Orange juice and its component, hesperidin, decrease the expression of multidrug resistance-associated protein 2 in rat small intestine and liver.

We investigated the effects of orange juice (OJ) or hesperidin, a component of OJ, on the pharmacokinetics of pravastatin (PRV) and the expression of both protein and mRNA of multidrug resistance-associated protein 2 (Mrp2) in the rat small intestine and liver. Eight-week-old male Sprague-Dawley rats were used in this study. OJ or a 0.079% hesperidin suspension was administered orally for 2 days. Tap water was given as a control. A single dose of PRV at 100 mg/kg p.o. was administered after 2 days of OJ, hesperidin, or tap water ingestion. The AUC, C(max), and t(1/2) values of PRV were significantly increased in OJ group. Mrp2 protein and mRNA levels in the small intestine and liver, respectively, were significantly decreased after the ingestion of OJ. The same results were obtained with hesperidin. These results suggest that the changes in PRV pharmacokinetic parameters and the decrease in Mrp2 expression caused by OJ are due to hesperidin in the juice.

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.

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.

Individual metabolic capacity evaluation of cytochrome P450 2C19 by protein and activity in the small intestinal mucosa of Japanese pancreatoduodenectomy patients.

Some P450 enzymes are expressed not only in the liver but also in the small intestine, and these enzymes play an important role in first-pass drug metabolism in the small intestine. Cytochrome P450 (CYP)2C19 has been confirmed to exist in the small intestine of white people, but not yet in Japanese. We investigated the mRNA level, protein level, and activity of CYP2C19 in the small intestine in a Japanese population. Samples were obtained from the healthy portions of resected small intestines from 18 patients who had undergone pancreatoduodenectomy. The microsomes were extracted from the epithelium of the small intestinal tissues. CYP2C19 mRNA and protein levels were analyzed using real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. CYP2C19 activity in the microsomes was evaluated based on the 5-hydroxylation of lansoprazole using HPLC. CYP2C19 mRNA and protein levels and activities in the small intestine showed interindividual differences. CYP2C19 mRNA levels were not correlated with protein levels or its activity. On the other hand, there was significant correlation between CYP2C19 protein levels and its activity. Further, CYP2C19 protein levels and activities in the small intestine were approximately equal to those in liver. These results suggest the metabolic capacity of CYP2C19 in Japanese small intestine may play as important a role as the liver in drug metabolism. Analyses of the protein level or protein activity of CYP2C19 rather than its mRNA level should be required for predicting the individual metabolic capacity of CYP2C19 in the small intestine.

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).

Limited effects of frequent CYP2D6*36-*10 tandem duplication allele on in vivo dextromethorphan metabolism in a Japanese population.

PURPOSE: although CYP2D6*36 was thought to be one of the alleles causing the poor metabolizer phenotype, several in vitro studies clarified that the enzyme produced by CYP2D6*36 showed enzymatic activities. However, the effects of CYP2D6*36 in tandem with CYP2D6*10 on the in vivo CYP2D6 activity have been unclear. In this study, we investigated in vivo metabolic capacities of CYP2D6 among the subjects carrying different numbers of CYP2D6*36 in tandem with CYP2D6*10. METHODS: we measured the metabolic ratio (MR) of dextromethorphan in 98 subjects. We determined the CYP2D6 genotype of these subjects, including allelic copy number of CYP2D6*10 and CYP2D6*36 by direct sequencing, TaqMan assay, and real-time Invader assay. RESULTS: single copies of CYP2D6*10 and tandem duplication of CYP2D6*36-*10 alleles were found at frequencies of 8.7 and 32.7%, respectively. Median dextromethorphan MRs of the subjects carrying CYP2D6*10 and CYP2D6*36-*10 were not significantly different (P = 0.24). CONCLUSIONS: CYP2D6*36 in tandem with CYP2D6*10 plays a minor role in interindividual variation of dextromethorphan metabolism in vivo.

Effects of a calcium-channel blocker (CV159) on hepatic ischemia/reperfusion injury in rats: evaluation with selective NO/pO2 electrodes and an electron paramagnetic resonance spin-trapping method.

Nitric oxide (NO) and the partial pressure of oxygen (pO(2)) in the liver were simultaneously quantified in rats with partial hepatic ischemia/reperfusion injury (PHIRI). Real-time NO/pO(2) monitoring and immunohistochemical analysis for superoxide dismutase and inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) were performed to evaluate the protective effects of a dihydropyridine-type calcium-channel blocker--CV159--on PHIRI. Serum high-mobility-group box-1 (HMGB-1) was measured to assess cellular necrosis. Moreover, we used in vitro/ex vivo electron paramagnetic resonance spin trapping to assess the hydroxyl radical (*OH)-scavenging activity (OHSA) of CV159 and the liver tissue. The NO levels were significantly higher in CV159-treated rats than in control rats throughout the ischemic phase. Immediately after reperfusion, the levels temporarily increased in waves and then gradually decreased in the treated rats but remained constant in the control rats. pO(2) was continually higher in the treated rats. In these rats, hepatic eNOS expression increased, whereas iNOS expression decreased. The treated rats exhibited significantly higher cytosolic and mitochondrial concentrations NOx (NO(2)+NO(3)). The serum HMGB-1 levels significantly decreased in the treated rats. Moreover, CV159 directly scavenged *OH and both mitochondrial and cytosolic OHSA were preserved in the treated rats. Thus, CV159-mediated inhibition of intracellular Ca(2+) overloading may effectively minimize organ damage and also have *OH-scavenging activity and the cytoprotective effects of eNOS-derived NO.

NF-kB signaling in cardiomyocytes is inhibited by sevoflurane and promoted by propofol.

Both inhalational and intravenous anesthetics affect myocardial remodeling, but the precise effect of each anesthetic on molecular signaling in myocardial remodeling is unknown. Here, we performed in silico analysis to investigate signaling alterations in cardiomyocytes induced by inhalational [sevoflurane (Sevo)] and intravenous [propofol (Prop)] anesthetics. Bioinformatics analysis revealed that nuclear factor-kappa B (NF-kB) signaling was inhibited by Sevo and promoted by Prop. Moreover, nuclear accumulation of p65 and transcription of NF-kB-regulated genes were suppressed in Sevo-administered mice, suggesting that Sevo inhibits the NF-kB signaling pathway. Our data demonstrate that NF-kB signaling is inhibited by Sevo and promoted by Prop. As NF-kB signaling plays an important role in myocardial remodeling, our results suggest that anesthetics may affect myocardial remodeling through NF-kB.

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.

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.

Potential of lansoprazole as a novel probe for cytochrome P450 3A activity by measuring lansoprazole sulfone in human liver microsomes.

Cytochrome P450 (CYP) 3A enzymes are responsible for the metabolism of many drugs. It is useful to know CYP3A activity in individual patients undergoing drug therapy so as to predict the efficacies or adverse events. Lansoprazole is metabolized to Lansoprazole sulfone (LS) by CYP3A, while to 5-hydroxylansoprasole by CYP2C19. The aim of this study was to evaluate whether lansoprazole can be used to assess CYP 3A activity in human liver. Lansoprazole sulfoxidation activity in 14 human liver microsomes was determined as the ratio of lansoprazole/LS, measuring these parameters by high-performance liquid chromatography. Testosterone 6beta-hydroxylation (T6beta-OH) activity, a known marker for CYP3A activity was also measured together with lansoprazole sulfoxidation activity. Lansoprazole sulfoxidation activity was also analyzed in microsomes preincubat-ed with anti-CYP2C19 antibody. Interindividual variation was observed in lansoprazole sulfoxidation activity and T6beta-OH activities of those microsomes, respectively. Lansoprazole sulfoxidation activity was significantly correlated with T6beta-OH activity and CYP3A protein level. Lansoprazole sulfoxidation activity in microsomes with anti-CYP2C19 antibody was closely correlated with T6beta-OH activity. In contrast, lansoprazole 5-hydroxylation activity was correlated with the CYP2C19 activity. These results suggest that metabolism of lansoprazole to LS by CYP3A occurs independently of metabolism by CYP2C19. LS can be used as a new marker of CYP3A activity.

Stress-related over-enhancement of the hypothalamic-pituitary-adrenal axis causes experimental neurolathyrism in rats.

Neurolathyrism is a motor neuron disease that is caused by the overconsumption of grass peas (Lathyrus sativus L.) under stressful conditions. The neuro-excitatory ß-N-oxalyl-L-α,ß-diaminopropionic acid present in grass peas was proposed the causative agent of spastic paraparesis of the legs. Historical reports of neurolathyrism epidemics, studies of neurolathyrism animal models, and in vitro studies on the mechanism of ß-N-oxalyl-L-α,ß-diaminopropionic acid toxicity support the hypothesis that stress increases susceptibility to neurolathyrism. To elucidate the role of stress in neurolathyrism-induced motor dysfunction, we focused on the hypothalamic-pituitary-adrenal axis in a rodent model of neurolathyrism. Our results implicated increased glucocorticoid and neuroinflammation in the motor dysfunction (paraparesis) exhibited by the stress loaded rat models of neurolathyrism.

Alterations of glucose-dependent and -independent bladder smooth muscle contraction in spontaneously hypertensive and hyperlipidemic rat.

Alteration of bladder contractility was examined in the spontaneously hypertensive and hyperlipidemic rat (SHHR; age, 9 months; systolic blood pressure, >150 mm Hg; plasma cholesterol, >150 mg/dl). Carbachol (CCh) induced time- and dose-dependent contractions in Sprague-Dawley (age-matched control) rats and SHHR; however, maximal levels differed significantly (13.3 +/- 2.2 and 5.4 +/- 1.9 microN/mm(2) following 10 microM CCh treatment, respectively; n = 5). This difference, which was maintained in calcium-replaced physiological salt solution (PSS), was suppressed by pretreatment with rho kinase inhibitor, 1 microM Y27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide]; moreover, total activity of rho kinase was also reduced in SHHR bladder. Pretreatment of bladders under high-glucose (HG) conditions (22.2 mM glucose-contained PSS for 30 min) led to enhancement of CCh-induced contraction solely in control animals. Under HG conditions, both protein kinase C (PKC) activity and production of diacylglycerol (DG) derived from incorporated glucose declined in SHHR bladder; however, sustained elevation of plasma glucose level was not detected in SHHR. These results suggested that bladder contractility dysfunction in SHHR is attributable to alteration of rho kinase activity and the DG-PKC pathway. This dysfunction may occur prior to chronic hyperglycemia onset in progressive hypertension and hyperlipidemia.

Matrix metalloproteinase-9 in spontaneously hypertensive hyperlipidemic rats.

The presence of hypertension and hyperlipidemia accelerates atherosclerosis and increases the risk of ocular disease. Since there were few rat models for atherosclerosis, spontaneously hypertensive rats (SHRs) and spontaneously hyperlipidemic rats (HLRs) were crossbred to obtain a new model: the spontaneously hypertensive hyperlipidemic rat (SHHR). Matrix metalloproteinases (MMPs) play an important role in ocular degeneration. The purpose of this study is to investigate changes in the MMP activities in vitreous and plasma as well as MMP expression in the retinas of SHHRs, which served as a model of vascular degeneration. We used 8-month-old Wistar-Kyoto (WKY) rats and Sprague-Dawley (SD) rats, SHRs, HLRs, and SHHRs. The MMP-2 and MMP-9 activities in plasma and vitreous were examined by zymography. The mRNA expression of MMP-2, MMP-9, and tissue inhibitor of metalloproteinases-3 (TIMP-3) in retina was examined by quantitative PCR. The localized expression of MMP-9 in the retinas was examined by immunostaining. The MMP-9 activity increased significantly in SHHRs compared with all other rats. MMP-9 was observed mainly at the superficial layer of the retina on immunostaining. The MMP-2, MMP-9, and TIMP-3 mRNA in retina was not significantly different in SHHRs as compared with all other rats. Increased MMP-9 activity in vitreous was influenced more intensely from plasma than retina because there was no change in MMP-9 expression in retina, and MMP-9 immunostaining was observed mainly at the surface of the retina, where blood vessels are present. In this study, the complications of hypertension and hyperlipidemia induced increased MMP-9 activity in vitreous and plasma. It is therefore suggested that MMP-9 may be involved in causing this result and in the development of retinal disease.

Calcium/calmodulin-dependent protein kinase II regulates the phosphorylation of CREB in NMDA-induced retinal neurotoxicity.

We examined the role of the phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII) and cyclic AMP-response element binding protein (CREB) in N-methyl-d-aspartate (NMDA)-induced neurotoxicity in the rat retina. Western blot analysis showed early elevation of phosphorylated CaMKII (p-CaMKII) protein levels and subsequential elevation of phosphorylated CREB (p-CREB) protein after NMDA injection. Immunohistochemistry showed that p-CaMKII was colocalized with Thy-1-positive retinal ganglion cells (RGCs) after NMDA injection. The increase in the p-CaMKII protein level was significantly inhibited by the preinjection of CaMKII small interfering RNA (siRNA), whereas negative control siRNA did not affect. Moreover, the increase in the p-CREB protein level after NMDA injection was also prevented by preinjection of CaMKII siRNA. In addition, our morphometric study of neurotracer retrograde labeling and Thy-1-positive cells showed that CaMKII siRNA significantly accelerated NMDA-induced RGC loss. Furthermore, the prevention of CREB binding by CRE decoy oligonucleotide also exacerbated RGC loss. These results suggest that the activation of CaMKII may regulate CREB phosphorylation and that the transient phosphorylation of CaMKII and CREB may be a neuroprotective response against NMDA-induced neurotoxicity.

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.

Fasudil attenuates sympathetic nervous activity in the adrenal medulla of spontaneously hypertensive rats.

We investigated the effects of fasudil, a Rho kinase inhibitor, on hypertension in spontaneously hypertensive rats and on the catecholamine synthetic pathway. Ten-week-old male SHR and Wistar-Kyoto rats were administered fasudil (10 mg/kg/day s.c.) for 4 days. Systolic blood pressure was measured using the tail-cuff method. Catecholamine levels were measured with high-performance liquid chromatography-ECD methods. Tyrosine hydroxylase protein levels were measured in Western blot analysis. The tyrosine hydroxylase mRNA level was measured using real-time PCR methods. Fasudil significantly decreased systolic blood pressure in spontaneously hypertensive rats, but not in Wistar-Kyoto rats. Fasudil also significantly decreased catecholamine, tyrosine hydroxylase protein, and tyrosine hydroxylase mRNA levels in the adrenal medulla of spontaneously hypertensive rats. These results suggest that the depressor effects of fasudil on hypertension in spontaneously hypertensive rats may be related to inhibition of the catecholamine synthetic pathway.

Development of hyperfibrinogenemia in spontaneously hypertensive and hyperlipidemic rats: a potentially useful animal model as a complication of hypertension and hyperlipidemia.

According to current concepts, hypertension and hyperlipidemia cause vascular damage that leads to a hypercoagulative state. In this study, we investigated whether spontaneously hypertensive and hyperlipidemic rats (SHHR) can be a useful experimental model for complications in combined hypertension and hyperlipidemia, by comparing coagulative and fibrinolytic activities in SHHR with those in spontaneously hypertensive rats (SHR) and spontaneously hyperlipidemic rats (HLR). We measured coagulation and fibrinolysis markers in plasma and levels of fibrinogen and prothrombin mRNA in livers of eight-month-old male Wistar Kyoto rats (WKY), Sprague-Dawley rats (SD), SHR, HLR and SHHR. The plasma levels of fibrinogen in SHR, HLR and SHHR were significantly higher than those in WKY and SD, and were highest in SHHR. Higher plasma levels of antithrombin III and plasminogen were detected in increasing order in SHR, HLR and SHHR as compared to those in WKY and SD. Hepatic mRNA expressions of fibrinogen chains and prothrombin were enhanced in SHR, HLR and SHHR, resulting in increased plasma fibrinogen levels in SHHR. These results suggest that hypertension and hyperlipidemia can each cause hypercoagulation, with hyperlipidemia being a stronger factor than hypertension. Since a greater hypercoagulative state is a complication of combined hypertension and hyperlipidemia, the SHHR model is a good system for studying the early stage of atherosclerosis ensuing from hyperfibrinogenemia.

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.

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.