Y-27632 Induces Neurite Outgrowth by Activating the NOX1-Mediated AKT and PAK1 Phosphorylation Cascades in PC12 Cells.

Y-27632 is known as a selective Rho-associated coiled coil-forming kinase (ROCK) inhibitor. Y-27632 has been shown to induce neurite outgrowth in several neuronal cells. However, the precise molecular mechanisms linking neurite outgrowth to Y-27632 are not completely understood. In this study, we examined the ability of Y-27632 to induce neurite outgrowth in PC12 cells and evaluated the signaling cascade. The effect of Y-27632 on the neurite outgrowth was inhibited by reactive oxygen species (ROS) scavengers such as N-acetyl cysteine (NAC) and trolox. Furthermore, Y-27632-induced neurite outgrowth was not triggered by NADPH oxidase 1 (NOX1) knockdown or diphenyleneiodonium (DPI), a NOX inhibitor. Suppression of the Rho-family GTPase Rac1, which is under the negative control of ROCK, with expression of the dominant negative Rac1 mutant (Rac1N17) prevented Y-27632-induced neurite outgrowth. Moreover, the Rac1 inhibitor NSC23766 prevented Y-27632-induced AKT and p21-activated kinase 1 (PAK1) activation. AKT inhibition with MK2206 suppressed Y-27632-induced PAK1 phosphorylation and neurite outgrowth. In conclusion, our results suggest that Rac1/NOX1-dependent ROS generation and subsequent activation of the AKT/PAK1 cascade contribute to Y-27632-induced neurite outgrowth in PC12 cells.

TLR4 Endogenous Ligand S100A8/A9 Levels in Adult-Onset Still's Disease and Their Association with Disease Activity and Clinical Manifestations.

S100A8/A9 has been suggested as a marker of disease activity in patients with adult-onset Still's disease (AOSD). We evaluated the clinical significance of S100A8/A9 as a biomarker and its pathogenic role in AOSD. Blood samples were collected prospectively from 20 AOSD patients and 20 healthy controls (HCs). Furthermore, skin and lymph node biopsy specimens of AOSD patients were investigated for S100A8/A9 expression levels via immunohistochemistry. Peripheral blood mononuclear cells (PBMCs) of active AOSD patients and HCs were investigated for S100A8/A9 cell signals. S100A8/A9, interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α) levels in active AOSD patients were higher than those of HCs. S100A8/A9 levels correlated positively with IL-1ß, TNF-α and C-reactive protein. The inflammatory cells expressing S100A8/A9 were graded from one to three in skin and lymph node biopsies of AOSD patients. The grading for S100A8/A9 was more intense in the skin lesions with karyorrhexis, mucin deposition, and neutrophil infiltration. Like lipopolysaccharide (LPS), S100A8/A9 induced phosphorylation of p38 and c-Jun amino-terminal kinase (JNK) in PBMCs, suggesting that S100A8/A9 activates Toll-like receptor 4 signaling pathways. These findings suggest that S100A8/A9 may be involved in the inflammatory response with induction of proinflammatory cytokines and may serve as a clinicopathological marker for disease activity in AOSD.

A dopamine-alpha-lipoic acid hybridization compound and its acetylated form inhibit LPS-mediated inflammation.

In the present study, we synthesized and evaluated the anti-inflammatory effects of dopamine and alpha-lipoic acid (ALA) hybrid compounds, ALA-dopamine (HBU-199) and its acetylated derivative, ALA-acetyl dopamine (HBU-200), in BV2 microglia and RAW264.7 macrophage cells. HBU-199 and HBU-200 both significantly and dose-dependently inhibited LPS-induced nitric oxide (NO) productions, NO synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-6 and interleukin-1ß mRNA expressions and iNOS and COX-2 protein expressions. Furthermore, HBU-199 and HBU-200 protected RAW264.7 cells from activation-induced cell death. However, at same concentrations, dopamine or ALA did not inhibit LPS-mediated production of inflammatory molecules and activation-induced cell death. HBU-199 and HBU-200 inhibited LPS-induced inhibition of inhibitory kappa-B-alpha (IκB-α) phosphorylation and nuclear factor-kappa B (NF-κB) activation. Furthermore, LPS-mediated DNA binding of p65 and p50 to the NF-κB binding site of the iNOS promoter was inhibited by HBU-199 and HBU-200, whereas dopamine and ALA did not inhibit LPS-induced NF-κB activation and IκB-α phosphorylation. Moreover, HBU-199 and HBU-200 suppressed LPS-stimulated phosphorylation of Akt, but not glycogen synthase kinase 3 beta. Overall, our data suggest that the ALA-dopamine hybrid compounds down-regulate inflammatory responses via inhibition of NF-κB and NF-κB-dependent gene expression, suggesting that it is a promising therapeutic agent for both systemic inflammatory diseases and inflammatory diseases of central nervous system.

Neuroprotective effect of 3-morpholinosydnonimine against Zn²âº-induced PC12 cell death.

Excessive intracellular accumulation of zinc (Zn(2+)) is neurotoxic and contributes to a number of neuropathological conditions. Here, we investigated the protective effect of 3-morpholinosydnonimine (SIN-1) against Zn(2+)-induced neuronal cell death in differentiated PC12 cells. We found that Zn(2+)-induced PC12 cell death was reduced in a concentration-dependent manner by pretreatment with SIN-1. The intracellular accumulation of Zn(2+) was not affected by pretreatment with SIN-1, indicating that SIN-1-induced neuroprotection was not attributable to reduced influx of Zn(2+) into cells. SIN-1C, the stable decomposition product of SIN-1, failed to prevent Zn(2+)-induced cell death. Furthermore, the protective effect of SIN-1 against Zn(2+)-induced PC12 cell death was almost completely abolished by uric acid, a free radical scavenger, suggesting that reactive oxygen and nitrogen species generated by SIN-1 may contribute to the protective effect. SIN-1 prevented the inactivation of glutathione reductase (GR) and the increase in the ratio of oxidized glutathione/total glutathione (GSSG/total GSH) induced by Zn(2+). Addition of membrane permeable GSH ethyl ester (GSH-EE) to PC12 cells prior to Zn(2+) treatment significantly increased cell viability. We therefore conclude that SIN-1 may exert neuroprotective effect against Zn(2+)-induced cell death in differentiated PC12 cells by preventing inhibition of GR and increase in GSSG/total GSH ratio.

Serum S100A12 may be a useful biomarker of disease activity in adult-onset Still's disease.

OBJECTIVE: S100A12 and soluble receptor for advanced glycation endproducts (sRAGE) have been suggested as biomarkers of disease activity in patients with systemic juvenile idiopathic arthritis. We investigated the clinical significance of these markers in adult-onset Still's disease (AOSD). METHODS: Blood samples were collected from 37 patients with active AOSD and 38 healthy controls (HC). Of the patients with AOSD, followup samples were collected from 19 patients after resolution of disease activity. RESULTS: Serum S100A12 (547.9 ± 148.4 ng/ml) in patients with AOSD was higher than those of HC (272.3 ± 133 ng/ml, p < 0.001). The sRAGE levels of AOSD (514.1 ± 273.6 pg/ml) were lower than those of HC (850.3 ± 405.8 pg/ml, p < 0.001). Serum S100A12 correlated with serum sRAGE (r = -0.228, p = 0.049). Serum S100A12 correlated with erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), ferritin, and systemic score, whereas sRAGE did not correlate with any disease activity markers. In addition, the level of S100A12 was decreased after disease activity was resolved in followed-up patients with AOSD (505.7 ± 161.3 ng/ml vs 361.3 ± 162.5 ng/ml, p = 0.01). Further, the change of S100A12 was well correlated with that of ESR, CRP, and systemic score. CONCLUSION: S100A12 levels showed strong correlations with known disease activity markers such as ESR, CRP, ferritin, and systemic score. In the followup patients with AOSD, most patients showed decreased S100A12 levels after resolution of disease activity. These results suggest that serum S100A12 can be a reliable clinical marker for monitoring disease activity and treatment response.

Serum growth arrest-specific protein 6 levels are elevated in adult-onset Still's disease.

We investigated the growth arrest-specific protein 6 in adult-onset Still's disease. Serums were collected from 52 adult-onset Still's disease patients with follow-up samples of 21 patients. The growth arrest-specific protein 6 levels in adult-onset Still's disease were higher compared to those in the normal controls (25.37±7.71 vs. 19.86±5.01 ng/mL, p<0.001). However, growth arrest-specific protein 6 did not correlate with disease activity. Also, growth arrest-specific protein 6 was not decreased after activity was resolved in the follow-up. The growth arrest-specific protein 6 in adult-onset Still's disease patients were higher than the normal controls. However, growth arrest-specific protein 6 was not correlated with disease activity.

"Chemical-pain sensor" based on nanovesicle-carbon nanotube hybrid structures.

We developed a "chemical-pain sensor" that could recognize chemical pain stimuli such as capsaicin and resiniferatoxin just like mammalian chemical pain sensory systems. Here, we first prepared nanovesicles containing rat pain sensory receptor, rat transient receptor potential vanilloid 1 (rTRPV1), which is activated by noxious heat and capsaicin. And the nanovesicles were immobilized on a single-walled carbon nanotube-based field effect transistor. The chemical-pain sensor could selectively detect chemical pain stimuli with a high sensitivity of a 1 pM detection limit. It also responded to different chemical pain stimuli in a manner similar as to that of mammalian chemical pain sensory systems. This sensor platform can be utilized for various practical applications such as food screening tools and artificial somesthetic sensors. Moreover, TRP families have been suggested as potential drug targets related to nerve and circulation disorders. Thus, the capability of monitoring TRP responses using our sensor platforms should provide a powerful means for the development of new drugs as well as the basic research about nerve and circulation systems.

Serum growth arrest-specific protein 6 levels are a reliable biomarker of disease activity in systemic lupus erythematosus.

PURPOSE: Growth arrest-specific protein 6 (Gas6) has been suggested to be a biomarker of disease activity in patients with systemic lupus erthematosus (SLE). We investigated the clinical significance of this protein in Korean SLE. METHODS: Blood samples were collected from 150 SLE patients and 50 normal controls (NC). In addition, follow-up samples were collected from 50 SLE patients. RESULTS: Serum Gas6 levels of SLE patients (43.01 ± 28.02 ng/mL) were higher than those of NC (20.15 ± 9.23 ng/mL, p<0.001). When evaluated sensitivity and specificity of the Gas6 for diagnosing SLE using ROC curves, the sensitivity and specificity were 72.7 % and 84 % with a cut-off value of 25.3 ng/mL. In the ROC analysis of Gas6, anti-dsDNA antibody, ESR, complement 3 and complement 4 to identify patients with active lupus, area under the curve (AUC) of Gas6 was highest with 0.763. Serum Gas6 levels were significantly higher in the patients with serositis (70.04 ± 30.85 ng/mL) and renal disorder (65.66 ± 32.28 ng/mL) compared to those without (41.88 ± 27.44 ng/mL, p=0.033, 40.3 ± 26.33 ng/mL, p=0.001, respectively). Gas6 levels were correlated positively with anti-dsDNA antibody (r=0.199, p=0.015), ESR (r=0.204, p=0.013) and SLEDAI (r=0.512, p<0.001). In addition, serum Gas6 levels were correlated negatively with hemoglobin (r= -0.165, p=0.043), lymphocyte count (r= -0.165, p=0.043), complement 3 (r= -0.343, p<0.001) and complement 4 (r= -0.316, p<0.001). Furthermore, change in serum Gas6 levels was correlated with change in SLEDAI levels in the SLE patients that were followed up (r=0.524, p<0.001). CONCLUSION: These results suggest that serum Gas6 can be a reliable clinical marker for monitoring disease activity and treatment response in SLE.

Xanthorrhizol induces apoptosis through ROS-mediated MAPK activation in human oral squamous cell carcinoma cells and inhibits DMBA-induced oral carcinogenesis in hamsters.

Xanthorrhizol, a natural sesquiterpenoid compound isolated from Curcuma xanthorrhiza Roxb, has been known to inhibit the growth of human colon, breast, liver and cervical cancer cells. In this study, xanthorrhizol decreased cell viability, induced apoptosis and decreased the level of full-length PARP in SCC-15 oral squamous cell carcinoma (OSCC) cells. A decrease in cell viability and PARP degradation was not prevented by treatment with the caspase inhibitor Z-VAD-fmk in xanthorrhizol-treated cells. Xanthorrhizol treatment elevated intracellular Ca(2+) and ROS levels in SCC-15 cells. Treatment with a Ca(2+) chelator, EGTA/AM, did not affect xanthorrhizol- induced cytotoxicity, but cell viability was partly recovered by treatment with endogenous antioxidant, GSH, or hydroxy radical trapper, MCI-186. Furthermore, the viability of xanthorrhizol-treated SCC-15 cells was significantly restored by treatment with SB203580 and/or SP600125 but not significantly by PD98059 treatment. Xanthorrhizol-induced activation of p38 MAPK and JNK was blocked by MCI-186. Finally, xanthorrhizol suppressed the number of tumors in buccal pouches and increased the survival rate in hamsters treated with 7,12-dimethylbenz[a]anthracene. In conclusion, xanthorrhizol may induce caspase-independent apoptosis through ROS-mediated p38 MAPK and JNK activation in SCC-15 OSCC cells and prevent chemical-induced oral carcinogenesis. Therefore, xanthorrhizol seems to be a promising chemopreventive agent.

Activation of Rac1-dependent redox signaling is critically involved in staurosporine-induced neurite outgrowth in PC12 cells.

Staurosporine, a non-specific protein kinase inhibitor, has been shown to induce neurite outgrowth in PC12 cells, but the mechanism by which staurosporine induces neurite outgrowth is still obscure. In the present study, we investigated whether the activation of Rac1 was responsible for the neurite outgrowth triggered by staurosporine. Staurosporine caused rapid neurite outgrowth independent of the ERK signaling pathways. In contrast, neurite outgrowth in response to staurosporine was accompanied by activation of Rac1, and the Rac1 inhibitor NSC23766 attenuated the staurosporine-induced neurite outgrowth in a concentration-dependent manner. In addition, suppression of Rac1 activity by expression of the dominant negative mutant Rac1N17 also blocked the staurosporine-induced morphological differentiation of PC12 cells. Staurosporine caused an activation of NADPH oxidase and increased the production of reactive oxygen species (ROS), which was prevented by NSC23766 and diphenyleneiodonium (DPI), an NADPH oxidase inhibitor. Staurosporine-induced neurite outgrowth was attenuated by pretreatment with DPI and exogenous addition of sublethal concentration of H2O2 accelerated neurite outgrowth triggered by staurosporine. These results indicate that activation of Rac1, which leads to ROS generation, is required for neurite outgrowth induced by staurosporine in PC12 cells.

Serum S100A8/A9, but not follistatin-like protein 1 and interleukin 18, may be a useful biomarker of disease activity in adult-onset Still's disease.

OBJECTIVE: S100A8/A9, follistatin-like protein 1, and interleukin 18 (IL-18) have been suggested as biomarkers of disease activity in patients with systemic juvenile idiopathic arthritis or adult-onset Still's disease (AOSD). We investigated the clinical significance of these factors in AOSD. METHODS: Blood samples were collected from 36 patients with AOSD, 40 patients with rheumatoid arthritis (RA), and 33 healthy controls. Of the patients with AOSD, followup samples were collected from 16 patients after resolution of disease activity. RESULTS: Serum levels of S100A8/A9 (11.77 ± 8.84 µg/ml) in AOSD patients were higher than those in RA patients (3.53 ± 3.43 µg/ml; p < 0.001) and controls (2.49 ± 1.83 µg/ml; p < 0.001). Follistatin-like protein 1 levels in AOSD were not different from those in RA and controls. IL-18 levels in AOSD (7560.3 ± 7577.6 pg/ml) were higher than those in RA (217.7 ± 292.1 pg/ml; p < 0.001) and controls (139.2 ± 86.2 pg/ml; p < 0.001). The sensitivity and specificity of IL-18 for diagnosing AOSD was highest with a cutoff value of 366.1 pg/ml. Serum S100A8/A9 correlated with leukocyte count, erythrocyte sedimentation rate, C-reactive protein, ferritin, and systemic disease score; however, IL-18 correlated only with ferritin and systemic disease score. S100A8/A9 was decreased after disease activity was resolved in followup of AOSD patients (9.96 ± 7.35 µg/ml in active AOSD vs 3.6 ± 4.77 µg/ml in resolved cases; p = 0.001). The change of S100A8/A9 was well correlated with that of systemic disease score. CONCLUSION: The data suggest that serum S100A8/A9 may be a useful biomarker for evaluating disease activity in patients with AOSD.

C-reactive protein is a more sensitive and specific marker for diagnosing bacterial infections in systemic lupus erythematosus compared to S100A8/A9 and procalcitonin.

OBJECTIVE: C-reactive protein (CRP), S100A8/A9, and procalcitonin have been suggested as markers of infection in patients with systemic lupus erythematosus (SLE). We investigated the clinical significance of these factors for indication of infection in SLE. METHODS: Blood samples were prospectively collected from 34 patients with SLE who had bacterial infections and 39 patients with SLE who had disease flares and no evidence of infection. A second set of serum samples was collected after the infections or flares were resolved. RESULTS: CRP levels of SLE patients with infections were higher than those with flares [5.9 mg/dl (IQR 2.42, 10.53) vs 0.06 mg/dl (IQR 0.03, 0.15), p < 0.001] and decreased after the infection was resolved. S100A8/A9 and procalcitonin levels of SLE patients with infection were also higher [4.69 µg/ml (IQR 2.25, 12.07) vs 1.07 (IQR 0.49, 3.05) (p < 0.001) and 0 ng/ml (IQR 0-0.38) vs 0 (0-0) (p < 0.001), respectively]; these levels were also reduced once the infection disappeared. In the receiver-operating characteristics analysis of CRP, S100A8/A9, and procalcitonin, the area under the curve was 0.966 (95% CI 0.925-1.007), 0.732 (95% CI 0.61-0.854), and 0.667 (95% CI 0.534-0.799), respectively. CRP indicated the presence of an infection with a sensitivity of 100% and a specificity of 90%, with a cutoff value of 1.35 mg/dl. CONCLUSION: Our data suggest that CRP is the most sensitive and specific marker for diagnosing bacterial infections in SLE.

Carmustine induces ERK- and JNK-dependent cell death of neuronally-differentiated PC12 cells via generation of reactive oxygen species.

Accumulation of reactive oxygen species (ROS) caused by the inhibition of glutathione reductase (GR) has been proposed as one of the mechanisms responsible for carmustine (1,3-bis(2-chloroethyl)-1-nitrosourea, BCNU)-induced cytotoxicity. Since mitogen-activated protein kinases (MAPKs) are known to mediate ROS-dependent cell death in multiple cell types, we examined whether redox-sensitive MAPK activation mediated the carmustine-induced cell death of neuronally differentiated PC12 cells. Carmustine induced a concentration- and time-dependent cell death, which was associated with increased caspase-3 activation, a reduction in GR activity accompanied by a concomitant decrease in reduced glutathione levels, and accumulation of ROS. Carmustine-induced caspase-3 activation and cell death were prevented by pretreatment with anti-oxidants or a reducing agent, indicating that carmustine-induced caspase-3 activation and cell death occur via redox-dependent processes. Carmustine induced phosphorylation of the MAPKs, such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. The activation of these kinases was inhibited by pretreatment with N-acetyl-L-cysteine (NAC). Although all the MAPKs were activated by carmustine, only the inhibitors of JNK and ERK prevented carmustine-induced cell death and caspase-3 activation. Our data suggest that carmustine-induced neurotoxicity is, at least in part, due to the activation of ROS-dependent JNK and ERK signaling.

1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one induces cell cycle arrest and apoptosis in HeLa cells by preventing microtubule polymerization.

1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) is known as a specific inhibitor of soluble guanylyl cyclase (sGC). Previously, however, ODQ was reported to induce cell death via sGC-dependent and sGC-independent means in a variety of cell types. The aim of this study was to investigate the mechanism by which ODQ induces cell death in HeLa cells. Treatment of HeLa cells with ODQ induced a concentration-dependent decrease in cell viability over the range from 10 to 100 µM. DNA fragmentation and fluorescence-activated cell sorting analysis using annexin V and propidium iodide staining revealed that ODQ triggered apoptosis at concentrations of 50 and 100 µM within 24 to 48 h. The addition of 8-Br-cGMP in the presence of ODQ failed to rescue HeLa cells from death, suggesting that the inhibition of sGC was not responsible for the pro-apoptotic action of ODQ. ODQ arrested the cell cycle at the G2/M phase and caused disassembly of the microtubule network. This process was reversed by dithiothreitol. In addition, ODQ was shown to inhibit the polymerization of purified tubulin, and this was also prevented by dithiothreitol. These results indicate that ODQ inhibits microtubule assembly by direct oxidation of tubulin, induces cell cycle arrest at the G2/M phase, and triggers apoptosis in HeLa cells.

Pharmacological characterization of rebamipide: its cholecystokinin CCK1 receptor binding profile and effects on Ca2+ mobilization and amylase release in rat pancreatic acinar cells.

We previously reported that rebamipide (2-(4-chlorobenzoylamino)-3-[2(1H)-quinolinon-4-yl]-propionic acid) generated oscillations of intracellular Ca2+ concentration ([Ca2+]i) probably through the activation of cholecystokinin type 1 (CCK1) receptors in rat pancreatic acinar cells. Therefore, in the present study, we aimed to establish the pharmacological characteristics of rebamipide in rat pancreatic acinar cells. CCK-8S and rebamipide inhibited [125I]BH-CCK-8S binding to rat pancreatic acinar cell membranes with IC50 values of 3.13 nM and 37.7 microM, respectively. CCK-8S usually evoked [Ca2+]i oscillations at concentrations lower than 50 pM, and it induced biphasic [Ca2+]i increases at higher concentrations. In contrast to CCK-8S, rebamipide only induced [Ca2+]i oscillations at all the concentrations we used in this study. In addition, rebamipide was shown to inhibit high concentrations of CCK-8S-induced biphasic increases in [Ca2+]i, suggesting that rebamipide might be a partial agonist at cholecystokinin CCK1 receptors. Although rebamipide induced [Ca2+]i oscillations by activating the cholecystokinin CCK1 receptors, rebamipide did not cause amylase release and only inhibited CCK-stimulated amylase release reversibly and dose-dependently. However, rebamipide did not inhibit carbachol-, vasoactive intestinal polypeptide (VIP)-, and forskolin-induced amylase releases. These data indicate that rebamipide functions as a partial agonist for Ca2+ -mobilizing action, and it is also an antagonist for the amylase-releasing action of CCK.

Partial inhibition of SERCA is responsible for extracellular Ca2+ dependence of AlF-4-induced [Ca2+]i oscillations in rat pancreatic.

AlF4-is known to generate oscillations in intracellular Ca2+ concentration ([Ca2+]i) by activating G proteins in many cell types. However, in rat pancreatic acinar cells, AlF4--evoked [Ca2+]i oscillations were reported to be dependent on extracellular Ca2+, which contrasts with the [Ca2+]i oscillations induced by cholecystokinin (CCK). Therefore, we investigated the mechanisms by which AlF4- generates extracellular Ca2+-dependent [Ca2+]i oscillations in rat pancreatic acinar cells. AlF4(-)-induced [Ca2+]i oscillations were stopped rapidly by the removal of extracellular Ca2+ and were abolished on the addition of 20 mM caffeine and 2 microM thapsigargin, indicating that Ca2+ influx plays a crucial role in maintenance of the oscillations and that an inositol 1,4,5-trisphosphate-sensitive Ca2+ store is also required. The amount of Ca2+ in the intracellular Ca2+ store was decreased as the AlF4--induced [Ca2+]i oscillations continued. Measurement of 45Ca2+ influx into isolated microsomes revealed that AlF4-directly inhibited sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). The activity of plasma membrane Ca2+-ATPase during AlF4- stimulation was not significantly different from that during CCK stimulation. After partial inhibition of SERCA with 1 nM thapsigargin, 20 pM CCK-evoked [Ca2+]i oscillations were dependent on extracellular Ca2+. This study shows that AlF4- induces [Ca2+]i oscillations, probably by inositol 1,4,5-trisphosphate production via G protein activation but that these oscillations are strongly dependent on extracellular Ca2+ as a result of the partial inhibition of SERCA.