15-deoxy-delta(12-14)-prostaglandin-j2 and Neuroblastoma

TDP-43 proteinopathy (amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions) is a newly categorized group of neurodegenerative disorders characterized by abnormal accumulation and mislocalization of nuclear TDP-43 protein in the neuronal cytoplasm. 15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) is non-enzymatically produced from PGD(2) and plays roles in inflammation and oxidative stress responses. Indeed, 15d-PGJ(2) is up-regulated in the spinal motor neurons in amyotrophic lateral sclerosis. In this study, biochemical and immunocytochemical analyses showed that 15d-PGJ(2) affects the proteolysis, solubility, and subcellular localization of TDP-43, similar to alterations found in TDP-43 proteinopathy. Further studies revealed that a cyclopentenone ring containing an electrophilic carbon of 15d-PGJ(2) is likely to influence these phenomena. These findings suggest that 15d-PGJ(2) is an endogenous modifier of TDP-43 protein in TDP-43 proteinopathy.

Topics: Aged; Aged, 80 and over; Amyotrophic Lateral Sclerosis; Cell Line; Cyclopentanes; Cytoplasm; DNA-Binding Proteins; Female; Gene Expression Regulation; Humans; Immunologic Factors; Male; Middle Aged; Neuroblastoma; Prostaglandin D2; Spinal Cord; Subcellular Fractions

We have previously shown that a prostaglandin D(2) metabolite, 15-deoxy-delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), is the potent inducer of intracellular oxidative stress on human neuroblastoma SH-SY5Y cells [Kondo, M., Oya-Ito, T., Kumagai, T., Osawa, T., and Uchida, K. (2001) Cyclopentenone prostaglandins as potential inducers of intracellular oxidative stress. J. Biol. Chem. 276, 12076-12083.]. In the present study, to investigate the correlation between the redox regulation and the 15d-PGJ(2)-induced oxidative stress and to establish the cellular mechanism for protection against the endogenous electrophile, we analyzed S-oxidized proteins using biotinylated cysteine as a molecular probe. In addition, the reversible regulation of protein function by S-oxidation/thiolation was characterized in vitro. When human neuroblastoma SH-SY5Y cells were exposed to 15d-PGJ(2), followed by treatment with biotinylated cysteine, 26 proteins, including glycolytic enzymes, cytoskeletal proteins, redox enzymes, and stress proteins, were identified as substrates for reversible cysteine-targeted oxidation. To investigate the regulatory mechanism of protein function by S-oxidation/thiolation, the binding of a low molecular weight thiol (glutathione) to a glycolytic enzyme alpha-enolase was characterized. Treatment of alpha-enolase with the thiol oxidant diamide in the presence of glutathione in vitro resulted in the binding of glutathione to the protein and concomitant loss of the enzymatic activity, whereas the glutathiolation and inactivation of alpha-enolase were fully reversed by dithiothreitol. Mass spectrometric analysis of the tryptic fragments from native and oxidized alpha-enolase identified two cysteine residues, Cys-118 and Cys-388, as the S-oxidation sites, which may play a role in the regulation of the biological activities of the protein and may be regulated by a reversible S-oxidation/thiolation reaction. These results suggest that cysteine-targeted oxidation/thiolation plays a critical role in the regulation of protein function under conditions of electrophile-induced oxidative stress.

Topics: Binding Sites; Biotinylation; Cell Line, Tumor; Cysteine; Glutathione; Glutathione Disulfide; Humans; Neuroblastoma; Oxidation-Reduction; Oxidative Stress; Phosphopyruvate Hydratase; Prostaglandin D2; Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sulfhydryl Compounds

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands have been demonstrated to inhibit growth of several cancer cells. Here, we investigated whether one of the PPAR-gamma ligands, 15-deoxy-Delta12,14-prostaglandin J2 (15-deoxy-PGJ2) inhibits cell growth of two human neuroblastoma cells (SK-N-SH and SK-N-MC) in a PPAR-gamma-dependent manner. PPAR-gamma was expressed in these cells, and 15-deoxy-PGJ2 increased expression, DNA binding activity, and transcriptional activity of PPAR-gamma. 15-Deoxy-PGJ2 also inhibited cell growth in time- and dose-dependent manners in both cells. Cells were arrested in G2/M phase after 15-deoxy-PGJ2 treatment with concomitant increase in the expression of G2/M phase regulatory protein cyclin B1 but decrease in the expression of cdk2, cdk4, cyclin A, cyclin D1, cyclin E, and cdc25C. Conversely, related to the growth inhibitory effect, 15-deoxy-PGJ2 increased the induction of apoptosis in a dose-dependent manner. Consistent with the induction of apoptosis, 15-deoxy-PGJ2 increased the expression of proapoptotic proteins caspase 3, caspase 9, and Bax but down-regulated antiapoptotic protein Bcl-2. 15-Deoxy-PGJ2 also activated extracellular signal-regulated kinase (ERK) 2. In addition, mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor PD98059 (2'-amino-3'-methoxyflavone) decreased 15-deoxy-PGJ2-induced ERK2 activation, and expression of PPAR-gamma, capase-3, and cyclin B1. Moreover, MEK1/2 inhibitor PD98059 significantly prevented against the 15-deoxy-PGJ2-induced cell growth inhibition. We also found that PPAR-gamma antagonist GW9662 (2-chloro-5-nitro-N-phenylbenzamide) reversed the 15-deoxy-PGJ2-induced cell growth inhibition, PPAR-gamma expression, and activation of ERK2. These results demonstrate that 15-deoxy-PGJ2 inhibits growth of human neuroblastoma cells via the induction of apoptosis in a PPAR-gamma-dependent manner through activation of ERK pathway and suggest that 15-deoxy-PGJ2 may have promising application as a therapeutic agent for neuroblastoma.

Topics: Anilides; Apoptosis; Caspase 3; Caspases; Cell Division; Cyclin B; Cyclin B1; Drug Interactions; G2 Phase; Humans; Immunohistochemistry; Immunologic Factors; Mitogen-Activated Protein Kinases; Mitosis; Neuroblastoma; NF-kappa B; Organic Chemicals; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Transcription Factors; Tumor Cells, Cultured

Prostaglandin D(2) (PGD(2)), a major cyclooxygenase product in a variety of tissues and cells, readily undergoes dehydration to yield the bioactive cyclopentenone-type PGs of the J(2)-series, such as 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)). The observation that the level of 15d-PGJ(2) increased in the tissue cells from patients with sporadic amyotrophic lateral sclerosis suggested that the formation of 15d-PGJ(2) may be closely associated with neuronal cell death during chronic inflammatory processes. In vitro experiments using SH-SY5Y human neuroblastoma cells revealed that 15d-PGJ(2) induced apoptotic cell death. An oligonucleotide microarray analysis demonstrated that, in addition to the heat shock-responsive and redox-responsive genes, the p53-responsive genes, such as gadd45, cyclin G1, and cathepsin D, were significantly up-regulated in the cells treated with 15d-PGJ(2). Indeed, the 15d-PGJ(2) induced accumulation and phosphorylation of p53, which was accompanied by a preferential redistribution of the p53 protein in the nuclei of the cells and by a time-dependent increase in p53 DNA binding activity, suggesting that p53 accumulated in response to the treatment with 15d-PGJ(2) was functional. The 15d-PGJ(2)-induced accumulation of p53 resulted in the activation of a death-inducing caspase cascade mediated by Fas and the Fas ligand.

Topics: Adult; Aged; Apoptosis; Female; Flow Cytometry; Gene Expression Regulation; Genes, p53; Humans; Immunologic Factors; Male; Middle Aged; Motor Neuron Disease; Motor Neurons; Neuroblastoma; Neurons; Oligonucleotide Array Sequence Analysis; Prostaglandin D2; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Although considerable research has shown a role for peroxisome proliferator-activated receptors (PPAR) in adipose differentiation and in the regulation of inflammation, little is known about its possible functions in neurons. We investigated the role of PPARgamma in primary cultures of cortical neurons and human neuroblastoma SH-SYSY cells. Incubation of cortical neurons with the specific PPARgamma ligand 15-Deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) induced morphological changes including neurite degeneration and nuclear condensation that were consistent with neurons dying by apoptosis. The morphological changes associated with incubation of cortical neurons with 15d-PGJ2 were prevented following pretreatment of neurons with the general caspase inhibitor, Z-VAD. These results highlight a novel role for PPARgamma in neurons and suggest that unwarranted activation of PPARgamma may contribute to the neuronal apoptosis associated with certain neurodegenerative disorders including Alzheimer's disease (AD).

Topics: Animals; Apoptosis; Cerebral Cortex; Humans; Immunologic Factors; Neuroblastoma; Neurons; Poly(ADP-ribose) Polymerases; Prostaglandin D2; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Transcription Factors; Tumor Cells, Cultured