4-hydroxy-2-nonenal and Astrocytoma

An NADP(+)-dependent dehydrogenase activity on 3-glutathionyl-4-hydroxynonanal (GSHNE) was purified to electrophoretic homogeneity from a line of human astrocytoma cells (ADF). Proteomic analysis identified this enzymatic activity as associated with carbonyl reductase 1 (EC 1.1.1.184). The enzyme is highly efficient at catalyzing the oxidation of GSHNE (KM 33 µM, kcat 405 min(-1)), as it is practically inactive toward trans-4-hydroxy-2-nonenal (HNE) and other HNE-adducted thiol-containing amino acid derivatives. Combined mass spectrometry and nuclear magnetic resonance spectroscopy analysis of the reaction products revealed that carbonyl reductase oxidizes the hydroxyl group of GSHNE in its hemiacetal form, with the formation of the corresponding 3-glutathionylnonanoic-δ-lactone. The relevance of this new reaction catalyzed by carbonyl reductase 1 is discussed in terms of HNE detoxification and the recovery of reducing power.

Topics: Alcohol Oxidoreductases; Aldehyde Reductase; Aldehydes; Astrocytoma; Glutathione; Humans; Inactivation, Metabolic; Lactones; Magnetic Resonance Spectroscopy; Mass Spectrometry; NADP; NADPH Dehydrogenase; Oxidation-Reduction; Proteomics; Substrate Specificity; Sulfhydryl Compounds; Tumor Cells, Cultured

To investigate the role of human aldo-keto reductase 1A1 (AKR1A1) in the resistance to oxidative stress and the metabolism of toxic aldehyde in astrocytoma cells.. The siRNA was transfected into 1321N1 astrocytoma cells using Lipofectamine(TM); RNAiMax. Western blotting and qRT-PCR were applied to evaluate the knock-down efficiency of AKR1A1. MTT assay was used to examine the cell viability after H2;O2; and 4-hydroxynonenal treatment in AKR1A1 knock-down cells. In addition, the effect of knocking down AKR1A1 on cellular reactive oxygen species (ROS) level in the presence of H2;O2; was measured using 2', 7'-dichlorofluorescein (DCFH-DA).. Western blotting and qRT-PCR showed that the AKR1A1-specific siRNA inhibited AKR1A1 gene expression by about 70% in 1321N1 cells. Cells with knock-down of AKR1A1 were more sensitive to H2;O2; and 4-hydroxynonenal-induced cytotoxicity. Furthermore, cellular ROS level in the cells with knock-down of AKR1A1 was much higher than that in the control cells in the presence of H2;O2;.. The specific siRNA could efficiently inhibit AKR1A1 expression in 1321N1 cells. AKR1A1 could be involved in the metabolism of 4-hydroxynonenal and play a role in the resistance to oxidative stress.

Topics: Aldehyde Reductase; Aldehydes; Astrocytoma; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Hydrogen Peroxide; Oxidative Stress; RNA Interference; RNA, Messenger

Gliomas are tumors originating from astrocytes, oligodendrocytes or ependimal cells. Those of astrocytic origin are the most widespread of primary brain tumors and account for more then 60% of all CNS neoplasms. The current state of knowledge on the associations between tumor etiology and oxidative stress suggests that environmental factors that cause oxidative stress could also induce and promote cancer, especially in case of hereditary predisposition. Among mediators of oxidative stress, lipid peroxidation product 4-hydroxynonenal (HNE) is of particular relevance in oncology, as it is known to act as a growth-regulating factor and a signaling molecule. The aim of present study was to investigate by immunohistochemistry the presence of HNE-modified proteins in different types of astrocytoma. Our study comprised 45 astrocytic tumors. These tumors were graded in accordance with the WHO classification as diffuse astrocytomas (DA), anaplastic astrocytomas (AA) and glioblastomas (GB), while each group comprised 15 tumors. Slides of paraffin-embedded tumor tissue were stained with hematoxylin-eosin or were prepared for immunohistochemistry with monoclonal antibodies to HNE-histidine conjugate. Positive immunohistochemical reaction to HNE was analyzed semi-quantitatively. HNE positivity was proportional with malignancy of astrocytomas. The weakest presence of HNE-histidine adducts was found in DA, followed by AA and GB. Lowest intensity of HNE immunopositivity was present in tumor cells of almost all DA, predominantly around blood vessels. In malignant variants of astrocytoma, AA and GB, HNE positivity was moderate to strong, and diffusely distributed in all tumors.

Topics: Aldehydes; Antibodies, Monoclonal; Astrocytoma; Blood Vessels; Brain Neoplasms; Endothelium, Vascular; Humans; Immunohistochemistry; Proteins

Protein modification is one of the important processes during oxidative stress. This modification of proteins is either due to direct oxidation of proteins by various oxidants or due to secondary modification by lipid peroxidation products, e.g. 4-hydroxynonenal. In the here presented work we compare the intracellular distribution of protein modification products after treatment of human U87 astrocytoma cells with hydrogen peroxide or HNE. The treatment with hydrogen peroxide leads mainly to a cytosolic formation of oxidized proteins whereas HNE treatment is forming HNE-adducts throughout the cell. Therefore, we concluded that HNE diffusion distance in cells enables this lipid peroxidation product to act as a second messenger within the cell and on the other hand is the reason for the genotoxic properties of this compound.

Topics: Aldehydes; Astrocytoma; Cell Line, Tumor; Humans; Hydrogen Peroxide; Lipid Peroxidation; Oxidants; Oxidation-Reduction; Oxidative Stress; Protein Carbonylation; Proteins