Edward Felix

Chronic stress promotes tumor growth and angiogenesis in a mouse model of ovarian carcinoma

Stress can alter immunological, neurochemical and endocrinological functions, but its role in cancer progression is not well understood. Here, we show that chronic behavioral stress results in higher levels of tissue catecholamines, greater tumor burden and more invasive growth of ovarian carcinoma cells in an orthotopic mouse model. These effects are mediated primarily through activation of the tumor cell cyclic AMP (cAMP)–protein kinase A (PKA) signaling pathway by the β2 adrenergic receptor (encoded by ADRB2). Tumors in stressed animals showed markedly increased vascularization and enhanced expression of VEGF, MMP2 and MMP9, and we found that angiogenic processes mediated the effects of stress on tumor growth in vivo. These data identify β-adrenergic activation of the cAMP–PKA signaling pathway as a major mechanism by which behavioral stress can enhance tumor angiogenesis in vivo and thereby promote malignant cell growth. These data also suggest that blocking ADRB-mediated angiogenesis could have therapeutic implications for the management of ovarian cancer.

Formation and antiproliferative effect of prostaglandin E3 from eicosapentaenoic acid in human lung cancer cells

We investigated the formation and pharmacology of prostaglandin E3 (PGE3) derived from fish oil eicosapentaenoic acid (EPA) in human lung cancer A549 cells. Exposure of A549 cells to EPA resulted in the rapid formation and export of PGE3. The extracellular ratio of PGE3 to PGE2 increased from 0.08 in control cells to 0.8 in cells exposed to EPA within 48 h. Incubation of EPA with cloned ovine or human recombinant cyclooxygenase 2 (COX-2) resulted in 13- and 18-fold greater formation of PGE3, respectively, than that produced by COX-1. Exposure of A549 cells to 1 μM PGE3 inhibited cell proliferation by 37.1% (P < 0.05). Exposure of normal human bronchial epithelial (NHBE) cells to PGE3, however, had no effect. When A549 cells were exposed to EPA (25 μM) or a combination of EPA and celecoxib (a selective COX-2 inhibitor), the inhibitory effect of EPA on the growth of A549 cells was reversed by the presence of celecoxib (at both 5 and 10 μM). This effect appears to be associated with a 50% reduction of PGE3 formation in cells treated with a combination of EPA and celecoxib compared with cells exposed to EPA alone. These data indicate that exposure of lung cancer cells to EPA results in a decrease in the COX-2-mediated formation of PGE2, an increase in the level of PGE3, and PGE3-mediated inhibition of tumor cell proliferation.

Acquired Resistance to Erlotinib in A-431 Epidermoid Cancer Cells Requires Down-regulation of MMAC1/PTEN and Up-regulation of Phosphorylated Akt

Erlotinib (Tarceva), an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, has clinical activity in advanced lung cancer, but disease that initially responds to erlotinib eventually progresses. The mechanism of this acquired resistance is unclear. We established two erlotinib-resistant pools of A-431 cells, a well-characterized epidermoid cancer cell line that constitutively overexpresses EGFR and is sensitive to erlotinib, by continuous exposure to erlotinib over a 6-month period. The extent of EGFR gene amplification or mutation of the EGFR tyrosine kinase domain was not altered in the resistant cells. Intracellular erlotinib concentrations, determined by liquid chromatography-tandem mass spectrometry, were almost the same in all three cell lines. Immunoprecipitation with EGFR antibody followed by detection with phosphotyrosine antibody revealed that erlotinib effectively reduced EGFR phosphorylation in both parental cells and resistant cells. Erlotinib induced mutated in multiple advanced cancers 1/phosphatase and tensin homologue (MMAC1/PTEN) and suppressed phosphorylated Akt (Ser(473)) but not in the erlotinib-resistant cells. Overexpression of MMAC1/PTEN by transfection with Ad.MMAC1/PTEN or by pharmacologic suppression of Akt activity restored erlotinib sensitivity in both resistant pools. Further, transfection of parental A-431 cells with constitutively active Akt was sufficient to cause resistance to erlotinib. We propose that acquired erlotinib resistance associated with MMAC1/PTEN down-regulation and Akt activation could be overcome by inhibitors of signaling through the phosphatidylinositol 3-kinase pathway.

Quantitative high-performance liquid chromatography/electrospray ionization tandem mass spectrometric analysis of 2- and 3-series prostaglandins in cultured tumor cells.

This paper describes a rapid and simple technique for the simultaneous quantitative analysis of PGE(2), PGE(3), and other closely related prostaglandins from cultured cells using liquid chromatography/electrospray ionization tandem mass spectrometry. This method permits quantification of selected individual prostaglandins derived either from arachidonic acid (AA) or eicosapentaenoic acid (EPA) from cell extracts without tedious derivatization, lengthy sample preparation, and separation required by GC-MS- or HPLC-UV-based methods. The validation assessment showed that the quantitative determination is linear (r(2)>0.999) for both PGE(2) and PGE(3) in the range tested (1-500 ng/ml, 0.0028-1.4 microM) and a coefficient of variation lower than 10% was obtained for samples analyzed on 3 separate days. The detection limit was 2.5 pg for both PGE(2) and PGE(3). Extraction efficiency of PGE(2) and PGE(3) from cell suspensions ranged from 89.4 to 98.2%. As an application of the method, prostaglandins formed by EPA in human lung cancer A549 cells were determined. A 62% reduction of PGE(2) formation was noted when A549 cells were treated with 10 microM of EPA. Concomitantly, EPA increased formation of PGE(3) by 10-fold in A549 cells. This is the first report that unequivocally demonstrates that EPA can be converted to PGE(3) by cyclooxygenase in human cancer cells.

Anticancer activity of fish oils against human lung cancer is associated with changes in formation of PGE2 and PGE3 and alteration of Akt phosphorylation.

The beneficial effects of omega‐3 fatty acids are believed to be due in part to selective alteration of arachidonate metabolism that involves cyclooxygenase (COX) enzymes. Here we investigated the effect of eicosapentaenoic acid (EPA) on the proliferation of human non‐small cell lung cancer A549 (COX‐2 over‐expressing) and H1299 (COX‐2 null) cells as well as their xenograft models. While EPA inhibited 50% of proliferation of A549 cells at 6.05 µM, almost 80 µM of EPA was needed to reach similar levels of inhibition of H1299 cells. The formation of prostaglandin (PG)E3 in A549 cells was almost threefold higher than that of H1299 cells when these cells were treated with EPA (25 µM). Intriguingly, when COX‐2 expression was reduced by siRNA or shRNA in A549 cells, the antiproliferative activity of EPA was reduced substantially compared to that of control siRNA or shRNA transfected A549 cells. In line with this, dietary menhaden oil significantly inhibited the growth of A549 tumors by reducing tumor weight by 58.8 ± 7.4%. In contrast, a similar diet did not suppress the development of H1299 xenograft. Interestingly, the ratio of PGE3 to PGE2 in A549 was about 0.16 versus only 0.06 in H1299 xenograft tissues. Furthermore, PGE2 up‐regulated expression of pAkt, whereas PGE3 downregulated expression of pAkt in A549 cells. Taken together, the results of our study suggest that the ability of EPA to generate PGE3 through the COX‐2 enzyme might be critical for EPA‐mediated tumor growth inhibition which is at least partly due to down‐regulation of Akt phosphorylation by PGE3.

Studies of the function and structure of in vitro propagated human granulocytes

ABSTRACT: In our study, hematopoietic progenitor cells isolated from human umbilical cord blood were grown in an in vitro liquid culture system using the recombinant colony stimulating factors IL-3 plus granulocyte-macrophage colony-stimulatory factor (GM-CSF) or IL-3 plus granulocyte colony stimulating factor (G-CSF). The morphology and function of the cells produced were then studied, and it was demonstrated that continuous exposure to IL-3 plus GM-CSF produced predominantly eosinophilic granulocytes, whereas IL-3 plus G-CSF produced neutrophilic granulocytes. Cells from IL-3/GM-CSF cultures showed progressively increasing oxygen metabolism and locomotive capabilities over time, which became equivalent to peripheral blood neutrophils at wk 4 and 3, respectively. Phagocytic activity of these cells was poor. IL-3/G-CSF cultures produced cells with progressive increases in oxygen metabolism, locomotion, and phagocytosis. These functions never became equivalent to those of peripheral blood neutrophils. Flow cytometric analysis of IL-3/G grown cells showed that they expressed CD11b on their surfaces and that surface expression increased 2-fold after secondary granule secretagogue exposure. Ultrastructurally, the eosinophilic granulocyte nature of the IL-3/GM grown cells was confirmed by immunogold-lectin staining and IL-3/G grown cells were shown to contain antigenic myeloperoxidase. The data demonstrate that human umbilical cord blood mononuclear cells can be used to propagate granulocytes in vitro, that the types of granulocytes produced in this culture system depend on the growth factors used, that the cells produced in vitro develop several of the functional characteristics of peripheral blood granulocytes, and that ultrastructural details of developing human granulocytes can be carefully examined in this model system.

Growth factors controlling interleukin-4 action on hematopoietic progenitors

SummaryWe investigated the effect of interleukin-4 (IL-4) on human hematopoietic progenitors using low-density bone marrow cells from 29 hematologically normal donors. We found that IL-4 could either inhibit or stimulate cell growth, depending upon the other constituents of the culture medium. At concentrations ranging from 0.1 to 10.0 μg/ml, it significantly inhibited colony-forming units granulocyte-macrophage (CFU-GM) in the presence of either fetal calf serum alone, erythropoietin, leukocyte-conditioned medium prepared with phytohemagglutinin, granulocyte-macro-phage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), or stem cell factor (SCF), in a dose-dependent fashion. In contrast, IL-4 stimulated CFU-GM colony multiplication in the presence of granulocyte colony-stimulating factor (G-CSF). Similar but less significant inhibitory effects were exerted by IL-4 on burst-forming units-erythroid (BFU-E). The growth suppressive effect of IL-4 was partially reversed by IL-1β, and to a lesser extent by IL-6. When tested by enzyme-linked immunosorbent assay (ELISA), IL-4 suppressed cellular IL-1β production, and, similar to IL-4, anti-IL-1β-neutralizing antibodies inhibited CFU-GM colony growth, suggesting that the inhibition of endogenous IL-1β is a factor in regulating the IL-4 effect. Furthermore, in the absence of exogenous growth factors, IL-4 inhibited CFU-GM colony growth when anti-G-CSF neutralizing antibodies were also present. Therefore, we tested the effect of IL-4 on G-CSF receptors and found that 6-or 24-h incubation of low-density marrow cells with 1.0 μg/ml IL-4 resulted in up-regulation of G-CSF receptors. Taken together, these results suggest that IL-4 possesses a dual modulatory role in the hematopoietic system via interaction with various cytokines.

8;20 Chromosomal translocation in a case of acute leukemia: Cytogenetic, immunophenotypic, ultrastructural, and molecular characteristics

An 8;20 chromosomal translocation was observed in the leukemia cells of a 3-year-old girl. To our knowledge, this is the first report of this translocation in de novo acute leukemia. This chromosomal defect was present in the leukemia cells at diagnosis and also at relapse, but remission bone marrow cells had the 46,XX karyotype. By morphologic and cytochemical criteria the leukemia was myeloid but these features were more lymphoid when the leukemia recurred. However, the immunophenotype was consistent with myeloid leukemia and did not change at relapse. No evidence for either immunoglobulin or TCR gene rearrangement was observed.

Reactive Oxygen Species Generation in Human Cells by a Novel Magnetic Resonance Imaging Contrast Agent

The novel positive-contrast magnetic resonance imaging (MRI) marker C4 consists of an aqueous solution of cobalt chloride (CoCl2) complexed with the chelator N-acetylcysteine (NAC). We evaluated whether the presence of C4 or its components would produce reactive oxygen species (ROS, including hydroxyl, peroxyl, or other reactive oxygen species) in cultured cells. Human cancer or normal cells were incubated with 1% (w/v) CoCl2·6H2O or 2% NAC or a combination of both (1% CoCl2·6H2O : 2% NAC in an aqueous solution, abbreviated as Co : NAC) in the presence or absence of H2O2. Intracellular ROS levels were measured and quantified by change in relative fluorescence units. Students t-tests were used. In all cell lines exposed to 1000 μM H2O2, the Co : NAC led to ≥94.7% suppression of ROS at 5 minutes and completely suppressed ROS at 60 and 90 minutes; NAC suppressed ROS by ≥76.6% at 5 minutes and by ≥94.5% at 90 minutes; and CoCl2·6H2O suppressed ROS by ≥37.2% at 30 minutes and by ≥48.6% at 90 minutes. These results demonstrate that neither Co : NAC nor its components generated ROS; rather, they suppressed ROS production in cultured cells, suggesting that C4 would not enhance ROS production in clinical use.

Abstract B48: Identification of potent, cell active MTH1 inhibitors and their use in target validation studies

MTH1 is a protein that sanitizes oxidized dNTPs in the cell. It preferentially hydrolyzes 8-oxo-dGTP and 2-OH-dATP to their corresponding monophosphates and thereby prevents the incorporation of oxidized nucleotides into DNA or RNA. The functional result is a reduction in down-stream mutations, or DNA damage, thus preventing cell death. Recent publications suggest that MTH1 is a non-essential enzyme in normal cells, but is required for the survival of cancer cells as a consequence of being subjected to high levels of oxidative stress; hence its relevance as an oncology target. Gad et al., 2014 The interesting target rationale combined with a perceived high chemical tractability of the target resulting from availability of x-ray crystallographic information for the protein, led to a decision to undertake, in parallel, target validation, assay development and drug discovery. This effort resulted in the structure-based design of potent, cell active MTH1 small molecule inhibitors. This poster will describe the discovery and optimization of these inhibitors, and their use to evaluate the potential of MTH1 as an oncology target. This work was complemented by parallel genetic studies. Pharmacodynamic evaluation of target engagement using proximal biomarkers will be presented, as will phenotypic responses across a range of cancer cell lines. Citation Format: Elisabetta Leo, Alessia Petrocchi, Jennifer Bardenhagen, Maria Alimova, Xi Shi, Connor Parker, Naphtali Reyna, Matthew Hamilton, Edward Felix, Andrzej Mazan, Christian Dillon, Faika Mseeh, Joseph R. Marszalek, Carlo Toniatti, Giulio Draetta, Phil Jones, Richard T. Lewis. Identification of potent, cell active MTH1 inhibitors and their use in target validation studies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B48.