Pathway-Focused PCR Array Profiling of Enriched Populations of Laser Capture Microdissected Hippocampal Cells after Traumatic Brain Injury

<div><p>Cognitive deficits in survivors of traumatic brain injury (TBI) are associated with irreversible neurodegeneration in brain regions such as the hippocampus. Comparative gene expression analysis of dying and surviving neurons could provide insight into potential therapeutic targets. We used two pathway-specific PCR arrays (RT2 Profiler Apoptosis and Neurotrophins & Receptors PCR arrays) to identify and validate TBI-induced gene expression in dying (Fluoro-Jade-positive) or surviving (Fluoro-Jade- negative) pyramidal neurons obtained by laser capture microdissection (LCM). In the Apoptosis PCR array, dying neurons showed significant increases in expression of genes associated with cell death, inflammation, and endoplasmic reticulum (ER) stress compared with adjacent, surviving neurons. Pro-survival genes with pleiotropic functions were also significantly increased in dying neurons compared to surviving neurons, suggesting that even irreversibly injured neurons are able to mount a protective response. In the Neurotrophins & Receptors PCR array, which consists of genes that are normally expected to be expressed in both groups of hippocampal neurons, only a few genes were expressed at significantly different levels between dying and surviving neurons. Immunohistochemical analysis of selected, differentially expressed proteins supported the gene expression data. This is the first demonstration of pathway-focused PCR array profiling of identified populations of dying and surviving neurons in the brain after TBI. Combining precise laser microdissection of identifiable cells with pathway-focused PCR array analysis is a practical, low-cost alternative to microarrays that provided insight into neuroprotective signals that could be therapeutically targeted to ameliorate TBI-induced neurodegeneration.</p></div

Apoptosis-related gene expression in dying and surviving neurons.

<p>A-H. Functional groups of genes involved in programmed cell death that are upregulated with significant (p<0.05) or borderline significance (0.05 < p < 0.1) in dying vs surviving neurons. (A) DNA Damage-induced Apoptosis. (B) TNF Ligand Family. (C) CARD Family. (D) CIDE Family. (E) Anti-apoptosis. (F) TNF Receptor Family. (G) Bcl-2 Family. (H) Caspase Family. Data are shown as fold changes in mRNA expression in dying compared to surviving cells, mean ± SEM (n = 3 biological pools each of Fluoro-Jade positive or Fluoro-Jade negative cells). Statistical analysis was performed using Student’s t-test, *p< 0.05, **p<0.1</p

Neurotrophins & Receptors PCR array gene expression in dying and surviving neurons.

<p>Data are shown as fold changes (mean ± SEM) (n = 3 biological pools each of Fluoro-Jade positive or Fluoro-Jade negative cells) in dying cells compared to surviving cells. Statistical analysis was performed using Student’s t test, *p< 0.05, **p<0.1</p

Laser capture microdissection of rat hippocampal neurons after fluid percussion brain injury.

<p>(A). Dying, Fluoro-Jade-positive neurons in the CA3 subfield of the rat hippocampus 24 hr after TBI are shown, before and after LCM on the capture caps. (B). Surviving, Fluoro-Jade negative neurons were captured immediately adjacent to FJ positive, dying neurons.</p

Immunohistochemical validation of gene expression data.

<p>Representative images of rat brain sections double-stained, using Fluoro-Jade C (FJ, in green) to identify injured neurons, and specific antibodies against CD40 (A), CASP 12 (B), CXCR4 (C) and active caspase 3 (D). Arrows point to cells co-expressing Fluoro-Jade C and the protein of interest. Arrowheads point to cells expressing low levels of CASP12 (B) and CXCR4 (C). (E) Gene expression in dying and surviving neurons, expressed as fold changes in dying to surviving neurons (normalized to a value of 1) are shown as a point of reference for the protein expression data. Calibration bars = 50 mm</p

Differential expression of Apoptosis and Neurotrophin genes in dying and surviving neurons.

<p>Differentially expressed genes that were deemed significant (p<0.05) or of borderline significance (p<0.1) are shown. The complete datasets are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127287#pone.0127287.s003" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127287#pone.0127287.s004" target="_blank">S2</a> Tables. Hyperlinks to the GeneCard entry for each gene and Digital Object Identifier (DOI) for at least one supporting published reference are included. GeneCards is a searchable, integrated, database of human genes that provides concise genomic related information, on all known and predicted human genes.</p><p>Fold changes are ratios of gene expression levels (dying/surviving neurons).</p><p>Differential expression of Apoptosis and Neurotrophin genes in dying and surviving neurons.</p

Cystathionine-β-Synthase Inhibition for Colon Cancer: Enhancement of the Efficacy of Aminooxyacetic Acid via the Prodrug Approach

Abstract Colon cancer cells contain high levels of cystathlonlne-β-synthase (CBS). Its product, hydrogen sulfide (H2S), promotes the growth and proliferation of colorectal tumor cells. To improve the antitumor efficacy of the prototypical CBS inhibitor aminooxyacetic acid (AOAA), we have designed and synthesized YD0171, a methyl ester derivative of AOAA. The antiproliferative effect of YD0171 exceeded the antiproliferative potency of AOAA in HCT116 human colon cancer cells. The esterase inhibitor paraoxon prevented the cellular inhibition of CBS activity by YD0171. YD0171 suppressed mitochondrial respiration and glycolytic function and induced G0/G1 arrest, but did not induce tumor cell apoptosis or necrosis. Metabolomic analysis in HCT116 cells showed that YD0171 affects multiple pathways of cell metabolism. The efficacy of YD0171 as an inhibitor of tumor growth was also tested in nude mice bearing subcutaneous HCT116 cancer cell xenografts. Animals were treated via subcutaneous injection of vehicle or AOAA (0.1, 0.5 or 1 mg/kg/d) for 3 wks. Tumor growth was significantly reduced by 9 mg/kg/d AOAA, but not at the lower doses. YD0171 was more potent: tumor volume was significantly inhibited at 0.5 and 1 mg/kg/d. Thus, the in vivo efficacy of YD0171 is nine times higher than that of AOAA. YD0171 (1 mg/kg/d) attenuated tumor growth and metastasis formation in the intracecal HCT116 tumor model. YD0171 (3 mg/kg/d) also reduced tumor growth in patient-derived tumor xenograft bearing athymic mice. YD0171 (3 mg/kg/d) induced the regression of established HCT116 tumors in vivo. A 5-d safety study in mice demonstrated that YD0171 at 20 mg/kg/d (given in two divided doses) does not increase plasma markers of organ injury, nor does it induce histological alterations in the liver or kidney. YD0171 caused a slight elevation in plasma homocysteine levels. In conclusion, the prodrug approach improves the pharmacological profile of AOAA; YD0171 represents a prototype for CBS inhibitory anticancer prodrugs. By targeting colorectal cancer bioenergetics, an emerging important hallmark of cancer, the approach exemplified herein may offer direct translational opportunities