Discovery of a novel neuroprotective compound, AS1219164, by high-throughput chemical screening of a newly identified apoptotic gene marker.

We have reported that tacrolimus (FK506), an immunosuppressive drug, and diclofenac, a non-steroidal anti-inflammatory drug, possess different modes of neuroprotective action. FK506 suppresses only thapsigargin-induced apoptosis in neuroblastoma SH-SY5Y cells while diclofenac reverses tunicamycin-induced as well as thapsigargin-induced apoptosis. The aim of this study is to discover novel compounds that exert neuroprotective properties by using the transcriptional response of a newly identified gene, which was regulated by both FK506 and diclofenac, as a surrogate screening marker in high-throughput chemical screening and characterize the compounds in comparison with FK506 and diclofenac. Using a microarray with 4504 human cDNAs and quantitative RT-PCR, two genes as apoptotic markers, transmembrane protein 100 (TMEM100) and limb-bud and heart (LBH), were identified because the thapsigargin-induced elevations in their mRNA levels were reversed by both FK506 and diclofenac. A luciferase reporter assay with a TMEM100 promoter region was applied to high-throughput chemical screening. AS1219164, {3-[(E)-2-{5-[(E)-2-pyridin-4-ylvinyl]pyridin-3-yl} vinyl]aniline}, suppressed thapsigargin-induced transactivation of the TMEM100 gene and reversed thapsigargin-induced increases in TMEM100 and LBH mRNA levels in SH-SY5Y cells, similar to the effects of FK506 and diclofenac. Furthermore, AS1219164 protected against SH-SY5Y cell death induced by four apoptotic agents including thapsigargin, similar to diclofenac, but was more potent than diclofenac, while FK506 only showed protective effects against thapsigargin-induced cell death. In conclusion, a novel neuroprotecitve compound, AS1219164, was discovered by high-throughput chemical screening using a reporter assay with the TMEM100 gene promoter regulated by both FK506 and diclofenac. Reporter assay using the promoter region of a gene under pharmacological and physiological transcriptional regulation would be well suit for use in high-throughput chemical screening.

Urinary trypsin inhibitor suppresses migration of malignant mesothelioma.

Urinary trypsin inhibitor (UTI), an inhibitor of urokinase plasminogen activator relevant to proteolytic processing from the inactive into the active form of platelet-derived growth factor-D (PDGF-D) to activate PDGF-betabeta receptor (PDGF-betabetaR), inhibited fetal bovine serum-stimulated migration of human malignant mesothelioma, with the extent varying among the cell types. The more effective inhibition was found in NCIH-2052 and -2452 cells, with the higher expression of PDGF-betabetaR. The results of the present study suggest that UTI suppresses malignant mesothelioma cell migration by neutralizing active dimer of PDGF-D (PDGF-DD)/PDGF-betabetaR-mediated signal transduction.

Neuroprotective efficacy of the peroxisome proliferator-activated receptor delta-selective agonists in vitro and in vivo.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily and function as ligand-modulated transcription factors that regulate gene expression in many important biological processes. The PPARdelta subtype has the highest expression in the brain and is postulated to play a major role in neuronal cell function; however, the precise physiological roles of this receptor remain to be elucidated. Herein, we show that the high-affinity PPARdelta agonists L-165041 [4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)-propoxyl]phenoxy]-acetic acid] and GW501516 [2-methyl4-((4-methyl-2-(4-trifluoromethylphenyl)-1,3-triazol-5-yl)-methylsulfanyl)phenoxy acetic acid] protect against cytotoxin-induced SH-SY5Y cell injury in vitro and both ischemic brain injury and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in vivo. In the SH-SY5Y studies, treatment with L-165041 or GW501516 significantly and concentration-dependently attenuated cell death following thapsigargin, 1-methyl-4-phenylpyridinium, or staurosporine exposure, with the extent of damage correlated with the level of caspase-3 inhibition. In the transient (90 min) middle cerebral artery occlusion model of ischemic brain injury in rats, i.c.v. infusion of L-165041 or GW501516 significantly attenuated the ischemic brain damage measured 24 h after reperfusion. Moreover, the PPARdelta agonists also significantly attenuated MPTP-induced depletion of striatal dopamine and related metabolite contents in mouse brain. These results demonstrate that subtype-selective PPARdelta agonists possess antiapoptotic properties in vitro, which may underlie their potential neuroprotective potential in in vivo experimental models of cerebral ischemia and Parkinson's disease (PD). These findings suggest that PPARdelta agonists could be useful tools for understanding the role of PPARdelta in other neurodegenerative disorders, as well as attractive therapeutic candidates for stroke and neurodegenerative diseases such as PD.

CyclinB2 and BIRC5 genes as surrogate biomarkers for neurite outgrowth in SH-SY5Y subclonal cells.

Neurite outgrowth plays a key role in neuronal development and regeneration, and is the hallmark assay for the effects of neurotrophic factors such as nerve growth factor (NGF). However, measuring neurite outgrowth is a slow and resource-intensive process. We therefore wanted to identify surrogate biomarkers for neurite outgrowth activity by gene expression analysis in SH-O10 cells, a subclone of the human SH-SY5Y neuroblastoma cell line but with much higher NGF-induced neurite outgrowth activity. Microarray analysis identified seven genes where mRNA levels were changed. NGF-induced decreases in levels of two genes, CyclinB2 and BIRC5, were confirmed by quantitative real-time RT-PCR. Levels of NGF-induced decreases in CyclinB2 and BIRC5 mRNA in several SH-SY5Y subclones with different neurite outgrowth responses correlated with their neurite outgrowth activities. Decreases in CyclinB2 and BIRC5 mRNA induced by FK506 or retinoic acid, both of which exert potentiation of NGF-induced neurite outgrowth effects but with different mechanisms, also correlated with their neurite outgrowth activities. In conclusion, decreasing levels of CyclinB2 and BIRC5 mRNA strongly correlate with neurite outgrowth activities in terms of NGF-related effect in SH-SY5Y subclonal cells, and have potential to become quantitative surrogate biomarkers for measuring NGF-related neurite outgrowth.

Diclofenac, a non-steroidal anti-inflammatory drug, suppresses apoptosis induced by endoplasmic reticulum stresses by inhibiting caspase signaling.

Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently used in the treatment of inflammation and pain. In many reports, NSAIDs have induced apoptosis in a variety of cell lines such as colon cancer cells. On the other hand, more recently a few reports have found that NSAIDs protect against apoptosis. Here we investigate endoplasmic reticulum (ER)-stress-induced apoptosis of neuronal cells. The aim of this study is to examine the involvement of NSAIDs, in particular diclofenac, on ER-stress-induced apoptosis of human neuroblastoma SH-SY5Y cells. Diclofenac significantly suppressed SH-SY5Y cell death induced by two types of ER-stress-inducing agents: thapsigargin, an inhibitor of Ca2+-ATPase on the endoplasmic reticulum membrane, and tunicamycin, a glycosylation blocker. Other NSAIDs, such as indomethacin, ibuprofen, aspirin, and ketoprofen, also suppressed ER-stress-induced SH-SY5Y cell death. The dose-dependent anti-apoptotic effect of diclofenac did not correlate with the reduction of prostaglandin release. Administration of prostaglandin E2, which was a primary product of arachidonic metabolism, showed no effects against anti-apoptotic effects produced by diclofenac. Thapsigargin and tunicamycin each significantly activated caspase-3, -9, and -2 in the intrinsic apoptotic pathway in SH-SY5Y cells. Diclofenac suppressed the activation of caspases induced by both ER stresses. Thapsigargin and tunicamycin decreased the mitochondrial membrane potential in SH-SY5Y cells. Diclofenac suppressed the mitochondrial depolarization induced by both ER stresses. Diclofenac inhibited ER-stress-induced apoptosis of SH-SY5Y cells by suppressing the activation of caspases in the intrinsic apoptotic pathway. This is the first report to find that diclofenac has protective effects against ER-stress-induced apoptosis.

Differences in gene expression profile among SH-SY5Y neuroblastoma subclones with different neurite outgrowth responses to nerve growth factor.

Nerve growth factor (NGF) plays a key role in the differentiation of neurons. In this study, we established three NGF-induced neurite-positive (NIN+) subclones that showed high responsiveness to NGF-induced neurite outgrowth and three NGF-induced neurite-negative (NIN-) subclones that abolished NGF-induced neurite outgrowth from parental SH-SY5Y cells, and analyzed differences in the NGF signaling cascade. The NIN+ subclones showed enhanced responsiveness to FK506-mediated neurite outgrowth as well. To clarify the mechanism behind the high frequency of NGF-induced neurite outgrowth, we investigated differences in NGF signaling cascade among subclones. Expression levels of the NGF receptor TrkA, and NGF-induced increases in mRNAs for the immediate-early genes (IEGs) c-fos and NGF inducible (NGFI) genes NGFI-A, NGFI-B and NGFI-C, were identical among subclones. Microarray analysis revealed that the NIN+ cell line showed a very different gene expression profile to the NIN- cell line, particularly in terms of axonal vesicle-related genes and growth cone guidance-related genes. Thus, the difference in NGF signaling cascade between the NIN+ and NIN- cell lines was demonstrated by the difference in gene expression profile. These differentially expressed genes might play a key role in neurite outgrowth of SH-SY5Y cells in a region downstream from the site of induction of IEGs, or in a novel NGF signaling cascade.

Protective effect of FK506 against apoptosis of SH-SY5Y cells correlates with regulation of the serum inducible kinase gene.

Recently, we established an in vitro model of apoptosis induced by exposure of neuroblastoma SH-SY5Y cells to thapsigargin, an endoplasmic reticular calcium-ATPase inhibitor, and demonstrated that FK506 (tacrolimus) protected against apoptosis. The purpose of this paper was to investigate a possible correlation between the protective effect of FK506 against apoptosis and the regulation of the serum inducible kinase (SNK) and fibroblast growth factor inducible kinase (FNK) genes-which are polo-like kinases expressed abundantly in the brain by FK506. Thapsigargin increased the mRNA level of SNK and FNK in SH-SY5Y cells. FK506 inhibited the increase in SNK mRNA but not FNK mRNA. Deletion analysis of the SNK promoter showed that the promoter site, which was regulated by thapsigargin and FK506 in a calcineurin-dependent manner, is a cAMP response element (CRE)/activating transcription factor (ATF)-like element located 84 base pairs (bp) proximal to the transcriptional initiation site. Although transcription of the SNK gene was also regulated by tunicamycin, etoposide, or staurosporine, FK506 did not show any effects on these regulations. We recently reported that FK506 did not protect against apoptosis induced by these agents. These results indicate that the induction of SNK mRNA by thapsigargin in SH-SY5Y cells is regulated by FK506 via an inhibition of calcineurin at the transcriptional stage, and the transcriptional regulation of the SNK gene by FK506 was well correlated with the protective effect of the compound against apoptosis. Thus, transcriptional regulation of the SNK gene may be a biological marker for analysis of apoptosis of SH-SY5Y cells.

Suppressive effects of FR167653, an inhibitor of p38 mitogen-activated kinase, on calreticulin mRNA expression induced by endoplasmic reticulum stresses.

Several endoplasmic reticulum chaperones are simultaneously transactivated in response to various forms of endoplasmic reticulum stresses. Calreticulin is one such chaperone. We here show that the compound FR167653 [1-[7-(4-fluorophenyl)-1,2,3,4-tetrahydro-8-(4-pyridyl)pyrazolo[5,1-c][1,2,4]triazin-2-yl]-2-phenylethanedione sulfate monohydrate] suppresses the transactivation of calreticulin following endoplasmic reticulum stress. FR167653, like SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-imidazole], has been reported to inhibit p38 mitogen-activated kinase (p38 MAPK). In this study, FR167653 concentration-dependently inhibited the up-regulation of the calreticulin mRNA level following an endoplasmic reticulum stress induced by thapsigargin in human embryonic kidney 293 (HEK293) cells and rat phechromocytoma PC12 cells. The compound concentration-dependently suppressed the transactivation of luciferase by thapsigargin in a reporter assay with a calreticulin promoter-luciferase conjugated reporter vector. SB203580 also significantly suppressed the transactivation of calreticulin by thapsigargin. Therefore, FR167653 regulated the mRNA expression of calreticulin at the transcriptional level without affecting the stability of the mRNA, as well as via inhibition of p38 MAPK activated by thapsigargin. FR167653 also inhibited the transactivation of calreticulin stimulated by two other endoplasmic reticulum stress inducers, tunicamycin and A23187. Moreover, the inhibitory action of the compound on the transactivation was observed in other cell lines. The calreticulin promoter region includes three sequential cis-acting endoplasmic reticulum stress response elements (ERSEs). As each of these ERSEs was sequentially deleted, there was an increasing loss of the transactivation by thapsigargin or tunicamycin. FR167653 inhibited the transactivation in all the reporter plasmid constructs containing the calreticulin promoter region with an ERSE/ERSEs. In conclusion, FR167653 is the first compound shown to inhibit the transactivation of calreticulin following various endoplasmic reticulum stresses. The suppressive effects of the compound were considered to be due to an inhibition of the signaling leading to ERSEs activation in the calreticulin promoter region via an inhibition of p38 MAPK, which is activated by endoplasmic reticulum stresses.

Detailed in vitro pharmacological analysis of FK506-induced neuroprotection.

FK506, a calcineurin inhibitor, shows potent neuroprotective effects in animal models such as those of stroke and neurodegenerative diseases. However, the mechanism underlying these neuroprotective effects is unclear. In this study, an in vitro model, in which FK506 protected the cells against cell death, was established and analyzed in detail by pharmacological experiments. Thapsigargin (TG), an inhibitor of endoplasmic reticulum calcium-ATPase, induced SH-SY5Y cell death. FK506 concentration-dependently protected the cells from this type of death. In contrast, FK506 did not suppress SH-SY5Y cell death caused by the following molecules: tunicamycin (TM), an inhibitor of N-linked glycosylation; etoposide (Eto), a topoisomerase II inhibitor; and staurosporine (STS), a phospholipid/calcium-dependent protein kinase inhibitor. Additionally, FK506 did not inhibit TG-induced cell death in either SK-N-MC or HeLa cell lines. FK506 completely inhibited caspase-3 activation and apoptosis caused by TG in a concentration-dependent manner, but not that caused by TM, Eto, and STS. TG did not activate caspase-3 in SK-N-MC cells, although it slightly activated caspase-3 in HeLa cells. FK506 did not change caspase-3 activity in either SK-N-MC or HeLa cell lines. Cyclosporin A, another calcineurin inhibitor, showed the same results as FK506 in this study, whereas rapamycin, an immunosuppressant not associated with calcineurin activity, did not have any effect in this context. Thus, the suppressive effects of FK506 on cell death are specific to SH-SY5Y cells treated with TG and are caused by the inhibition of calcineurin and subsequent suppression of caspase-3 activation. Therefore, an in vitro system using SH-SY5Y cells treated with TG could provide a model reflective of certain aspects of the neuroprotective activity of FK506.

Discovery of novel inhibitors of inducible nitric oxide synthase.

We have discovered three compounds, 5-chloro-1,3-dihydro-2H-benzimidazol-2-one (FR038251), 1,3(2H,4H)-isoquinolinedione (FR038470) and 5-chloro-2,4(1H,3H)-quinazolonedione (FR191863), which show inhibition of inducible nitric oxide synthase (iNOS). The iNOS inhibitory activity of the compounds was examined in comparison with that of aminoguanidine, a representative iNOS inhibitor. FR038251, FR038470 and FR191863 inhibited mouse iNOS, with IC50 values of 1.7, 8.8 and 1.9 microM, respectively, in an in-vitro enzyme assay. These inhibitory activities are comparable with that of aminoguanidine (IC50 = 2.1 microM). The three compounds had iNOS selectivity 38- and 8-times, > 11- and 3-times, and 53- and 3-times compared with rat neuronal NOS and bovine endothelial NOS, respectively. These compounds concentration-dependently inhibited NO production in intact RAW264.7 mouse macrophages stimulated by lipopolysaccharide (LPS)/interferon-gamma. At 100 microM, FR038251, FR038470, FR191863 and aminoguanidine showed 81, 44, 54 and 78% inhibition of NO production, respectively. In an in-vivo experiment, FR038251, FR038470, FR191863 and aminoguanidine inhibited NO production in LPS-treated mice by 68, 40, 5 and 68% at an oral dose of 100 mg kg-1. The in-vivo inhibitory activity of FR038251 was almost identical to that of aminoguanidine. In conclusion, the three FR compounds are iNOS inhibitors with novel structures and may be candidate compounds leading to discovery of more iNOS inhibitors in the future.