bluuebunny/crossref_metadata_2025_split huggingface dataset:
Depletion of the Intracellular Akt Kinase, and Its Downstream Signaling, Mediates Synergy between the Proteasome Inhibitor MG-132 and the Heat Shock Protein 90 Inhibitor 17-AAG in the Multiple Myeloma Cell Line U266.
Authors: Maisel Christopher , Bahlis Nizar , Miao Yanling , Liu Lili , Gerson Stanton 
Published: October 2019

Abstract
While proteasome inhibitors are effective therapy for multiple myeloma (MM), their efficacy could be improved by synergistic targeting of apoptosis. The intracellular serine/threonine kinase Akt has been demonstrated to have a central role in MM cell growth, survival, and drug resistance. Akt is activated by extracellular cytokines such as IGF-1 and IL-6, and contributes to MM resistance by ameliorating the apoptotic effects of proteasome inhibition. Akt requires chaperone proteins for proper stability and function, including the 90 kD molecule Heat Shock Protein 90 (HSP-90). HSP-90 function is abrogated by geldanamycin and its derivative, 17-allylamino-17-demethoxygeldanamycin (17-AAG).
In the U266 MM cell line, the IC50 of the proteasome inhibitor MG-132 and 17-AAG was 100 nM and 800 nM, respectively. Following exposure of U266 MM cells to either drug alone, or the combination at a fixed-ratio of their IC50s (1:8), apoptosis was determined by Annexin V staining and FACScan analysis. Synergy analysis was performed using Calcusyn (Biosoft, Cambridge, UK). We found that the combination index (CI) was synergistic (CI<1) throughout the dose range, with a CI = 0.449 ± 0.298 at the combination IC50 (highly significant). For example, the apoptotic effect of 50 nM MG-132 and 400 nM 17-AAG was 6 ±2 % and 23 ±3 %, respectively, whereas the 50:400 nM combination produced apoptosis in 68 ± 2 % of the cells.
To analyze effects on Akt and its substrates, we incubated U266 MM cells with MG-132 (50 nM), 17-AAG (400 nM), or the combination. We harvested lysates after zero, two, six, and 24 hours incubation, and Western blot analysis was performed. Co-incubation with MG-132 and 17-AAG, but not either alone, depleted Akt by 24 hours post-therapy. Co-treatment also produced significantly greater upregulation of HSP-90 and HSP-70 than 17-AAG alone, thus demonstrating greater functional inhibition of Akt. The combination also demonstrated the greatest abrogation of Akt-mediated effects on mitochondrial apoptosis: Co-treatment produced the greatest expression of BAD, decreased BCL-XL expression, reduced phosphorylation of GSK-3, and produced the greatest activation of caspase 3.
Monotherapy with 17-AAG upregulated HSP-90 and HSP-70, reduced BCL-XL expression, and activated caspase 9. MG-132 monotherapy produced none of these effects. These findings demonstrate that synergy between proteasome inhibitors and 17-AAG is mediated by Akt depletion and abrogation of Akt signaling, predominantly by MG-132 augmentation of 17-AAG-mediated decay of Akt. Down-regulation of Akt-mediated resistance allows dual-apoptotic signaling and synergistic effect of combination therapy. These findings demonstrate a mechanistic rationale for utilizing heat shock protein inhibitors in combination with proteasome inhibitors as therapy for MM.
Homeodomain Expression in AML and T-ALL Cell Lines.
Authors: Drabkin Harry , Gadgil Sharvari , Zeng Chan , Baron Anna , Bernard Olivier 
Published: October 2019

Abstract
HOX genes are frequent targets of chromosomal translocations and retroviral integrations in human and murine acute leukemia, often involving genes at the 5′-end of the HOX clusters. We previously reported that HOX expression patterns in AML were related to prognostic cytogenetic subsets. We also identified a distinct subset of patients with intermediate cytogenetics based on high levels of HOX and FLT3 expression, frequent FLT3 mutations and a low incidence of C/EBPa mutations. Certain cases of T-ALL also have rearrangements of homeodomain genes and some T-ALLs express limited myeloid markers. To further explore the spectrum of homeodomain gene expression, we developed qRT-PCR assays for nearly all clustered HOXA-D genes, selected homeodomain genes on chromosomes often altered in AML, and selected polycomb (Pc) genes, FLT3 and MLL. Altogether, 52 genes were analyzed in 32 AML and T-ALL cell lines. FLT3 expression was confined to a subset of AMLs. HOX11, HOX11L2 and NKX2.5 were expressed only in cases involving rearrangements of these genes. The Pc and MLL genes were uniformly expressed. Among HOX clusters, the frequency of gene expression was HOXA>B>C>D. Genes more highly expressed in the HOXC and D clusters were those at the 5′-ends (e.g., D13, C10). Marked or selective overexpression of individual genes suggests their possible involvement in the disease process, immortalization or differentiation. Examples include EN1 (SUPT1), D13 (MEGA1, B9 (PEER). A hierarchical cluster analysis based on homeodomain genes successfully identified subsets of related cell lines. Thus, the analysis of quantitative HOX expression may provide an important new tool to better understand the biology of acute leukemia.
Induction of Cell Cycle Arrest and Apoptosis by Dephosphorylation of ERK, Akt, and Bcl-2 and Phosphorylation of JNK by LY293111 in Human Anaplastic Large Cell Lymphoma (ALCL).
Authors: Zhang Weiguo , Konopleva Marina , McQueen Teresa , Schober Wendy , Andreeff Michael 
Published: October 2019

Abstract
The prognosis of patients with anaplastic large cell lymphoma (ALCL) treated with standard chemotherapy remains poor. Leukotriene B4 (LTB4) receptor has been reported to exhibit a growth-promoting activity in lymphoma, since it augments DNA synthesis and increases cell proliferation of lymphocytes. LY293111 (2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]-propoxy]-phenoxy] benzoic acid sodium salt) is a Leukotriene B4 receptor antagonist, which was found to be safe and tolerable in Phase I clinical trials. LY293111 inhibits pancreatic cancer cell growth, induces apoptosis, and reduces growth of colon cancer in vivo. In the present study, we investigated the potential therapeutic effects and mechanisms of action of LY293111 in an ALCL cell line. We utilized the Sup/M2 cell line derived from human anaplastic large cell lymphoma as a model. Dose-response studies demonstrated that the IC50 of LY293111 was 4μM after 5 days of treatment. LY293111 caused retardation of Sup/M2 cell entry into S phase in a dose-dependent fashion as measured by BrdU/propidium iodide flow cytometry. LY293111 at 2.5μM induced complete G1-S cell cycle arrest in cells synchronized by serum starvation. LY293111 upregulated p21 and p27 protein expression and reduced cyclin E mRNA and protein. Pre-treatment with LY293111 for 4 hours resulted in profound inhibition of serum-induced phosphorylation of ERK1/2, Akt, and Bcl-2. Concomitantly, phosphorylation of stress-activated kinase JNK dramatically increased following LY293111 treatment. LY293111 induced pronounced apoptosis of Sup/M2 cells and caspase inhibition by Benzyloxycarbonyl -Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK) abrogated this effect. LY293111 induced cleavage of the caspase-9, -3, PARP and XIAP, but had no effect on caspase-8, suggesting activation of the intrinsic apoptotic pathway. Accordingly, early loss of mitochondrial membrane potential was observed in a dose-dependent fashion. At 72 hrs, LY293111 decreased Bcl-2 expression levels. These findings provide the first evidence that LY293111 inhibits ALCL proliferation by arresting cells in G1 phase of the cell cycle, as a result of cyclin E downregulation and induction of cell-cycle inhibitory proteins p21 and p27. LY293111 induces apoptosis in Sup/M2 lymphoma cells, with activation of the mitochondrial apoptosis pathway, including cleavage of caspase 9, caspase 3, PARP, XIAP, and Bcl-2. Furthermore, LY293111 treatment markedly shifts signaling toward the JNK stress-related pathway and away from cytoprotective MEK/ERK and AKT signaling. These results suggest that LY293111 may have a utility in the management of aggressive non-Hodgkin’s lymphomas.
Motexafin Gadolinium Induces Apoptosis in Lymphoid Cell Lines and Demonstrates Enhanced Biological Activity with Akt Kinase Inhibitors.
Authors: Naumovski Louie , Ramos Jason , Chen Jun , Sirisawad Mint , Lucas David , Byrd John C. , Miller Richard A. 
Published: October 2019

Abstract
Motexafin gadolinium (MGd, Xcytrin®) is a tumor-selective redox mediator that catalytically oxidizes intracellular reducing metabolites and produces reactive oxygen species (ROS). In this report, we demonstrate that MGd induces apoptosis or growth inhibition in several hematopoietic tumor-derived cell lines and tumor cells from patients with chronic lymphocytic leukemia. Lymphoma (HF-1, Ramos, DHL-4, DB, Hut78 and Raji) and leukemia (Jurkat, HL-60) cell lines were cultured in RPMI 1640 media with 10% heat inactivated fetal bovine serum with or without 50 uM MGd. MGd inhibited the growth of 6 of the cell lines (HF-1, Ramos, HL-60, DHL-4, Jurkat and DB) and was cytotoxic to HF-1. ROS were implicated in MGd-induced cell death since their presence was detected by dichlorofluorescein diacetate staining and peroxiredoxin oxidation in MGd treated HF-1 cells that undergo apoptosis, but not in Jurkat cells that do not undergo MGd-induced apoptosis. MGd triggered an apoptotic pathway in HF-1 cells as demonstrated by loss of mitochondrial membrane potential, release of cytochrome c from mitochondria, activation of caspases, cleavage of PARP and annexin-V binding. MGd also induced cell death and activated caspases in vitro in primary chronic lymphocytic leukemia cells. Protein lysates from cultured cell lines (HF-1, Ramos) were subjected to immunoblot analysis to determine caspase cleavage patterns, and the phosphorylation status of Akt, a kinase that regulates survival pathways. In MGd treated HF-1, phospho-Akt protein levels initially increased 2–3 fold between 30 min and 1 hr (n=4) and then decreased to 40–50% of control levels by 24–48 hrs (n=4). The drop in phospho-Akt protein coincided with an increase in apoptotic cell death as indicated by morphology, staining with Annexin-V and activation of caspases. Addition of a specific inhibitor of Akt phosphorylation (SH-5) reduced Akt phosphorylation in MGd treated HF-1 cells by 90% and enhanced the cytotoxic effect of MGd. In Ramos cells, which do not undergo apoptosis when treated with MGd, co-treatment with MGd and SH-5 decreased phospho Akt levels by only 15% and did not result in cytotoxicity. These data point to a potential role for Akt in MGd-induced apoptosis and suggest that MGd activity may be enhanced by inhibition of Akt. These data show that the pro-apoptotic effects of MGd involve caspase activation and provide a rationale to evaluate MGd in the treatment of lymphoma and leukemia patients.
Differential Targeting of Pro-Survival Bcl-2 Proteins by Their Pro-Apoptotic BH3-Only Ligands: Implications for Targeted Therapies.
Authors: Wei Andrew , Chen Lin , Roberts Andrew , Huang David 
Published: October 2019

Abstract
Chemoresistance is a significant barrier in the management of hematological malignancies. Despite some striking successes, many malignancies remain incurable. The efficacy of cytotoxic drugs is often blunted by loss of p53 or overexpression of pro-survival proteins like Bcl-2 or its functional homologs Bcl-xL and Mcl-1. Bcl-2-like proteins promote cell survival until antagonized by BH3-only proteins such as Bim. A potential way of overcoming tumor resistance is to engineer BH3-only mimetics to trigger cell death. Although such drugs may have a useful role in overcoming chemoresistance, a serious concern is the potential deleterious effects on normal cells. To assess the feasibility of such an approach, we studied the binding of mammalian pro-apoptotic BH3-only proteins to their pro-survival Bcl-2-like targets. The targeting of pro-survival Bcl-2-like proteins has been considered to be promiscuous, but unexpected selectivity emerged from our analysis. The interactions varied over 10,000-fold in affinity and, accordingly, certain protein pairs associated selectively inside cells. Some death ligands that bound promiscuously were potent killers when overexpressed, whereas Noxa which only targets some pro-survival proteins, but not Bcl-2, had weak activity. Importantly, although expression of Noxa by itself caused limited cytotoxicity, it markedly enhanced sensitivity to cytotoxic drugs such as etoposide, even in those cells normally highly chemoresistant because of Bcl-2 overexpression. Thus, selective targeting of key pro-survival proteins in a malignancy may improve the efficacy of currently used cytotoxic agents with reduced undesirable side effects. In particular, our results suggest that it should be feasible to discover BH3 mimetic drugs with better therapeutic profiles and with the potential to overcome chemoresistant cancers overexpressing pro-survival proteins.
Characterization of the Resistance Mechanisms of HL60 Leukemia Cell Derivatives to Apo2L/TRAIL.
Authors: Cheng Jinrong , Hylander Bonnie L. , Baer Maria R. , Repasky Elizabeth A. 
Published: October 2019

Abstract
Tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) is a recently identified member of the TNF superfamily. Recombinant Apo2L/TRAIL is a promising immunotherapeutic agent for treating malignant diseases since this molecule preferentially induces apoptosis in a variety of tumor cells with apparently little toxicity to normal cells. However, it has also been shown that some tumor cells are resistant to this molecule. We hypothesized that resistance to Apo2L/TRAIL occurs through defects in the Apo2L/TRAIL-mediated apoptotic signaling pathway. To address this hypothesis, we developed several Apo2L/TRAIL-resistant HL60 derivatives (HL60/TR) by exposure of Apo2L/TRAIL-sensitive HL60 human leukemia cells to escalating levels of Apo2L/TRAIL, followed by subcloning. Two of these resistant clones (a moderately resistant clone-R1 and a highly resistant clone-R3) were selected for further study. Molecules in the Apo2L/TRAIL-mediated apoptotic pathway of R1 and R3 cells were analyzed by Western blot analysis, flow cytometry and gene sequencing and compared to those in parental HL60 cells. In the R1 cells, the activation of caspase-8 and -10 by Apo2L/TRAIL was significantly inhibited. However, R1 cells were still sensitive to Fas agonistic monoclonal antibody treatment, indicating that the FAS-mediated apoptosis-inducing pathway was intact. In the R3 cells, caspase-8 expression was completely lost and activation of caspase-10 in response to Apo2L/TRAIL was totally inhibited; R3 cells were therefore also resistant to FAS antibody treatment. Although the total protein level of death receptors DR4 and DR5 was equal in HL60 cells and in the Apo2L/TRAIL-resistant derivatives, the cell surface levels of DR4 were significantly decreased in both R1 and R3 cells, while the surface expression level of DR5 in these two clones was comparable to that on HL60 cells. No mutation in either the DR4 or DR5 genes was found in these cells. These results suggest that defective targeting of DR4 molecules to the cell surface occurs in these Apo2L/TRAIL resistant cells. Blocking cell surface DR4 significantly attenuated the sensitivity of parental HL60 cells to Apo2L/TRAIL, indicating that cell surface expression of DR4 plays a crucial role in regulating susceptibility of tumor cells to Apo2L/TRAIL. Taken together, our results demonstrate that malignant cells can develop resistance to Apo2L/TRAIL by several different mechanisms and multiple resistance mechanisms may develop in a single tumor cell (such as R3 cells). Understanding the basis of Apo2L/TRAIL resistance will help to predict sensitivity and to develop strategies to circumvent resistance.
JS-K, a Novel Nitric Oxide (NO) Generator, Shows Potent Anti-Angiogenic Activity.
Authors: Shami Paul J. , Kaur Gurmeet , Thillainathan Jagadambal , Jia Lee , Saavedra Joseph E. , Keefer Larry K. 
Published: October 2019

Abstract
NO induces differentiation and apoptosis in Acute Myelogenous Leukemia (AML) cells. Glutathione S-Transferases (GST) play an important role in multidrug resistance and are upregulated in 90% of AML cells. We have designed a novel prodrug class that releases NO on metabolism by GST. O2-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K, a member of this class) has potent antileukemic activity in vitro and in vivo (Molecular Cancer Therapeutics 2:409-417,2003). The purpose of this study was to determine the effect of JS-K on angiogenesis. The anti-angiogenic properties of JS-K were tested in 3 different in vitro assays: proliferation, cord formation (reflecting new vessel formation) and migration using Human Umbilical Vein Endothelial Cells (HUVEC). JS-K inhibited the proliferation of HUVEC’s with a 50% inhibitory concentration (IC50) of 0.432, 0.466, and 0.505 μM at 24, 48, and 72 hours, respectively. At concentrations of 1 μM or above, HUVEC proliferation was totally inhibited. In the cord formation assay, treatment with JS-K lad to a decrease in both the number of cord junctions and cord length with an IC50 of 0.637 and 0.696 μM, respectively. At a concentration of 1 μM, JS-K inhibited cord formation completely. JS-K inhibited cell migration at 5 hours using 10 ng/mL VEGF as a chemoattractant. At that time point, migration inhibition occurred at JS-K concentrations that did not affect cell growth with an IC50 of 0.493 μM. We conclude that JS-K is a potent inhibitor of 3 important elements of angiogenesis, namely endothelial cell proliferation, cord formation, and endothelial cell migration. These experiments identify a new mechanism by which JS-K and similar compounds may inhibit leukemia and solid tumor cell growth in vivo. Determining whether the anti-angiogenic effects of JS-K are NO-dependent will require further studies. (NO1-CO-12400).
The ERG Gene in Normal and Malignant Megakaryopoiesis.
Authors: Rainis Liat , Rosenthal Esther , Strehl Sabine , Haas Oskar A. , Izraeli Shai 
Published: October 2019

Abstract
About 10% of patients with Down syndrome (DS) are born with a transient megakaryoblastic leukemia. We (Rainis et al. Blood2003; 102:981) and others have demonstrated acquired intrauterine inactivating mutations in the X-linked gene GATA1 in these leukemias. The gene(s) on chromosome 21 that promote the proliferation of these abnormal megakaryoblasts are presently unknown. We hypothesize such a role to the ets transcription factor ERG that is located at the critical DS region on chromosome 21. This hypothesis is based on the close homology between ERG and FLI-1, a transcription factor that induces megakaryopoiesis through cooperation with GATA1. ERG occasionally replaces FLI-1 in the translocation with EWS in Ewing sarcoma and is also involved in the leukemogenic translocation FUS-ERG. However no role for ERG in hematopoiesis has been demonstrated so far. Here we show that (a) ERG is expressed in platelets and in megakaryoblastic cell lines, including those derived from Down Syndrome. (b) ERG is expressed in primary leukemic cells from DS patients. (c) Its expression is induced upon megakaryocytic differentiation (d) ERG collaborates with GATA1 in activation of megakaryocytic promoters in reporter assays; and (e) Forced ectopic expression of ERG in K562 cells induces megakaryocytic differentiation. Thus we provide the first evidence that the ERG gene participate in megakaryopoiesis. Promotion of megakaryopoiesis caused by its overexpression in DS coupled with the differentiation arrest induced by the acquired mutation in GATA1 may explain the high incidence of a congenital megakaryoblastic proliferation disorder in Down Syndrome.
The Kinase Inhibitor Imatinib Mesylate Potently Inhibits TNF-α Production by Myeloid Cells and Prevents T-Cell-Mediated Hepatic Injury in Mice.
Authors: Wolf Anna M. , Wolf Dominik G.F. , Rumpold Holger F. , Enrich Barabara , Gastl Guenther A. , Eberhard Gunsilius , Tilg Herbert 
Published: October 2019

Abstract
Imanitib mesylate (Gleevec®) exhibits potent anti-leukemic effects in vitro and in vivo. Despite of it`s well known anti-leukemic effects, the potential of Imatinib in the treatment of inflammation remains elusive so far. Our current report provides strong evidence that Imatinib indeed exerts anti-inflammatory effects. It potently inhibits LPS- and ConA-induced TNF-α and IFN-γ production of human myeloid cells in vitro (PBMNC, CD14-selected monocytes and monocyte-derived macrophages). Of note, the production of the anti-inflammatory cytokine IL-10 was only slightly affected by Imatinib. In line with this observation, the activation of NF-κB, which has recently been shown to be critically involved in TNF-α but not IL-10 expression, was significantly impaired by Imatinib. In addition, Imatinib reduced either LPS or ConA-induced intracellular phophotyrosine content. For in vivo testing of the anti-inflammatory role of Imatinib in a murine model of inflammation, we injected BALB/c mice with either 75 mg/kg body weight Imatinib or solvent before administration of ConA. The latter has recently been shown to induce T-cell, macrophage and TNF-α-dependent inflammatory damage of the liver. Imatinib pre-treatment prevented the development of acute hepatic injury, which was paralleled by reduced intrahepatic TNF-α mRNA and circulating TNF-α protein levels. Improvement of liver pathology by Imatinib was further assessed by light microscopy and TUNEL staining. Of note, Imatinib protected mice also from GalN/LPS- but not from GalN/TNF-induced liver pathology, which corroborates our in vitro findings that Imatinib potently inhibits TNF-α production of myeloid cells. These findings point out to a potent anti-inflammatory role of the tyrosine kinase inhibitor Imatinib, which might be of therapeutic benefit for the treatment of TNF-α-mediated immune-pathologies, such as inflammatory bowel disease (IBD), aGvHD or immune-mediated liver injury.