Novel predictive epigenetic signature for temozolomide in non-G-CIMP glioblastomas.

OBJECTIVE: To identify novel epigenetic signatures that could provide predictive information that is complementary to promoter methylation status of the O-6-methylguanine-DNA methyltransferase (MGMT) gene for predicting temozolomide (TMZ) response, among glioblastomas (GBMs) without glioma-CpGs island methylator phenotype (G-CIMP) METHODS: Different cohorts of primary non-G-CIMP GBMs with genome-wide DNA methylation microarray data were included for discovery and validation of a multimarker signature, combined using a RISK score model. Different statistical analyses and functional experiments were performed for clinical and biological validation. RESULTS: By employing discovery cohorts with radiotherapy (RT) and TMZ versus RT alone and a strict multistep selection strategy, we identified seven CpGs, each of which was significantly correlated with overall survival (OS) of non-G-CIMP GBMs with RT/TMZ, independent of age, MGMT promoter methylation status, and other identified CpGs. A RISK score signature of the 7 CpGs was developed and validated to distinguish non-G-CIMP GBMs with differential survival outcomes to RT/TMZ, but not to RT alone. The interaction analyses also showed differential outcomes to RT/TMZ versus RT alone within the RISK score-based subgroups. The signature could also improve the risk classification by age and MGMT promoter methylation status. Functional experiments showed that HSBP2 appeared to be epigenetically regulated by one identified CpG and was associated with TMZ resistance, but it was not associated with cell proliferation or apoptosis in GBM cell lines. The predictive value of the single CpG methylation of HSBP2 by pyrosequencing was observed in a local cohort of isocitrate dehydrogenase 1 (IDH1) R132H wild-type GBMs. CONCLUSIONS: This novel epigenetic signature might be a promising predictive (but not a general prognostic) biomarker and be helpful for refining the MGMT-based guiding approach to TMZ usage in non-G-CIMP GBMs.

Effects of RNA binding protein QKI on pancreatic cancer ductal epithelial cells and surrounding activation fibroblasts.

To determine the correlation between QKI and pancreatic cancer tissues, the QKI expression of pancreatic cancer cells and fibroblasts in the tumor-surrounding microenvironment were detected. Then, QKI overexpression and interference with QKI short hairpin RNA in LX-2 (a fibroblast cell line) were established in vitro. Meanwhile, to observe the cell proliferation, invasion, migration, and other changes, QKI, and related epithelial-mesenchymal transition (EMT) molecules were detected by a polymerase chain reaction and Western blot analysis. In addition, an in vivo tumorigenicity test in node mice was performed to confirm whether QKI expression can promote the proliferation, invasion, and metastasis of pancreatic cancer ductal epithelial cells. Finally, the autophagy levels of fibroblasts with QKI overexpression were observed by electron microscopy to further explore the QKI pathogenic mechanism. It was found that cell proliferation, invasion, migration, and EMT-related markers were increased in QKI-overexpressed fibroblasts LX-2. Furthermore, in vivo, liver and peritoneal metastasis decreased overall survival rate and increasing autophagy levels in QKI-overexpressing nude mice were observed. Meanwhile, knock down QKI with small interfering RNA can reverse all the above effects. QKI can promote the proliferation, metastasis, and invasion of pancreatic cancer through activating fibroblasts surrounding pancreatic cancer and accelerating EMT and increasing the autophagy in pancreatic cancer. QKI may become a potential target for the treatment of pancreatic cancer.

Hyperglycemia promotes vasculogenesis in choroidal neovascularization in diabetic mice by stimulating VEGF and SDF-1 expression in retinal pigment epithelial cells.

To investigate the influence of hyperglycemia on the severity of choroidal neovascularization (CNV) in diabetic mice, especially the involvement of bone marrow-derived cells (BMCs) and underlying molecular mechanisms. The mice were randomly divided into control group, diabetes group and diabetes treated with insulin group, which were laser treated to induce CNV. The CNV severity was evaluated by fundus fluorescein angiography, HE staining and choroidal flatmount. The BMCs recruitment and differentiation in CNV were examined in GFP chimeric mice by choroidal flatmount and immunofluorescence. The bone marrow-derived mesenchymal stem cells (BMSCs) recruitment and migration were tested in vivo and in vitro. VEGF and SDF-1 production in vivo and in vitro were tested by realtime PCR and ELISA. The CNV severity and expression of VEGF and SDF-1 were enhanced in DM mice compared with control mice and that insulin treatment decreased CNV severity in DM mice. The DM mice demonstrated more BMCs and bone marrow-derived mesenchymal stem cells (BMSCs) recruited and incorporated into CNV, increased ratio of BMCs expressing endothelial cell marker or macrophage marker, and up-regulated expression of VEGF and SDF-1 in CNV. Human BMSCs migration and expression of VEGF and SDF-1 in retinal pigment epithelial (RPE) cells increased when cultured under high glucose. This study suggested that hyperglycemia enhanced the expression of VEGF and SDF-1 in RPE cells, and promoted recruitment and incorporation of BMCs and affected differentiation of BMCs in CNV, which led to more severe CNV in diabetic mice.

[Effects of nuclear factor of activated T cells on the promoter activity of constitutively activated SV40].

AIM: To check if the common used constitutively promoters, such as CMV, TK and SV40 could be responded to the nuclear factor of activated T cell (NFAT), and to explore the strategies to choose rational internal control in the dual luciferase reporter assay. METHODS: pCMV-luc vector, in which luciferase activity is driven by CMV promoter, was cloned by amplifying the CMV promoter fragment from the pCDNA3.1 vector and then inserting the CMV promoter region into the pGL3-basic vector using the standard protocol. pTK-Luc reporter was similarly constructed, with the TK promoter from the pRL-TK vector. The constructed pCMV-Luc or pTK-Luc was co-transfected with pBIND or pRL-TK respectively, together with NFAT or constitutively active form named NFATCA. Relative luciferase activity was calculated as instructed by the manual instruction. RESULTS: Both pCMV-Luc and pTK-Luc vectors were successfully constructed. Luciferase activity assay revealed that SV40 promoter responded to active NFAT. CONCLUSION: The common used internal control promoter SV40 could respond to active NFAT, which should be kept in mind for selection of the rational internal control vector in the dual luciferase reporter assay. In addition, our study here also provides a practical strategy for rational selection of the internal control.

Inhibition of glucocorticoid-induced alteration of vimentin by a glucocorticoid receptor antagonist RU486 in the organ-cultured rat lens.

PURPOSE: Long-term application of glucocorticoids as a treatment for conditions such as allergy, autoimmune diseases, and transplantation presents a high risk of development of steroid-induced cataract. The presence of a functional glucocorticoid receptor (GR) in human and rat lens epithelial cells suggests a direct and specific targeting of these lens cells by glucocorticoids. One important cytoskeletal protein in lens epithelial cells is vimentin, which plays an important role in maintaining the normal lens morphology and function. Previous studies have shown that vimentin is involved in signal transduction, changes in cell structure and differentiation, and apoptosis. Based on a model of steroid-induced cataract from our previous study, the present study focuses on whether changes in vimentin can be induced in vitro through specific GR activation in glucocorticoid-induced cataracts of the rat lens. METHODS: Clear rat lenses, cultured in vitro, were treated with or without dexamethasone (Dex) or RU486 (a glucocorticoid receptor antagonist). Lenses were cultured for 7 days at 37 °C under 5% CO2, and were observed daily with an inverted microscope. Changes in morphology were followed by Hematoxylin-eosin (HE) staining, transmission electron microscopy, and immunohistochemistry. The expression of vimentin mRNA and protein was examined by reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis, respectively, in the capsule-epithelium and fiber tissue of the lenses. RESULTS: Opacity was obviously present at day 7 in the Dex group. The lenses of the untreated group and the RU486+Dex group remained transparent throughout the incubation. Electron microscopy showed an orderly arrangement of fiber cells and normal cell junctions in the control group and the RU486+Dex group. However, in the Dex group, fiber cells were disarranged and the cell-cell junctions exhibited lacunae. The expression of vimentin protein in the lens capsule-epithelium and fiber tissue decreased in the Dex-treated group, but normal expression of vimentin mRNA was maintained. CONCLUSIONS: These results suggest that the GR-mediated reduction in vimentin may be involved in the formation of steroid-induced cataract.

Glycyrrhizin treatment is associated with attenuation of lipopolysaccharide-induced acute lung injury by inhibiting cyclooxygenase-2 and inducible nitric oxide synthase expression.

Glycyrrhizin (GL), a major active constituent of licorice root, has been attributed numerous pharmacologic effects, including anti-inflammatory, anti-viral, anti-tumor, and hepatoprotective activities. In this study, we investigated the anti-inflammatory effect of GL on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. ALI was induced in Balb/c mice by intratracheal instillation of LPS (1 mg/kg). Before 1 h of LPS administration, the mice received intraperitoneal injection of GL at varied doses (10, 25, and 50 mg/kg). The severity of pulmonary injury was evaluated 12 h after LPS administration. GL pretreatment led to significant attenuation of LPS induced evident lung histopathologic changes, alveolar hemorrhage, and neutrophil infiltration with evidence of reduced myeloperoxidase (MPO) activity. The lung wet/dry weight ratios, as an index of lung edema, were markedly reduced by GL pretreatment. The concentrations of pro-inflammatory cytokines interleukin (IL)-1ß and tumor necrosis factor (TNF)-α were elevated in bronchoalveolar lavage fluid (BALF) after LPS administration, which were significantly inhibited by GL pretreatment. GL pretreatment also reduced the concentrations of nitric oxide (NO) in lung tissues. Furthermore, the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) was suppressed by GL pretreatment. In conclusion, GL potently protected against LPS-induced ALI, and the protective effects of GL may attribute partly to the suppression of COX-2 and iNOS expression.

Protective effect of nicotine on lipopolysaccharide-induced acute lung injury in mice.

BACKGROUND: Recently, nicotine administration has been shown to be a potent inhibitor of a variety of innate immune responses, including endotoxin-induced sepsis. OBJECTIVE: It was the aim of this study to evaluate the effect of nicotine on attenuating lung injury and improving the survival in mice with lipopolysaccharide (LPS)-induced acute lung injury (ALI). METHODS: ALI was induced in mice by intratracheal instillation of LPS (3 mg/ml). The mice received intratracheal instillation of nicotine (50, 250 and 500 µg/kg) before or after LPS administration. Pulmonary histological changes were evaluated by hematoxylin-eosin stain, and lung wet/dry weight ratios were observed. Concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and high mobility group box (HMGB)-1, as well as myeloperoxidase (MPO) activity were measured by enzyme-linked immunosorbent assay. The mortality rate was recorded and analyzed by the Kaplan-Meier method. RESULTS: Nicotine pretreatment significantly attenuated the severity of lung injury and inhibited the production of TNF-α, IL-1ß and HMGB-1 in mice with ALI. After LPS administration, the lung wet/dry weight ratios, as an index of lung edema, and MPO activity were also markedly reduced by nicotine pretreatment. Early treatment with a high dose of nicotine (500 µg/kg) after LPS administration decreased the mortality in mice with ALI, even when treatment was started 24 h after LPS administration. CONCLUSION: Nicotine attenuated the lung injury and reduced mortality in mice with LPS-induced ALI.

Inhibition of glucocorticoid-induced changes of Na(+), K(+)-ATPase in rat lens by a glucocorticoid receptor antagonist RU486.

Cataract formation can be induced by prolonged use of glucocorticoids. The underlying mechanism is not fully understood yet. The presence of the functional glucocorticoid receptor (GR) in human and rat lens epithelial cells suggests that glucocorticoids target lens epithelial cells directly and specifically. Na(+), K(+)-ATPase has long been recognized for its role in regulating electrolyte concentration in the lens, contributing to lens transparency. We previously reported that the inactivation of Na(+), K(+)-ATPase induced by a glucocorticoid in rat lens. Therefore, the question is whether the changes of Na(+), K(+)-ATPase can be induced through the specific GR activation in glucocorticoid-induced cataract formation. Clear rat lenses were cultured in vitro and were treated with or without dexamethasone (Dex) or RU486 (a GR antagonist). The lenses were cultured for 7 days and photographed daily to record the development of opacity. The activity of Na(+), K(+)-ATPase was determined by using spectrophotometric analysis. The mRNA and protein level expressions of Na(+), K(+)-ATPase α1 were examined by reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry analysis, respectively. Our findings are presented in this study and show that mist-like opacity of the lens was observed as early as 5 days after incubation with dexamethasone. The opacity was more obvious at day 7 in the Dex group. The lenses of the untreated group and the RU486+Dex group remained transparent throughout the incubation. The activity of Na(+), K(+)-ATPase in the Dex-treated group decreased in a time-dependent manner. There was no significant loss of enzyme activity in either the control or the RU486+Dex group throughout the incubation period. Both the protein and mRNA expression levels of Na(+), K(+)-ATPase α1 in the capsule-epithelium of lenses decreased in the Dex-treated group. The GR antagonist RU486 inhibited the decrease of the expression of Na(+), K(+)-ATPase α1 induced by Dex. All of the above results suggested that the GR-mediated reduction of Na(+), K(+)-ATPase may contribute to the formation of steroid-induced cataract. Intervention in this pathway maybe helpful to avoid glucocorticoids-cataract formation.

Simvastatin inhibits angiotensin II-induced cardiac cell hypertrophy: role of Homer 1a.

1. The scaffolding protein Homer 1a is constitutively expressed in the myocardium, although its function in cardiomyocytes remains poorly understood. The aim of the present study was to investigate Homer 1a expression in hypertrophic cardiac cells and its role in angiotensin (Ang) II-induced cardiac hypertrophy. 2. After serum starvation for 24 h, cells were treated with 1 micromol/L simvastatin, 100 nmol/L angiotensin (Ang) II or their combination added to Dulbecco's modified Eagle's medium containing 0.5% serum. For combination treatment with AngII plus simvastatin, cells were exposed to simvastatin 12 h before the addition of AngII to the medium and cells were then incubated in the presence of both drugs for a further 24 h. Western blotting was used to determine Homer 1a protein expression. Hypertrophy was evaluated by determining the protein content per cell. 3. Homer 1a protein levels were upregulated following AngII-induced hypertrophy in H9C2 cells and neonatal rat cardiomyocytes, and these increases were augmented by simvastatin pretreatment. Concomitantly, simvastatin pretreatment inhibited extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and AngII-induced hypertrophy. 4. The inhibitory effects of simvastatin against AngII-induced hypertrophy were attenuated by Homer 1a silencing, suggesting that simvastatin suppresses cardiac hypertrophy in a Homer 1a-dependent manner. Furthermore, AngII-induced hypertrophy and ERK1/2 phosphorylation in neonatal rat cardiomyocytes were significantly inhibited following the overexpression of Homer 1a using an adenovirus. 5. These results suggest a possible role for Homer 1a in inhibiting cardiac hypertrophy perhaps in part through inhibition of ERK1/2 activation.