Kyoji Furuta

Role of heme oxygenase‐1 protein in the neuroprotective effects of cyclopentenone prostaglandin derivatives under oxidative stress

Previously we found that some cyclopenteone prostaglandin derivatives (PGs), referred to as neurite outgrowth‐promoting PGs (NEPPs), have dual biological activities of promoting neurite outgrowth and preventing neuronal death [Satoh et al. (2000) J. Neurochem., 75, 1092‐1102; Satoh et al. (2001) J. Neurochem., 77, 50‐62; Satoh et al. (2002) In Kikuchi, II. (ed.), Strategenic Medical Science Against Brain Attack. Springer‐Verlag, Tokyo, pp. 78‐93]. To investigate possible cellular mechanisms of the neuroprotective effects, we performed oligo hybridization‐based DNA array analysis with mRNA isolated from HT22, a cell line that originated from a mouse hippocampal neuron. Several transcripts up‐regulated by NEPP11 were identified. Because heme oxygenaseu20031 (HO‐1) mRNA was the most prominently induced and was earlier reported to protect neuronal and non‐neuronal cells against oxidative stress, we focused on it as a possible candidate responsible for the neuroprotective effects. We found NEPP11 to induce HO‐1 protein (32u2003kDa) in HT22 cells in both the presence and the absence of glutamate, whereas non‐neuroprotective prostaglandins (PGs) Δ12‐PGJ2 or PGA2 did not. Overexpression of HO‐1‐green fluorescence protein (GFP) fusion protein significantly protected HT22 cells against oxidative glutamate toxicity, whereas that of GFP alone did not. Furthermore, biliverdin and bilirubin, products of HO‐1 enzymatic activity on heme, protected HT22 cells from oxidative glutamate toxicity. These results, together with our previous results, suggest that NEPP11 activates the expression of HO‐1 and that HO‐1 produces biliverdin and bilirubin, which result in the inhibition of neuronal death induced by oxidative stress. NEPP11 is the first molecular probe reported to have a neuroprotective action through induction of HO‐1 in neuronal cells.

The human multidrug resistance-associated protein (MRP) gene family: from biological function to drug molecular design.

Abstract The ATP-binding cassette transmembrane proteins play an important role in transport of drugs as well as of biologically active endogenous substances. The human multidrug resistance-associated protein (MRP) subfamily consists of at least six members, exhibiting a wide spectrum of biological functions. MRP1 operates as an ATP-dependent primary active transporter for substrates conjugated with glucuronide, sulfate or glutathione. Leukotriene C4 is an important endogenous substrate for MRP1. Glutathione serves as a cofactor in MRP1-mediated drug transport as well. Genes encoding both MRP1 and the catalytic subunit of γ-glutamylcysteine synthetase (γ-GCS) are coordinately regulated in cultured cancer cell lines as well as colorectal cancer tissues from colon cancer patients. The induction of MRP1 and γ-GCS expression by oxidative stress varies among different cell lines, and p53 mutations are associated with elevated levels of induction. To modulate the transport function of MRP1, we have synthesized novel glutathione derivatives as photoreactive biochemical probes targeting the transporter protein. GIF-0019 restored the cellular sensitivity of MRP1-overexpressing drug-resistant cancer cells to anticancer prostaglandins in vitro, which was characterized by enhanced mRNA levels of the cyclin-dependent kinase inhibitor p21, suppressed c-myc expression and G1 arrest.

Facilitatory Roles of Novel Compounds Designed from Cyclopentenone Prostaglandins on Neurite Outgrowth-Promoting Activities of Nerve Growth Factor

Abstract: Cyclopentenone prostaglandins (PGs) are known to arrest the cell cycle at the G1 phase in vitro and to suppress tumor growth in vivo. However, their effects on neurons are unclear. Here, we report that some cyclopentenone PGs function as neurite outgrowth‐promoting factors. They promoted neurite outgrowth from PC12 cells and from dorsal root ganglion explants but only in the presence of nerve growth factor (NGF). We refer to these PGs as neurite outgrowth‐promoting PGs (NEPPs). Through study of the structure‐function relationship of NEPP1‐10 and related compounds, we found that the cross‐conjugated dienone moiety of NEPPs was essential for promoting neurite outgrowth, and NEPP10 was concluded to be the best candidate for drug development. We also investigated the intracellular mechanism of the promotion by NEPPs and obtained evidence that immunoglobulin heavy chain binding protein/glucose‐regulated protein 78 (BiP/GRP78) plays a role in the promotion, based on the following observations: Antisense nucleotides for BiP/GRP78 gene blocked the promotion of neurite outgrowth; BiP/GRP78 protein level increased in response to NEPPs; and overexpression of BiP/GRP78 protein by adenoviral gene transfer promoted the neurite outgrowth by NGF.

Potent Prostaglandin A1 Analogs That Suppress Tumor Cell Growth through Induction of p21 and Reduction of Cyclin E

Although the cyclopentenone prostaglandin A1 (PGA1) is known to arrest the cell cycle at the G1 phase in vitro and to suppress tumor growth in vivo, its relatively weak activity limits its usefulness in cancer chemotherapy. In an attempt to develop antitumor drugs of greater potency and conspicuous biological specificity, we synthesized novel analogs based on the structure of PGA1. Of the newly synthesized analogs, 15-epi-Δ7-PGA1 methyl ester (NAG-0092), 12-iso-Δ7-PGA1 methyl ester (NAG-0093), and ent-Δ7-PGA1methyl ester (NAG-0022) possess a cross-conjugated dienone structure around the five-member ring with unnatural configurations at C(12) and/or C(15) and were found to be far more potent than native PGA1 in inhibiting cell growth and causing G1arrest in A172 human glioma cells. These three analogs induced the expression of p21 at both RNA and protein levels in a time- and dose-dependent fashion. Kinase assays with A172 cells treated with these analogs revealed that both cyclin A- and E-dependent kinase activities were markedly reduced, although cyclin D1-dependent kinase activity was unaffected. Immunoprecipitation-Western blot analysis showed that the decrease in cyclin A-dependent kinase activity was due to an increased association of p21 with cyclin A-cyclin-dependent kinase 2 complexes, whereas the decrease in cyclin E-dependent activity was due to a combined mechanism involving reduction in cyclin E protein itself and increased association of p21. Thus, these newly synthesized PGA1analogs may prove to be powerful tools in cancer chemotherapy as well as in investigations of the structural basis of the antiproliferative activity of A series prostaglandins.

Neurotrophic actions of novel compounds designed from cyclopentenone prostaglandins

Previously we found that some cyclopentenone prostaglandin derivatives promoted neurite outgrowth from PC12 cells and dorsal root ganglia explants in the presence of nerve growth factor; and so we referred to them as neurite outgrowth‐promoting prostaglandins (NEPPs). In this study, NEPPs protected HT22 cells against oxidative glutamate toxicity. NEPP6, one of the most effective promoters of neurite outgrowth in PC12 cells, protected the cells most potently among NEPPs 1–10. Several derivatives, NEPPs 11–19, were newly synthesized based on the chemical structure of NEPP6. NEPP11 had a more potent neuroprotective effect than NEPP6. NEPP11 also prevented the death of cortical neurons induced by various stimuli and reduced ischemic brain damage in mice. Biotinylated compounds of NEPPs were synthesized to investigate their cellular accumulation. NEPP6‐biotin protected the cells and emitted potent signals from the cells. In contrast, biotinylated non‐neuroprotective derivatives emitted much weaker signals. These results suggest that NEPPs are novel types of neurotrophic compounds characterized by their dual biological activities of promoting neurite outgrowth and preventing neuronal death and that their accumulation in the cells is closely associated with their neuroprotective actions.

Anti-apoptotic and pro-apoptotic effect of NEPP11 on manganese-induced apoptosis and JNK pathway activation in PC12 cells

Neurite outgrowth-promoting prostaglandins (NEPPs), cyclopentenone prostaglandin derivatives, are found to be neurotrophic. These small organic compounds promote neurite outgrowth of PC12 cells and dorsal root ganglion explants in the presence of nerve growth factor, and prevent neuronal cell death of HT22 cells and cortical neurons induced by various stimuli. In this study, we examined whether NEPP11 prevents manganese-induced apoptosis of PC12 cells. NEPP11 (5 microM) attenuated manganese-induced DNA fragmentation by approximately 50%. In addition, NEPP11 partially prevented manganese-induced c-Jun phosphorylation and c-Jun N-terminal kinase (JNK) phosphorylation determined by Western blotting. Inhibition of the JNK signaling pathway by NEPP11 appeared to be selective, because NEPP11 did not inhibit manganese-induced activation of p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase1/2 (ERK1/2), MEK1/2 and p70 S6 kinase (p70S6K) in PC12 cells. In contrast, NEPP11 alone was toxic at higher concentrations (>10 microM) producing DNA fragmentation and activation of the JNK pathway. Molecular modifications of NEPP11 may strengthen its inhibitory effects on the JNK pathway while preventing its cytotoxicity, and thus may become a useful small molecule reagent for the treatment of manganese toxicity and other similar neurodegenerative processes.

MOLECULAR DESIGN OF GLUTATHIONE-DERIVED BIOCHEMICAL PROBES TARGETING THE GS-X PUMP

Abstract The syntheses of novel glutathione derivatives as photoaffinity and fluorescent biochemical probes for the GS-X pump are described. GIF-0017, an S -alkylated glutathione derivative with a benzophenone photophore, competitively inhibits the transport of leukotriene C 4 by the GS-X pump with an IC 50 value of 0.40 μM, indicating the existence of strong interaction between the probe molecule and the GS-X pump.

Synthesis of an acromelic acid A analog-based 11C-labeled PET tracer for exploration of the site of action of acromelic acid A in allodynia induction

A novel (11)C-labeled PET (positron emission tomography) tracer, which was designed based on the (phenylthio)pyrrolidine derivative that can competitively block the acromelic acid A-induced allodynia, was synthesized. A protocol in which methylation by palladium-mediated coupling of the boronate derivative with [(11)C]CH(3)I and deprotection of the protected amino acid moiety are successively performed in one-pot within 5 min was established for the synthesis of the tracer. The tracer is potentially useful as a tool to investigate the mode of action of acromelic acid A in the induction of allodynia.

Neuroprotective effects of (arylthio)cyclopentenone derivatives on manganese-induced apoptosis in PC12 cells.

Parkinsons disease is characterized by degeneration of dopaminergic neurones in the substantia nigra. Chronic manganese poisoning shares many features of Parkinsons disease, and also induces extrapyramidal syndromes that resemble those of Parkinsons disease due to dopamine depletion in the central nervous system. This study was undertaken to develop novel neuroprotective drugs via the identification of compounds that inhibit manganese-induced apoptosis. Here, we report that (arylthio)cyclopentenone derivatives, which are synthetic analogs of cyclopentenone prostaglandins, prevent manganese-induced apoptosis in PC12 cells. A highly sensitive assay of caspase-3/7 activity was used for screening newly synthesized prostaglandin analogs. The results showed that some cyclopentenone derivatives (GIF-0642, GIF-0643, GIF-0644, GIF-0745, and GIF-0747) inhibit manganese-induced caspase-3/7 activation in a concentration-dependent manner. Effective compounds all have an arylthio group, indicating that this structure plays an important role in the anti-apoptotic effects of (arylthio)cyclopentenone derivatives. The anti-apoptotic effects of these compounds were confirmed by verifying their ability to inhibit the DNA fragmentation and caspase-9 activation induced by manganese. Furthermore, GIF-0747 prevented manganese-induced cytochrome c release from mitochondria. These results suggest that (arylthio)cyclopentenone derivatives may be good candidates for treating neurodegenerative diseases.

Synthesis of a F-18 labeled analog of antitumor prostaglandin Δ7-pGA1 methyl ester using p-[18F]Fluorobenzylamine

A fluorine-18 labeled analog of an antitumor prostaglandin delta 7-PGA1 methyl ester, 15-deoxy-13,14-dihydro-delta 7-PGA1 4-[18F]fluorobenzyl amide ([18F]3), was synthesized as a tracer candidate for detecting tumors with positron emission tomography. p-[18F]Fluorobenzylamine (p-[18F]FBnA) used as a labeled precursor for the synthesis of [18F]3 was prepared by fluorination of a 4-N, N, N-trimethylammonium-benzonitrile triflate with [18F]fluoride and subsequent reduction with borane-dimethylsulfide. Radiochemical yield and purity of p-[18F]FBnA obtained were 39-49% (decay uncorrected) and 91-96%, respectively, after C18 Sep-Pak purification. Treatment of p-[18F]FBnA with a 15-deoxy-13,14-dihydro-delta 7-PGA1 N-succinimidyl ester in acetonitrile and subsequent HPLC purification gave radiochemically pure (> 99%) [18F]3 with a 58% decay uncorrected yield. The total synthesis time was 70 min from the start of the radiosynthesis of p-[18F]FBnA.