significantly increased in the cells cultured on both col. I- and gelatin-coated dishes, accompanying decreased levels of antioxidant enzyme catalase (CAT) and anti-oxidant glutathione (GSH), and enhanced nuclear translocation of NF-κB. ConclusionCol I - or gelatin-coated culture induced the formation of multicellular aggregates and increased production of NF-κB-associated pro-inflammatory molecules in MPMs through up-regulation of ROS levels.

Anti-proliferative activity of bovine blood hydrolysates towards cancer cells in culture

Four proteins, α/β globulin, serum albumin, γ-globulin and fibrinogen, were isolated from bovine blood and hydrolysed using papain. Hydrolysates were assessed for non-cellular and cellular antioxidant activity. The anti-proliferative activity of hydrolysed fractions was assessed in a number of cancer cell lines including U937 lymphoma cells, MCF-7 breast cancer cells, HepG2 hepatocytes and Caco-2 epithelial colorectal adenocarcinoma cells. Anti-inflammatory activity of the hydrolysates was also assessed. Hydrolysates generated from γ-globulin or fibrinogen had significant antioxidant activity in non-cellular assays. Hydrolysates were also found to be highly toxic to different cancer cell lines, in particular U937 lymphoma cells when assessed using the MTT assay. The fibrinogen hydrolysate was the most toxic sample and toxicity appeared to correlate with its non-cellular antioxidant activity. None of the hydrolysates had significant anti-inflammatory activity. The high cytotoxicity of the γ-globulin and the fibrinogen hydrolysates towards cancer cells may indicate a potential use as anti-proliferative agents.

Apoptotic Effect of Nigella sativa on Human Lymphoma U937 Cells.

Objective:Nigella sativa is from botanical Ranunculaceae family and commonly known as black seed. Apoptotic effect of N. sativa and its apoptotic signaling pathways on U937 lymphoma cells are unknown.Materials and Methods:In this study, we investigated selective cytotoxic and apoptotic effects of N. sativa extract and its apoptotic mechanisms on U937 cells. In addition, we also studied selective cytotoxic activity of thymoquinone that is the most active essential oil of N. sativa.Results:Our results showed that N. sativa extract has selective cytotoxicity and apoptotic effects on U937 cells but not ECV304 control cells. However, thymoquinone had no significant cytotoxicity against on both cells. N. sativa extract increased significantly caspase-3, BAD, and p53 gene expressions in U937 cells.Conclusions:N. sativa may have anticancer drug potential and trigger p53-induced apoptosis in U937 lymphoma cells.SUMMARY This is the first study showing the apoptotic effect of Nigella sativa extract on U937 cells. Abbreviations used: CI: Cytotoxicity index, DMEM: Dulbecco's Modified Eagle Medium, HL: Hodgkin's lymphoma, MTT: 3-(4,5-dimethy lthiazol-2yl)-2,5-diphenyl tetrazolium bromide, RPMI: Roswell Park Memorial Institute medium.

Ethyl acetate extract of Peperomia tetraphylla induces cytotoxicity, cell cycle arrest, and apoptosis in lymphoma U937 cells.

The current study evaluated the cytotoxicity and the mechanism of apoptotic induction by Peperomia tetraphylla in U937 lymphoma cells. The results showed that P. tetraphylla ethyl acetate extract (EAEPT) inhibited the cell growth in U937 cells by MTT assay. After the U937 cells were treated with EAEPT, the cells exhibited marked morphological features of apoptosis (Hoechst 33342 staining) and the number of apoptotic cell (Annexin V-FITC/PI staining) increased. The treatment of EAEPT could induce loss of mitochondrial membrane potential (MMP) and increase the ROS level. Moreover, EAEPT treatment resulted in the accumulation of cells at S phase. We found that EAEPT could induce the cleavage of the caspase 3, caspase 8, caspase 9 and Bid. And the treatment of EAEPT could increase expression of Bax and down-regulate the expression of CCNB1, CCND1 and CDK1. The sub-fraction of EAEPT, namely EASub1 demonstrated the highest cytotoxicity activity on U937 cells. It was confirmed that EAEPT could inhibit the growth of U937 cells by blocking the cell cycle and prompted apoptosis via the ROS-medicated mitochondria pathway in vitro.

Cladribine, gemcitabine, busulfan, and SAHA combination as a potential pretransplant conditioning regimen for lymphomas: A preclinical study.

Hematopoietic stem cell transplantation (HSCT) is an effective treatment for patients with refractory lymphomas. Nucleoside analogs (NAs) and DNA alkylating agents are efficacious in treating hematologic malignancies. To design an efficacious and more economical pretransplant regimen for lymphoma patients, we analyzed the cytotoxicity of cladribine (Clad), gemcitabine (Gem), busulfan (Bu), and suberoylanilide hydroxamic acid (SAHA) in lymphoma cell lines. J45.01 and U937 lymphoma cell lines were exposed to drugs, alone or in combination, for 48hours and analyzed with the MTT and annexin V assays, Western blotting, and flow cytometry. On the basis of the IC5-10 values of the drugs, the Clad+Gem+Bu combination inhibited the proliferation of both cell lines to ∼55%-60%. Addition of SAHA to this combination decreased proliferation further to ∼30%. Exposure to the Clad+Gem+Bu+SAHA combination activated the DNA damage response and ATM-CHK2 pathway; modified histones; decreased mitochondrial membrane potential, which caused leakage of apoptosis-inducing factors; and activated apoptosis. Pretreatment of cells with the pan-caspase inhibitor Z-VAD-FMK blocked the phosphorylation of histone 2AX and cleavage of PARP-1 and caspases. The Clad+Gem+Bu+SAHA combination provides synergistic cytotoxicity in lymphoma cell lines. Our results may be a basis for using this combination as a pretransplant conditioning regimen in a clinical trial for lymphoma patients undergoing hematopoietic stem cell transplantation, replacing the more expensive nucleoside analog clofarabine.

The elimination of miR-23a in heat-stressed cells promotes NOXA-induced cell death and is prevented by HSP70.

Protein-damaging stress stimulates cell destruction through apoptosis; however, non-lethal proteotoxic stress induces an adaptive response leading to the increased synthesis of heat shock proteins, which inhibit apoptosis. In this study, we sought to determine the mechanism responsible for the accumulation of the BH3-only protein NOXA in heat-stressed cells and its prevention by the heat shock protein HSP70. Analysis of transcript levels by RT-qPCR revealed that miR-23a levels decreased in heat-stressed cells and that this was correlated with an increased abundance of NOXA mRNA, which contains a miR-23a binding site in its 3′ untranslated region. Cells overexpressing HSP70 had higher levels of miR-23a, maintained these levels after heat shock and accumulated lower levels of NOXA mRNA and protein. The enhanced abundance of mir-23a in these HSP70-expressing cells is primarily due to its increased stability although higher levels of pri/pre-miR-23a expression, nuclear export and maturation were also contributing factors. Stable overexpression of miR-23a in the acute lymphoblastic T-cell line PEER resulted in reduced basal and heat-induced levels of NOXA mRNA and significantly inhibited heat-induced apoptosis. Additionally, stable overexpression of an shRNA targeting miR-23a in U937 lymphoma cells produced stable knockdown of miR-23a and resulted in increased NOXA mRNA and an increased sensitivity to heat-induced apoptosis. These results demonstrate the novel finding that hyperthermia affects the abundance of a microRNA that targets the expression of a pro-apoptotic protein and that HSP70 protects cells from heat-induced apoptosis by regulating the abundance of this microRNA. We speculate that the inhibition of miRNA transcription in heat-stressed cells could represent a general mechanism for apoptosis induction that is regulated by the molecular chaperone protein HSP70. Furthermore, we propose that HSP70 could be beneficial to tumor cells by helping to maintain the expression of oncogenic miRNAs under conditions of cellular stress.

Bioactivity screening and mass spectrometric confirmation for the detection of PPARδ agonists that increase type 1 muscle fibres

Sensitive and robust bioassays able to detect nuclear receptor activation are very useful for veterinary and doping control, pharmaceutical industry and environmental scientists. Here, we used bioassays based on human leukemic monocyte lymphoma U937 and human liver hepatocellular carcinoma HepG2 cell lines to detect the ligand-induced activation of the peroxisome proliferator-activated receptor delta (PPARδ). Exposure of U937 cells to the PPARδ agonist GW501516 resulted in a marked increase in mRNA expression of the PPARδ target gene Angptl4 which was quantified by qRT-PCR analysis. Exposure of HepG2 cells transiently transfected with a PPARδ expression plasmid and a PPAR-response element-driven luciferase reporter plasmid to PPARδ agonists GW501516, GW610742 and L-165041 resulted in clear dose-response curves. Although the qRT-PCR resulted in higher fold inductions, the luciferase assay with transfected HepG2 cells is cheaper and quicker and about ten times more sensitive to GW501516 compared to analysis of Angptl4 mRNA expression in U937 cells by qRT-PCR. The HepG2-based luciferase assay was therefore used to screen GW501516-spiked supplements and feed and water samples. After liquid extraction and clean-up by solid phase extraction using a weak anion exchange column, extracts were screened in the HepG2 bioassay followed by confirmation with a newly developed UPLC-MS/MS method, using two transitions for each compound, i.e., for GW501516, 454.07>188.15 (collision energy (CE) 46V) and 454.07>257.08 (CE 30V); for GW610742, 472.07>206.2 (CE 48V) and 472.07>275.08 (CE 30V); and for L-165041, 401.2>193.15 (CE 26V) and 401.2>343.2 (CE 20V).

Acidosis Decreases c-Myc Oncogene Expression in Human Lymphoma Cells: A Role for the Proton-Sensing G Protein-Coupled Receptor TDAG8

Acidosis is a biochemical hallmark of the tumor microenvironment. Here, we report that acute acidosis decreases c-Myc oncogene expression in U937 human lymphoma cells. The level of c-Myc transcripts, but not mRNA or protein stability, contributes to c-Myc protein reduction under acidosis. The pH-sensing receptor TDAG8 (GPR65) is involved in acidosis-induced c-Myc downregulation. TDAG8 is expressed in U937 lymphoma cells, and the overexpression or knockdown of TDAG8 further decreases or partially rescues c-Myc expression, respectively. Acidic pH alone is insufficient to reduce c-Myc expression, as it does not decrease c-Myc in H1299 lung cancer cells expressing very low levels of pH-sensing G protein-coupled receptors (GPCRs). Instead, c-Myc is slightly increased by acidosis in H1299 cells, but this increase is completely inhibited by ectopic overexpression of TDAG8. Interestingly, TDAG8 expression is decreased by more than 50% in human lymphoma samples in comparison to non-tumorous lymph nodes and spleens, suggesting a potential tumor suppressor function of TDAG8 in lymphoma. Collectively, our results identify a novel mechanism of c-Myc regulation by acidosis in the tumor microenvironment and indicate that modulation of TDAG8 and related pH-sensing receptor pathways may be exploited as a new approach to inhibit Myc expression.

Tumor Necrosis Factor-stimulated Gene-6 (TSG-6) Amplifies Hyaluronan Synthesis by Airway Smooth Muscle Cells

We tested the hypothesis that the artificial addition of heavy chains from inter-α-inhibitor to hyaluronan (HA), by adding recombinant TSG-6 (TNF-stimulated gene-6) to the culture medium of murine airway smooth muscle (MASM) cells, would enhance leukocyte binding to HA cables produced in response to poly(I:C). As predicted, the addition of heavy chains to HA cables enhanced leukocyte adhesion to these cables, but it also had several unexpected effects. (i) It produced thicker, more pronounced HA cables. (ii) It increased the accumulation of HA in the cell-associated matrix. (iii) It decreased the amount of HA in the conditioned medium. Importantly, these effects were observed only when TSG-6 was administered in the presence of poly(I:C), and TSG-6 did not exert any effect on its own. Increased HA synthesis occurred during active, poly(I:C)-induced HA synthesis and did not occur when TSG-6 was added after poly(I:C)-induced HA synthesis was complete. MASM cells derived from TSG-6(-/-), HAS1/3(-/-), and CD44(-/-) mice amplified HA synthesis in response to poly(I:C) + TSG-6 in a manner similar to WT MASM cells, demonstrating that they are expendable in this process. We conclude that TSG-6 increases the accumulation of HA in the cell-associated matrix, partially by preventing its dissolution from the cell-associated matrix into the conditioned medium, but primarily by inducing HA synthesis.

Changes in Free Amino Acid, Protein, and Flavonoid Content in Jujube (Ziziphus jujube) Fruit during Eight Stages of Growth and Antioxidative and Cancer Cell Inhibitory Effects by Extracts

Jujube (Ziziphus jujube) was analyzed at eight stages of ripeness (S1-8) for protein, by HPLC and mass spectroscopy for free amino acids and flavonoids, and by colorimetry for total flavonoids and antioxidative activity. The ripe fruit had lower levels of protein, flavonoids, and antioxidative activity than that of the unripe fruit. Free amino acids levels peaked at S5, due mainly to an increase in free asparagine. Extracts were also tested against four cell lines using the MTT cell viability assay. All growth stages dose-dependently inhibited HeLa cervical cancer cells, whereas the inhibition of Hel299 normal lung and A549 lung cancer cells decreased as the fruit matured and was well correlated with the flavonoid content and antioxidative activity. Chang normal liver cells were inhibited by only the S5 extract. U937 lymphoma cells were unaffected by the extracts. These results show the effect of fruit maturity on nutritional and health-promoting components.

Synthesis and anti-leukaemic activity of pyrrolo[3,2,1-hi]indole-1,2-diones, pyrrolo[3,2,1-ij]quinoline-1,2-diones and other polycyclic isatin derivatives

To further expand the structure–cytotoxic activity relationships of isatin derivatives and to reduce flexibility in substituent groups at nitrogen, 20 analogues incorporating a ring system between the N1 and C7 atoms of isatin were prepared using a variety of synthetic strategies. This yielded pyrroloindole-, pyrroloquinoline-, pyrroloacridine-, pyrrolophenanthridine- and benzopyrrolophenanthridine-based systems with embedded isatin moieties, the latter possessing a novel carbon skeleton. These compounds were subsequently assessed for their in vitro cytotoxicity against human U937 lymphoma cells, with the brominated pyrroloacridine dione 27 showing the most promising activity (IC50 3.01 μM) after 24 h.

Rapid synthesis, characterization, anticancer and antimicrobial activity studies of substituted thiadiazoles and their dinucleating ligand metal complexes

Synthesis of 2,5-disubstituted thiadiazoles was accomplished via a conventional method as well as microwave irradiation method. These substituted thiadiazoles were diazotized and coupled with 2,4-pentanedione (AcAc), ethylcyanoacetate (ECA) and malanodinitrile (MN) to get dinucleating ligands. The ligands were isolated, characterized and condensed with Ni (II), Cu (II) and Ru(III) chlorides. These compounds were screened on HL-60 Human leukemia cell Line and U-937 Lymphoma cell lines for anticancer activities. The antimicrobial activity of the ligands and their complexes against bacteria and fungi was also investigated. The effect of metal on the ligand activity is discussed.

SOD/catalase mimetic platinum nanoparticles inhibit heat-induced apoptosis in human lymphoma U937 and HH cells

Platinum nanoparticles (Pt-NPs) are known to possess anti-tumouric activity and the ability to scavenge superoxides and peroxides indicating that they can act as superoxide dismutase (SOD)/catalase mimetics. These potentials seem useful in the protection and/or amelioration of oxidative stress-associated pathologies, but, when they are combined with a therapeutic modality that depends upon the mediation of reactive oxygen species in cell killing induction, the effect of Pt-NPs might be questionable. Here, the effects of polyacrylic acid-capped Pt-NPs (nano-Pts) on hyperthermia (HT)-induced apoptosis and the underlying molecular mechanisms were investigated in human myelomonocytic lymphoma U937 and human cutaneous T-cell lymphoma HH cells. The results showed that the pre-treatment with nano-Pts significantly inhibited HT-induced apoptosis in a dose-dependent manner. Superoxide, but not peroxides, was suppressed to varying extents. All pathways involved in apoptosis execution were also negatively affected. The results reveal that the combination of nano-Pts and HT could result in HT-desensitization.

Changes in Free Amino Acid, Phenolic, Chlorophyll, Carotenoid, and Glycoalkaloid Contents in Tomatoes during 11 Stages of Growth and Inhibition of Cervical and Lung Human Cancer Cells by Green Tomato Extracts

Tomato ( Solanum lycopersicum ) plants synthesize nutrients, pigments, and secondary metabolites that benefit nutrition and human health. The concentrations of these compounds are strongly influenced by the maturity of the tomato fruit on the vine. Widely consumed Korean tomatoes of the variety Doturakworld were analyzed for changes in the content of free amino acids, phenolic compounds, chlorophylls, carotenoids, and glycoalkaloids at 11 stages (S1-S11) of ripeness. The results show that (a) the total content (in mg/100 g of FW) of the free amino acids and other nitrogen-containing compounds in the extracts ranged from about 41 to 85 in the green tomato extracts S1-S7 and then increased to 251 (S9) in the red extracts, followed by a decrease to 124 in S11 red extracts; (b) the total initial concentration and composition of up to 12 phenolic compounds of approximately 2000 microg/100 g of FW varied throughout the ripening process, with the quantity decreasing and the number of individual compounds increasing in the red tomato; (c) chlorophyll a and b content of tomatoes harvested during S1 was 5.73 mg/100 g of fresh pericarp and then decreased continuously to 1.14 mg/100 g for S11; (d) the concentration (in mg/100 g of FW) of lycopene in the S8 red extract of 0.32 increased to 1.27 in S11; and (e) tomatoes harvested during S1 contained 48.2 mg of dehydrotomatine/100 g of FW, and this value continually decreased to 1.5 in S7, with no detectable levels in S8-S11. The corresponding alpha-tomatine content decreased from S1 (361) to S8 (13.8). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell assay IC(50) values showed that Hel299 lung cells, A549 lung cancer cells, and HeLa cervical carcinoma cells were highly susceptible to inactivation by glycoalkaloid-rich green tomato extracts. Chang normal liver cells and U937 lymphoma cells were less susceptible. The possible significance of the results for plant physiology and the diet is discussed.

Abstract 3791: Sulphoraphane inhibits STAT3 and its related genes in Du 145 cancer cells

Abstract Sulforaphane is a naturally occurring isothiocyanate compound mainly found in cruciferous vegetables. Sulforaphane has demonstrated cancer chemopreventive activities in a number of organ sites, including prostate. Mechanistic investigations have shown that SFN induces caspase-mediated apoptosis that is mediated through a rapid intracellular ROS generation. Since STAT3 signaling is crucial for cancer cell survival and ROS has been linked to inactivation of this transcriptional factor, we hypothesized that STAT3 may be an important target for SFN. Here, we investigated the effect of sulforaphane on the regulation of STAT3 in association of ROS in Du145 prostate cancer cells. We first confirmed that sulforaphane induced apoptosis by activation of caspase 8, 9, and 3, PARP cleavage and increase of TUNEL positive cells in Du145 cells, which were preceded for many hours by the increase of intracellular ROS, peaking at 30 minutes of exposure. We demonstrate for the first time that sulforaphane suppressed constitutive STAT3 activation and DNA-binding of STAT3 in a time- and dose-dependent manner in Du145 cells. These actions were also found in U266 multiple myeloma cells and U937 lymphoma cells. In addition, sulforaphane down-regulated the expression of STAT3-related genes such as bcl-2, bcl-xL, survivin, cyclin D1, and VEGF in Du145 cells. Furthermore, sulforaphane cross-linked JAK2, the upstream kinase of STAT3, in a time- and dose-dependent manner. Thiol-reducing agent dithiothreitol (DTT) reversed the inactivation of STAT3 and JAK2 induced by sulforaphane. Interestingly, N-acetyl cysteine (NAC), which leads to increased intracellular GSH synthesis, reversed the molecular patterns of PARP cleavage and inactivation of JAK2 and STAT3 induced by sulphoraphane. Taken together, these findings suggest that sulforaphane exposure leads to a rapid generation of ROS in Du145 cells and inhibition of JAK2/STAT3 pathway, followed by caspase-mediated apoptosis. Our data suggest that the JAK2/STAT3 pathway may be a novel target of SFN contributing to apoptosis. 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 3791.

Abstract 2500: ON 01910. Na, a clinical stage anticancer mitotic inhibitor, produces prolonged hyperphosphorylation of RanGAP1•SUMO1 as a potential mechanism of G2/M arrest and apoptosis

Abstract INTRODUCTION: The benzyl styryl sulfone ON 01910. Na (abbreviated as 1910) is a novel anticancer agent that inhibits mitotic progression and induces apoptosis in most cancer cell cultures. The compound is currently in Phase 1 and 2 trials. Available data show the drug produces three major abnormalities in tumor cell lines: (a) aberrant cell division, including irregular chromosomal segregation and cytokinesis; (b) G2/M arrest and apoptosis in many tumor cells; and (c) decreased expression of Cdc25C phosphatase. Early data suggested that 1910 was a PLK1 inhibitor. Subsequent studies did not confirm this, although 1910 was found to inhibit the PLK pathway. Precise mechanism of action continues to be investigated. METHODS and RESULTS: We assessed DNA damage checkpoints throughout the cell cycle and effects on signaling molecules upstream of Cdc25C following treatment of DU145 prostate cancer, Bel7404 hepatoma, U937 lymphoma or MOLT-3 ALL cells with 1910. Camptothecin (CPT) and doxorubicin (DOX) served as positive controls. Cell lysates after drug exposure for 4, 16 or 24 h were resolved on SDS-PAGE and analyzed by Western blot for Chk1, Chk2, ATM, RanGAP1•SUMO1 and their phosphorylated forms. CPT and DOX exposure resulted in activation/phosphorylation of DNA damage-responsive molecules Chk1, Chk2 and ATM by 4 h, whereas 1910 did not. However, hyperphosphorylation of RanGAP1•SUMO1 was observed within 4 h and sustained for more than 24 h during 1910 exposure. This was also seen with the anti-tubulin agent, nocodazole, but not with ON 01911, an inactive analog of 1910. Mild phosphorylation of Chk2 was observed only after 24 h exposure, suggesting that DNA damage response is not a primary effect of 1910. MOLT-3 cells, synchronized by double thymidine block, were released into medium supplemented with 1910, which resulted in peak accumulation of G2/M cells by 9-12 h. The G2/M cell fraction remained in plateau for more than 20 h. This timeframe correlated with the hyperphosphorylation of RanGAP1•SUMO1. Cleaved Lamin B, detected by 16 h and thereafter in 1910-containing release medium, confirmed an active apoptotic process during this period. MOLT-3 cells released into drug-free medium reached G2/M peak at 9-10 h and continued to cycle with synchronized transition into G1 at 15-16 h. RanGAP1•SUMO1 was not hyperphosphorylated and no cleaved Lamin B was detected. Tubulin polymerization assay revealed that 1910 and ON 01911 had little or no effect, whereas nocodazole inhibited the process. CONCLUSION: These findings show that 1910 is neither a direct DNA damage response inducer nor a tubulin toxin, and suggest that 1910 is an inhibitor of RanGAP1•SUMO1 phosphatase. Its mechanism of action appears to rely on prolonged hyperphosphorylation of RanGAP1•SUMO1, leading to G2/M arrest and induction of apoptosis. 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 2500.

The Anti‐tumor Effects of Androstene Steroids Exhibit a Strict Structure–Activity Relationship Dependent upon the Orientation of the Hydroxyl Group on Carbon‐17

Androstene steroids are metabolites of dehydroepiandrosterone and exist as androstene-diols or -triols in alpha- and beta-epimeric forms based upon the placement of the hydroxyl groups relative to the plane of the Delta(5)cycloperhydrophenanthrene ring. 5-Androstene-3beta,17beta-diol (3beta,17beta-AED) functions to upregulate immunity and the addition of a third hydroxyl group at C-7 in the alpha- or beta-orientation (3beta,7alpha,17beta-AET and 3beta,7beta,17beta-AET, respectively) enhances the immunological activity of the molecule. In contrast, 5-androstene-3beta,17alpha-diol (3beta,17alpha-AED) possesses potent anti-tumor activity. We synthesized a new androstene by adding a third hydroxyl group at C-7 to make 5-androstene-3beta,7alpha,17alpha-triol (3beta,7alpha,17alpha-AET) and compared the anti-tumor activity of this steroid to the four existing androstenes. The results showed that this modification reduced the activity of 3beta,17alpha-AED. The ranking of the anti-tumor activities of these steroids and their IC50 on human glioblastoma and lymphoma cells was: 3beta,17alpha-AED ( approximately 10 microm) > 3beta,7alpha,17alpha-AET ( approximately 30 microm) " 3beta,7alpha,17beta-AET ( approximately 150 microm)> 3beta,7beta,17beta-AET (not achievable) >or= 3beta,17beta-AED (not achievable). 3beta,17alpha-AED and 3beta,7alpha,17alpha-AET induced autophagy in T98G glioblastoma cells and apoptosis in U937 lymphoma cells. These results indicate that the position of the hydroxyl group on C-17 dictates the anti-tumor activity of the androstenes and must be in the alpha-configuration, demonstrating a strict structure-activity relationship.