Epigenetic and Glucocorticoid Receptor-Mediated Regulation of Glutathione Peroxidase 3 in Lung Cancer Cells.

Glutathione peroxidase 3 (GPx3), an antioxidant enzyme, acts as a modulator of redox signaling, has immunomodulatory function, and catalyzes the detoxification of reactive oxygen species (ROS). GPx3 has been identified as a tumor suppressor in many cancers. Although hyper-methylation of the GPx3 promoter has been shown to down-regulate its expression, other mechanisms by which GPx3 expression is regulated have not been reported. The aim of this study was to further elucidate the mechanisms of GPx3 regulation. GPx3 gene analysis predicted the presence of ten glucocorticoid response elements (GREs) on the GPx3 gene. This result prompted us to investigate whether GPx3 expression is regulated by the glucocorticoid receptor (GR), which is implicated in tumor response to chemotherapy. The corticosteroid dexamethasone (Dex) was used to examine the possible relationship between GR and GPx3 expression. Dex significantly induced GPx3 expression in H1299, H1650, and H1975 cell lines, which exhibit low levels of GPx3 expression under normal conditions. The results of EMSA and ChIP-PCR suggest that GR binds directly to GRE 6 and 7, both of which are located near the GPx3 promoter. Assessment of GPx3 transcription efficiency using a luciferase reporter system showed that blocking formation of the GR-GRE complexes reduced luciferase activity by 7-8-fold. Suppression of GR expression by siRNA transfection also induced down-regulation of GPx3. These data indicate that GPx3 expression can be regulated independently via epigenetic or GR-mediated mechanisms in lung cancer cells, and suggest that GPx3 could potentiate glucocorticoid (GC)-mediated anti-inflammatory signaling in lung cancer cells.

Quantitative pattern analysis of the N-terminally processed isoforms of platelet factor-4 in serum.

RATIONALE: Platelet factor 4 (PF4) is a small cytokine belonging to the CXC chemokine family which has been shown to play a role in inflammation and in the regulation of angiogenesis. In general, chemokines are susceptible to proteolytic cleavage in amino and carboxy terminal regions, which usually results in dramatic changes to the chemokine bioactivity. The purpose of this study was to identify various platelet factor-4 (PF4) isoforms caused by proteolytic processing and to quantify their levels in normal serum. METHODS: First, we identified the N-terminally truncated PF4 isoforms from a standard purified PF4 protein sample by using mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analysis. Then, we used high-performance liquid chromatography (HPLC) to semi-purify PF4 from serum samples, and the levels of the four most abundant PF4 isoforms were quantitatively determined using selected reaction monitoring (SRM) assays on a nano-LC/triple-quadrupole mass spectrometer. RESULTS: We have identified seven N-terminally processed PF4 isoforms and compared the levels of major PF4 isoforms from nine serum samples. Pro-p1 (EAEEDGDLQCLCVK-; average MW, 7765.2) is the major PF4 isoform in serum whereas the PF4 isoforms, designated Prot-p4 (FASAEAEEDGDLQCLCVK-;average MW, 8141.5), Prot-p3 (SAEAEEDGDLQCLCVK-; average MW, 7923.3), and Prot-p2 (AEEDGDLQCLCVK- ; average MW, 7836.3), are at about 16%, 3%, and 2% levels of the major one, respectively. CONCLUSIONS: This study is the first report on the levels of N-terminally processed PF4 isoforms in serum. Also, this study shows the usefulness of SRM in determining concentrations of protein isoform variants, which can be often overlooked in immunoassay analysis.

Periovulatory expression of hydrogen peroxide-induced sulfiredoxin and peroxiredoxin 2 in the rat ovary: gonadotropin regulation and potential modification.

Reactive oxygen species are involved in ovulation. The aim of this study was to examine gonadotropin regulation of antioxidant enzyme sulfiredoxin (Srx) and peroxiredoxin 2 (PRDX2) expressions and modification during the ovulatory process in rats. Administration of antioxidants in vivo reduced ovulation rate and cumulus expansion. LH treatment increased H(2)O(2) levels within 15 min, which, in turn, induced Srx gene expression in cultured preovulatory follicles. Treatment of preovulatory follicles with catalase suppressed the stimulatory effect of LH on Akt phosphorylation. LH- or H(2)O(2)-stimulated Srx mRNA levels were suppressed by inhibitors of antioxidant agents and MAPK kinase. An in vivo injection of equine chorionic gonadotropin-human chorionic gonadotropin (hCG) stimulated Srx mRNA within 1 h in granulosa but not thecal cells of preovulatory follicles. Srx protein levels were stimulated from 3 h post-hCG injection. Immunofluorescence analysis revealed that oocytes expressed the Srx protein. Furthermore, hCG treatment increased Srx expression in mural granulosa, theca and cumulus cells, but the Srx protein was not detected in corpora lutea. Gene expression of PRDX2, identified as an Srx-dependent modified enzyme, was stimulated by gonadotropins. In situ hybridization analysis demonstrated that PRDX2 mRNA was detected in oocytes and theca cells as well as granulosa cells of some antral and preovulatory follicles. High levels of PRDX2 mRNA were detected in corpora lutea. Total levels of PRDX2 protein were not changed by gonadotropins. However, levels of hyperoxidized PRDX2 increased within 2-3 h after the hCG injection. Taken together, gonadotropin stimulation of Srx expression and PRDX2 modification in the ovary suggest the existence of an antioxidant system to maintain H(2)O(2) production and elimination during the periovulatory period.

Anti-cancer properties of glucosamine-hydrochloride in YD-8 human oral cancer cells: Induction of the caspase-dependent apoptosis and down-regulation of HIF-1α.

Evidence suggests anti-tumor activities of glucosamine-hydrochloride (GS-HCl). In the present study, we investigated anti-proliferative, growth suppressive and/or pro-apoptotic effects of GS-HCl on YD-8 human oral squamous cell carcinoma (OSCC) cells. Fundamentally, treatment with GS-HCl strongly inhibited proliferation and induced apoptosis in YD-8 cells, as determined by MTS and DNA fragmentation analyses. Of further note, as measured by Western analyses, GS-HCl treatment led to activation of caspase-3, cytosolic accumulation of cytochrome c, down-regulation of Mcl-1 and HIF-1α, up-regulation of GRP78, an indicator of ER stress, and generation of ROS in YD-8 cells. Importantly, results of pharmacological inhibition studies showed that treatment with z-VAD-fmk, a pan-caspase inhibitor, but not with vitamin E, an anti-oxidant strongly blocked the GS-HCl-induced apoptosis in YD-8 cells. Analyses of additional cell culture works further revealed that GS-HCl had a strong growth suppressive effect on not only YD-8 but also YD-10B and YD-38, two other human OSCC cell lines. These findings collectively demonstrate that GS-HCl has anti-proliferative, anti-survival, and pro-apoptotic effects on YD-8 cells and the effects appear to be mediated via mechanisms associated with the mitochondrial-dependent activation of caspases, down-regulation of Mcl-1, and induction of ER stress. Considering HIF-1α as a tumor angiogenic transcription factor, the ability of GS-HCl to down-regulate HIF-1α in YD-8 cells may further support its anti-cancer property. It is thus suggested that GS-HCl may be used as a potential anti-cancer drug against human OSCC.

Overexpression of cyclooxygenase-2 in NCI-H292 human alveolar epithelial carcinoma cells: roles of p38 MAPK, ERK-1/2, and PI3K/PKB signaling proteins.

Evidence suggests overexpression of COX-2 and its role in many human cancers, including lung. However, the regulatory mechanism underlying COX-2 overexpression in lung cancer is not fully understood. We herein investigated whether COX-2 is overexpressed in human airway cancer cell lines, including A549 (lung), Hep-2 (bronchial), and NCI-H292 (alveolar). When grown in cell culture medium containing 10% FBS (serum), of note, there was strong and transient induction of COX-2 protein and mRNA in NCI-H292 cells, but little or low COX-2 expression is seen in A549 or Hep-2 cells. Interestingly, strong and sustained activities of ERK-1/2, JNK-1/2, p38 MAPK, and PKB were also shown in NCI-H292 cells grown in presence of serum. Profoundly, results of pharmacological inhibition studies demonstrated that the serum-dependent COX-2 up-regulation in NCI-H292 cells is attributed to not only the p38 MAPK-, PI3K/PKB-, and ERK-1/2-mediated COX-2 transcriptional up-regulation but also the p38 MAPK- and ERK-1/2-mediated post-transcriptional COX-2 mRNA stabilization. Of further note, it was shown that the ERK-1/2 and PI3K/PKB (but not COX-2, p38 MAPK, and JNK-1/2) activities are necessary for growth of NCI-H292 cells. These findings collectively demonstrate for the first time that COX-2 expression is transiently up-regulated by serum addition in NCI-H292 cells and the serum-induced COX-2 expression is closely linked to the p38 MAPK-, ERK-1/2-, and PI3K/PKB-mediated COX-2 transcriptional and post-transcriptional up-regulation.

Attenuation of cell cycle progression by 2,3,7,8-tetrachlorodibenzo-p-dioxin eliciting ovulatory blockade in gonadotropin-primed immature rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) reduces ovulation rate in rats. The present study was to investigate whether TCDD alters the progression of cell cycle, and thus resulting in the blockade of ovulation in gonadotropin-primed, immature rats. The ovulation rate and ovarian weight were reduced in intact rats given TCDD (32 µg/kg BW in corn oil) by gavage one day before pregnant mare's serum gonadotropin (PMSG; 5 IU/rat) injection. Flow cytometry demonstrated that the percentage of granulosa cells in S-phase was increased at 24 h following PMSG treatment, but declined at 8 h following hCG treatment in corn oil-treated rats. Interestingly, the number of S-phase cells in TCDD-treated rats was reduced 24 and 48 h following PMSG treatment. TCDD, however, increased the percentage of cells in G2/M-phase at 24 h following PMSG treatment. TCDD inhibited the mRNA levels of Cdk2 at 0 h and 24 h, and cyclin D2 at 24 h and 48 h following PMSG treatment. Protein levels of aryl hydrocarbon receptor in granulosa cells were elevated in TCDD-treated rats at 12 h and 24 h following PMSG treatment. The present study indicates that TCDD reduces S-phase cells and inhibits levels of Cdk2 and cyclin D2 at 24 h following PMSG treatment, implying the ovulation-inhibiting action of TCDD may be exerted through the attenuation of cell cycle progression via AhR-mediated cascade.