Somatic Pairing of Chromosome 19 in Renal Oncocytoma Is Associated with Deregulated ELGN2-Mediated Oxygen-Sensing Response

Chromosomal abnormalities, such as structural and numerical abnormalities, are a common occurrence in cancer. The close association of homologous chromosomes during interphase, a phenomenon termed somatic chromosome pairing, has been observed in cancerous cells, but the functional consequences of somatic pairing have not been established. Gene expression profiling studies revealed that somatic pairing of chromosome 19 is a recurrent chromosomal abnormality in renal oncocytoma, a neoplasia of the adult kidney. Somatic pairing was associated with significant disruption of gene expression within the paired regions and resulted in the deregulation of the prolyl-hydroxylase ELGN2, a key protein that regulates the oxygen-dependent degradation of hypoxia-inducible factor (HIF). Overexpression of ELGN2 in renal oncocytoma increased ubiquitin-mediated destruction of HIF and concomitantly suppressed the expression of several HIF-target genes, including the pro-death BNIP3L gene. The transcriptional changes that are associated with somatic pairing of chromosome 19 mimic the transcriptional changes that occur following DNA amplification. Therefore, in addition to numerical and structural chromosomal abnormalities, alterations in chromosomal spatial dynamics should be considered as genomic events that are associated with tumorigenesis. The identification of EGLN2 as a significantly deregulated gene that maps within the paired chromosome region directly implicates defects in the oxygen-sensing network to the biology of renal oncocytoma

Oral administration of nano-emulsion curcumin in mice suppresses inflammatory-induced NFκB signaling and macrophage migration.

Despite the widespread use of curcumin for centuries in Eastern medicine as an anti-inflammatory agent, its molecular actions and therapeutic viability have only recently been explored. While curcumin does have potential therapeutic efficacy, both solubility and bioavailability must be improved before it can be more successfully translated to clinical care. We have previously reported a novel formulation of nano-emulsion curcumin (NEC) that achieves significantly greater plasma concentrations in mice after oral administration. Here, we confirm the immunosuppressive effects of NEC in vivo and further examine its molecular mechanisms to better understand therapeutic potential. Using transgenic mice harboring an NFκB-luciferase reporter gene, we demonstrate a novel application of this in vivo inflammatory model to test the efficacy of NEC administration by bioluminescent imaging and show that LPS-induced NFκB activity was suppressed with NEC compared to an equivalent amount of curcumin in aqueous suspension. Administration of NEC by oral gavage resulted in a reduction of blood monocytes, decreased levels of both TLR4 and RAGE expression, and inhibited secretion of MCP-1. Mechanistically, curcumin blocked LPS-induced phosphorylation of the p65 subunit of NFκB and IκBα in murine macrophages. In a mouse model of peritonitis, NEC significantly reduced macrophage recruitment, but not T-cell or B-cell levels. In addition, curcumin treatment of monocyte derived cell lines and primary human macrophages in vitro significantly inhibited cell migration. These data demonstrate that curcumin can suppress inflammation by inhibiting macrophage migration via NFκB and MCP-1 inhibition and establish that NEC is an effective therapeutic formulation to increase the bioavailability of curcumin in order to facilitate this response

Curcumin labels monocytes and inhibits macrophage migration in human cells.

<p><b>A</b>, Human cell lines Jurkat, Clone E6-1 and THP-1 were cultured in complete RPMI medium, supplemented with various amounts of curcumin or vehicle, and analyzed by FACS. Representative results are shown. <b>B</b>, THP-1 (top) and primary human monocyte derived macrophages (bottom) were treated with the indicated concentrations of curcumin and cell viability was measured over time by MTT assay. Values are expressed as fold changes relative to initial baseline levels. <b>C</b>, THP-1 cells were differentiated into macrophages and isolated by adherence to culture plates. Scratch assays were performed on adherent cells with or without curcumin to measure cell migration. Representative images (left) and migrated cell counts (right). <b>D</b>, Primary macrophages were isolated from healthy human blood samples (<i>n</i> = 4) and subjected to migration assays with and without curcumin (20 µM) for the indicated time. Representative images (left) and relative fold changes of migrated cells (right) are shown. Original magnification X 40. *  =  <i>p</i>≤0.05 versus vehicle. n.s.  =  not significant versus vehicle.</p

Schematic showing the cellular pathways by which curcumin (NEC) inhibits macrophage inflammatory signaling.

<p>Curcumin regulates inflammatory pathways at multiple levels: i) downregulation of TLR4 expression on the surface of cells and ii) inhibition of p65 and IκBα phosphorylation in the activation of NFκB. Thus, NEC inhibits transcriptional regulation <i>in vivo</i> of NFκB target genes, including RAGE, MCP-1, and NOS2, which leads to suppressed macrophage-mediated inflammatory responses.</p

LPS-induced NFκB reporter gene expression is suppressed with nano-emulsion curcumin (NEC) in mice when compared to equivalent concentration of suspension curcumin (SC).

<p>Transgenic BALB/C-Tg(NFκB-RE-luc)-Xen mice (<i>n</i> = 13) were treated with 1 g/kg NEC, equivalent component concentrations of the nano-emulsion vehicle, or SC by oral gavage 10 min prior to LPS injection (IP, 2 mg/kg), and imaged (IVIS 200; 150 mg luciferin/kg IP) at 0 h, 2 h, 4 h and 6 h. The whole animal, including areas covering the thymus, lymph nodes, and the abdominal region were outlined and subjected to relative photon counting. <b>A,</b> Representative whole body bioluminescent images; <b>B,</b> Digitization of emitted light photons. The fold change in luminescent intensity is expressed relative to the photon counting in the mice at 0 h. Data were analyzed using the mixed effect model, as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111559#s2" target="_blank">methods</a>. *  =  <i>p</i><0.0001 versus SC. #  =  <i>p</i><0.0001 versus vehicle. IP  =  intraperitoneal.</p

FACS analyses show that NEC selectively diminishes levels of blood monocytes.

<p>Leukocytes were isolated from whole blood collected from BALB/c mice (<i>n</i> = 6) before and 30 min after oral administration of NEC (1 g/kg). <b>A</b>, FACS analyses of leukocytes measuring the expression of cell surface markers: T-cells (CD3, CD4, or CD8), B-cells (B220), or monocytes (F4/80). * <i>p</i>≤0.05 versus time zero. <b>B</b>, Representative FACS analysis of F4/80⁺ expression.</p