SERPINE1 and SERPINB7 as potential biomarkers for intravenous vitamin C treatment in non-small-cell lung cancer.

High dose intravenous vitamin C (IVC) has been proposed as a pro-oxidant anticancer agent. However, there is a lack of biomarkers that are specific for this treatment. Here, we explored profiles of gene expression responding to IVC treatment in non-small cell lung cancer (NSCLC) cells as an effort for potential biomarker discovery. Genome-wide RNA-seq was performed in human NSCLC cell lines treated with pharmacological concentrations of vitamin C(VitC) for differential expression of genes. The identified genes were analyzed for correlations with patient prognosis using data from the Kaplan-Meier Plotter and the Human Protein Atlas databases. Further, tumor samples from a retrospective study of 153 NSCLC patients were analyzed with immunohistochemistry for expression of targeted genes, and patient prognosis was correlated to these genes. Two genes, namely SERPINE1 and SERPINB7 were found to be downregulated in NSCLC cells following VitC treatment. Combined patient data from the cohort analysis and online databases revealed that these 2 genes presented an unfavorable prognostic prediction of overall survival (OS) in NSCLC patients receiving standard of care. However, high expression level of these 2 genes were associated with prolonged OS in NSCLC patients receiving IVC in addition to standard of care. These data revealed that SERPINE1 and SERPINB7 have the potential to serve as predictive factors indicating favorable responses to IVC treatment in patients with NSCLC. Further validations are warranted.

Telomere length correlates with subtelomeric DNA methylation in long-term mindfulness practitioners.

Mindfulness and meditation techniques have proven successful for the reduction of stress and improvement in general health. In addition, meditation is linked to longevity and longer telomere length, a proposed biomarker of human aging. Interestingly, DNA methylation changes have been described at specific subtelomeric regions in long-term meditators compared to controls. However, the molecular basis underlying these beneficial effects of meditation on human health still remains unclear. Here we show that DNA methylation levels, measured by the Infinium HumanMethylation450 BeadChip (Illumina) array, at specific subtelomeric regions containing GPR31 and SERPINB9 genes were associated with telomere length in long-term meditators with a strong statistical trend when correcting for multiple testing. Notably, age showed no association with telomere length in the group of long-term meditators. These results may suggest that long-term meditation could be related to epigenetic mechanisms, in particular gene-specific DNA methylation changes at distinct subtelomeric regions.

The contrary intracellular and extracellular functions of PEDF in HCC development.

Pigment epithelium-derived factor (PEDF), a classic angiogenic inhibitor, has been reported to function as a tumor suppression protein and to downregulate in many types of solid tumors. However, the expression level of PEDF and its role in hepatocellular carcinoma (HCC) are contradictory. The present study investigates the expression and different activities of secreted and intracellular PEDF during HCC development, as well as the underlying mechanism of PEDF on HCC lipid disorders. We found that PEDF had no association with patients' prognosis, although PEDF was highly expressed and inhibited angiogenesis in HCC tumor tissues. The animal experiments indicated that full-length PEDF exhibited equalizing effects on tumor growth activation and tumor angiogenesis inhibition in the late stage of HCC progression. Importantly, the pro-tumor activity was mediated by the intracellular PEDF, which causes accumulation of free fatty acids (FFAs) in vivo and in vitro. Based on the correlation analysis of PEDF and lipid metabolic indexes in human HCC tissues, we demonstrated that the intracellular PEDF led to the accumulation of FFA and eventually promoted HCC cell growth by inhibiting the activation of AMPK via ubiquitin-proteasome-mediated degradation, which causes increased de novo fatty acid synthesis and decreased FFA oxidation. Our findings revealed why elevated PEDF did not improve the patients' prognosis as the offsetting intracellular and extracellular activities. This study will lead to a comprehensive understanding of the diverse role of PEDF in HCC and provide a new selective strategy by supplement of extracellular PEDF and downregulation of intracellular PEDF for the prevention and treatment of liver cancer.

Astragalus root extract inhibits retinal cell apoptosis and repairs damaged retinal neovascularization in retinopathy of prematurity.

Since the functions of Astragalus root extract in retinopathy remain to be unraveled, this study is performed to elucidate whether Astragalus root extract functions in retinal cell apoptosis and angiogenesis in retinopathy of prematurity (ROP). Newborn mice were selected for establishing mice models of oxygen-induced retinopathy (OIR), which were treated with high-, medium- or low-Astragalus root extract. Evans Blue (EB) was perfused to detect the blood retinal barrier. Additionally, the vascular morphology, number of endothelial cell nuclei of neovascularization, proliferation of blood vessels, ultrastructural changes were determined via a series of assays. Moreover, levels of reactive oxygen species (ROS), expression of other factors such as VEGF, PEDF, IGF-1, HIF-1α, Bax, Bcl-2, eNOS, nNOS, and iNOS were detected. Astragalus root extract was found to protect blood-retinal barrier in the OIR model mice through repairing the structure and morphology of retina, inhibiting ROS production, retinal cell apoptosis, as well as improving retinal vascular angiogenesis. Astragalus root extract was also found to decrease VEGF and HIF-1α expression, but enhance PEDF and IGF-1 expression in the OIR model mice, thereby protecting retinas in ROP. This study highlights that Astragalus root extract is able to suppress retinal cell apoptosis and repair damaged retinal neovascularization in ROP, which provides basis for ROP therapy.

Diet induced obesity is independent of metabolic endotoxemia and TLR4 signalling, but markedly increases hypothalamic expression of the acute phase protein, SerpinA3N.

Hypothalamic inflammation is thought to contribute to obesity. One potential mechanism is via gut microbiota derived bacterial lipopolysaccharide (LPS) entering into the circulation and activation of Toll-like receptor-4. This is called metabolic endotoxemia. Another potential mechanism is systemic inflammation arising from sustained exposure to high-fat diet (HFD) over more than 12 weeks. In this study we show that mice fed HFD over 8 weeks become obese and show elevated plasma LPS binding protein, yet body weight gain and adiposity is not attenuated in mice lacking Tlr4 or its co-receptor Cd14. In addition, caecal microbiota composition remained unchanged by diet. Exposure of mice to HFD over a more prolonged period (20 weeks) to drive systemic inflammation also caused obesity. RNAseq used to assess hypothalamic inflammation in these mice showed increased hypothalamic expression of Serpina3n and Socs3 in response to HFD, with few other genes altered. In situ hybridisation confirmed increased Serpina3n and Socs3 expression in the ARC and DMH at 20-weeks, but also at 8-weeks and increased SerpinA3N protein could be detected as early as 1 week on HFD. Overall these data show lack of hypothalamic inflammation in response to HFD and that metabolic endotoxemia does not link HFD to obesity.

Nanosecond Laser Treatment for Age-Related Macular Degeneration Does Not Induce Focal Vision Loss or New Vessel Growth in the Retina.

Purpose: Subthreshold, nanosecond pulsed laser treatment shows promise as a treatment for age-related macular degeneration (AMD); however, the safety profile needs to be robustly examined. The aim of this study was to investigate the effects of laser treatment in humans and mice. Methods: Patients with AMD were treated with nanosecond pulsed laser at subthreshold (no visible retinal effect) energy doses (0.15-0.45 mJ) and retinal sensitivity was assessed with microperimetry. Adult C57BL6J mice were treated at subthreshold (0.065 mJ) and suprathreshold (photoreceptor loss, 0.5 mJ) energy settings. The retinal and vascular responses were analyzed by fundus imaging, histologic assessment, and quantitative PCR. Results: Microperimetry analysis showed laser treatment had no effect on retinal sensitivity under treated areas in patients 6 months to 7 years after treatment. In mice, subthreshold laser treatment induced RPE loss at 5 hours, and by 7 days the RPE had retiled. Fundus imaging showed reduced RPE pigmentation but no change in retinal thickness up to 3 months. Electron microscopy revealed changes in melanosomes in the RPE, but Bruch's membrane was intact across the laser regions. Histologic analysis showed normal vasculature and no neovascularization. Suprathreshold laser treatment did not induce changes in angiogenic genes associated with neovascularization. Instead pigment epithelium-derived factor, an antiangiogenic factor, was upregulated. Conclusions: In humans, low-energy, nanosecond pulsed laser treatment is not damaging to local retinal sensitivity. In mice, treatment does not damage Bruch's membrane or induce neovascularization, highlighting a reduced side effect profile of this nanosecond laser when used in a subthreshold manner.

Antiapoptotic serine protease inhibitors contribute to survival of allergenic TH2 cells.

BACKGROUND: The mechanisms that regulate maintenance of persistent TH2 cells and potentiate allergic inflammation are not well understood. OBJECTIVE: The function of serine protease inhibitor 2A (Spi2A) was studied in mouse TH2 cells, and the serine protease inhibitor B3 (SERPINB3) and SERPINB4 genes were studied in TH2 cells from patients with grass pollen allergy. METHODS: Spi2A-deficient TH2 cells were studied in in vitro culture or in vivo after challenge of Spi2A knockout mice with ovalbumin in alum. Expression of SERPINB3 and SERPINB4 mRNA was measured in in vitro-cultured TH2 cells and in ex vivo CD27-CD4+ cells and innate lymphoid cell (ILC) 2 from patients with grass pollen allergy by using quantitative PCR. SERPINB3 and SERPINB4 mRNA levels were knocked down in cultured CD27-CD4+ cells with small hairpin RNA. RESULTS: There were lower levels of in vitro-polarized TH2 cells from Spi2A knockout mice (P < .005) and in vivo after ovalbumin challenge (P < .05), higher levels of apoptosis (Annexin V positivity, P < .005), and less lung allergic inflammation (number of lung eosinophils, P < .005). In vitro-polarized TH2 cells from patients with grass pollen allergy expressed higher levels of both SERPINB3 and SERPINB4 mRNA (both P < .05) compared with unpolarized CD4 T cells. CD27-CD4+ from patients with grass pollen allergy expressed higher levels of both SERPINB3 and SERPINB4 mRNA (both P < .0005) compared with CD27+CD4+ cells. ILC2 expressed higher levels of both SERPINB3 and SERPINB4 mRNA (both P < .0005) compared with ILC1. Knockdown of either SERPINB3 or SERPINB4 mRNA (both P < .005) levels resulted in decreased viability of CD27-CD4+ compared with control transduced cells. CONCLUSION: The Serpins Spi2A in mice and SERPINB3 and SERPINB4 in allergic patients control the viability of TH2 cells. This provides proof of principle for a therapeutic approach for allergic disease through ablation of allergic memory TH2 cells through SERPINB3 and SERPINB4 mRNA downregulation.

Genome-wide association analysis reveals genetic loci and candidate genes for feeding behavior and eating efficiency in Duroc boars.

Efficient use of feed resources is a challenge in the pork industry because the largest variability in expenditure is attributed to the cost of fodder. Efficiency of feeding is directly related to feeding behavior. In order to identify genomic regions controlling feeding behavior and eating efficiency traits, 338 Duroc boars were used in this study. The Illumina Porcine SNP60K BeadChip was used for genotyping. Data pertaining to individual daily feed intake (DFI), total daily time spent in feeder (TPD), number of daily visits to feeder (NVD), average duration of each visit (TPV), mean feed intake per visit (FPV), mean feed intake rate (FR), and feed conversion ratio (FCR) were collected for these pigs. Despite the limited sample size, the genome-wide association study was acceptable to detect candidate regions association with feeding behavior and eating efficiency traits in pigs. We detected three genome-wide (P < 1.40E-06) and 11 suggestive (P < 2.79E-05) single nucleotide polymorphism (SNP)-trait associations. Six SNPs were located in genomic regions where quantitative trait loci (QTLs) have previously been reported for feeding behavior and eating efficiency traits in pigs. Five candidate genes (SERPINA3, MYC, LEF1, PITX2, and MAP3K14) with biochemical and physiological roles that were relevant to feeding behavior and eating efficiency were discovered proximal to significant or suggestive markers. Gene ontology analysis indicated that most of the candidate genes were involved in the development of the hypothalamus (GO:0021854, P < 0.0398). Our results provide new insights into the genetic basis of feeding behavior and eating efficiency in pigs. Furthermore, some significant SNPs identified in this study could be incorporated into artificial selection programs for Duroc-related pigs to select for increased feeding efficiency.

[Inhibitory effect and the mechanism of Astragalus polysaccharide combined with cisplatin on growth of inplanted Lewis lung carcinoma in mice].

Objective To observe the effect of Astragalus polysaccharides (APS) combined with cisplatin (DDP) on the expressions of cytochrome C (CytC) and high temperature required serine protease A2 (Omi/HtrA2) in the mice with Lewis lung carcinoma (LLC) transplantated tumors. Methods Ninty C57BL/6J mice were randomly divided into normal control group, model group, and (50, 100, 200) µg/mL APS groups, 6 mg/kg DDP group, 3 mg/kg DDP combined with (50, 100, 200) µg/mL APS groups. Each group included 10 mice. Except the mice in the normal group, the rest mice were inoculated subcutaneously with LLC cells (1×107 mL) at the right fore axillary fossa to establish tumor-bearing mouse models. In the second day of building models, the mice in the treatment group were given intraperitoneal injection of 0.3 mL of the drug. DDP was given once a week, and the other drugs once a day. The mice in the normal group and the model group were administrated the same amount of saline injection for continuous 20 days. All mice were killed at the 21st day. The pathological changes of tumor tissues were observed by HE staining. The expressions and location of CytC and Omi/HtrA2 proteins in the transplanted tumor tissues were detected by immunohistochemical staining and image analysis. Results The mass of tumor decreased in the mice of (100, 200) µg/mL APS group and 3 mg/kg DDP combined with (100, 200) µg/mL APS group. Compared with the model group, the necrosis of tumor tissues in 200 µg/mL APS combined with 3 mg/kg DDP group was the most obvious. The expressions of CytC and Omi/HtrA2 increased in the treatment groups, and the increase was the most remarkable in 200 µg/mL APS combined with 3 mg/kg DDP group. Conclusion APS and APS combined with DDP can restrain the growth of Lewis Lung cancer in C57BL/6J mice, which may be related to the increased expressions of CytC and Omi/HtrA2.

Pigment Epithelium-Derived Factor (PEDF) Prevents Hepatic Fat Storage, Inflammation, and Fibrosis in Dietary Steatohepatitis of Mice.

BACKGROUND AND AIMS: Pigment epithelium-derived factor (PEDF) has been shown to be a potent inhibitor of inflammation through its anti-oxidative property. Since oxidative response is considered to play the pivotal role of the development and progression of nonalcoholic steatohepatitis (NASH), it is conceivable that PEDF may play a protective role against NASH. In this study, we examined whether administration of PEDF slowed the progression of NASH in mice models. METHODS: Mice were fed methionine- and choline-deficient (MCD) diet with or without intramuscular administration of adenovirus-expressing PEDF (Ad-PEDF). Effects of PEDF administration on NASH were histologically and biochemically evaluated. RESULTS: Administration of Ad-PEDF significantly decreased hepatic fat storage as well as serum levels of ALT in MCD diet-fed mice. Dihydroethidium staining showed that MCD diet-triggered oxidative stress was reduced in the liver of Ad-PEDF-administered mice compared to that of PBS- or Ad-LacZ-administered mice. Activation of Kupffer cells and hepatic fibrosis was also inhibited by Ad-PEDF administration. Quantitative real-time RT-PCR revealed that MCD diet up-regulated expressions of TNF-α, IL-1ß, IL-6, TGF-ß, collagen-1, and collagen-3 mRNA, which were also attenuated with Ad-PEDF administration, whereas MCD diet-induced down-regulation of expressions of PPAR-γ mRNA was restored with Ad-PEDF administration. Furthermore, immunoblotting analysis showed that MCD diet-induced up-regulation of NADPH oxidase components was significantly decreased in Ad-PEDF-administered mice. CONCLUSIONS: The present results demonstrated for the first time that PEDF could slow the development and progression of steatohepatitis through the suppression of steatosis and inflammatory response in MCD diet-fed mice. Our study suggests that PEDF supplementation may be a novel therapeutic strategy for the treatment of NASH.

Genetic and Targeted eQTL Mapping Reveals Strong Candidate Genes Modulating the Stress Response During Chicken Domestication.

The stress response has been largely modified in all domesticated animals, offering a strong tool for genetic mapping. In chickens, ancestral Red Junglefowl react stronger both in terms of physiology and behavior to a brief restraint stress than domesticated White Leghorn, demonstrating modified functions of the hypothalamic-pituitary-adrenal (HPA) axis. We mapped quantitative trait loci (QTL) underlying variations in stress-induced hormone levels using 232 birds from the 12th generation of an advanced intercross between White Leghorn and Red Junglefowl, genotyped for 739 genetic markers. Plasma levels of corticosterone, dehydroepiandrosterone (DHEA), and pregnenolone (PREG) were measured using LC-MS/MS in all genotyped birds. Transcription levels of the candidate genes were measured in the adrenal glands or hypothalamus of 88 out of the 232 birds used for hormone assessment. Genes were targeted for expression analysis when they were located in a hormone QTL region and were differentially expressed in the pure breed birds. One genome-wide significant QTL on chromosome 5 and two suggestive QTL together explained 20% of the variance in corticosterone response. Two significant QTL for aldosterone on chromosome 2 and 5 (explaining 19% of the variance), and one QTL for DHEA on chromosome 4 (explaining 5% of the variance), were detected. Orthologous DNA regions to the significant corticosterone QTL have been previously associated with the physiological stress response in other species but, to our knowledge, the underlying gene(s) have not been identified. SERPINA10 had an expression QTL (eQTL) colocalized with the corticosterone QTL on chromosome 5 and PDE1C had an eQTL colocalized with the aldosterone QTL on chromosome 2. Furthermore, in both cases, the expression levels of the genes were correlated with the plasma levels of the hormones. Hence, both these genes are strong putative candidates for the domestication-induced modifications of the stress response in chickens. Improved understanding of the genes associated with HPA-axis reactivity can provide insights into the pathways and mechanisms causing stress-related pathologies.

Biological Modulation of Mouse RPE Cells in Response to Subthreshold Diode Micropulse Laser Treatment.

Many clinical trials have demonstrated the effectiveness of subthreshold phototherapy with no visible damage in retinal vascular diseases, such as diabetic retinopathy. We aimed primarily to investigate the effect of subthreshold diode micropulse laser (SDM) treatment on mouse retinal pigmented epithelium (RPE) cells. The expression of angiogenesis-modulating cytokines in response to SDM was also explored. The least toxic laser dose was selected by measuring cell viability with MTT assay and 5 % duty cycle (DC) was chosen for use in further experiments. RPE cells were treated with laser-induced radiation ranging from 0 to 400 mW for 24 h. The apoptotic rate of RPE cells was assessed by flow cytometry. Expressions of vascular endothelial growth factor A (VEGF-A), transforming growth factor beta (TGF-ß), basic fibroblast growth factor (bFGF), and pigment epithelium-derived factor (PEDF) were determined by Western Blotting and real-time PCR, respectively. After 24 h of laser irradiation, cell viability was reduced dose dependently and the effect was significant compared to the controls (P < 0.05). In addition, laser treatment with intensities of 100 and 200 mW with DC of 5 % produced no significant effect on cell viability and apoptosis as compared with the control group (P > 0.05). The protein and mRNA expressions of angiogenic stimulators (VEGF-A, TGF-ß, and bFGF) were significantly down-regulated (P < 0.05), whereas those of the angiogenic inhibitor (PEDF) were up-regulated (P < 0.05). No significant difference was found between the cells treated with different intensities of laser radiation (P > 0.05). Our results showed that SDM treatment of the RPE cells suppressed the expression of choroid neovasculization-promoting cytokines and up-regulated the angiogenic inhibitor, PEDF without damaging the cells. Further investigation is needed to understand the mechanism and to optimize the use of SDM as a novel method of treatment for retinal vascular diseases.

Synergistic antitumor effect of recombinant adeno-associated virus-mediated pigment epithelium-derived factor with hyperthermia on solid tumor.

Adeno-associated virus (AAV) is an ideal choice for gene delivery; however, its further development has been limited owing to its low transduction efficiency. DNA-damaging agents can improve AAV-mediated transgene expression. Hyperthermia, as one of the oldest documented tumor treatment modalities, can cause DNA damage as well. However, combined treatment consisting of hyperthermia and AAV-mediated gene therapy has not been reported yet. In this work we investigated whether therapy consisting of AAV-mediated pigment epithelium-derived factor (PEDF) delivery combined with hyperthermia has synergistic antitumor effect on established solid tumors. We produced the recombinant AAV encoding PEDF (rAAV-PEDF). The therapeutic effect of rAAV-PEDF plus hyperthermia was evaluated in a subcutaneous fibrosarcoma mouse model, and the possible mechanism of antitumor effect was investigated. We found that rAAV-PEDF could infect a murine fibrosarcoma cell line (Meth-A) and express PEDF protein with bioactivity in vitro. In addition, in vivo experiments suggested that the combination of rAAV-PEDF with hyperthermia could significantly suppress tumor growth and prolong survival time of treated mice. Immunofluorescence studies indicated that the combination therapy could inhibit angiogenesis and induce apoptosis in tumor tissues. An immunohistochemistry assay of tumor tissue showed that PEDF expression in the combined treatment group was significantly higher than in the rAAV-PEDF group, which implied that hyperthermia could improve the expression of PEDF protein in vivo. No significant differences were observed in each group by hematoxylin-eosin staining of major organs, serum chemistry test, and complete blood assay. These results indicate that the combination of rAAV-PEDF with hyperthermia has synergistic therapeutic effects on established solid tumors, with no side effects. In addition, hyperthermia could improve AAV-mediated transgene expression, which suggests that hyperthermia could serve as a promising adjunctive therapy for AAV-mediated gene therapy.

Characterization of synergistic anti-cancer effects of docosahexaenoic acid and curcumin on DMBA-induced mammary tumorigenesis in mice.

BACKGROUND: The major obstacles to the successful use of individual nutritional compounds as preventive or therapeutic agents are their efficacy and bioavailability. One approach to overcoming this problem is to use combinations of nutrients to induce synergistic effects. The objective of this research was to investigate the synergistic effects of two dietary components: docosahexaenoic acid (DHA), an omega-3 fatty acid present in cold-water fish, and curcumin (CCM), an herbal nutrient present in turmeric, in an in vivo model of DMBA-induced mammary tumorigenesis in mice. METHODS: We used the carcinogen DMBA to induce breast tumors in SENCAR mice on control, CCM, DHA, or DHA + CCM diets. Appearance and tumor progression were monitored daily. The tumors were harvested 15 days following their first appearance for morphological and immunohistological analysis. Western analysis was performed to determine expression of maspin and survivin in the tumor tissues. Characterization of tumor growth was analyzed using appropriate statistical methods. Otherwise all other results are reported as mean ± SD and analyzed with one-way ANOVA and Tukey's post hoc procedure. RESULTS: Analysis of gene microarray data indicates that combined treatment with DHA + CCM altered the profile of "PAM50" genes in the SK-BR-3 cell line from an ER⁻/Her-2⁺ to that resembling a "normal-like" phenotype. The in vivo studies demonstrated that DHA + CCM treatment reduced the incidence of breast tumors, delayed tumor initiation, and reduced progression of tumor growth. Dietary treatment had no effect on breast size development, but tumors from mice on a control diet (untreated) were less differentiated than tumors from mice fed CCM or DHA + CCM diets. The synergistic effects also led to increased expression of the pro-apoptotic protein, maspin, but reduced expression of the anti-apoptotic protein, survivin. CONCLUSIONS: The SK-BR-3 cells and DMBA-induced tumors, both with an ER⁻ and Her-2⁺ phenotype, were affected by the synergistic interaction of DHA and CCM. This suggests that the specific breast cancer phenotype is an important factor for predicting efficacy of these nutraceuticals. The combination of DHA and CCM is potentially a dietary supplemental treatment for some breast cancers, likely dependent upon the molecular phenotype of the cancer.

The diuretic torasemide does not prevent aldosterone-mediated mineralocorticoid receptor activation in cardiomyocytes.

Aldosterone binds to the mineralocorticoid receptor (MR) and exerts pleiotropic effects beyond enhancing renal sodium reabsorption. Excessive mineralocorticoid signaling is deleterious during the evolution of cardiac failure, as evidenced by the benefits provided by adding MR antagonists (MRA) to standard care in humans. In animal models of cardiovascular diseases, MRA reduce cardiac fibrosis. Interestingly diuretics such as torasemide also appear efficient to improve cardiovascular morbidity and mortality, through several mechanisms. Among them, it has been suggested that torasemide could block aldosterone binding to the MR. To evaluate whether torasemide acts as a MRA in cardiomyocytes, we compared its effects with a classic MRA such as spironolactone. We monitored ligand-induced nuclear translocation of MR-GFP and MR transactivation activity in the cardiac-like cell line H9C2 using a reporter gene assay and known endogenous aldosterone-regulated cardiac genes. Torasemide did not modify MR nuclear translocation. Aldosterone-induced MR transactivation activity was reduced by the MRA spironolactone, not by torasemide. Spironolactone blocked the induction by aldosterone of endogenous MR-responsive genes (Sgk-1, PAI-1, Orosomucoid-1, Rgs-2, Serpina-3, Tenascin-X), while torasemide was ineffective. These results show that torasemide is not an MR antagonist; its association with MRA in heart failure may however be beneficial, through actions on complementary pathways.

Wogonin, an active ingredient of Chinese herb medicine Scutellaria baicalensis, inhibits the mobility and invasion of human gallbladder carcinoma GBC-SD cells by inducing the expression of maspin.

ETHNOPHARMACOLOGICAL RELEVANCE: A traditional Chinese medicine Scutellaria baicalensis is prescribed for the treatment of a variety of inflammatory diseases and tumors in clinic in China. However, the detailed mechanism of anti-metastasis effect of wogonin, a main active ingredient of Scutellaria baicalensis, remains elusive. AIM OF THE STUDY: The present study was designed to investigate the action and mechanism of wogonin on the mobility and invasion of human gallbladder carcinoma GBC-SD cells. MATERIALS AND METHODS: Viability, apoptosis, mRNA and protein expression of tumor cells were analyzed by MTT, Annexin V staining, real-time PCR and Western blot, respectively. The migration and invasion assay was used to evaluate the anti-metastasis effect of wogonin. Knockdown of maspin was performed by specific small interference RNA. RESULTS: Wogonin at the dose of 1-10 µM, which did not induce apoptosis, significantly inhibited the mobility and invasion activity of human gallbladder carcinoma GBC-SD cells. In addition, the expressions of matrix metalloproteinase (MMP)-2, MMP-9 and phosphorylated extracellular regulated protein kinase 1/2 (ERK1/2) but not phosphorylated Akt were dramatically suppressed by wogonin in a concentration-dependent manner. Furthermore, the metastasis suppressor maspin was confirmed as the downstream target of wogonin. Both maspin mRNA and protein were upregulated by wogonin. Interestingly, the knockdown of maspin resulted in almost completely blocking of wogonin-induced inhibition of MMP-2, MMP-9 and phosphorylated ERK1/2 as well as the mobility and invasion activity of GBC-SD cells. CONCLUSIONS: These findings suggest that wogonin inhibits cell mobility and invasion by upregulating the metastasis suppressor maspin. Together, these data provide novel insights into the chemoprotective effect of wogonin, a main active ingredient of Chinese medicine Scutellaria baicalensis.

Plants and the study of serpin biology.

Serpins appear to be ubiquitous in the Plant Kingdom and have several unique properties when compared to the substantial number of other families of protease inhibitors in plants. Serpins in plants are likely to have functions distinct from those of animal serpins, partly because plants and animals developed multicellularity independently and partly because most animal serpins are involved in animal-specific processes, such as blood coagulation and the activation of complement. To encourage and facilitate the discovery of plant serpin functions, here we provide a set of protocols for detection of serpins in plant extracts, localization of serpins in plant tissues and cells, purification of serpins from a range of organs from monocot and eudicot plants, production and purification of recombinant plant serpins, and analysis of plant-protease interactions including identification of in vivo target proteases.

Vitamin B6 suppresses serine protease inhibitor 3 expression in the colon of rats and in TNF-α-stimulated HT-29 cells.

SCOPE: Previous reports in the areas of animal studies and, recently epidemiology, have linked anti-tumorigenic and anti-inflammatory effects to dietary vitamin B6. This study investigated the molecular mechanism of these effects of vitamin B6. METHODS AND RESULTS: DNA microarray analysis was used to obtain information on changes in colon gene expression from vitamin B6 (pyridoxine) repletion in vitamin B6-deficient rats. Pyridoxine supplementation down-regulated the inflammatory molecule, serine protease inhibitor clade A member 3 (SPI-3) mRNA expression in the colon. This study also showed that tumor necrosis factor α (TNF-α) induced SPI-3 mRNA expression in HT-29 human colon cancer cells, and vitamin B6 (pyridoxal hydrochloride) pretreatment of HT-29 cells inhibited TNF -induced mRNA expression of SPI-3. Vitamin B6 inhibited TNF-α-induced NF-κB activation via suppression of IκBα degradation in HT-29 cells. HT-29 cells stably expressing epitope-tagged ubiquitin were generated and vitamin B6 pretreatment was shown to inhibit ubiquitination of the IkB protein in response to TNF-α-i. CONCLUSION: Vitamin B6 suppressed SPI-3 expression in the colon of rats and in TNF-α-stimulated HT-29 cells. Further, this study showed a possible role of vitamin B6 in the regulation of protein ubiquitination.

The yin and yang of VEGF and PEDF: multifaceted neurotrophic factors and their potential in the treatment of Parkinson's Disease.

Over the last few decades, vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) have emerged as multifaceted players in not only the pathogenesis, but potential treatment, of numerous diseases. They activate diverse intracellular signaling cascades known to have extensive crosstalk, and have been best studied for their effects in cardiology and cancer biology. Recent work with the two factors indicates that the activity of one growth factor is often directly related to the action of the other. Their respective neuroprotective effects, in particular, raise important questions regarding the treatment of neurodegenerative disorders, including Parkinson's disease.

Acute toxicity study of a simian immunodeficiency virus-based lentiviral vector for retinal gene transfer in nonhuman primates.

A phase 1 clinical trial evaluating the safety of gene therapy for patients with wet age-related macular degeneration (AMD) or retinoblastoma has been completed without problems. The efficacy of gene therapy for Leber's congenital amaurosis (LCA) was reported by three groups. Gene therapy may thus hold promise as a therapeutic method for the treatment of intractable ocular diseases. However, it will first be important to precisely evaluate the efficiency and safety of alternative gene transfer vectors in a preclinical study using large animals. In the present study, we evaluated the acute local (ophthalmic) and systemic toxicity of our simian immunodeficiency virus from African green monkeys (SIVagm)-based lentiviral vectors carrying human pigment epithelium-derived factor (SIV-hPEDF) for transferring genes into nonhuman primate retinas. Transient inflammation and elevation of intraocular pressure were observed in some animals, but these effects were not dose dependent. Electroretinograms (ERGs), including multifocal ERGs, revealed no remarkable change in retinal function. Histopathologically, SIV-hPEDF administration resulted in a certain degree of inflammatory reaction and no apparent structural destruction in retinal tissue. Regarding systemic toxicity, none of the animals died, and none showed any serious side effects during the experimental course. No vector leakage was detected in serum or urine samples. We thus propose that SIVagm-mediated stable gene transfer might be useful and safe for ocular gene transfer in a clinical setting.