The ATP-dependent RNA helicase, DDX42 interacts with paxillin and regulates apoptosis and polarization of Ba/F3 cells.

Paxillin is a focal adhesion adaptor protein, heavily phosphorylated at multiple tyrosine residues, as well as at serine 273 (S273), and is known to be critical for cytoskeleton rearrangement and cell migration. We previously found that paxillin plays a regulatory role in IL-3-dependent survival of Ba/F3 cells, a mouse pro-B cell line. In this study, by using overexpressed His6 tagged-paxillin as a bait, we found that DDX42, a DEAD-box RNA helicase, interacted with paxillin, inhibited apoptosis, and promoted polarization of Ba/F3 cells. His6 tagged-paxillin was stably overexpressed in Ba/F3 cells, pulled-down from cell lysates with Ni+-NTA beads, and analyzed by one-dimensional SDS-PAGE followed by LC-MS. We found that DDX42 co-precipitated with paxillin, as demonstrated by western blotting analysis of His6 tagged-paxillin precipitates with anti-DDX42 antibodies and His6 tagged-DDX42 precipitates with anti-paxillin antibodies. In addition, we observed a preferential interaction of DDX42 with the paxillin mutant, S273A, compared to the S273D mutant. Furthermore, DDX42 overexpression in Ba/F3 cells delayed the apoptosis induced by IL-3 deprivation and promoted restoration of the elongated shape in Ba/F3 cells induced by IL-3 re-supply after a 6 h-deprivation. These results suggested that DDX42 interacts with paxillin and participates in IL-3-dependent cell survival, as well as in the cytoskeletal rearrangements underlying polarization of Ba/F3 cells.

Roles of paxillin phosphorylation in IL-3 withdrawal-induced Ba/F3 cell apoptosis.

BACKGROUND: Ba/F3, a mouse pro-B cell line, is dependent on IL-3 for its survival and proliferation. IL-3 withdrawal causes cells to round, stop in G1 phase, then undergo apoptosis. Additionally, IL-3 is known to induce tyrosine phosphorylation of paxillin, a scaffold and signaling protein. We previously determined that overexpression of paxillin prohibited Ba/F3 cell apoptosis induced by IL-3 withdrawal. OBJECTIVE: Address whether phosphorylation is essential for the anti-apoptotic effect of overexpressed paxillin. METHODS: Mutations were introduced into paxillin cDNA at five phosphorylation sites-Y31F, Y40F, Y118F, Y181F, S273A, or S273D. After overexpression of paxillin mutants in Ba/F3 cells, the apoptotic proportions of cell populations were measured by an annexin V conjugation assay while cells were undergoing IL-3 withdrawal. RESULTS: The anti-apoptotic effect of paxillin overexpression was abolished by site-directed mutagenesis replacing Y31, Y40, Y118, and Y181 with phenylalanine, and S273 with aspartic acid. In contrast, the mutation replacing S273 with alanine had no effect on the anti-apoptotic effect. CONCLUSION: The above results suggest that paxillin-mediated phosphorylation at Y31, Y40, Y118, and Y181 is essential for the anti-apoptotic effect of paxillin overexpression in Ba/F3 cells and contributes to the cell survival signaling pathway triggered by IL-3. Conversely, phosphorylation at S273 is involved in the negative regulation of the anti-apoptotic action of overexpressed paxillin.

Native low density lipoprotein increases the production of both nitric oxide and reactive oxygen species in the human umbilical vein endothelial cells.

BACKGROUND: Nitric oxide synthases (NOSs) are a unique family of enzymes that catalyze the production of nitric oxide (NO) from L-arginine. Atherogenic action of oxidized low-density lipoproteins (oxLDL) may be mediated partly by the formation of NO in endothelial cells. OBJECTIVE: The objective of this study was to identify sources of reactive oxygen species (ROS) causing native LDL (nLDL)-induced senescence of cultured human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were treated with nLDL and NO production was assessed using Griess reagent as substrate and spectrophotometry in the absence or presence of specific inhibitors of endothelial NOS (eNOS) and inducible NOS (iNOS). In addition, expression levels of eNOS and iNOS were measured with ELISA and western blotting, and ROS was evaluated using 2',7'-dichlorofluorescin diacetate (DCF-DA) and a fluorescence microplate reader. RESULTS: NO formation in nLDL-treated HUVECs was significantly increased. Long-term treatment with nLDL up-regulated both eNOS and iNOS proteins. Such increase of NO production in HUVECs induced by nLDL was significantly suppressed by treatment with iNOS-selective inhibitor 1400 W, but not by the eNOS-selective inhibitor L-NIO. Native LDL treatment uncoupled Hsp90, the regulatory binding protein of eNOS, from the enzyme in HUVECs. Native LDL also significantly increased ROS production in HUVECs. CONCLUSION: These findings suggest that oxidative stress originated from induction of iNOS and eNOS could be a causative factor for nLDL-induced senescence of HUVECs.

DPIE [2-(1,2-diphenyl-1H-indol-3-yl)ethanamine] Augments Pro-Inflammatory Cytokine Production in IL-1ß-Stimulated Primary Human Oral Cells.

Interleukin-1β (IL-1β) is a prominent pro-inflammatory cytokine that is implicated in a variety of autoimmune diseases and plays an important role in host defense against infections. IL-1β activity increases with its increasing binding capacity to IL-1 receptors (IL-1Rs). Thus, numerous studies have targeted the discovery of molecules modulating the interactions between IL-1β and IL-1R1. We have conducted an IL-1R1 structure-based virtual screening to identify small molecules that could alter IL-1β activity, using in silico computational analysis. Sixty compounds from commercial libraries were predicted to bind to IL-1R1, and their influence on cytokine production in IL-1β-stimulated gingival fibroblasts (GFs) was determined. Of these, only (2-(1,2-diphenyl-1H-indol-3-yl)ethanamine (DPIE) showed a synergistic increase in inflammatory molecules and cytokine production (IL-6, IL-8, and COX-2) at both mRNA and protein levels in IL-1β-stimulated GFs. The enhancing activity of DPIE in IL-1β-induced cytokine production increased in a dose-dependent manner without cytotoxicity. This pattern was also observed in IL-1β-stimulated primary human periodontal ligament cells (PDLs). Furthermore, we measured the impact of DPIE on the IL-1β⁻IL-1R1 system using surface plasmon resonance and demonstrated that DPIE increased the binding affinity of IL-1β to IL-1R1. These data indicate that DPIE boosts IL-1β signaling by enhancing the binding of IL-1β to IL-1R1 in oral primary cells.

NADPH Oxidase Mediates ß-Amyloid Peptide-Induced Neuronal Death in Mouse Cortical Cultures.

ß-Amyloid peptide (Aß) is the main component of senile plaques in patients with Alzheimer's disease, and is known to be a main pathogenic factor of the disease. Recent evidence indicates that activation of NADPH oxidase (NOX) in microglia or astrocytes may be a source of Aß-induced reactive oxygen species (ROS). We investigated the role of neuronal NOX in Aß-induced neuronal death in mouse mixed cortical cultures. Cell death was assessed by measuring lactate dehydrogenase efflux to bathing media 24 or 48 hr after exposure to Aß25-35, a fragment of Aß with an equivalent neurotoxic effect. Aß25-35 induced neuronal death in concentration- and time- dependent manners with apoptotic features. Neuronal death was significantly attenuated, not only by anti-apoptotic drugs, such as z-VAD-fmk and cycloheximide, but also by antioxidants, such as trolox, ascorbic acid, and epigallocatethin gallate. We also demonstrated that treatment with 20 µM Aß25-35 increased fluorescent signals in mixed cortical cultures, but produced only weak signals in pure astrocyte cultures in the presence of 2',7'-dichlorofluorescin diacetate (DCF-DA), an indicator for intracellular ROS. Increased DCF-DA fluorescence was markedly inhibited, not only by trolox, but also by selective NOX inhibitors, such as apocynin and AEBSF. Western blot analyses revealed that Aß25-35 increased the expression of gp91phox, a main subunit of NOX in cells. The above antioxidants, apocynin, and AEBSF significantly attenuated neuronal death induced by Aß25-35. Furthermore, the gp91phox-specific siRNA-based knockdown of NOX significantly inhibited neuronal death. These results suggest that activation of neuronal NOX is involved in Aß25-35-induced neuronal death.

Functional validation of novel MKS3/TMEM67 mutations in COACH syndrome.

COACH syndrome is an autosomal recessive developmental disorder, a subtype of Joubert syndrome and related disorders, characterized by cerebellar vermis hypoplasia, oligophrenia, ataxia, coloboma, and hepatic fibrosis. Although mutations in TMEM67 (transmembrane protein 67)/MKS3 (Meckel-Gruber syndrome, type 3) were reported to cause COACH syndrome, this causality has not verified by functional studies. In a 20-year-old Korean man, we found cerebellar ataxia, isolated elevation in serum γ-glutamyl transpeptidase (γ-GTP) activity, oligophrenia, the molar tooth sign (MTS) in the brain MR images and congenital hepatic fibrosis (CHF). Two novel compound heterozygous mutations were found in TMEM67 in the patient: i) missense mutation (c.395 G > C and p.Gly132Ala) in exon 3, and ii) deletion in exon 26 (c.2758delT and p.Tyr920ThrfsX40). Western blotting showed that the p.Tyr920ThrfsX40 mutation accelerates turnover of the TMEM67 protein. Although wild-type human TMEM67 RNA rescued phenotypes of zebrafish embryos injected with anti-sense oligonucleotide morpholinos against tmem67, the two human TMEM67 RNAs individually harboring the two mutations did not. Finally, Wnt signaling, but not Hedgehog signaling, was suppressed in tmem67 morphants. To the best of our knowledge, this is the first report verifying the causality between COACH syndrome and TMEM67, which will further our understanding of molecular pathogenesis of the syndrome.

Redox regulation of the tumor suppressor PTEN by the thioredoxin system and cumene hydroperoxide.

Intracellular redox status influences the oxidation and enzyme activity of the tumor suppressor phosphatase and tensin homolog on chromosome 10 (PTEN). Cumene hydroperoxide (CuHP), an organic hydroperoxide, is a known tumor promoter. However, molecular targets and action mechanism of CuHP in tumor promotion have not been well characterized. In this study, we investigated the effect of CuHP on the redox state of PTEN in HeLa cells. In addition, the intracellular reducing system of oxidized PTEN was analyzed using a biochemical approach and the effect of CuHP on this reducing system was also analyzed. While PTEN oxidized by hydrogen peroxide is progressively converted to its reduced form, PTEN was irreversibly oxidized by exposure to CuHP in HeLa cells. A combination of protein fractionation and mass analysis showed that the reducing system of PTEN was comprised of NADPH, thioredoxin reductase (TrxR), and thioredoxin (Trx). Although CuHP-mediated PTEN oxidation was not reversible in cells, CuHP-oxidized PTEN was reactivated by the exogenous Trx system, indicating that the cellular Trx redox system for PTEN is inactivated by CuHP. We present evidence that PTEN oxidation and the concomitant inhibition of thioredoxin by CuHP results in irreversible oxidation of PTEN in HeLa cells. In addition, ablation of peroxiredoxin (Prdx) enhanced CuHP-induced PTEN oxidation in cells. These results provide a new line of evidence that PTEN might be a crucial determinant of cell fate in response to cellular oxidative stress induced by organic hydroperoxides.

Redox Regulation of the Tumor Suppressor PTEN by Hydrogen Peroxide and Tert-Butyl Hydroperoxide.

Organic peroxides and hydroperoxides are skin tumor promoters. Free radical derivatives from these compounds are presumed to be the prominent mediators of tumor promotion. However, the molecular targets of these species are unknown. Phosphatase and tensin homologs deleted on chromosome 10 (PTEN) are tumor suppressors that play important roles in cell growth, proliferation, and cell survival by negative regulation of phosphoinositol-3-kinase/protein kinase B signaling. PTEN is reversibly oxidized in various cells by exogenous and endogenous hydrogen peroxide. Oxidized PTEN is converted back to the reduced form by cellular reducing agents, predominantly by the thioredoxin (Trx) system. Here, the role of tert-butyl hydroperoxide (t-BHP) in redox regulation of PTEN was analyzed by using cell-based and in vitro assays. Exposure to t-BHP led to oxidation of recombinant PTEN. In contrast to H2O2, PTEN oxidation by t-BHP was irreversible in HeLa cells. However, oxidized PTEN was reduced by exogenous Trx system. Taken together, these results indicate that t-BHP induces PTEN oxidation and inhibits Trx system, which results in irreversible PTEN oxidation in HeLa cells. Collectively, these results suggest a novel mechanism of t-BHP in the promotion of tumorigenesis.

Chrysin inhibits tumor promoter-induced MMP-9 expression by blocking AP-1 via suppression of ERK and JNK pathways in gastric cancer cells.

Cell invasion is a crucial mechanism of cancer metastasis and malignancy. Matrix metalloproteinase-9 (MMP-9) is an important proteolytic enzyme involved in the cancer cell invasion process. High expression levels of MMP-9 in gastric cancer positively correlate with tumor aggressiveness and have a significant negative correlation with patients' survival times. Recently, mechanisms suppressing MMP-9 by phytochemicals have become increasingly investigated. Chrysin, a naturally occurring chemical in plants, has been reported to suppress tumor metastasis. However, the effects of chrysin on MMP-9 expression in gastric cancer have not been well studied. In the present study, we tested the effects of chrysin on MMP-9 expression in gastric cancer cells, and determined its underlying mechanism. We examined the effects of chrysin on MMP-9 expression and activity via RT-PCR, zymography, promoter study, and western blotting in human gastric cancer AGS cells. Chrysin inhibited phorbol-12-myristate 13-acetate (PMA)-induced MMP-9 expression in a dose-dependent manner. Using AP-1 decoy oligodeoxynucleotides, we confirmed that AP-1 was the crucial transcriptional factor for MMP-9 expression. Chrysin blocked AP-1 via suppression of the phosphorylation of c-Jun and c-Fos through blocking the JNK1/2 and ERK1/2 pathways. Furthermore, AGS cells pretreated with PMA showed markedly enhanced invasiveness, which was partially abrogated by chrysin and MMP-9 antibody. Our results suggest that chrysin may exert at least part of its anticancer effect by controlling MMP-9 expression through suppression of AP-1 activity via a block of the JNK1/2 and ERK1/2 signaling pathways in gastric cancer AGS cells.

Assay of the redox state of the tumor suppressor PTEN by mobility shift.

PTEN is reversibly oxidized in various cells by exogenous hydrogen peroxide as well as by endogenous hydrogen peroxide generated when cells are stimulated with growth factors, cytokines and hormones. A gel mobility shift assay showed that oxidized PTEN migrated more rapidly than reduced PTEN on a non-reducing SDS-PAGE gel. Oxidized PTEN was reduced when treated with dithiothreitol. Supplementation of N-ethylmaleimide in the cell lysis buffer was critical for the apparent bands of oxidized and reduced PTEN. Formation of oxidized PTEN was abolished when the active site Cys(124) or nearby Cys(71) was replaced with Ser suggesting that Cys(124) and Cys(71) are involved in the formation of an intramolecular disulfide bond. These results show that the mobility shift assay is a convenient method to analyze the redox state of PTEN in cells.

Chrysin inhibits cell invasion by inhibition of Recepteur d'origine Nantais via suppressing early growth response-1 and NF-κB transcription factor activities in gastric cancer cells.

Cell invasion is one of crucial reasons for cancer metastasis and malignancy. Recepteur d'origine Nantais (RON) has been reported to play an important role in the cancer cell invasion process. High accumulation and activation of RON has been implicated in gastric adenocarcinoma AGS cells. Chrysin is a naturally occurring phytochemical, a type of flavonoid, which has been reported to suppress tumor metastasis. However, the effects of chrysin on RON expression in gastric cancer are not well studied. In the present study, we examined whether chrysin affects RON expression in gastric cancer, and if so, its underlying mechanism. We examined the effect of chrysin on RON expression and activity, via RT-PCR, promoter study, and western blotting in human gastric cancer AGS cells. Chrysin significantly inhibited endogenous and inducible RON expression in a dose-dependent manner. After demonstrating that Egr-1 and NF-κB are the critically required transcription factors for RON expression, we discovered that chrysin suppressed Egr-1 and NF-κB transcription factor activities. Additionally, the phorbol-12-myristate-13-acetate- (PMA) induced cell invasion was partially abrogated by chrysin and an RON antibody. Our results suggest that chrysin has anticancer effects at least by suppressing RON expression through blocking Egr-1 and NF-κB in gastric cancer AGS cells.

Piperine inhibits IL-1ß-induced IL-6 expression by suppressing p38 MAPK and STAT3 activation in gastric cancer cells.

Piperine, a kind of natural alkaloid found in peppers, has been reported to exhibit anti-oxidative and anti-tumor activities, both in vitro and in vivo. Interleukin-6 (IL-6) is an important cytokine that activates the signal transduction, promotes tumor cell metastasis, and induces malignancy, including in gastric cancer. However, the effects of piperine on IL-6 expression in gastric cancer cells have not yet been well defined. In this study, we investigated the effects of piperine on the IL-6 expression, and examined the underlying signaling pathways via RT-PCR, promoter studies and Western blotting in human gastric cancer TMK-1 cells. Our results showed that piperine inhibited interleukin-1ß (IL-1ß)-induced IL-6 expression in a dose-dependent manner. In addition, piperine also inhibited IL-6 promoter activity. Experiments with mitogen-activated protein kinase (MAPK) inhibitors and dominant negative mutant p38 MAPK indicated that p38 MAPK was essential for IL-6 expression in the TMK-1 cells. Additionally, signal transducer and activator of transcription 3 (STAT3) was also involved in the IL-1ß-induced IL-6 expression in gastric cancer cells. Piperine inhibited IL-1ß-induced p38 MAPK and STAT3 activation and, in turn, blocked the IL-1ß-induced IL-6 expression. Furthermore, gastric cancer cells pretreated with IL-1ß showed markedly enhanced invasiveness, which was partially abrogated by treatment with IL-6 siRNA, piperine, and inhibitors of p38 MAPK and STAT3. These results suggest that piperine may exert at least part of its anti-cancer effect by controlling IL-6 expression through the suppression of p38 MAPK and STAT3.

Cadmium induces urokinase-type plasminogen activator receptor expression and the cell invasiveness of human gastric cancer cells via the ERK-1/2, NF-κB, and AP-1 signaling pathways.

Cadmium exposure has been linked to human cancers, including stomach cancer. In this study, the effects of cadmium on urokinase-type plasminogen activator receptor (uPAR) expression in human gastric cancer cells and the underlying signal transduction pathways were investigated. Cadmium induced uPAR expression in a time- and concentration-dependent manner. Cadmium also induced uPAR promoter activity. Additionally, cadmium induced the activation of extracellular signal regulated kinase-1/2 (ERK-1/2), p38 mitogen-activated protein kinase (MAPK), and the activation of c-Jun amino terminal kinase (JNK). A specific inhibitor of MEK-1 (PD98059) inhibited cadmium-induced uPAR expression, while JNK and p38 MAPK inhibitors did not. Expression vectors encoding dominant-negative MEK-1 (pMCL-K97M) also prevented cadmium-induced uPAR promoter activity. Site-directed mutagenesis and electrophoretic mobility shift studies showed that sites for the transcription factors nuclear factor (NF)-κB and activator protein-1 (AP-1) were involved in cadmium-induced uPAR transcription. Suppression of the cadmium-induced uPAR promoter activity by a mutated-type NF-κB-inducing kinase and I-κB and an AP-1 decoy oligonucleotide confirmed that the activation of NF-κB and AP-1 are essential for cadmium-induced uPAR upregulation. Cells pretreated with cadmium showed markedly enhanced invasiveness and this effect was partially abrogated by uPAR-neutralizing antibodies and by inhibitors of ERK-1/2, NF-κB, and AP-1. These results suggest that cadmium induces uPAR expression via ERK-1/2, NF-κB, and AP-1 signaling pathways and, in turn, stimulates cell invasiveness in human gastric cancer AGS cells.

Gastroprotective Effects of Glutinous Rice Extract against Ethanol-, Indomethacin-, and Stress-induced Ulcers in Rats.

This study was designed to evaluate the efficacy of an orally administered aqueous extract of glutinous rice (GRE) to protect against acute gastric mucosal lesions induced by ethanol, indomethacin, and water immersion restraint stress in rats and to characterize the active substances responsible for the protection. GRE was shown to dose-dependently prevent the gastric lesions induced by the above ulcerogenic treatments at doses of 30 to 300 mg/kg. GRE treatment increased the gastric mucin content and partially blocked the ethanol-induced depletion of the gastric mucus layer. Also, it increased the nonprotein sulfhydryl concentration in the gastric mucosa. The gastroprotective action of GRE was markedly enhanced by co-treatment with 4-8 mg/kg tea extracts. The activity of GRE was completely lost by heat treatment at 80℃ for 3 min or treatment with 0.01% pepsin at 37℃ for 1 h. Protein extraction studies indicated that prolamins are involved in the gastroprotective activity of GRE. Our results suggest that glutinous rice proteins are useful for the prevention and treatment of gastritis and peptic ulcer.

Comparative proteomic analysis of cysteine oxidation in colorectal cancer patients.

Oxidative stress promotes damage to cellular proteins, lipids, membranes and DNA, and plays a key role in the development of cancer. Reactive oxygen species disrupt redox homeostasis and promote tumor formation by initiating aberrant activation of signaling pathways that lead to tumorigenesis. We used shotgun proteomics to identify proteins containing oxidation-sensitive cysteines in tissue specimens from colorectal cancer patients. We then compared the patterns of cysteine oxidation in the membrane fractions between the tumor and non-tumor tissues. Using nano-UPLC-MS(E) proteomics, we identified 31 proteins containing 37 oxidation-sensitive cysteines. These proteins were observed with IAM-binding cysteines in non-tumoral region more than tumoral region of CRC patients. Then using the Ingenuity pathway program, we evaluated the cellular canonical networks connecting those proteins. Within the networks, proteins with multiple connections were related with organ morphology, cellular metabolism, and various disorders. We have thus identified networks of proteins whose redox status is altered by oxidative stress, perhaps leading to changes in cellular functionality that promotes tumorigenesis.

Medical students' failure experiences and their related factors.

PURPOSE: A considerable number of medical students drop out due to low academic achievement, and these students have a high probability of repeated failure experiences. This study investigated the personal and academic problems of these students to help develop student support systems. METHODS: First-year (n=146) and second-year (n=119) medical students were asked to complete questionnaires. The questionnaires consisted of personality traits and the students' management of/satisfaction with school life. RESULTS: Students who had already dropped out accounted for 17.4% of the study subjects. The most common reason for dropping out was low academic achievement, and the most difficult part of taking a leave of absence from school was psychological anxiety. The group who dropped out had significantly lower levels of emotional stability, sociability, responsibility, dominance, masculinity, and superiority and more vulnerable mental states compared with those who did not drop out. They also expressed less motivation with regard to medical science and less satisfaction with school life than did the group that did not drop out. Those who dropped out tended not to prepare for exams, and they managed their time ineffectively. They also tried to resolve their difficulties alone and rarely sought help from teachers. CONCLUSION: More intimate student-teacher relationships should be established, and teachers should be encouraged to meet and interact with their students on a regular basis. Additionally, personality inventories should be used to assist in efforts to understand students, especially to identify hidden social and emotional problems.

Tyr740 and Tyr751 residues of platelet-derived growth factor beta receptor are responsible for the redox regulation of phosphatase and tensin homolog in the cells stimulated with platelet-derived growth factor.

Exposure of cells to hydrogen peroxide or platelet-derived growth factor (PDGF) induced Akt phosphorylation and oxidation of phosphatase and tensin homolog (PTEN). The Cys124 and Cys71 residues of PTEN were critical for the formation of a disulfide bond and the intermediate glutathionylation in the process of reduction of the disulfide bond. To determine which specific tyrosine residues of the PDGF beta receptor (PDGFßR) is involved in PDGF-induced PTEN oxidation and Akt phosphorylation, we investigated a kinase activity-deficient mutant and PDGFßR mutants where the tyrosine residues in the binding site for phosphoinositide 3-kinase (PI3K), GTPase-activating protein of Ras, Src homology 2 domain containing protein-tyrosine phosphatase-2, and phospholipase C-1 were replaced by Phe. Both PTEN oxidation and Akt phosphorylation did not occur in response to PDGF in the kinase-deficient mutant and in the PDGFßR mutant with a mutation in the PI3K binding site (Tyr740 and Tyr751). Thus, the kinase activity and the constituent Tyr740 and Tyr751 residues of PDGFßR in the cells stimulated with PDGF are responsible for the oxidation of PTEN and the Akt phosphorylation.

Estrogen receptor α induces down-regulation of PTEN through PI3-kinase activation in breast cancer cells.

Estrogen receptor α (ERα) mediates most of the biological effects of estrogen in mammary epithelial cells and stimulates growth signals involving phosphoinositide-3-OH kinase (PI3K)/Akt in breast cancer cells. Phosphatase and tensin homologue (PTEN) is a critical counter-regulator of PI3K signaling and is thus one of the major tumor suppressors in breast cancer. Inhibition of PI3K with an inhibitor, wortmannin, increased the level of PTEN protein in ERα-positive MCF-7 cells, while levels in ERα-negative MDA-MB 231 cells were not altered. In addition, the level of PTEN protein in MCF-7 cells was significantly lower than that in MDA-MB 231 cells, which correlated with high levels of phospho-Akt and phosphatidylinositol-3,4,5,-trisphosphate (PIP3). However, PTEN mRNA expression as measured by real-time PCR showed no differences in either cell line. Notably, the levels of casein kinase 2 (CK2) and phospho-PTEN (Ser380/Thr382/383) in MCF-7 cells were lower than those in MDA-MB 231 cells, indicating that the down-regulation of PTEN protein in MCF-7 cells is caused by low levels of CK2 expression, leading to accelerated PTEN degradation. Collectively, these results suggest that ERα induces the down-regulation of PTEN through PI3K activation in breast cancer cells.

Redox regulation of the tumor suppressor PTEN by glutaredoxin 5 and Ycp4.

Human PTEN (phosphatase and tensin homolog deleted on chromosome 10; a phosphatidylinositol 3-phosphatase) expressed in Saccharomyces cerevisiae was oxidized in a time- and H(2)O(2)-concentration-dependent manner. Oxidized hPTEN was reduced by cellular reductants as in human cells. The reduction rate of oxidized hPTEN was monitored in S. cerevisiae mutants in which the genes involved in redox homeostasis had been disrupted. Reduction of hPTEN was delayed in each of S. cerevisiae grx5Δ and ycp4Δ mutants. Expression of Grx5 and Ycp4 in each of the mutants rescued the reduction rate of oxidized hPTEN. Furthermore, an in vitro assay revealed that the human Grx5/GSH system efficiently catalyzed the reduction of oxidized hPTEN. These results suggest that the reduction of oxidized hPTEN is regulated by Grx5 and Ycp4.

Effect of light and reductones on differentiation of Pleurotus ostreatus.

Vegetative mycelia of Pleurotus ostreatus were differentiated into primordia and subsequently into fruit bodies in synthetic sucrose-asparagine medium when exposed to light at low temperature. During photo-morphogenesis, L-ascorbic acid-like substances called reductones were produced. L-ascorbic acid, D-erythroascorbic acid, 5-O-(α-D-glucopyranosyl)-D-erythroascorbic acid, 5-O-(α-D-xylopyranosyl)-D-erythroascorbic acid, 5-methyl-5-O-(α-D-glucopyranosyl)-D-erythroascorbic acid and 5-methyl-5-O-(α-D-xylopyranosyl)-D-erythroascorbic acid were accumulated initially in the illuminated mycelia before the initiation of fruiting. The content of glycosides of erythroascorbic acid and their methylated compounds increased again in the primordia and the fruit bodies. Exogenous L-ascorbic acid induced the formation of primordia from the mycelia in the dark in a dose-dependent manner. Thus, this suggests that these reductones might play a role in mediating the light stimulus in photomorphogenesis.