A new bioinformatics approach identifies overexpression of GRB2 as a poor prognostic biomarker for prostate cancer.

A subset of prostate cancer displays a poor clinical outcome. Therefore, identifying this poor prognostic subset within clinically aggressive groups (defined as a Gleason score (GS) ≧8) and developing effective treatments are essential if we are to improve prostate cancer survival. Here, we performed a bioinformatics analysis of a TCGA dataset (GS ≧8) to identify pathways upregulated in a prostate cancer cohort with short survival. When conducting bioinformatics analyses, the definition of factors such as "overexpression" and "shorter survival" is vital, as poor definition may lead to mis-estimations. To eliminate this possibility, we defined an expression cutoff value using an algorithm calculated by a Cox regression model, and the hazard ratio for each gene was set so as to identify genes whose expression levels were associated with shorter survival. Next, genes associated with shorter survival were entered into pathway analysis to identify pathways that were altered in a shorter survival cohort. We identified pathways involving upregulation of GRB2. Overexpression of GRB2 was linked to shorter survival in the TCGA dataset, a finding validated by histological examination of biopsy samples taken from the patients for diagnostic purposes. Thus, GRB2 is a novel biomarker that predicts shorter survival of patients with aggressive prostate cancer (GS ≧8).

A bioinformatics-to-clinic sequential approach to analysis of prostate cancer biomarkers using TCGA datasets and clinical samples: a new method for precision oncology?

Biomarker-driven cancer therapy has met with significant clinical success. Identification of a biomarker implicated in a malignant phenotype and linked to poor clinical outcome is required if we are to develop these types of therapies. A subset of prostate adenocarcinoma (PACa) cases are treatment-resistant, making them an attractive target for such an approach. To identify target molecules implicated in shorter survival of patients with PACa, we established a bioinformatics-to-clinic sequential analysis approach, beginning with 2-step in silico analysis of a TCGA dataset for localized PACa. The effect of candidate genes identified by in silico analysis on survival was then assessed using biopsy specimens taken at the time of initial diagnosis of localized and metastatic PACa. We identified PEG10 as a candidate biomarker. Data from clinical samples suggested that increased expression of PEG10 at the time of initial diagnosis was linked to shorter survival time. Interestingly, PEG10 overexpression also correlated with expression of chromogranin A and synaptophysin, markers for neuroendocrine prostate cancer, a type of treatment-resistant prostate cancer. These results indicate that PEG10 is a novel biomarker for shorter survival of patients with PACa. Also, PEG10 expression at the time of initial diagnosis may predict focal neuroendocrine differentiation of PACa. Thus, PEG10 may be an attractive target for biomarker-driven cancer therapy. Thus, bioinformatics-to-clinic sequential analysis is a valid tool for identifying targets for precision oncology.

Proteomic analysis of differential protein expression by brain metastases of gynecological malignancies.

Brain metastases of gynecological malignancies are rare, but the incidence is increasing. Patients with brain metastases have a poor prognosis, therefore early detection and optimal management is necessary. In order to determine a new biomarker, we aimed to identify proteins that associated with brain metastases. We investigated proteins associated with brain metastases of gynecological malignancies in three patients who underwent surgical resection (stage IIb cervical cancer, stage Ib endometrial cancer, and stage IIIb ovarian cancer). Proteomic analysis was performed on formalin-fixed paraffin-embedded (FFPE) samples of the primary tumors and brain metastases, which were analyzed by liquid chromatography with tandem mass spectrometry. Thereafter, candidate proteins were identified by the Scaffold system and Mascot search program, and were analyzed using western blotting and immunohistochemistry. As a result, a total of 129 proteins were identified. In endometrial and ovarian cancers, western blotting revealed that the expression of alpha-enolase (ENO1) and triosephosphate isomerase (TPI-1) was higher and the expression of Transgelin-2 (TAGLN2) was lower in metastatic tumors than in primary tumors. On the other hand, the expression of TPI-1 and TAGLN2 was lower in metastatic tumors than in primary tumors in cervical cancer. Immunohistochemistry confirmed that ENO1 expression was elevated in the metastatic tumors compared with the primary tumors. In conclusion, the present study showed that FFPE tissue-based proteomics analysis can be powerful tool, and these findings suggested that ENO1, TPI-1, and TAGLN2 may have a role in the development and progression of brain metastasis from gynecological malignancies.

The Rho kinase inhibitor fasudil is involved in p53-mediated apoptosis in human hepatocellular carcinoma cells.

PURPOSE: Rho kinase is an important factor in tumor progression. We demonstrated that Rho kinase-associated coil-containing protein kinase (ROCK) is expressed in hepatic tissues in hepatocellular carcinoma (HCC) and confirmed its roles in cell survival in HCC cells using the ROCK inhibitor, fasudil. METHODS: ROCK protein levels were estimated in hepatic tissues with HCC compared with healthy liver tissues or hepatic hemangioma tissues using immunohistochemistry. Furthermore, HepG2 and Huh7 cells were cultured with ROCK inhibitor, fasudil for 24 h in vitro. Cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt assay, and apoptotic cells were detected by cell death ELISA. The expression apoptosis-related proteins were analyzed using Western blotting. RESULTS: Fasudil significantly decreased cell proliferation and induced apoptosis mediated by increases in p53, Bax, caspase-9, and caspase-3 in HepG2 and Huh7 cells. The induction of apoptosis was inhibited in HCC cells precultured with p53 decoy oligodeoxynucleotide. CONCLUSION: These results suggest that ROCK inhibits the p53-mediated apoptosis pathway in HCC. Fasudil may thus be a beneficial approach to HCC therapy.

17ß-estradiol prevents reduction of retinal phosphorylated 14-3-3 zeta protein levels following a neurotoxic insult.

Previous studies demonstrated the substantial protective role of 17ß-estradiol (E2) in several types of neuron, although its mechanism of action remains to be elucidated. In this study, we found that the levels of 14-3-3 zeta mRNA and phosphorylated and total 14-3-3 zeta proteins were significantly decreased in the rat retina after intravitreal injection of N-methyl-d-aspartate (NMDA). 17ß-E2 implantation significantly inhibited NMDA-induced decreases in phosphorylated but not in total 14-3-3 zeta protein levels in the retina. There was a decrease in both phosphorylated and total 14-3-3 protein levels in RGC-5 cells, a retinal ganglion cell line, after glutamate and buthionine sulfoximine (BSO) exposure, and 17ß-E2 treatment significantly inhibited only the decrease in phosphorylated but not in total 14-3-3 zeta protein levels. The cell viability assay showed substantial cell death after glutamate and BSO exposure and that 17ß-E2 treatment significantly protects against this cell death. 17ß-E2 treatment also significantly increased the level of phosphorylated 14-3-3 protein in RGC-5 cells without other treatments. These results suggest that a decrease in 14-3-3 zeta expression may be associated with retinal neurotoxicity induced by NMDA or the combination of glutamate and BSO. The regulation of 14-3-3 zeta phosphorylation is one possible mechanism of the protective effect of 17ß-E2 in the retina.

Comparison of the effects of omeprazole and rabeprazole on ticlopidine metabolism in vitro.

The thienopyridine derivative ticlopidine (TCL) is an inhibitor of adenosine diphosphate-induced platelet aggregation. Combination therapy with a thienopyridine derivative and aspirin is standard after coronary stenting, although more hemorrhagic complications occur with the combination therapy than with aspirin alone. A proton pump inhibitor (PPI) is required for prevention or treatment of upper gastrointestinal bleeding in such cases. We examined the effects of PPIs [omeprazole (OPZ) and rabeprazole (RPZ)] on TCL metabolism using pooled human liver microsomes prepared from various human liver blocks and 12 individual human liver microsomes. We calculated the K(i) values of each PPI for TCL metabolic activity and compared the inhibitory effect of each PPI on TCL metabolism. The K(i) values of OPZ and RPZ were 1.4 and 12.7 µM, respectively. The inhibitory effect of OPZ (78.6 ± 0.05%) was significantly greater than that of RPZ (24.2 ± 0.05%) (P < 0.001). Interestingly, a negative correlation existed between the inhibitory effect of OPZ and CYP2C19 activity (r = -0.909, P < 0.001). These results suggest that the inhibitory effect of OPZ is more potent than that of RPZ in vitro. In conclusion, RPZ appears preferable when administering TCL, aspirin, and a PPI in combination.

Axonal protection by 17ß-estradiol through thioredoxin-1 in tumor necrosis factor-induced optic neuropathy.

Axonal degeneration often leads to the death of neuronal cell bodies. Previous studies demonstrated the substantial protective role of 17ß-estradiol (E2) in several types of neuron. However, most studies examined cell body protection, and the role of 17ß-E2 in axonal degeneration of retinal ganglion cells (RGC) remains unclear. In this study, we showed the presence of thioredoxin-1 (Trx1) in the optic nerve axons and found that the levels of Trx1 protein were significantly decreased in isolated RGC and the optic nerve after intravitreal injection of TNF, which was shown previously to induce optic nerve degeneration and subsequent loss of RGC. These changes were concomitant with disorganization of the microtubules with neurofilament accumulation, which were blocked by 17ß-E2 implantation. 17ß-E2 treatment also totally abolished TNF-induced decreases in Trx1 protein levels in isolated RGC and the optic nerve. The induction of Trx1 by 17ß-E2 in the optic nerve was significantly inhibited by simultaneous injection of Trx1 small interfering RNA (siRNA) with TNF. Up-regulation of Trx1 by 17ß-E2 in RGC-5 cells was prevented by Trx1 siRNA treatment. 17ß-E2 significantly prevented TNF-induced axonal loss, and this axonal-protective effect was inhibited by intravitreal injection of Trx1 siRNA. This finding was also supported by the quantification of microtubules and neurofilaments. These results suggest that a Trx1 decrease in RGC bodies and their axons may be associated with TNF-induced optic nerve axonal degeneration. Axonal protection by 17ß-E2 may be related to its regulatory effect on Trx1 induction.

Comparative study of culture conditions for maintaining CYP3A4 and ATP-binding cassette transporters activity in primary cultured human hepatocytes.

The aim of this study was to determine suitable culture conditions for maintaining the activity of cytochrome p450 (CYP) 3A4 and drug transporters in primary cultured human hepatocytes. Human hepatocytes were isolated using the two-step collagenase perfusion technique and were cultured with four different media, serum-free William's E medium (serum-free WEM), WEM containing fetal calf serum (FCS-WEM), WEM with human serum (HS-WEM), and Lanford's medium. The albumin levels were maintained for 7 days in hepatocytes. Although CYP3A4 mRNA levels gradually decreased from 3 days, CYP3A4 and hepatocyte nuclear factor-4α alpha protein levels and activities were maintained for 7 days in hepatocytes cultured with serum-free WEM and Lanford's but not in those with FCS-WEM and HS-WEM. Furthermore, CYP3A4 protein levels were significantly increased by the addition of rifampicin and dexamethasone to the culture media, indicating that the induction potential was maintained. The protein levels of P-glycoprotein, multi-drug-resistance-2, and breast cancer-resistance protein were maintained for 7 days in all media. Serum-free WEM and Lanford's also maintained protein levels of CYP2C19, CYP2D6, and organic anion transporter polypeptide in the hepatocytes. Serum-free WEM and Lanford's may be appropriate culture media for maintaining CYP3A4 and drug transporter protein levels in primary cultured hepatocytes.

Axonal and cell body protection by nicotinamide adenine dinucleotide in tumor necrosis factor-induced optic neuropathy.

Axonal degeneration often leads to the death of neuronal cell bodies. Previous studies have demonstrated the crucial role of nicotinamide adenine dinucleotide (NAD) biosynthesis in axonal protection of motor neurons, but the role of nicotinamide mononucleotide adenylyltransferase 1 and NAD in optic nerve degeneration is unclear. Intravitreal injection of tumor necrosis factor (TNF) induces optic nerve degeneration and subsequent loss of retinal ganglion cells. We found that the levels of nicotinamide mononucleotide adenylyltransferase 1 mRNA and protein and of NAD were significantly decreased in the optic nerve after intravitreal injection of TNF in rats. The concomitant disorganization of microtubules with vacuoles and neurofilament accumulations in the axons were blocked by exogenous NAD treatment. Nicotinamide adenine dinucleotide also prevented TNF-induced axonal loss and delayed retinal ganglion cell loss 2 months after TNF injection. Microglia identified by immunohistochemistry were increased in the optic nerves after TNF injection; this increase was inhibited by NAD treatment. These results suggest that axonal nicotinamide mononucleotide adenylyltransferase 1 and NAD declines are associated with TNF-induced optic nerve axonal degeneration and that axonal protection of NAD may be related to its inhibitory effect on microglial activation.

Antenatal glucocorticoid therapy increase cardiac alpha-enolase levels in fetus and neonate rats.

AIMS: Antenatal glucocorticoid therapy has been shown to prevent acute diseases including infant respiratory distress syndrome and reduce mortality, although little is known about the effects on cardiac function-related proteins in the fetus or neonate. We investigated whether cardiac function-related proteins were altered in cardiac tissues of fetuses and neonates born to pregnant rats treated by glucocorticoid. MAIN METHODS: Dexamethasone (DEX) was administered to pregnant rats for 2 days on day 17 and 18 or day 19 and 20 of gestation to simulate antenatal DEX therapy, and cardiac tissues of 19- and 21-day fetuses and 1-, 3-, and 5-day neonates were analyzed using a proteomic technique with liquid chromatography-mass spectrometry/mass spectrometry. KEY FINDINGS: The identified five proteins; alpha-enolase, creatine kinase-M type, beta-tubulin, troponin T, and ATP synthase beta-chain, were significantly increased in fetal cardiac tissues with DEX administration. We observed that significant increase of alpha-enolase in the 19-day fetuses by DEX using Western blotting and immunohistochemistry. ATP and cAMP levels were also increased in the fetal heart tissue. In addition, pyruvate levels were significantly increased in the fetus groups by DEX. SIGNIFICANCE: These results suggest that increased alpha-enolase may contribute to acceleration of glycolysis in the preterm heart.

Axonal protection by brain-derived neurotrophic factor associated with CREB phosphorylation in tumor necrosis factor-alpha-induced optic nerve degeneration.

Brain-derived neurotrophic factor (BDNF) is a potent survival and developmental factor that is regulated by cyclic AMP-response element binding protein (CREB) and has a protective effect against retinal ganglion cell (RGC) death. However, the effect of BDNF on the optic nerve axonal degeneration remains to be examined. In this study, we show that intravitreal injection of tumor necrosis factor (TNF)-alpha induces transient increases in phosphorylated-CREB (p-CREB) and BDNF expression in the optic nerve. Administration of exogenous BDNF further increased the p-CREB and endogenous BDNF level and exerted a neuroprotective effect against TNF-alpha-induced axonal loss. The increases in BDNF mRNA and protein induced by TNF-alpha were inhibited significantly by a CRE decoy oligonucleotide. The protective effect of exogenous BDNF on axons was also inhibited by the CRE decoy oligonucleotide. These results suggest that the protective effect of exogenous BDNF may be associated with increases in CREB phosphorylation and endogenous BDNF in the optic nerve.

Overexpression of heat shock protein 27 in squamous cell carcinoma of the uterine cervix: a proteomic analysis using archival formalin-fixed, paraffin-embedded tissues.

Proteomic analysis of squamous cell carcinoma of the uterine cervix was performed using total protein from archival formalin-fixed, paraffin-embedded tissues. A wide range of proteins with molecular weights of 10 to greater than 200 kd was extracted from formalin-fixed, paraffin-embedded tissues using a recently developed protocol based on the heat-induced antigen retrieval technique. The extracted proteins from normal squamous epithelium (n = 53) and squamous cell carcinoma (n = 21) were fluorescently labeled and separated using 2-dimensional gel electrophoresis. We identified 728 differentially expressed proteins, with 144 up-regulated and 584 down-regulated as compared with normal squamous epithelial tissue samples. Nine proteins showing pronounced up-regulation in squamous cell carcinoma were analyzed on liquid chromatography-tandem mass spectrometry. Among the candidate proteins identified, minichromosome maintenance 8, a disintegrin and metalloproteinase domain 18, and heat shock protein 27 were analyzed in Western blotting, resulting in significant overexpression of heat shock protein 27 in squamous cell carcinoma over normal mucosa (P < .05). Furthermore, immunostaining revealed heat shock protein 27 overexpression not only in squamous cell carcinoma but in various stages of cervical intraepithelial neoplasia (grades 1-3, n = 90), including dysplasia and carcinoma in situ. The expression levels of heat shock protein 27 in cervical intraepithelial neoplasia grades 1 to 3 and squamous cell carcinoma were significantly higher than that in normal mucosa (P < .05). In the neoplastic lesions, heat shock protein 27 expression levels in cervical intraepithelial neoplasia grade 3 and squamous cell carcinoma were significantly higher than that in cervical intraepithelial neoplasia grade 1 (P < .05). These results may suggest a role of heat shock protein 27 in tumor development and progression in the cervical intraepithelial neoplasia-squamous cell carcinoma sequence. Future experiments using formalin-fixed, paraffin-embedded tissue-based proteomic analysis will be a powerful tool for various pathologic studies.

Irinotecan-induced apoptosis is inhibited by increased P-glycoprotein expression and decreased p53 in human hepatocellular carcinoma cells.

Irinotecan, a DNA topoisomerase I inhibitor, is widely used in cancer chemotherapy. However, little is known of the mechanisms of its antitumor effects and the development of drug resistance in human hepatocellular carcinoma (HCC). In this study, we investigated the effects of short-term culture with SN-38, the active metabolite of irinotecan, on apoptosis in Huh7 cells. The cells were cultured with SN-38 for 24, 72, and 120 h, and apoptosis was determined using the terminal dUTP nick-end labeling (TUNEL) assay. The expressions of p53, apoptosis-related proteins, and P-glycoprotein (P-gp), a protein conferring the multidrug-resistant phenotype, were analyzed using Western blotting. Induced expression of P-gp was detected using fluorescence microscopy. SN-38 significantly induced apoptosis in Huh7 cells at 24 h. SN-38 also increased the expression of p53, Bax, and caspase-9 and decreased Bcl-xL expression in Huh7 cells. SN-38 decreased p53 expression and increased P-gp expression after 120 h, resulting in inhibition of apoptosis. This inhibition was reversed by the addition of verapamil to the culture medium during 120 h incubation. SN-38-induced P-gp expression was additionally enhanced by p53 decoy oligodeoxynucleotide. The changes in P-gp expression were directly moderated by p53 gene downregulation, suggesting that it plays a role in the mechanism of drug resistance. These results suggest that the accumulation of irinotecan in HCC leads to the development of drug resistance.

Irinotecan activates p53 with its active metabolite, resulting in human hepatocellular carcinoma apoptosis.

The topoisomerase I inhibitor irinotecan is widely used in anticancer therapy, although the detailed mechanism is still unclear. We investigated the apoptotic mechanisms of irinotecan in human hepatocellular carcinoma (HCC) cell lines (Huh7). SN-38 caused a significant decrease in cell proliferation and induced apoptosis in Huh7 cells and HepG2 cells. SN-38 significantly increased the expression of p53 protein and its phosphorylation at Ser(15) in the nucleus and apoptosis-inducing proteins Bax, caspase-9, and caspase-3, while it significantly decreased the antiapoptosis protein Bcl-xL of Huh7 cells. SN-38-induced apoptosis was recovered after p53 antisense oligodeoxynucleotide (AS ODN) pretreatment, while Huh7 cells were precultured with p53 AS ODN, followed by the addition of SN-38 for 24 h. Furthermore, increases in p53 DNA-binding activity were observed in the nuclei of Huh7 cells after SN-38 treatment as shown by electrophoretic mobility shift analysis. SN-38 binding motifs were detected in the proximal promoter of p53 (bases -433 to -317 and -814 to -711). These results suggest that the p53-mediated apoptosis pathway is important in the anticancer effects of irinotecan in hepatocellular carcinoma.

Hypertension aggravates glomerular dysfunction with oxidative stress in a rat model of diabetic nephropathy.

Oxidative stress may contribute to the pathogenesis of diabetic nephropathy (DN), although the detailed mechanism of reactive oxygen species (ROS) regulation is still unclear. This study examined the effect of high-salt diet on ROS production and expression of antioxidant enzymes in control and experimentally diabetic rats. Wistar fatty rats (WFR) as a type 2 diabetes mellitus model and Wistar lean rats (WLR) as a control were fed a normal-salt diet (NS) and high-salt diet (HS) from the age of 6 to 14 weeks. We then examined the blood pressure, urinary albumin excretion (UAE), and urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels. The expression of antioxidant enzymes including alpha-catalase (CAT), Cu-Zn superoxide dismutase (SOD), Mn SOD, and glutathione peroxidase (GPx) were analyzed in the glomeruli of the rats using Western blotting. The expression of NAD(P)H oxidase p47(phox) and NFkappaB p65 was evaluated using immunohistochemical staining. By 14 weeks of age, the WFR-HS group exhibited hypertension and markedly increased UAE. The level of 8-OHdG, a marker of oxidative damage, in the WFR-HS group was also higher than that in the WLR groups or WFR-NS group. The expression of alpha-CAT and Mn SOD proteins was significantly decreased in isolated glomeruli in the WFR-HS group. GPx and Cu-Zn SOD expression did not differ between the WFR and WLR groups. High expression of ROS and decreases in antioxidants were seen in the glomeruli of diabetic rats with hypertension, suggesting that oxidative stress may be involved in the development of DN.

The elucidation of the mechanism of weight gain and glucose tolerance abnormalities induced by chlorpromazine.

Antipsychotic drugs induce weight gain and metabolic abnormalities. Recently, the role of adipocytokines secreted from adipocytes in the development of metabolic syndrome has received attention. The aim of this study was to investigate the effects of chlorpromazine (Cp) on body weight, glucose, lipid metabolism, and adipocytokine secretion in rats fed sucrose. Wistar rats received 15% sucrose (Suc group), 15% sucrose and Cp at 7.5 mg/kg per day (Suc + Cp group), or Cp alone (Cp group) in water for 10 weeks. Fasting glucose levels in the Suc and Suc + Cp groups were significantly higher than in the control (Cont) group. Fasting insulin levels in the Suc, Suc + Cp, and Cp groups were also significantly higher than in the Cont group. The adiponectin level in the Suc group was significantly higher than in the Cont group, although the adiponectin level in the Suc + Cp group was not. Furthermore, the plasma tumor necrosis factor (TNF)-alpha level in the Suc + Cp group was significantly higher than in the Suc group. These data suggest that Cp inhibits the compensatory response of adiponectin with respect to obesity due to increased expression of plasma TNF-alpha level. Cp may exert more harmful effects on the glucose level and insulin resistance than on other factors, which may be one of the mechanisms responsible for the metabolic syndrome induced by antipsychotic agents.

Involvement of TNF-alpha in glutamate-induced apoptosis in a differentiated neuronal cell line.

We examined the involvement of tumor necrosis factor (TNF)-alpha on glutamate-induced cytotoxicity in a differentiated neuronal cell line. In this study, we used nerve growth factor (NGF)-differentiated PC12h cells. Glutamate cytotoxicity was assessed using the MTS and TUNEL assays. To detect TNF-alpha levels in culture supernatants after glutamate exposure, we used ELISA methods. The involvement of caspase-8, which is downstream from TNF receptor 1 (TNF-R1) in glutamate-induced cytotoxicity, was determined by Western blot analysis. The MTS assay showed that the addition of glutamate resulted in dose-dependent cell death, while the TUNEL assay showed that glutamate induced apoptosis in differentiated PC12h cells in a dose-dependent manner. TNF-alpha levels in the supernatant of glutamate-exposed cells were significantly increased compared with those in unexposed cells. In addition, glutamate caused increases in the levels of caspase-8 protein. The increases in caspase-8 levels were ameliorated by pretreatment with soluble TNF-R1. Moreover, soluble TNF-R1 significantly ameliorated the cell death induced by glutamate. These results suggest that TNF-alpha released from neuronal cells may be associated with glutamate-induced neuronal cell death.

Pro-apoptotic role of c-Jun in NMDA-induced neurotoxicity in the rat retina.

We examined the role of c-Jun on N-methyl-D-aspartate (NMDA)-induced neurotoxicity in the rat retina. An increase in c-Jun mRNA, c-Jun protein and phosphorylated c-Jun (p-c-Jun) levels in the retina was detected 3 hr after intravitreal injection of NMDA (20 nmol). These levels peaked after 12 hr, and then returned to their control levels by 24 hr. c-Jun and p-c-Jun immunoreactivities were observed in the retinal ganglion cell layer (RGCL), especially in retinal ganglion cells (RGCs), and in the inner nuclear layer (INL) 12 hr after NMDA injection, and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL)-positive cells were immunopositive for c-Jun and p-c-Jun. A c-Jun antisense oligodeoxynucleotide (AS ODN), which was simultaneously injected with NMDA, penetrated the cells in the RGCL and the INL, suppressed the NMDA-induced increase in c-Jun and p-c-Jun protein levels and reduced the number of TUNEL-positive cells in the RGCL 12 hr after the injection. The protective effect of c-Jun AS ODN on the NMDA-treated retina was also shown by the RGCL cell count and measurement of the IPL thickness, as well as by quantitative real-time PCR analysis of Thy-1 mRNA 7 days after the injection. These results suggest that c-Jun synthesis and phosphorylation participate in NMDA-induced neuronal cell death.

Neuroprotective effect of atrial natriuretic peptide against NMDA-induced neurotoxicity in the rat retina.

Atrial natriuretic peptide (ANP) can regulate aqueous humor production in the eye and has recently been suggested to play some functional roles in the retina. It has also been reported that ANP increases tyrosine hydroxylase (TH) mRNA levels and intracellular dopamine levels in PC12 cells. The effect of ANP on TH levels and the role of ANP in retinal excitotoxicity remain unknown. In this study, we investigated the effects of ANP on TH expression and dopamine levels in rat retina after intravitreal injection of NMDA. Immunohistochemistry localized natriuretic peptide receptor-A (NPRA) in the ganglion cell layer (GCL), the inner nuclear layer (INL) and the outer nuclear layer (ONL) in the rat retina. Quantitative real-time PCR and Western blot analysis showed a dramatic reduction in retinal TH levels 5 days after NMDA injection, while ANP, at a concentration of 10(-4) M, ameliorated this reduction in TH mRNA and TH protein levels. High-performance liquid chromatography (HPLC) analysis showed that NMDA reduced dopamine levels in the retina, and that ANP attenuated this reduction. Moreover, morphological analysis showed that ANP ameliorated NMDA-induced neurotoxicity through NPRA. The ameliorative effect of ANP was inhibited by a dopamine D(1) receptor antagonist. These results suggest that ANP may have a neuroprotective effect through possible involvement of dopamine induction.

Orange juice increased the bioavailability of pravastatin, 3-hydroxy-3-methylglutaryl CoA reductase inhibitor, in rats and healthy human subjects.

Our objective was to investigate the effects of orange juice on the pharmacokinetics of pravastatin in rats and healthy volunteers. The pharmacokinetics of pravastatin (100 mg/kg p.o.) were assessed with water, orange juice, and carbohydrates (12.5 ml/kg over 30 min) and with acetic acid (0.1 M, pH 3.44). The pharmacokinetics of simvastatin (100 mg/kg p.o.) were assessed with water and orange juice. In addition, the pharmacokinetics (based on plasma levels) of pravastatin 80 mg/kg i.v. were assessed with water and orange juice (5 ml/kg) in rats. The pharmacokinetics of oral pravastatin (10 mg) were assessed when administered with water and orange juice (800 ml over 3 h) in a two-way crossover study in 14 healthy volunteers. Orange juice significantly increased the area under the curve (0-150 min) of pravastatin in rats. Orange juice had no effects on the pharmacokinetic parameters of intravenously administered pravastatin in rats. Carbohydrates and acetic acid with pH and concentration equivalent to those of orange juice also resulted in no statistically significant differences in pravastatin pharmacokinetic parameters in rats. Orange juice did not result in any significant differences in the pharmacokinetic parameters of simvastatin in rats. Orange juice significantly increased oatp1 and oatp2 mRNA and protein in the intestine of rats. Orange juice significantly increased the area under the curve (0-240 min) of pravastatin in healthy volunteers. In conclusion, orange juice increases the bioavailability of pravastatin administered orally. Oatp1 and oatp2 may be related to increases of pharmacokinetics of pravastatin by orange juice.