FSH1 overexpression triggers apoptosis in Saccharomyces cerevisiae.

FSH1 belongs to the family of serine hydrolases in yeast and is homologous to the human ovarian tumor suppressor gene (OVAC2). Our preliminary results showed that cells lacking Fsh1p exhibit an increase in cell growth, and a decrease in the expression of AIF1 and NUC1 (apoptosis responsive genes) when compared to the wild type cells. Growth inhibition of cells overexpressing FSH1 is due to induction of cell death associated with cell death markers typical of mammalian apoptosis namely DNA fragmentation, phosphatidylserine externalization, ROS accumulation, Cytochrome c release, and altered mitochondrial membrane potential. When wild type cells were overexpressed with FSH1 there was up regulation of AIF1 level when compared to control cells suggesting that overexpression of FSH1 regulated cell death in yeast.

Grim-19 expressed by recombinant adenovirus for esophageal neoplasm target therapy.

Esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EA) are the two most common types of esophageal cancer, which is the sixth highest cause of cancer­associated mortality and the eighth most common cancer worldwide. Gene associated with retinoid­interferon (IFN)­induced mortality­19 (Grim­19) is reported to be a cell death activator that may be used to define mechanisms involved in IFN­ß­ and retinoic acid­induced cell death and apoptosis in a number of tumor cell lines. The present study constructed a recombinant adenovirus expressing Grim­19 (rAd­Grim­19) and investigated its therapeutic outcomes in ESCC cells and tumor­bearing mice. Grim­19 expression was detected in EC­109 (ESCC) cells by reverse transcription­quantitative polymerase chain reaction and western blot analysis. Tumor cell death and apoptosis induced by rAd­Grim­19 in EC­109 cells were analyzed by flow cytometry. The inhibitory effects of rAd­Grim­19 on EC­109 growth were determined by MTT assays. Furthermore, the therapeutic effects of rAd­Grim­19 were investigated in EC­109­bearing mice. The results demonstrated that Grim­19 mRNA and protein expression was downregulated in EC­109 esophageal carcinoma cells compared with Het­1A normal esophageal epithelial cells. In addition, EC­109 cells exhibited a significant reduction in tumor cell growth in the rAd­Grim­19 group compared with the control groups. Furthermore, rAd­Grim­19 increased EC­109 cell apoptosis compared with the control group. These results indicated that rAd-Grim-19 may regulate tumor cell growth and apoptosis. Additionally, the results demonstrated that rAd­Grim­19 led to beneficial outcomes and prolonged the survival of esophageal tumor­bearing mice. In conclusion, the present study demonstrated that rAd­Grim­19 may have potential as an antitumor agent for esophageal neoplasms and may therefore be beneficial for patients with esophageal neoplasms.

Overexpression of CaAPX Induces Orchestrated Reactive Oxygen Scavenging and Enhances Cold and Heat Tolerances in Tobacco.

Ascorbate peroxidase (APX) acts indispensably in synthesizing L-ascorbate (AsA) which is pivotal to plant stress tolerance by detoxifying reactive oxygen species (ROS). Enhanced activity of APX has been shown to be a key step for genetic engineering of improving plant tolerance. However it needs a deeper understanding on the maintenance of cellular ROS homeostasis in response to stress. In this study, we identified and characterized an APX (CaAPX) gene from Camellia azalea. Quantitative real-time PCR (qRT-PCR) analysis showed that CaAPX was expressed in all tissues and peaked in immature green fruits; the expression levels were significantly upregulated upon cold and hot stresses. Transgenic plants displayed marked enhancements of tolerance under both cold and heat treatments, and plant growth was correlated with CaAPX expression levels. Furthermore, we monitored the activities of several ROS-scavenging enzymes including Cu/Zn-SOD, CAT, DHAR, and MDHAR, and we showed that stress tolerance was synchronized with elevated activities of ROS-scavenging. Moreover, gene expression analysis of ROS-scavenging enzymes revealed a role of CaAPX to orchestrate ROS signaling in response to temperature stresses. Overall, this study presents a comprehensive characterization of cellular response related to CaAPX expression and provides insights to breed crops with high temperature tolerances.

Capsaicin Inhibited Aggressive Phenotypes through Downregulation of Tumor-Associated NADH Oxidase (tNOX) by POU Domain Transcription Factor POU3F2.

Capsaicin has been reported to preferentially inhibit the activity of tumor-associated NADH oxidase (tNOX), which belongs to a family of growth-related plasma membrane hydroquinone oxidases in cancer/transformed cells. The inhibitory effect of capsaicin on tNOX is associated with cell growth attenuation and apoptosis. However, no previous study has examined the transcriptional regulation of tNOX protein expression. Bioinformatic analysis has indicated that the tNOX promoter sequence harbors a binding motif for POU3F2, which is thought to play important roles in neuronal differentiation, melanocytes growth/differentiation and tumorigenesis. In this study, we found that capsaicin-mediated tNOX downregulation and cell migration inhibition were through POU3F2. The protein expression levels of POU3F2 and tNOX are positively correlated, and that overexpression of POU3F2 (and the corresponding upregulation of tNOX) enhanced the proliferation, migration and invasion in AGS (human gastric carcinoma) cells. In contrast, knockdown of POU3F2 downregulates tNOX, and the cancer phenotypes are affected. These findings not only shed light on the molecular mechanism of the anticancer properties of capsaicin, but also the transcription regulation of tNOX expression that may potentially explain how POU3F2 is associated with tumorigenesis.

Resveratrol Protects against Titanium Particle-Induced Aseptic Loosening Through Reduction of Oxidative Stress and Inactivation of NF-κB.

Aseptic implant loosening is closely associated with chronic inflammation induced by implant wear debris, and reactive oxygen species (ROS) play an important role in this process. Resveratrol, a plant compound, has been reported to act as an antioxidant in many inflammatory conditions; however, its protective effect and mechanism against wear particle-induced oxidative stress remain unknown. In this study, we evaluated resveratrol's protective effects against wear particle-induced oxidative stress in RAW 264.7 macrophages. At non-toxic concentrations, resveratrol showed dose-dependent inhibition of nitric oxide (NO) production, ROS generation, and lipid peroxidation. It also downregulated the gene expression of oxidative enzymes, including inducible nitric oxide synthase (iNOS) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)-1 and NOX-2, and promoted the gene expression and activities of antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), and glutathione peroxidase (GPx). This protective effect against wear particle-induced oxidative stress was accompanied by a reduction of gene expression and release of tumor necrosis factor-α (TNF-α), and decreased gene expression and phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). These findings demonstrate that resveratrol can inhibit wear particle-induced oxidative stress in macrophages, and may exert its antioxidant effect and protect against aseptic implant loosening.

Expression of GRIM-19 in adenomyosis and its possible role in pathogenesis.

OBJECTIVE: To study the expression of the gene associated with retinoid-interferon (IFN)-induced mortality 19 (GRIM-19) in the endometrial tissue of patients with adenomyosis and to describe the possible pathogenic mechanisms of this phenomenon. DESIGN: Experimental study using human samples and cell lines. SETTING: University-affiliated hospital. PATIENT(S): Ectopic and eutopic endometrial tissues were obtained from 30 patients with adenomyosis, whereas normal endometrial specimens were obtained from 10 control patients without adenomyosis. INTERVENTION(S): Patients with rapid pathology report-confirmed adenomyosis were recruited, and eutopic and ectopic endometrial tissue samples were collected from patients who had undergone hysterectomies by either the transabdominal or laparoscopic method at Qilu Hospital. Normal endometrial tissue was collected from a group of control patients without adenomyosis. MAIN OUTCOME MEASURE(S): Immunohistochemistry (IHC) was performed to evaluate the expression of GRIM-19, phospho-signal transducer and activator of transcription 3 (Y705) (Y705) (pSTAT3(Y705)), and vascular endothelial growth factor (VEGF) in endometrial tissue samples. The protein levels of GRIM-19, pSTAT3(Y705), STAT3, and VEGF were detected by Western blot. Apoptosis in endometrial specimens was assayed by TUNEL. Immunohistochemistry with an antibody directed against CD34 was performed to detect new blood vessels in the endometrial tissue. GRIM-19 small interfering RNA and a recombinant plasmid carrying GRIM-19 were constructed to evaluate the effects of GRIM-19 on the downstream factors pSTAT3(Y705), STAT3, and VEGF in Ishikawa cells. RESULT(S): The expression of GRIM-19 was down-regulated in the eutopic endometria of patients with adenomyosis compared with the endometria of patients in the control group, and it was further reduced in the endometrial glandular epithelial cells of adenomyotic lesions. Apoptosis was reduced in the eutopic endometrium compared with the control group, and it was significantly reduced in ectopic endometrial tissues. In addition, the ectopic and eutopic endometria of patients with adenomyosis displayed a much higher microvessel density. In the eutopic and ectopic endometria of patients with adenomyosis, the expression levels of pSTAT3(Y705) and VEGF were significantly higher than in the controls. Furthermore, down-regulation of GRIM-19 in Ishikawa cells significantly promoted the activation of both pSTAT3(Y705) and its dependent gene VEGF. CONCLUSION(S): Aberrant expression of GRIM-19 may be associated with adenomyosis through the regulation of apoptosis and angiogenesis.

Decreased reactive oxygen species production and NOX1, NOX2, NOX4 expressions contribute to hyporeactivity to phenylephrine in aortas of pregnant SHR.

AIMS: We determined whether decreased reactive oxygen species (ROS) production in the aorta of pregnant spontaneously hypertensive rats (SHR) resulted in increased nitric oxide (NO) bioavailability and hyporeactivity to phenylephrine (PE). MAIN METHODS: Systemic and aortic oxidative stress were measured in pregnant and non-pregnant Wistar rats and SHR. Furthermore, the hypotensive effects of apocynin (30 mg/kg) and Tempol (30 mg/kg) were analyzed. Intact aortic rings of pregnant and non-pregnant rats were stimulated with PE in the absence of or after incubation (30 min) with apocynin (100 µmol/L). The effect of apocynin on the concentrations of NO and ROS were measured in aortic endothelial cells (AEC) using DAF-2DA (10 mmol/L) and DHE (2.5 mmol/L), respectively. Western blotting was performed to analyze eNOS, NOX1, NOX2, NOX4 and SOD expression. ROS production was analyzed by the lucigenin chemiluminescence method. KEY FINDINGS: Aortic oxidative stress and ROS concentration in AEC were reduced in pregnant Wistar rats and SHR, when compared to non-pregnant rats. ROS production and NOX1, NOX2 and NOX4 expression in the aortas were decreased in pregnant SHR, but not in pregnant Wistar rats. Increased eNOS expression in aortas and NO concentration in AEC were observed in pregnant Wistar rats and SHR. Apocynin reduced PE-induced vasoconstriction in the aortas of non-pregnant Wistar rats and SHR, and pregnant Wistar rats, but not in the aortas of pregnant SHR. SIGNIFICANCE: Taken together, these results suggest that ROS production was decreased in the aortas of pregnant SHR and could contribute to higher NO bioavailability and hyporeactivity to PE in the aortas of pregnant SHR.

GRIM-19 expression is a potent prognostic marker in colorectal cancer.

Retinoid-interferon-induced mortality-19 (GRIM-19), a recently discovered cell death regulatory gene, may function as a tumor suppressor in many human malignancies. However, the expression of GRIM-19 in and its prognostic value for patients with colorectal cancer (CRC) have not been well investigated to date. Here, GRIM-19 expression was measured immunohistochemically in 94 colon samples and by quantitative real-time reverse transcriptase polymerase chain reaction in 15 paired CRC tissues and adjacent normal tissues. The prognostic significance was assessed using Kaplan-Meier survival estimates and log-rank tests. Our results showed that GRIM-19 mRNA and protein levels in adenoma tissues were similar to those in adjacent normal tissues. However, GRIM-19 expression was severely depressed in carcinomas compared to matched normal tissues (P = .000). Additionally, we found GRIM-19 to be located in both the cytoplasm and nucleus in normal tissues but only in the cytoplasm in CRC tissues. Alteration in GRIM-19 expression occurs early in the pathogenesis of CRC; moreover, low GRIM-19 expression was associated with poor tumor differentiation (P = .013), the presence of lymph nodes (P = .000), metastasis to other organs (P = .045) and vascular invasion (P = .010). During a mean period of 40 months follow-up, patients without GRIM-19 had a statistically significantly lower rate of recurrence/metastasis (P < .05) and a shorter overall survival time (P < .01) than the patients with GRIM-19 expression. Taken together, GRIM-19 expression is closely associated with CRC progression and might be a very promising prognostic biomarker for CRC patients.

Antioxidant Supplementation Modulates Age-Related Placental Bed Morphology and Reproductive Outcome in Mice.

The number of women who delay their first childbirth is increasing. This demographic shift is an important health issue because advanced maternal age is a risk factor for reproductive capacity loss and the occurrence of placental bed disorders that may lead to placenta abruption, preeclampsia, and placenta insufficiency. A redox imbalance status, resulting from the enhanced production of reactive oxygen species or their deficient neutralization, is proposed to occur in this setting. Thus, uterine redox status was evaluated in young (8- to 12-wk-old) and reproductively aged (38- to 42-wk-old) mice. In addition, it was hypothesized that specific dietary antioxidant supplementation would restore the balance and improve the reproductive outcome of aging female mice. To test this hypothesis, two different antioxidants, the nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor apocynin and the superoxide dismutase mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (TEMPOL), were added to the drinking water of female mice prior to and during pregnancy. Compared to younger females, uteri from reproductively aged nonpregnant mice exhibited areas of endometrial cystic dilation, increased level of NOX1 expression, and enhanced protein carbonylation, especially in the apical surface of the luminal epithelium. Both antioxidants decreased protein carbonylation level in the uterus of reproductively aged mice. When reproductively aged females became pregnant, the litter size was smaller and fetuses were heavier. The change was accompanied by a significant decrease in decidua thickness. Provision of apocynin significantly increased litter size and restored decidua thickness. Reproductively aged mice provided with TEMPOL did not evidence such benefits, but whereas apocynin normalized fetal birth weight, TEMPOL further increased it. These findings emphasize that uterine redox balance is important for reproductive success and suggest that age-related redox imbalance might be compensated by specific antioxidant supplementation.

ROS-Induced Nuclear Translocation of Calpain-2 Facilitates Cardiomyocyte Apoptosis in Tail-Suspended Rats.

Isoproterenol (ISO) induced nuclear translocation of calpain-2 which further increased susceptibility of cardiomyocyte apoptosis in tail-suspended rats. The underlying mechanisms remain elusive. In the present study, the results showed that ISO (10 nM) significantly elevated NADPH oxidases (NOXs) activity and NOXs-derived ROS productions which induced nuclear translocation of calpain-2 in cardiomyocytes of tail-suspended rats. In contrast, the inhibition of NADPH oxidase or cleavage of ROS not only reduced ROS productions, but also resisted nuclear translocation of calpain-2 and decreased ISO-induced apoptosis of cardiomyocyte in tail-suspended rats. ISO also increased the constitutive binding between calpain-2 and Ca(2+)/calmodulin-dependent protein kinase II δB (CaMK II δB) in nuclei, concomitant with the promotion of CaMK II δB degradation and subsequent down-regulation of Bcl-2 mRNA expression and the ratio of Bcl-2 to Bax protein in tail-suspended rat cardiomyocytes. These effects of ISO on cardiomyocytes were abolished by a calpain inhibitor PD150606. Inhibition of calpain significantly reduced ISO-induced loss of the mitochondrial membrane potential, cytochrome c release into the cytoplasm, as well as the activation of caspase-3 and caspase-9 in mitochondrial apoptotic pathway. In summary, the above results suggest that ISO increased NOXs-derived ROS which activated nuclear translocation of calpain-2, subsequently nuclear calpain-2 degraded CaMK II δB which reduced the ratio of Bcl-2 to Bax, and finally the mitochondria apoptosis pathway was triggered in tail-suspended rat cardiomyocytes. Therefore, calpain-2 may represent a potentially therapeutic target for prevention of oxidative stress-associated cardiomyocyte apoptosis.

Scoparone attenuates RANKL-induced osteoclastic differentiation through controlling reactive oxygen species production and scavenging.

Scoparone, one of the bioactive components of Artemisia capillaris Thunb, has various biological properties including immunosuppressive, hepatoprotective, anti-allergic, anti-inflammatory, and antioxidant effects. This study aims at evaluating the anti-osteoporotic effect of scoparone and its underlying mechanism in vitro. Scoparone demonstrated potent cellular antioxidant capacity. It was also found that scoparone inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and suppressed cathepsin K and tartrate-resistant acid phosphatase (TRAP) expression via c-jun N-terminal kinase (JNK)/extracellular signal-regulated kinase (ERK)/p38-mediated c-Fos-nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) signaling pathway. During osteoclast differentiation, the production of general reactive oxygen species (ROS) and superoxide anions was dose-dependently attenuated by scoparone. In addition, scoparone diminished NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 1 (Nox1) expression and activation via the tumor necrosis factor receptor-associated factor 6 (TRAF6)-cSrc-phosphatidylinositol 3-kinase (PI3k) signaling pathway and prevented the disruption of mitochondrial electron transport chain system. Furthermore, scoparone augmented the expression of superoxide dismutase 1 (SOD1) and catalase (CAT). The overall results indicate that the inhibitory effect of scoparone on RANKL-induced osteoclast differentiation is attributed to the suppressive effect on ROS and superoxide anion production by inhibiting Nox1 expression and activation and protecting the mitochondrial electron transport chain system and the scavenging effect of ROS resulting from elevated SOD1 and CAT expression.

Molecular cloning, computational and expression analysis of anthocyanidin reductase in tea (Camellia sinensis).

Tea [Camellia sinensis (L.) O. Kuntze] is one of the most popular non-alcoholic beverages rich in phenolic compounds, which includes epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), epicatechin (EC) and catechin (C). Anthocyanidin reductase (ANR) is responsible for catechin biosynthesis in plants, and analysis of its protein sequences and structures will be valuable for further research in the field. We have screened our dormant bud-specific complementary DNA (cDNA) library and reported 1,322-bp cDNA encoding CsANR. Analysis of the sequence revealed the presence of 1,011-bp open reading frame with coding capacity for a polypeptide of 337 amino acids, flanked by 1,123- and 196-bp 5' and 3' untranslated regions, respectively. Theoretical molecular weight (MW) and isoelectric point (pI) of the deduced ANR protein were predicted (using ProtParam) to be 36.4 kDa and 6.54. For the first time, we have reported 3D model of ANR from C. sinensis. Quality of the predicted model was analysed with PROCHECK analysis. Molecular docking of modelled ANR revealed similar binding pockets for both substrates and products. Expression analyses of CsANR and accumulation pattern of catechins were observed to be varied with developmental age of tissue and seasonal condition. Variation in accumulation pattern of catechins and its fractions was found to be correlated with expression pattern of ANR.

Glutathione transferase omega 1 is required for the lipopolysaccharide-stimulated induction of NADPH oxidase 1 and the production of reactive oxygen species in macrophages.

Bacterial lipopolysaccharide (LPS) stimulation of macrophages and inflammation via the Toll-like receptor 4 (TLR4) signaling pathway through NF-κΒ generates reactive oxygen species (ROS) and proinflammatory cytokines such as IL-1ß, IL-6, and TNFα. Because glutathione transferase Omega 1-1 (GSTO1-1) can catalyze redox reactions such as the deglutathionylation of proteins and has also been implicated in the release of IL-1ß we investigated its role in the development of LPS-mediated inflammation. Our data show that shRNA knockdown of GSTO1-1 in macrophage-like J774.1A cells blocks the expression of NADPH oxidase 1 and the generation of ROS after LPS stimulation. Similar results were obtained with a GSTO1-1 inhibitor. To maintain high ROS levels during an inflammatory response, LPS stimulation causes the suppression of enzymes such as catalase and glutathione peroxidase that protect against oxidative stress. The knockdown of GSTO1-1 also attenuates this response. Our data indicate that GSTO1-1 needs to be catalytically active and mediates its effects on the LPS/TLR4 inflammatory pathway upstream of NF-κΒ. These data suggest that GSTO1-1 is a novel target for anti-inflammatory intervention.

Pre-treatment with cardamonin protects against cisplatin-induced nephrotoxicity in rats: impact on NOX-1, inflammation and apoptosis.

Cisplatin is an effective anti-cancer drug; however, its clinical use is usually associated with nephrotoxicity as a dose-limiting side effect. Several molecular mechanisms have been found to be involved in this nephrotoxicity such as oxidative stress, inflammation and apoptosis. The aim of this study was to explore the potential nephroprotective effect of cardamonin, a flavone found in Alpinia plant, in a rat model of cisplatin-induced nephrotoxicity. The possible mechanisms underlying this nephroprotective effect were investigated. Cardamonin was given at two different doses; 10 and 30mg/kg orally for two weeks, starting one week before giving a single nephrotoxic dose of cisplatin (7mg/kg). Acute nephrtoxicity was evident by significantly increased blood urea nitrogen and serum creatinine levels. Also, cisplatin increased lipid peroxidation and depleted reduced glutathione level and superoxide dismutase. Additionally, cisplatin showed a marked pro-inflammatory response as evidenced by significant increase in tissue levels of IL-1ß, TNF-α, NF-kB, iNOS, ICAM-1 and MCP-1. Pre-treatment with cardamonin significantly attenuated the nephrotoxic effects, oxidative stress and inflammation induced by cisplatin, in a dose-dependent manner. Also, cardamonin decreased caspase-3 expression and Bax/Bcl-2 ratio as compared to cisplatin group. Besides, cradamonin reversed cisplatin-induced decrease in EGF. Furthermore, up-regulation of NOX-1 was found to be involved in cisplatin-induced nephrotoxicity and its expression was significantly reduced by cardamonin. Histopathological examination further confirmed the nephroprotective effect of cardamonin. Moreover, pre-treatment with subtoxic concentration of cardamonin has significantly enhanced cisplatin cytotoxic activity in four different human cancer cell lines; hela, hepG2, PC3 and HCT116 cancer cell lines. In conclusion, these findings suggest that cardamonin improves therapeutic index of cisplatin and that NOX-1 is partially involved in the pathogenesis of cispaltin-induced nephrotoxicity.

Nox1 causes ileocolitis in mice deficient in glutathione peroxidase-1 and -2.

We previously reported that mice deficient in two Se-dependent glutathione peroxidases, GPx1 and GPx2, have spontaneous ileocolitis. Disease severity depends on mouse genetic background. Whereas C57BL/6J (B6) GPx1/2-double-knockout (DKO) mice have moderate ileitis and mild colitis, 129S1Svlm/J (129) DKO mice have severe ileocolitis. Because GPx's are antioxidant enzymes, we hypothesized that elevated reactive oxygen species trigger inflammation in these DKO mice. To test whether NADPH oxidase 1 (Nox1) contributes to colitis, we generated B6 triple-KO (TKO) mice to study their phenotype. Because the Nox1 gene is X-linked, we analyzed the effects of Nox1 on male B6 TKO mice and female B6 DKO mice with the Nox1(+/-) (het-TKO) genotype. We found that the male TKO and female het-TKO mice are virtually disease-free when monitored from 8 through 50 days of age. Male TKO and female het-TKO mice have nearly no signs of disease (e.g., lethargy and perianal alopecia) that are often exhibited in the DKO mice; further, the slower growth rate of DKO mice is almost completely eliminated in male TKO and female het-TKO mice. Male TKO and female het-TKO mice no longer have the shortened small intestine present in the DKO mice. Finally, the pathological characteristics of the DKO ileum, including the high level of crypt apoptosis (analyzed by apoptotic figures, TUNEL, and cleaved caspase-3 immunohistochemical staining), high numbers of Ki-67-positive crypt epithelium cells, and elevated levels of monocytes expressing myeloperoxidase, are all significantly decreased in male TKO mice. The attenuated ileal and colonic pathology is also evident in female het-DKO mice. Furthermore, the male DKO ileum has eightfold higher TNF cytokine levels than TKO ileum. Nox1 mRNA is highly elevated in both B6 and 129 DKO ileum compared to wild-type mouse ileum. Taking these results together, we propose that ileocolitis in the DKO mice is caused by Nox1, which is induced by TNF. The milder disease in female het-TKO intestine is probably due to random or imprinted X-chromosome inactivation, which produces mosaic Nox1 expression.

ATP stimulates PGE(2)/cyclin D1-dependent VSMCs proliferation via STAT3 activation: role of PKCs-dependent NADPH oxidase/ROS generation.

Vascular smooth muscle cells (VSMCs) that function as synthetic units play important roles in cardiovascular diseases. Extracellular nucleotides, such as ATP, have been shown to act via activation of P2 purinoceptors implicated in various inflammatory diseases, we hypothesized that extracellular nucleotides contribute to vascular diseases via up-regulation of inflammatory proteins, including cyclooxygenase-2 (COX-2) and cytosolic phospholipase A2 (cPLA2) in VSMCs. However, the mechanisms of ATP-induced cPLA2 and COX-2 expression and PGE2 synthesis remain largely unclear. We showed that pretreatment with the inhibitors of STAT3 (CBE), NADPH oxidase [diphenyleneiodonium chloride (DPI) or apocynin (APO)], ROS [N-acetyl-l-cysteine (NAC)], and PKC (Ro-318220, Gö6983, or Rottlerin) or transfection with siRNAs of STAT3 and p47(phox) markedly inhibited ATPγS-induced cPLA2 and COX-2 mRNA/protein expression and promoter activity and PGE2 secretion. ATPγS further stimulated PKC, p47(phox), and STAT3 translocation. Moreover, ATPγS-induced STAT3 phosphorylation and translocation was inhibited by pretreatment with the inhibitors of PKC, NADPH oxidase, and ROS. ATPγS enhanced NADPH oxidase activity and ROS generation in VSMCs, which were reduced by pretreatment with Ro-318220, Gö6983, or Rottlerin. Finally, we found that ATPγS significantly induced cyclin D1 expression and VSMCs proliferation, which were inhibited by pretreatment with NAC, APO, DPI, Ro-318220, Gö6983, Rottlerin, or CBE or transfection with siRNAs of COX-2 and cyclin D1. We also demonstrated that ATPγS induced cyclin D1 expression via a PGE2-dependent pathway. These results suggested that ATPγS-induced cPLA2/COX-2 expression and PGE2 secretion is mediated through a PKC/NADPH oxidase/ROS/STAT3-dependent pathway in VSMCs.

Regulation of NADPH oxidase gene expression with PKA and cytokine IL-4 in neurons and microglia.

Neuronal excitation is mediated by the activation of NMDA receptor and associated with the formation of reactive oxygen species due to the activation of NADPH oxidase complex proteins. The activation of Gs protein coupled receptors (GPCRs) induces neuronal activation in the cAMP-dependent protein kinase A (PKA)-mediated signal cascade and regulates NADPH oxidase activity. However, it is unknown whether PKA regulates NADPH oxidase gene expression in neurons and microglia. In the present research, the NADPH oxidase gene expression was studied in rat cortical neurons and microglia in vitro. Purified microglial cells were identified with OX-42 antibody and they also expressed apolipoprotein E (ApoE). The time-dependent effect of cytokine interleukin-4 (IL-4) (20 ng/ml) in NADPH oxidase gene expression was studied in microglial cells. The levels of mRNA were determined by quantitative RT-PCR. The expression of NOX1, NOX2, and NCF2 was upregulated after IL-4 treatment for 4 h, but it was downregulated after 8-24 h. The expression of NCF1 was suppressed during any time of cytokine effect. IL-4 upregulated arginase1 (Arg1) and serine racemase1 (SRR1) gene expressions in microglia. Amyloid beta (Ab) suppressed NOX2, NCF1, and NCF2 gene expressions and upregulated glutamate cystine transporter (xCT), although IL-4 attenuated the effect of Ab (500 µM) in the upregulation of xCT gene expression. The activation of PKA with agonist dibutyryl cAMP (dbcAMP) (100 µM) induced the upregulation of Arg1 gene expression in microglia involving in the process of microglial activation. The transcription of NOX1, NOX2, and NCF1 was suppressed in microglial cells after dbcAMP treatment within 24 h. Neurons were identified with the microtubule-associated protein tau. The uniform distribution of tau along axons was established in normal neurons. Tau protein was redistributed after PKA agonist dbcAMP treatment for 24 h. L-glutamate (50 µM) caused the apoptotic processes and the accumulation of tau in the soma of neurons and along axons. The activation of PKA for 24 h induced the transcriptional upregulation of NOX1 and NCF1 in cortical neurons. However, L-glutamate suppressed NOX1 gene expression in neurons. These data demonstrate that the effects of IL-4 and dbcAMP are similar in the regulation of SRR1, Arg1, and NADPH oxidase complex gene expressions in neurons and microglia. IL-4 prevents glutamate release from microglia suppressing xCT expression induced by Ab. These findings suggest that the activation of GPCR in PKA-mediated pathway leads to transcriptional regulation of NADPH oxidase complex. The modulation of GPCR activation may inhibit the oxidative stress in neurons.

GRIM-19 inhibits the STAT3 signaling pathway and sensitizes gastric cancer cells to radiation.

Gastric cancer is one of the most common malignancies, and radiation resistance is one of the key obstacles in gastric cancer treatment. In this study, we demonstrate that "genes associated retinoid-IFN induced mortality-19" (GRIM-19) expression was lower in patients with radiotherapy-resistant tumors compared to patients with radiotherapy-sensitive tumors. In order to further investigate the effects of GRIM-19 expression on the radiation response in gastric cancer cells, we established BGC-803 clones stably expressing exogenous GRIM-19. We found that the percentage of apoptotic cells was higher in cells expressing GRIM-19 than untransfected cells post-radiation treatment. Furthermore, caspase-3, -8, and -9 activity was significantly increased in GRIM-19-expressing cells compared to untransfected cells after radiation. Finally, we demonstrate that expression of GRIM-19 in BGC-803 cells suppresses accumulation of STAT3. Collectively, these data show that GRIM-19 expression sensitizes BGC-803 cells to radiation, and this is likely due to suppression of STAT3 accumulation. In summary, our results indicate that GRIM-19 expression might be a useful therapy to enhance apoptosis in gastric cancer cells in response to radiation treatment.

Powerful vascular protection by combining cilnidipine with valsartan in stroke-prone, spontaneously hypertensive rats.

Cilnidipine is an L- and N-type calcium channel blocker (CCB), and amlodipine is an L-type CCB. Valsartan (10 mg kg(-1)), valsartan (10 mg kg(-1)) and amlodipine (1 mg kg(-1)), and valsartan (10 mg kg(-1)) and cilnidipine (1 mg kg(-1)) were administered once daily for 2 weeks to stroke-prone, spontaneously hypertensive rats (SHR-SPs). Blood pressure was significantly reduced by valsartan, and it was further reduced by the combination therapies. Vascular endothelial dysfunction was significantly attenuated in all therapeutic groups, and further significant attenuation was observed in the valsartan+cilnidipine-treated group, but not in the valsartan+amlodipine-treated group. Vascular nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit NOX1 gene expression was significantly attenuated in all therapeutic groups, and significantly greater attenuation was observed in the valsartan+cilnidipine-treated group than in the valsartan-treated group. Compared with the valsartan-treated group, the positive areas for 4-hydroxy-2-nonenal were significantly lower only in the valsartan+cilnidipine-treated group. Plasma renin activity was significantly augmented in the valsartan-treated group, and it was significantly attenuated in the valsartan+cilnidipine-treated group. A significant increase in the ratio of plasma angiotensin-(1-7) to angiotensin II was observed only in the valsartan+cilnidipine-treated group. Vascular angiotensin-converting enzyme (ACE) gene expression was significantly attenuated only in the valsartan+cilnidipine-treated group, but ACE2 gene expression was significantly higher in all of the therapeutic groups. Thus, valsartan and cilnidipine combination therapy might have a powerful protective effect in the vascular tissues via increases in the angiotensin-(1-7)/angiotensin II ratio in plasma.