Effects of 3-Tetrazolyl Methyl-3-Hydroxy-Oxindole Hybrid (THOH) on Cell Proliferation, Apoptosis, and G2/M Cell Cycle Arrest Occurs by Targeting Platelet-Derived Growth Factor D (PDGF-D) and the MEK/ERK Signaling Pathway in Human Lung Cell Lines SK-LU-1, A549, and A-427.

BACKGROUND The aim of this study was to evaluate the effects of 3-tetrazolyl methyl-3-hydroxy-oxindole hybrid (THOH) on cell proliferation, apoptosis, and the cell cycle in human lung cancer cell lines SK-LU-1, A549, and A-427, and the normal lung fibroblast cell line, MRC-5, in vitro. MATERIAL AND METHODS Human lung adenocarcinoma cells SK-LU-1, A549, and A-427, and the normal lung fibroblast cells, MRC-5 were cultured and treated with increasing concentrations of 10 mM of a stock solution of THOH in dimethyl sulfoxide (DMSO). An MTT cell proliferation assay was used. Cell apoptosis and the cell cycle were studied using fluorescence-activated cell sorting (FACs) with fluorescein isothiocyanate (FITC), Annexin-V, propidium iodide (PI), and nuclear staining with 4',6-diamidino-2-phenylindole (DAPI). DNA damage was measured using the comet (single-cell gel electrophoresis) assay. Cell migration was evaluated using a wound healing assay, and Western blotting was used to measure protein expression levels. RESULTS Treatment of SK-LU-1 cells with THOH inhibited cell migration. Treatment of lung cancer cells, SK-LU-1, A549, and A-427, with THOH inhibited cell proliferation, with the most marked inhibition found in the SK-LU-1 lung cancer cells (IC50, 12 µM). Treatment of lung cancer cells, SK-LU-1, A549, and A-427, with THOH increased cell apoptosis, resulted in G2/M cell cycle arrest, and inhibited both the platelet-derived growth factor D (PDGF-D) and MEK/ERK signaling pathways. CONCLUSIONS Treatment of adenocarcinoma cells, SK-LU-1, A549, and A-427, with THOH inhibited cell proliferation, apoptosis, and resulted in G2/M cell cycle arrest by targeting PDGF-D and the MEK/ERK signaling pathway.

Susceptibility to corticosteroid-induced adrenal suppression: a genome-wide association study.

BACKGROUND: A serious adverse effect of corticosteroid therapy is adrenal suppression. Our aim was to identify genetic variants affecting susceptibility to corticosteroid-induced adrenal suppression. METHODS: We enrolled children with asthma who used inhaled corticosteroids as part of their treatment from 25 sites across the UK (discovery cohort), as part of the Pharmacogenetics of Adrenal Suppression with Inhaled Steroids (PASS) study. We included two validation cohorts, one comprising children with asthma (PASS study) and the other consisting of adults with chronic obstructive pulmonary disorder (COPD) who were recruited from two UK centres for the Pharmacogenomics of Adrenal Suppression in COPD (PASIC) study. Participants underwent a low-dose short synacthen test. Adrenal suppression was defined as peak cortisol less than 350 nmol/L (in children) and less than 500 nmol/L (in adults). A case-control genome-wide association study was done with the control subset augmented by Wellcome Trust Case Control Consortium 2 (WTCCC2) participants. Single nucleotide polymorphisms (SNPs) that fulfilled criteria to be advanced to replication were tested by a random-effects inverse variance meta-analysis. This report presents the primary analysis. The PASS study is registered in the European Genome-phenome Archive (EGA). The PASS study is complete whereas the PASIC study is ongoing. FINDINGS: Between November, 2008, and September, 2011, 499 children were enrolled to the discovery cohort. Between October, 2011, and December, 2012, 81 children were enrolled to the paediatric validation cohort, and from February, 2010, to June, 2015, 78 adults were enrolled to the adult validation cohort. Adrenal suppression was present in 35 (7%) children in the discovery cohort and six (7%) children and 17 (22%) adults in the validation cohorts. In the discovery cohort, 40 SNPs were found to be associated with adrenal suppression (genome-wide significance p<1 × 10-6), including an intronic SNP within the PDGFD gene locus (rs591118; odds ratio [OR] 7·32, 95% CI 3·15-16·99; p=5·8 × 10-8). This finding for rs591118 was validated successfully in both the paediatric asthma (OR 3·86, 95% CI 1·19-12·50; p=0·02) and adult COPD (2·41, 1·10-5·28; p=0·03) cohorts. The proportions of patients with adrenal suppression by rs591118 genotype were six (3%) of 214 patients with the GG genotype, 15 (6%) of 244 with the AG genotype, and 22 (25%) of 87 with the AA genotype. Meta-analysis of the paediatric cohorts (discovery and validation) and all three cohorts showed genome-wide significance of rs591118 (respectively, OR 5·89, 95% CI 2·97-11·68; p=4·3 × 10-9; and 4·05, 2·00-8·21; p=3·5 × 10-10). INTERPRETATION: Our findings suggest that genetic variation in the PDGFD gene locus increases the risk of adrenal suppression in children and adults who use corticosteroids to treat asthma and COPD, respectively. FUNDING: Department of Health Chair in Pharmacogenetics.

[The antioxidant therapy modulates intracellular lymphokine expression in children on dialysis]. / Leczenie antyoksydacyjne moduluje wewnatrzkomórkowa ekspresje limfokin u dzieci dializowanych.

UNLABELLED: Oxidative stress (SOX) is believed to be responsible for functional disabilities of lymphocytes in end-stage renal disease (ESRD). Therefore, we investigated the effect of antioxidant therapy with vitamin E and N-acetylcysteine (NAC) on SOX and cytokine synthesis in T cells in dialyzed children. Eighteen children (aged 2-20, mean 10.9 yr) treated with hemodialysis (n=5) and peritoneal dialysis (n=13) were enrolled into the study. Vitamin E and NAC were given orally for six months. Throughout the study, intracellular lymphokines [interleukin (IL)-2, interferon (IFN)-gamma, IL-4, IL-6] and SOX in T cells were measured by means of flow cytometry. In dialyzed children, mean fluorescence intensity (MFI), which reflected intracellular SOX, was significantly higher than in the controls in both CD3+ and CD3+CD4+ cells (p<0.05). We also found a cytokine dysregulation with a trend toward a predominant T helper (Th)-1 response compared to the controls. After 6 months of treatment with antioxidants, a significant reduction in MFI was noted compared to baseline values in CD3+ and CD3+CD4- cells (p<0.001). Interestingly, the therapy led to a decrease in IFN-gamma as well as an increase in IL-4 and IL-6 production. In addition, a gradual decline in IFN-gamma/IL-4 ratio in Th cells was noted. CONCLUSIONS: Vitamin E and NAC used in combination are effective in reducing the intracellular SOX, and besides their action on cellular redox state, they modulate the cytokine profile in children on dialysis.

Nuclear localisation of endogenous SUMO-1-modified PDGF-C in human thyroid tissue and cell lines.

We investigated post-translational modification and subcellular localisation of endogenous platelet-derived growth factor-C (PDGF-C) in human thyroid papillary carcinomas (PTC), non-neoplastic thyroid tissues, and a selection of cultured cell lines. PDGF-C expressed nuclear localisation in 95% of all tested cell types in culture and in 10% of the thyrocytes from both PTC and non-neoplastic tissue. The cell lines expressed two forms of full-length PDGF-C, approximately 39 and approximately 55 kDa, in cell membrane and cytosol, while the approximately 55 kDa form dominated in the nucleus where it was partly chromatin-associated. The approximately 55 kDa form was post-translationally modified by SUMO-1. The putative PDGF-C SUMOylation site is the surface exposed (314)lysine part of a positively charged loop ((312)RPKTGVRGLHK(322)) with characteristics of a nuclear localisation signal. The tissue thyrocytes expressed a non-SUMOylated approximately 43 kDa and the 55 kDa PDGF-C. The SUMO-1 modified approximately 55 kDa PDGF-C expression was low in PTC where the approximately 43 kDa PDGF-C dominated. This is in contrast to non-neoplastic tissue and cultured cells where the SUMOylated approximately 55 kDa PDGF-C was strongly expressed. Our data provide novel evidence for nuclear localisation of PDGF-C, post-translational modification by SUMOylation and the expression of a novel form of PDGF-C in human papillary thyroid carcinomas.

Inducible nitric oxide synthase inhibitors prolonged the survival of skin xenografts through selective down-regulation of pro-inflammatory cytokine and CC-chemokine expressions.

To elucidate the possible immunoregulatory role of nitric oxide (NO) in cellular xenograft rejection we performed rat-to-mouse skin xenotransplantation. The rat skin engrafted mice were treated with the inducible NO synthase (iNOS) inhibitors, aminoguanidine (AMG, 200 mg/kg) and NG-nitro-L-arginine methyl ester (L-NAME, 60 mg/kg) every other day until rejection. Skin xenograft survival was monitored and immune cell infiltration and intragraft cytokine and chemokine mRNA expressions were analyzed 7 days after grafting. Compared with the control mice, the AMG- and L-NAME treated mice showed delayed xenograft rejection by approximately 3 days (8.9 +/- 0.7 days vs. 11.7 +/- 1.2 and 12.0 +/- 0.9 days, respectively). Infiltrations of CD11b+, MOMA-2+ cells and neutrophils were significantly reduced in both AMG- and L-NAME treated graft but CD4+ and CD8+ cells were not. The expression of cytokines such as IL-1beta, IL-2, IL-6, IL-12 and IFN-gamma in AMG- and L-NAME treated grafts were significantly decreased (P<0.01), whereas IL-10, TNF-alpha and TGF-beta1 were unchanged or enhanced. Additionally, the expressions of CC-chemokines, such as RANTES and MIP-1alpha, were significantly reduced (P<0.01) whereas the expressions of CXC-chemokines, such as IP-10 and MIG, were unchanged. These results imply that prolonged rat-to-mouse skin xenograft survival by iNOS inhibitors may be due to the selective inhibition of pro-inflammatory cytokines and chemokines and suggest the possible regulatory role of NO in cytokine and chemokine expressions during xenotransplant rejection.

Regulation of the pro-angiogenic microenvironment by carboxyamido-triazole.

Anti-angiogenic agents regulate tumor growth by inhibiting endothelial cell proliferation and invasion. Carboxyamido-triazole (CAI), an inhibitor of non-voltage-operated calcium entry and calcium influx-mediated pathways, has angiogenesis and invasion inhibitory activity. We hypothesized that CAI may express its anti-angiogenic effects through negative regulation of pro-angiogenic cytokine production and/or function. In vivo, orally administered CAI prevented A2058 human melanoma xenograft growth and concomitantly resulted in a marked reduction in circulating vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8). In vitro, A2058 cell secretion of VEGF was inhibited by CAI treatment under limiting micronutrient conditions that approximate the tumor microenvironment, media restriction, and acidification to pH 6.8 (P=0.0003 and P=0.0006, respectively). VEGF and HIF-1alpha message and protein were also reduced by CAI treatment. Oral CAI treatment reduced vascular ingrowth in vivo into VEGF-containing Matrigel plugs. Commensurate with those findings, human umbilical vein endothelial cell (HUVEC) migration towards VEGF was reduced below background by exposure to CAI in the migration chamber (P<0.0001). An 88% reduction in circulating IL-8 concentration was measured in CAI-treated animals. However, IL-8 protein secretion and gene expression were increased by CAI treatment in culture (P< or =0.01), where CAI caused a dose-dependent acidification of the culture milieu (P< or =0.005). This paradox suggests that IL-8 production in vitro may be more sensitive to ambient pH than cytosolic calcium. These observations suggest that CAI inhibition of tumor cell VEGF production and endothelial cell response to VEGF results in disruption of signaling between the tumor and its microenvironment, causing a net anti-angiogenic effect.

Anti-angiogenic potential of Gleditsia sinensis fruit extract.

Blood supply plays a crucial role in solid tumour development and leukaemogenesis. It has been suggested that blocking of angiogenesis could be possible in cancer therapy. We have demonstrated the antiproliferative activity of Gleditsia sinensis fruit extract (GSE) on various human solid tumour cancer cell lines as well as leukaemia cell lines and primary cultured leukaemia cells obtained from leukaemia patients. However, the antiangiogenic potential of GSE has not been demonstrated. Here we demonstrated that GSE could reduce vascular endothelial growth factor (VEGF) mRNA expression in dose- and time course-dependently in MDA-MB231 breast cancer and HepG2 hepatoblastoma cell lines as measured by reverse transcriptase polymerase chain reaction. Enzyme-linked immunosorbent assay further showed that GSE could reduce the VEGF secretion from various cancer cell lines including MDA-MB231, HepG2, HL-60 (acute promyelocytic leukaemia) and eleven primary cultured leukaemia cells obtained from acute myelogenous leukaemia patients. In vivo chick chorioallantoic membrane assay illustrated that GSE could reduce the angiogenic activity of basic fibroblast growth factor. Taken together, the information suggested that GSE could be potentially used as an angiogenic inhibitor in both solid tumour and leukaemia therapy.

Rethinking nutritional support for persons with cancer cachexia.

Cancer cachexia is a poorly understood syndrome of anorexia, weight loss, and muscle wasting that negatively impacts quality of life and survival in cancer patients. Research has clearly implicated pro-inflammatory cytokines in the biology of cancer cachexia. More recent research implicates products of arachidonic acid and suggests that cachexia may be a chronic inflammatory condition rather than a nutritional aberration. To date, nutritional support to slow weight loss has focused primarily on increasing calorie intake. Alternatively, many foods contain factors that can modulate the synthesis or activity of pro-inflammatory mediators, especially the synthesis of prostaglandin E2 from arachidonic acid. These factors and foods are sometimes called nutraceuticals, and research is needed to evaluate their efficacy in combating cancer cachexia.

[Down-regulation of vascular endothelial growth factor expression in ovarian cancer cell line SKOV3 by interferon-gamma].

OBJECTIVE: To determine whether interferon-gamma (IFN-gamma)inhibit the expression of vascular endothelial growth factor (VEGF) in ovarian cancer cell line SKOV3 with the level of mRNA and protein. METHODS: Cultured SKOV3 was treated by IFN-gamma with different concentration and time. The morphological changes of SKOV3 treated by IFN-gamma through microscope and proliferation by methyl thiazolyl tetrazolium (MTT). The expression of VEGF mRNA in SKOV3 culture was determined by relative quantitative reverse-transcription polymerase chain reaction. The VEGF protein level in supernatants was determined by enzyme-linked immunosorbent assay (ELISA) and in cytoplasma by immunocytochemical staining. RESULTS: There is a small changes in shape on SKOV3, but no alter of cell proliferation during treating with different concentration and time. Expression of VEGF mRNA and protein decreased with the INF-gamma concentration increasement, but no time-effect pattern exist. The effect peak appeared when the concentration was 1,000 U/ml. CONCLUSION: IFN-gamma can inhibit the expression of VEGF in ovarian cancer cell line SKOV3 with the level of mRNA and protein.

Preventing ovarian hyperstimulation syndrome by inhibiting the effects of vascular endothelial growth factor.

OBJECTIVE: To evaluate whether inhibiting the effects of vascular endothelial growth factor can prevent the ovarian hyperstimulation syndrome. STUDY DESIGN: Rates were hyperstimulated with follicle-stimulating hormone injections. On the final day of stimulation, the rats were randomized to receive or not receive exogenous soluble fmslike tyrosine kinase (sFlt-1). Forty-eight hours later capillary permeability was determined by measuring the concentration of Evans blue dye (EB) in peritoneal irrigation fluid 3 minutes after an intravenous injection of EB. RESULTS: The peritoneal EB level was statistically significantly lower in the hyperstimulated group, which received the sFlt-1, than in the stimulation-only group. CONCLUSION: sFlt-1 may play a therapeutic role in management of the ovarian hyperstimulation syndrome by specifically inhibiting the effects of vascular endothelial growth factor.

2ME2 inhibits tumor growth and angiogenesis by disrupting microtubules and dysregulating HIF.

Inhibition of angiogenesis is an important new modality for cancer treatment. 2-methoxyestradiol (2ME2) is a novel antitumor and antiangiogenic agent, currently in clinical trials, whose molecular mechanism of action remains unclear. Herein, we report that 2ME2 inhibits tumor growth and angiogenesis at concentrations that efficiently disrupt tumor microtubules (MTs) in vivo. Mechanistically, we found that 2ME2 downregulates hypoxia-inducible factor-1 (HIF) at the posttranscriptional level and inhibits HIF-1-induced transcriptional activation of VEGF expression. Inhibition of HIF-1 occurs downstream of the 2ME2/tubulin interaction, as disruption of interphase MTs is required for HIF-alpha downregulation. These data establish 2ME2 as a small molecule inhibitor of HIF-1 and provide a mechanistic link between the disruption of the MT cytoskeleton and inhibition of angiogenesis.

Midazolam stimulates vascular endothelial growth factor release in aortic smooth muscle cells: role of the mitogen-activated protein kinase superfamily.

BACKGROUND: Intravenous anesthetics used during perioperative periods affect the vascular signaling molecules and the vascular reactivity. Vascular endothelial growth factor (VEGF), an angiogenesis factor produced in and secreted from aortic smooth muscle cells, is a specific mitogen for vascular endothelial cells. This study investigated the effects of various intravenous anesthetics on VEGF release, and the underlying mechanism, in a rat aortic smooth muscle cell line, A10 cells. METHODS: Intravenous anesthetics (midazolam and propofol) were continuously administered to rats by infusion. Cultured A10 cells were stimulated by intravenous anesthetics (midazolam, propofol, and ketamine). VEGF was evaluated by immunoassay. The phosphorylation of mitogen-activated protein (MAP) kinases was evaluated by Western blotting. RESULTS: Continuous infusion of midazolam, but not propofol, increased the VEGF concentration in rat plasma. In cultured cells, midazolam stimulated VEGF release, but propofol and ketamine did not. Midazolam induced phosphorylation of p44/p42 MAP kinase and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), without affecting p38 MAP kinase. PD98059 and U0126, specific inhibitors of MAP kinase kinase, significantly reduced the midazolam-stimulated release of VEGF. SP600125, a specific inhibitor of SAPK/JNK, significantly reduced midazolam-stimulated VEGF release. Applied together, PD98059 and SP600125 produced an additive reduction in midazolam-stimulated VEGF release. Moreover, a bolus injection of PD98059 truly inhibited the midazolam-increased VEGF concentration in rat plasma in vivo. CONCLUSIONS: Midazolam, but not propofol or ketamine, stimulates VEGF release in aortic smooth muscle cells. Its effect is mediated at least in part via activation of p44/p42 MAP kinase and SAPK/JNK.

Effects of interferon alpha on vascular endothelial growth factor gene transcription and tumor angiogenesis.

BACKGROUND: Interferon alpha (IFN-alpha) has antiangiogenic activity, although the underlying mechanism of action is unclear. Because human neuroendocrine (NE) tumors are highly vascularized and sensitive to IFN-alpha, we investigated whether the therapeutic effects of IFN-alpha result from an inhibition of angiogenesis mediated by a decrease in vascular endothelial growth factor (VEGF) gene expression. METHODS: VEGF gene and protein expression was analyzed in NE tumors by immunohistochemistry and in NE tumor cell lines by quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). VEGF promoter-reporter gene constructs containing various deletions or mutations and gel shift assays were used to identify minimal promoter requirements and potential transcription factors. A xenograft nude mouse model (five mice per group) was used to determine the effect of IFN-alpha on tumor growth (NE Bon cells and pancreatic Capan-1 cells) and microvessel density. Liver metastases from eight patients with NE tumors were analyzed for microvessel density, VEGF mRNA content, and VEGF plasma levels before and after initiation of IFN-alpha therapy. RESULTS: NE tumors and cell lines expressed VEGF mRNA and secreted VEGF protein. In vitro, IFN-alpha decreased transcription of VEGF gene expression through an Sp1- and/or Sp3-dependent inhibition of VEGF promoter activity. Compared with vehicle treatment in mice, IFN-alpha inhibited tumor growth by 36% and reduced microvessel density from 56 (95% confidence interval [CI] = 49 to 69) to 37 per x400 Field (95% CI = 32 to 41, P =.015). Patients with NE tumors had lower VEGF plasma levels and reduced VEGF mRNA levels and microvessel density in liver metastasis biopsy material after IFN-alpha treatment. CONCLUSION: IFN-alpha confers its antitumor activity, at least in part, by its antiangiogenic activity, which results from Sp1- and/or Sp3-mediated inhibition of VEGF gene transcription.

Interleukin-6 promotes cervical tumor growth by VEGF-dependent angiogenesis via a STAT3 pathway.

Interleukin-6 (IL-6) has received particular attention in the pathogenesis of cervical cancer, although the underlying mechanism remains elusive. This study revealed that IL-6 promotes in vivo tumor growth of human cervical cancer C33A cells, but does not substantially alter their in vitro growth kinetics. The in vivo angiogenic assays showed that IL-6 increases angiogenic activity in human cervical cancer cells, an effect that is specifically associated with upregulation of vascular endothelial growth factor (VEGF). Also, using anti-VEGF antibody to block VEGF function significantly inhibited IL-6-mediated angiogenesis and tumor growth in nude mice, strongly supporting the critical role of VEGF in the IL-6-mediated cervical tumorigenesis. Accordingly, the signaling pathway downstream of IL-6/IL-6R responsible for the regulation of VEGF was investigated. Notably, pharmacological inhibition of PI3-K or MAPK failed to inhibit IL-6-mediated transcriptional upregulation of VEGF. Meanwhile, blocking STAT3 pathway with dominant-negative mutant STAT3D effectively abolished IL-6-induced VEGF mRNA. In transient transfections, a luciferase reporter construct containing the full-length 1.5-kb VEGF promoter or a 1.2-kb fragment lacking the known hypoxic-response element also exhibited the same degree of response to IL-6. Additionally, transient transfection of STAT3D downregulated the 1.2-kb VEGF promoter luciferase reporter stimulated by IL-6. Based on the above phenomenon combined with the concomitant increased tumor expression of IL-6 and VEGF in cervical cancer tissues, we conclude that IL-6 may promote cervical tumorigenesis by activating VEGF-mediated angiogenesis via a STAT3 pathway.

Inhibitory effect of clotrimazole on angiogenesis associated with bladder epithelium proliferation in rats.

BACKGROUND: Up-regulation of epithelial vascular endothelial growth factor (VEGF) expression was recently reported at an early stage of bladder carcinogenesis in rats. Subsequently, clotrimazole (CLT) was reported to exert an inhibitory effect on the in vitro proliferation of vascular endothelial cells stimulated by VEGF. The present study was performed to evaluate the effect of CLT on the initial stage of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced carcinogenesis of the urinary bladder in rats. METHODS: Seven-week-old-female Wistar rats were divided into both a control group and the other four groups that were allowed free access to drinking water containing 0.05%BBN. Two groups of rats (10-CLT and 20-CLT) received CLT (100 mg/kg per day) once daily by gavage for 10 and 20 weeks, respectively. The other two groups of rats (10-BBN and 20-BBN) were maintained on the same BBN-containing drinking water for 10 and 20 weeks, respectively, without receiving CLT. After removing the urinary bladder of each rat, expression of VEGF and VEGF receptor FLT1, and new capillaries were examined by immunohistochemical staining. VEGF mRNA was detected by the reverse transcription polymerase chain reaction. RESULTS: The ratio of VEGF-positive cells and the expression of VEGF mRNA were lower in the 20-CLT group than in the 10-BBN group. Newly formed capillaries in the 20-CLT group were significantly decreased compared with those in the 10-BBN group. CONCLUSION: These quantitative data suggested that CLT can inhibit VEGF expression in vivo and hence inhibit angiogenesis in the early stages of bladder carcinoma.