Histiocytic Lymphoma Research Articles

Quinoline-2-carboxaldehyde thiosemicarbazones and their Cu(II) and Ni(II) complexes as topoisomerase IIa inhibitors

A series of quinoline-2-carboxaldehyde thiosemicarbazones and their copper(II) and nickel(II) complexes were synthesized and characterized. In all complexes the ligands are in the E configuration with respect to the imino bond and behave as terdentate. The copper(II) complexes form square planar derivatives with one molecule of terdentate ligand and chloride ion. A further non-coordinated chloride ion compensates the overall charge. Nickel(II) ions form instead octahedral complexes with two ligands for each metal ion, independently from the stoichiometric metal:ligand ratio used in the synthesis. Ligands and complexes were tested for their antiproliferative properties on histiocytic lymphoma cell line U937. Copper(II) derivatives are systematically more active than the ligands and the nickel complexes. All copper derivatives result in inhibiting topoisomerase IIa in vitro. Computational methods were used to propose a model to explain the different extent of inhibition presented by these compounds. The positive charge of the dissociated form of the copper complexes may play a key role in their action.

Assessment of the antioxidant properties of tomato extracts: A synergistic approach using in vitro chemical tests and cell-based assays

The aim of this research was to assess the total antioxidant activity (TAA) of lipophilic (Lextr) and hydrophilic (Hextr) tomato extracts using in vitro chemical tests and cell-based assays, focusing on possible synergistic actions between tomato antioxidants. Both Hextr and Lextr were HPLC analysed for their carotenoids, phenolic compounds, and ascorbic acid contents. For the evaluation of TAA, extracts were assayed alone or in combination using in vitro chemical tests (TEAC, FRAP) and cell-based (CAA) assays using human hepatoma (HepG2) and human histiocytic lymphoma (U937) cells. The only carotenoid detected in Lextr was lycopene, while a mixture of phenolic compounds (chlorogenic acid, caffeic acid, and rutin) was identified in Hextr. Ascorbic acid was not found either in Hextr or in Lextr. Upon extract combination (1:1, v/v), the FRAP assay revealed additive action between Lextr and Hextr, whilst a slight synergistic action was observed in TAA as measured by the TEAC assay. Synergistic action was better revealed when TAA was analysed using either U937 or HepG2 cells. This could be explained by the presence of a multiphase media (cell membrane and extra- and intracellular media) that might facilitate the distribution and interaction of antioxidants with different polarities and different mechanisms of action.

Liposome-bound TRAIL induces superior DR5 clustering and enhanced DISC recruitment in histiocytic lymphoma U937 cells

Human Apo2-Ligand/TRAIL is a promising antitumor agent. Our group demonstrated that TRAIL was physiologically released to the extracellular medium inserted in lipid vesicles, known as exosomes. Recently we demonstrated that artificial lipid nanoparticles coated with bioactive TRAIL (LUV-TRAIL), which resemble the natural exosomes, greatly improved TRAIL activity compared with the soluble form of this death ligand and were able to induce apoptosis in hematological malignancies. In this study we have deepened the underlying mechanism of action of LUV-TRAIL in hematologic cells. Using histiocytic lymphoma U937 cells, we demonstrated that TRAIL signaling almost exclusively depends on DR5 despite these cells expressing high amounts of DR4, and proved that LUV-TRAIL's higher pro-apoptotic effect relies on its superior ability to induce DR5 clustering on cell surface, therefore enhancing DISC recruitment and triggering caspase activation more efficiently than the soluble form of TRAIL.

Structure-activity relationship studies and in vivo activity of guanidine-based sphingosine kinase inhibitors: discovery of SphK1- and SphK2-selective inhibitors.

Sphingosine 1-phosphate (S1P) is a pleiotropic signaling molecule that acts as a ligand for five G-protein coupled receptors (S1P1-5) whose downstream effects are implicated in a variety of important pathologies including sickle cell disease, cancer, inflammation, and fibrosis. The synthesis of S1P is catalyzed by sphingosine kinase (SphK) isoforms 1 and 2, and hence, inhibitors of this phosphorylation step are pivotal in understanding the physiological functions of SphKs. To date, SphK1 and 2 inhibitors with the potency, selectivity, and in vivo stability necessary to determine the potential of these kinases as therapeutic targets are lacking. Herein, we report the design, synthesis, and structure-activity relationship studies of guanidine-based SphK inhibitors bearing an oxadiazole ring in the scaffold. Our studies demonstrate that SLP120701, a SphK2-selective inhibitor (Ki = 1 μM), decreases S1P levels in histiocytic lymphoma (U937) cells. Surprisingly, homologation with a single methylene unit between the oxadiazole and heterocyclic ring afforded a SphK1-selective inhibitor in SLP7111228 (Ki = 48 nM), which also decreased S1P levels in cultured U937 cells. In vivo application of both compounds, however, resulted in contrasting effect in circulating levels of S1P. Administration of SLP7111228 depressed blood S1P levels while SLP120701 increased levels of S1P. Taken together, these compounds provide an in vivo chemical toolkit to interrogate the effect of increasing or decreasing S1P levels and whether such a maneuver can have implications in disease states.

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.

Quantifying Molecular Transport through Lipid Electropores Induced by Nanosecond Pulsed Electric Fields

High amplitude, nanosecond-duration, pulsed electric fields (nsPEFs) induce the formation of nanometer-diameter pores in cell membranes. These transient lipid electropores facilitate the transport of normally impermeant ions and small molecules across the membrane. Quantitative measurements of molecular transport through lipid electropores are essential for the validation of electroporation models and for optimizing the nsPEF dose for application-specific responses. We calibrated the fluorescence intensity of the membrane integrity marker dye YO-PRO-1 in dense cell lysates using a fast confocal microscope in order to quantify the time-dependent molecular influx of YO-PRO-1 into living cells after nsPEF exposure. Using this method we were able to measure the number of YO-PRO-1 molecules that enter U-937 human histiocytic lymphoma cells following electropermeabilization by a single 6 ns, 20 MV/m pulse.

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.

Abstract 1090: Type 2 endometrial cancer is associated with an M1 subtype, tumor associated macrophage polarization in the stromal compartment

Abstract Objectives: The more aggressive behavior of type 2 than type 1 endometrial cancers may be due to differences in their tumor microenvironments. In other solid cancers, tumor associated macrophages (TAMs) play a pivotal role in orchestrating the microenvironment. TAMs differentially polarize into M1 or M2 macrophages with distinct cytokine secretion patterns and actions. The aim of our work is to characterize the density, subtype and distribution of TAMs in endometrial hyperplasia and cancer. Methods: Five patients with complex atypical hyperplasia (hyperplasia), 5 patients with grade 1, non-invasive endometrioid adenocarcinoma (type 1) and 5 patients with uterine papillary serous carcinoma (type 2) were included. Formalin-fixed, paraffin-embedded sections from each specimen were stained with anti-CD68 and anti-CD163 monoclonal antibodies as markers for total TAMs and M2 TAMs, respectively. M1 macrophages were estimated by subtraction of CD163+ from CD68+ densities. Macrophages were counted at 40x magnification in 10 fields per slide by 4 observers. Repeated measure models, accounting for intra- and inter-observer correlation were contstructed and the compound symmetry covariance method was used to determine the relationship between macrophages and cancer. To establish an in vitro model, immortalized histiocytic lymphoma (U937) cells were differentiated to macrophages and treated with LPS to induce a M1 phenotype. Supernatants were applied to Cytokine C1000 microarray (RayBiotech) to screen for differentially expressed cytokines. Results: The correlation between the 4 observers was robust (r=0.71). Most TAMs were located in the stromal (mean=41.0/field) compared to epithelial (mean=11.0) or luminal (mean=11.6) compartments (p<0.0001). The mean total TAM density was highest in patients with type 2 cancers (mean=90.1/field), followed by type 1 cancers (mean=51.4) and hyperplasias (mean=41.1, p<0.0001). The calculated density of M1 macrophages was higher in type 2 cancers (mean=71.7) compared to type 1 cancers (mean =36.4, p<0.0001). This difference was only observed in the stromal compartment (p<0.0001). In the in vitro M1 macrophage model, ENA-78, a neutrophil chemo-attractant, and IL-6 were expressed at the highest concentrations. Conclusions: Type 2 cancers have nearly twice the TAM density of type 1 cancers. This difference is due to M1 macrophage predominance in the stroma of type 2 cancers. We postulate that cytokines produced by M1 TAMs within the tumor stroma contribute to the aggressive biology of these cancers. Future experiments will determine how well the in vitro model mimics M1 TAMs in the tumor microenvironment. Citation Format: Michael G. Kelly, Antonio M.C. Francisco, Adela Cimic, Anne Wofford, Nora Fitzgerald, Jie Yu, Robert N. Taylor. Type 2 endometrial cancer is associated with an M1 subtype, tumor associated macrophage polarization in the stromal compartment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1090. doi:10.1158/1538-7445.AM2014-1090

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.

A Transformative Approach to Cancer Metastasis: Primo Vascular System as a Novel Microenvironment for Cancer Stem Cells

Metastasis is the principal cause of death among cancer patients. Despite intensive research with the application of genetic and molecular biology, its prevention and management remain far from satisfactory. More innovative approaches are needed. The major routes for tumor dissemination are the two circulatory systems, lymphatic vessels and blood vessels, so the metastases occurring through them are referred to as lymphatic and hematogenous metastases, respectively. Surprisingly, a new circulatory system, named the primo vascular system (PVS), which is composed of very thin primo vessels (PVs) and primo nodes (PNs), has recently emerged as a third circulatory system. Thus, a natural conjecture would be that the PVS might be associated with carcinogenesis and metastasis, and, indeed, that conjecture has been supported by strong evidence of metastasis in rodents with xenograft tumors. In fact, a study showed that migration of tumor cells to secondary sites was more efficient in the PVS than in the lymphatic system. In addition, the PVS is also a conduit for a fluid, which, according to proteomic analyses contains remarkably high levels of carbohydrate metabolic derivatives that are usually associated with stem cells, cancer cells and differentiated myeloid cells. Thus, the PVS has the potential to transport growth and communication factors between primary and secondary tumor sites, thereby enhancing the oncogenicity of tumor cells at secondary sites. Furthermore, as tudy of a tumor-derived PVS in murine xenografts of human histiocytic lymphoma has suggested that the PVS may provide a safe haven for a select population of cancer stem cells. These pieces of evidences for the involvement of the PVS in cancer metastas is call for more intensive investigations of its possible role as an incubating microenvironment for tumor formation, propagation, sustenance, and relapse.

Characterization and inhibition of AF10‐mediated interaction

The non-random chromosomal translocations t(10;11)(p13;q23) and t(10;11)(p13;q14-21) result in leukemogenic fusion proteins comprising the coiled coil domain of the transcription factor AF10 and the proteins MLL or CALM, respectively, and subsequently cause certain types of acute leukemia. The AF10 coiled-coil domain, which is crucial for the leukemogenic effect, has been shown to interact with GAS41, a protein previously identified as the product of an amplified gene in glioblastoma. Using sequential synthetic peptides, we mapped the potential AF10/GAS41 interaction site, which was subsequently be used as scaffold for a library targeting the AF10 coiled-coil domain. Using phage display, we selected a peptide that binds the AF10 coiled-coil domain with higher affinity than the respective coiled-coil region of wild-type GAS41, as demonstrated by phage ELISA, CD, and PCAs. Furthermore, we were able to successfully deploy the inhibitory peptide in a mammalian cell line to lower the expression of Hoxa genes that have been described to be overexpressed in these leukemias. This work dissects molecular determinants mediating AF10-directed interactions in leukemic fusions comprising the N-terminal parts of the proteins MLL or CALM and the C-terminal coiled-coil domain of AF10. Furthermore, it outlines the first steps in recognizing and blocking the leukemia-associated AF10 interaction in histiocytic lymphoma cells and therefore, may have significant implications in future diagnostics and therapeutics.

Effects of the Absorption Behaviour of ZnO Nanoparticles on Cytotoxicity Measurements

ZnO absorbs certain wavelengths of light and this behavior is more pronounced for nanoparticles of ZnO. As many toxicity measurements rely on measuring light transmission in cell lines, it is essential to determine how far this light absorption influences experimental toxicity measurements. The main objective was to study the ZnO absorption and how this influenced the cytotoxicity measurements. The cytotoxicity of differently sized ZnO nanoparticles in normal and cancer cell lines derived from lung tissue (Hs888Lu), neuron‐phenotypic cells (SH‐SY5Y), neuroblastoma (SH‐SY5Y), human histiocytic lymphoma (U937), and lung cancer (A549) was investigated. Our results demonstrate that the presence of ZnO affected the cytotoxicity measurements due to the absorption characteristic of ZnO nanoparticles. The data revealed that the ZnO nanoparticles with an average particle size of around 85.7 nm and 190 nm showed cytotoxicity towards U937, SH‐SY5Y, differentiated SH‐SY5Y, and Hs888Lu cell lines. No effect on the A549 cells was observed. It was also found that the cytotoxicity of ZnO was particle size, concentration, and time dependent. These studies are the first to quantify the influence of ZnO nanoparticles on cytotoxicity assays. Corrections for absorption effects were carried out which gave an accurate estimation of the concentrations that produce the cytotoxic effects.

Targeted Therapy Of Acute Myeloid Leukemia By A CD117-Specific Aptamer-Drug-Conjugate

Abstract The current treatment paradigm for acute myeloid leukemia (AML) is remission induction chemotherapy, followed by either consolidation chemotherapy or allogeneic stem cell transplantation. As most patients diagnosed with AML are in their sixth or seventh decade of life, many are not candidates for standard remission induction chemotherapy because of the risk of toxicities, such as profound myelosuppression, life-threatening infections, and cardiotoxicity. The development of new effective and safe treatments for AML is therefore needed. The “ideal” therapy should specifically target AML tumor cells with no side-effect on normal cells. Since CD117 (c-Kit) is a transmembrane receptor on tumor cells surface and expresses in 70% cases of AML, it is a potential molecule for developing new targeted therapy. Aptamers are single-stranded oligonucleotides (DNA or RNA), which have the ability to specifically bind to their targets with high affinity. As a “chemical antibody”, aptamers can be chemically synthesized, easily conjugated with therapeutic drugs, and more importantly, less or not immunogenic. In this study, we developed single strand DNA (ssDNA) aptamer specific for CD117 by using a unique hybrid SELEX approach with both cell-selection and protein-enrichment. When sequencing the enriched ssDNA library with million reads, one dominant sequence had over 80% copies of total sequence reads. Cell binding analysis of the aptamer by flow cytometry and fluorescent microscopy (figure 1) demonstrated that, the aptamer molecule specifically bound to CD117-expressing AML cells, but did not react to CD117-negative control cells including Histiocytic lymphoma Cell line: U937, Burkitt's lymphoma cell line: CA46, Breast adenocarcinoma cell line: 468 and human prostate carcinoma cell line: LNCap. In addition, the presence of aptamers inhibited cell binding anti-CD117 antibody, indicating that aptamer targeted CD117 receptor on leukemic cells. Notably, this aptamer specifically targeted CD117-positve AML cells of clinical specimens with an identical staining pattern to that observed with anti-CD117 antibody (figure 2), indicating potential for in clinical use. For targeted therapy, an aptamer -drug-conjugate was formulated by chemical conjugation of ssDNA CD117 aptamer (Apt) to chemotherapeutic drug, Methotrexate (MTX). For treatment study, cell mixture of leukemic cells HEL (CD117+) and U937 (CD117-), which showed the same sensitivity to free MTX, were used (figure 3A). Exposure of cells to the Apt-MTX revealed that the formed aptamer-drug-conjugate specifically killed CD117 positive cells, but had no effect on the growth of the off-target cells in the same cultures. For therapeutic effect, Apt-MTX killed 60% of positive cells at as low as 10nM final concentration (figure 3B). In contrast, under the same condition, free MTX had no effect on cell growth (figure 3A). Our study demonstrated that the aptamer-drug-conjugate could be a new targeted therapeutics in addition to current antibody-drug-conjugate. Disclosures: No relevant conflicts of interest to declare.

Changes in the frequencies of human hematopoietic stem and progenitor cells with age and site

This study enumerated CD45(hi)/CD34(+) and CD45(hi)/CD133(+) human hematopoietic stem cells (HSCs) and progenitor granulocyte-macrophage colony forming cells (GM-CFCs) in blood and trochanteric and femoral bone marrow in 233 individuals. Stem cell frequencies were determined with multiparameter flow cytometry and using an internal control to determine the intrinsic variance of the assays. Progenitor cell frequency was determined using a standard colony assay technique. The frequency of outliers from undetermined methodological causes was highest for blood, but less than 5% for all values. The frequency of CD45(hi)/CD133(+) cells correlated highly with the frequency of CD45(hi)/CD34(+) cells in trochanteric and femoral bone marrow. The frequency of these HSC populations in trochanteric and femoral bone marrow rose significantly with age. In contrast, there was no significant trend of either of these cell populations with age in the blood. Trochanteric marrow progenitor GM-CFCs showed no significant trends with age, but femoral marrow GM-CFCs trended downward with age, potentially because of the reported conversion of red marrow at this site to fat with age. Hematopoietic stem and progenitor cells exhibited changes in frequencies with age that differed between blood and bone marrow. We previously reported that side population (SP) multipotential HSC, which includes the precursors of CD45(hi)/CD133(+) and CD45(hi)/CD34(+), decline with age. Potentially the increases in stem cell frequencies in the intermediate compartment between SP and GM progenitor cells observed in this study represent a compensatory increase for the loss of more potent members of the HSC hierarchy.

Resistance To Vorinostat In Hematological Malignancies May Involve Cytoprotective UPR and Correlates With Increased Sensitivity To Bortezomib-Induced Cell Death

Histone deacetylase inhibitors (HDACi) have shown promising activity against hematological malignancies in clinical trials and have led to the approval of vorinostat for the treatment of cutaneous T-cell lymphoma. However, as with many cancer therapies, de novoresistance is common and acquired resistance inevitably follows sensitivity. This issue is particularly difficult to resolve in HDACi therapy, as the mechanism of action is still unclear and may involve several components.Our objective was to understand the molecular mechanisms underlying resistance to HDACi in order to design better combination strategies and to identify predictive biomarkers for response to HDACi therapy.To gain insight into HDACi resistance, we developed vorinostat-resistant clones using a dose escalation protocol in the monocytic-like, histiocytic lymphoma cell line U937 and the diffuse large B-cell lymphoma SUDHL6. Indeed, resistant cells grow in 4 µM vorinostat without induction of cell death. Using a variety of targeted drugs, we evaluated the lethal dose (LD)50in the resistant cells versus their parental counterpart in order to screen for potential pathways involved in resistance to vorinostat.We found that the vorinostat-resistant cells are cross-resistant to other HDACi but not all. Interestingly, the resistant cells exhibit increased sensitivity toward bortezomib, an inhibitor of the proteasome and to chloroquine, an inhibitor of autophagy. Both these drugs target protein processing, suggesting its importance in driving resistance to HDACi. We found that, in addition to elevated autophagy, vorinostat-resistant cells exhibit marks of ER stress, such as dilated ER when visualized by electron microscopy. Moreover, resistant cells have an increased protein synthesis rate and an accumulation of ubiquitinated proteins compared to their parental counterparts. Consistent with this, we observe activation of the UPR in the resistant cells.We hypothesized that activation of UPR could be a mechanism of vorinostat resistance, because the UPR induces the upregulation of pro-survival genes and may induce autophagy. Understanding vorinostat resistance holds clinical relevance in terms of improving HDACi therapy and being able to identify subsets of patients who would benefit most from combination therapy, such as vorinostat with bortezomib. Disclosures:No relevant conflicts of interest to declare.

Abstract C41: Chc1L is a tumor suppressor involved in development of histiocyte-rich neoplasms

Abstract Human chromosome 13q14 is one of the hotspots of chromosomal deletion in prostate cancer, multiple myeloma, and chronic lymphocytic leukemia. Chromosome Condensation 1-like (CHC1L) is an uncharacterized gene in this region. CHC1L is found within the smallest common region of loss of heterozygosity in prostate cancer, and its decreased expression is linked to pathogenesis and progression of both prostate cancer and multiple myeloma. In the present study, we describe the generation and characterization of Chc1L gene knockout mice. Knockout mice develop normally and display an exaggerated proliferative response to LPS stimulation of cultured bone marrow and spleen cells at ten weeks of age. At approximately two years of age, both homozygous and heterozygous mice have markedly increased incidence of tumorigenesis, 80% and 56% respectively, compared to wild-type mice, 26%. The tumors are distributed most commonly to the spleen, mesenteric lymph nodes, liver and small intestine. The morphologic and immunophenotypic features of the tumor cells are consistent with those of tissue histiocytes, and most Chc1L knockout and heterozygote mice succumb to either Histiocytic Sarcoma or Histiocyte-Associated Lymphoma. These data provide the first direct evidence that Chc1L is a tumor suppressor gene involved in suppression of histiocyte-rich neoplasms. Citation Format: David Spillane, Ding Yan Wang, Changxin Shi, Youdong Wang, Anthony Gramolini, Keith Stewart, Susan Newbigging, Mingyao Liu, Xiao-Yan Wen. Chc1L is a tumor suppressor involved in development of histiocyte-rich neoplasms. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C41.

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.

Red wines attenuate TNFα production in human histiocytic lymphoma cell line: An NMR spectroscopy and chemometrics based study

Nuclear magnetic resonance (NMR) spectroscopy and multivariate data analyses methods are applied to the metabolic profiling of different red wines from Portugal. The water, methanol–water (1:1), and methanol fractions from solid phase extraction (SPE) with C18 resin were subjected to in vitro TNFα activity assay. Principal component analysis allowed the clear separation among the different SPE fractions according to the activity. Various supervised data reduction algorithms were tested and compared to highlight the TNFα inhibition by SPE fractions of wines. Partial least squares-discriminant analysis and orthogonal bidirectional PLS-DA were found most effective in discriminating the samples with different activities. By calculating variable importance in the projection, the active ingredients in the high activity samples have been identified as caftaric acid, quercetin, and catechin. Among the different vintages, samples from 2010 vintage were found with maximum anti-TNFα activity. The effectiveness of NMR spectroscopy in combination with chemometrics to identify the possible bioactivity in the several crude extracts is highlighted.

P03.15 - Development of Therapeutic Cytotoxic Immunotoxin to Histocytic Lymphoma Cells

A large number of cancers at advanced stage express human chorionic gonadotropin (hCG) or its subunits. It is observed that hCG expressing cancers are not only drug refractory but also have poor prognosis than those of non-hCG expressing cancers. PiPP, a high affinity anti-hCG&bgr; monoclonal antibody, was used to develop a immunotoxin for the management of hCG/hCG&bgr; based cancer cells. The efficacy of the recombinant immunotoxin was tested on hCG secreting histiocytic lymphoma U937 cells which is a rare type of non-Hodgkin's lymphoma characterized by the presence of large tumor cells resembling histiocytes.