Doxycycline-inducible expression of SPARC/osteonectin/BM40 in MDA-MB-231 human breast cancer cells results in growth inhibition

SPARC (secreted protein acidic and rich in cysteine)/BM40/Osteonectin is a matricellular protein with multiple effects on cell behaviour. In vitro, its major known functions are anti-adhesive and anti-proliferative, and it is associated with tissue remodelling and cancer in vivo. SPARC is overexpressed in many cancers, including breast cancer, and the effects of SPARC seem to be cell type-specific. To study the effects of SPARC on breast cancer, we transfected SPARC into the MDA-MB-231 BAG, human breast cancer cell line using the Tet-On inducible system. By western analysis, we found low background levels in the MDA-MB-231 BAG and clone X parental cells, and prominent induction of SPARC protein expression after doxycycline treatment in SPARC transfected clones X5, X21, X24 and X75. Induction of SPARC expression did not affect cell morphology or adhesiveness to collagens type I and IV, but it slowed the rate of proliferation in adherent cultures. Cell cycle analysis showed that SPARC slowed the progression to S phase. Doxycycline induction of SPARC also slowed the rate of monolayer wound closure in the cultured wound healing assay. Thymidine inhibition of proliferation abrogated this effect, confirming that it was due to anti-proliferation rather than inhibition of migration. Consistent with this, we were unable to detect any differences in migration and Matrigel outgrowth analysis of doxycycline-stimulated cells. We conclude that SPARC is inhibitory to human breast cancer cell proliferation, and does not stimulate migration, in contrast to its stimulatory effects reported for melanoma (proliferation and migration) and glioma (migration) cells. Similar growth repression by SPARC has been reported for ovarian cancer cells, and this may be a common feature among carcinoma.

Differential (18)F-FDG and 3'-deoxy-3'-(18)F-fluorothymidine PET responses to pharmacologic inhibition of the c-MET receptor in preclinical tumor models.

The ability of PET to image functional changes in tumors is increasingly being used to evaluate response and predict clinical benefit to conventional and novel cancer therapies. Although the use of (18)F-FDG PET is well established, 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) PET has potential advantages as a more specific marker of cellular proliferation. c-MET signaling is frequently dysregulated in cancer and is therefore an attractive therapeutic target. Crizotinib (PF-2341066) is a novel adenosine triphosphate-competitive c-MET kinase inhibitor with antitumor activity in a range of tumor models. The aim of this study was to investigate the utility of PET of glucose metabolism and cell proliferation to monitor tumor response to crizotinib in 2 cell lines with aberrant c-MET signaling.

Inhibition of matrix metalloproteinase activity in the chick sclera and its effect on myopia development

To investigate the contribution of matrix degradation in the two-layer avian sclera to the development of myopia. Tissue inhibitor of metalloproteinase-2 (TIMP-2) was used to inhibit chick scleral collagen degradation with (3)H-proline, a marker for this effect. Ex vivo scleral culture experiments confirmed TIMP-2 doses for in vivo experimentation. Ocular growth and refractive response to exogenous TIMP-2 (11.25, 2.25, and 0.45 picomoles, plus vehicle only) were monitored in 7-day-old chicks during the induction of myopia over 4 days with a translucent occluder. Collagen degradation was assessed, in whole sclera and in separated scleral layers by using the same paradigm (11.25 picomoles TIMP-2; vehicle only).Approximately 60% of collagen degradation was inhibited with low (2 nM) doses of TIMP-2 in the ex vivo sclera. Degradative activity in the in vivo chick sclera increased significantly (46%) during myopia development, with all the altered activity confined to the fibrous layer. Addition of TIMP-2 significantly reduced (by 46%) this accelerated scleral collagen degradation, also by acting in the fibrous layer. TIMP-2 had no significant effect on (3)H-proline incorporated in the cartilaginous scleral layer and cornea. Despite inhibiting collagen degradation TIMP-2 had no significant effect on myopia development. Increased collagen degradation is a feature of scleral remodeling in chick myopia development, but is confined to the fibrous scleral layer. Significant inhibition of this collagenolytic activity with TIMP-2 has little effect on refractive error development, suggesting that collagen degradation in the sclera contributes little to the development of myopia in the chick.

Maternal dietary supplementation with saturated, but not monounsaturated or polyunsaturated fatty acids, leads to tissue-specific inhibition of offspring Na+,K+-ATPase

In rats, a maternal diet rich in lard is associated with reduced Na+,K+-ATPase activity in adult offspring kidney. We have addressed the role of different fatty acids by evaluating Na+,K+-ATPase activity in offspring of dams fed diets rich in saturated (SFA), monounsaturated (MUFA) or polyunsaturated (PUFA) fatty acids. Female Sprague–Dawley rats were fed, during pregnancy and suckling, a control diet (4% w/w corn oil) or a fatty acid supplemented diet (24% w/w). Offspring were reared on chow (4% PUFA) and studied at 6 months. mRNA expression (real-time PCR) of Na+,K+-ATPase α subunit and protein expression of Na+,K+-ATPase subunits (Western blot) were assessed in kidney and brain. Na+,K+-ATPase activity was reduced in kidney (P < 0.05 versus all groups) and brain (P < 0.05 versus control and MUFA offspring) of the SFA group. Neither Na+,K+-ATPase α1 subunit mRNA expression, nor protein expression of total α, α1, α2, α3 or β1 subunits were significantly altered in kidney in any dietary group. In brains of SFA offspring α1 mRNA expression (P < 0.05) was reduced compared with MUFA and PUFA offspring, but not controls. Also in brain, SFA offspring demonstrated reduced (P < 0.05) α1 subunit protein and increased phosphorylation (P < 0.05) of the Na+,K+-ATPase modulating protein phospholemman at serine residue 63 (S63 PLM). Na+,K+-ATPase activity was similar to controls in heart and liver. In utero and neonatal exposure to a maternal diet rich in saturated fatty acids is associated with altered activity and expression of Na+,K+-ATPase in adulthood, but mechanisms appear tissue specific.

The corrosion inhibition of aluminium alloy 7075-T651 with cerium and Mischmetal di phenyl phosphate

Previous studies have shown that cerium diphenyl phosphate (Cedpp) 3 is a very effective inhibitor of corrosion of aluminium alloys in chloride solutions. This paper describes the results of further studies using electrochemical and constant immersion corrosion tests to compare the effectiveness of Ce(dpp) 3 and Mischmetal diphenyl phosphate Mm(dpp) 3 as inhibitors of corrosion pitting on AA7075-T651 aluminium alloy. The results shows that both Ce(dpp) 3 and Mm(dpp) 3 are excellent inhibitors of pitting corrosion of this alloy in very aggressive environments of continuously aerated 0.1M and 1.0M sodium chloride (NaCl) solutions. Polarisation tests indicate that these compounds act as a cathodic inhibitors by reducing the rate of the oxygen reduction reaction, which results in a decreased corrosion current density and a separation of the corrosion potential from the pitting potential. This inhibition is thought to be due to the formation of a surface film consisting of rare earth metal oxide, aluminium oxide and a cerium-aluminium organo-phosphate complex. Surface analysis data from scanning electron microscopy and X-ray Energy Dispersive Spectroscopy show the complex nature of this protective film. This work further develops our understanding about the mechanisms through which these complex films form, and how inhibition occurs in the presence of these compounds.

HDAC inhibition as a therapeutic potential to treat metabolic diseases

Type 2 diabetes is one of the leading threats to human health. The research focused to identify new drugs that could partly mimic the positive effects of exercise and to test whether these drugs could have the potential to treat metabolic diseases such as obesity and type 2 diabetes

Response of BRAF-mutant melanoma to BRAF inhibition is mediated by a network of transcriptional regulators of glycolysis.

Deregulated glucose metabolism fulfills the energetic and biosynthetic requirements for tumor growth driven by oncogenes. Because inhibition of oncogenic BRAF causes profound reductions in glucose uptake and a strong clinical benefit in BRAF-mutant melanoma, we examined the role of energy metabolism in responses to BRAF inhibition. We observed pronounced and consistent decreases in glycolytic activity in BRAF-mutant melanoma cells. Moreover, we identified a network of BRAF-regulated transcription factors that control glycolysis in melanoma cells. Remarkably, this network of transcription factors, including hypoxia-inducible factor-1α, MYC, and MONDOA (MLXIP), drives glycolysis downstream of BRAF(V600), is critical for responses to BRAF inhibition, and is modulated by BRAF inhibition in clinical melanoma specimens. Furthermore, we show that concurrent inhibition of BRAF and glycolysis induces cell death in BRAF inhibitor (BRAFi)-resistant melanoma cells. Thus, we provide a proof-of-principle for treatment of melanoma with combinations of BRAFis and glycolysis inhibitors.

Plumbagin induces apoptotic and autophagic cell death through inhibition of the PI3K/Akt/mTOR pathway in human non-small cell lung cancer cells.

Plumbagin (PLB) has shown anti-cancer activity but the mechanism is unclear. This study has found that PLB has a potent pro-apoptotic and pro-autophagic effect on A549 and H23 cells. PLB arrests cells in G2/M phase, and increases the intracellular level of reactive oxygen species in both cell lines. PLB dose-dependently induces autophagy through inhibition of PI3K/Akt/mTOR pathway as indicated by reduced phosphorylation of Akt and mTOR. Inhibition or induction of autophagy enhances PLB-induced apoptosis. There is crosstalk between PLB-induced apoptosis and autophagy. These findings indicate that PLB initiates both apoptosis and autophagy in NSCLC cells through coordinated pathways.

Methylmercury-induced inhibition of paraoxonase-1 (PON1)-implications for cardiovascular risk

Methylmercury (MeHg) has been associated with increased risk for cardiovascular disease in some but not all epidemiology studies. These inconsistent results may stem from the fact that exposure typically occurs in the context of fish consumption, which is also associated with cardioprotective factors such as omega-3 fatty acids. Mechanistic information may help to understand whether MeHg represents a risk to cardiovascular health. MeHg is a pro-oxidant that inactivates protein sulfhydryls. These biochemical effects may diminish critical antioxidant defense mechanism(s) involved in protecting against atherosclerosis. One such defense mechanism is paraoxonase-1 (PON1), an enzyme present on high-density lipoproteins and that prevents the oxidation of blood lipids and their deposition in vascular endothelium. PON1 is potentially useful as a clinical biomarker of cardiovascular risk, as well as a critical enzyme in the detoxification of certain organophosphate oxons. MeHg and other metals are known to inhibit PON1 activity in vitro. MeHg is associated with lowered serum PON1 activity in a fish-eating population. The implications of lowering PON1 are evaluated by predicting the shift in PON1 population distribution induced by various doses of MeHg. An MeHg dose of 0.3 μg/kg/d is estimated to decrease the population average PON1 level by 6.1% and to increase population risk of acute cardiovascular events by 9.7%. This evaluation provides a plausible mechanism for MeHg-induced cardiovascular risk and suggests means to quantify the risk. This case study exemplifies the use of upstream disease biomarkers to evaluate the additive effect of chemical toxicity with background disease processes in assessing human risk.

Dietary repletion with ω3 fatty acid or with COX inhibition reverses cognitive effects in F3 ω3 fatty-acid-deficient mice

Dietary deficiency of ω3 fatty acid during development leads to impaired cognitive function. However, the effects of multiple generations of ω3 fatty-acid deficiency on cognitive impairment remain unclear. In addition, we sought to test the hypothesis that the cognitive impairments of ω3 fatty-acid-deficient mice are mediated through the arachidonic acid-cyclooxygenase (COX) pathway. To address these issues, C57BL/6J mice were bred for 3 generations and fed diets either deficient (DEF) or sufficient (SUF) in ω3 fatty acids. At postnatal day 21, the F3 offspring remained on the dam's diet or were switched to the opposite diet, creating 4 groups. In addition, 2 groups that remained on the dam's diet were treated with a COX inhibitor. At 19 wk of age, spatial-recognition memory was tested on a Y-maze. Results showed that 16 wk of SUF diet reversed the cognitive impairment of F3 DEF mice. However, 16 wk of ω3 fatty-acid-deficient diet impaired the cognitive performance of the F3 SUF mice, which did not differ from that of the F3 DEF mice. These findings suggest that the cognitive deficits after multigenerational maintenance on ω3 fatty-acid-deficient diet are not any greater than are those after deficiency during a single generation. In addition, treatment with a COX inhibitor prevented spatial-recognition deficits in F3 DEF mice. Therefore, cognitive impairment due to dietary ω3 fatty-acid deficiency appears to be mediated by the arachidonic acid-COX pathway and can be prevented by 16 wk of dietary repletion with ω3 fatty acids or COX inhibition.

Inhibition of reactive oxygen species production ameliorates inflammation induced by influenza A viruses via upregulation of SOCS1 and SOCS3.

Highly pathogenic avian influenza virus infection is associated with severe mortality in both humans and poultry. The mechanisms of disease pathogenesis and immunity are poorly understood although recent evidence suggests that cytokine/chemokine dysregulation contributes to disease severity following H5N1 infection. Influenza A virus infection causes a rapid influx of inflammatory cells, resulting in increased reactive oxygen species production, cytokine expression, and acute lung injury. Proinflammatory stimuli are known to induce intracellular reactive oxygen species by activating NADPH oxidase activity. We therefore hypothesized that inhibition of this activity would restore host cytokine homeostasis following avian influenza virus infection. A panel of airway epithelial and immune cells from mammalian and avian species were infected with A/Puerto Rico/8/1934 H1N1 virus, low-pathogenicity avian influenza H5N3 virus (A/duck/Victoria/0305-2/2012), highly pathogenic avian influenza H5N1 virus (A/chicken/Vietnam/0008/2004), or low-pathogenicity avian influenza H7N9 virus (A/Anhui/1/2013). Quantitative real-time reverse transcriptase PCR showed that H5N1 and H7N9 viruses significantly stimulated cytokine (interleukin-6, beta interferon, CXCL10, and CCL5) production. Among the influenza-induced cytokines, CCL5 was identified as a potential marker for overactive immunity. Apocynin, a Nox2 inhibitor, inhibited influenza-induced cytokines and reactive oxygen species production, although viral replication was not significantly altered in vitro. Interestingly, apocynin treatment significantly increased influenza virus-induced mRNA and protein expression of SOCS1 and SOCS3, enhancing negative regulation of cytokine signaling. These findings suggest that apocynin or its derivatives (targeting host responses) could be used in combination with antiviral strategies (targeting viruses) as therapeutic agents to ameliorate disease severity in susceptible species.

Pro-oxidative synergic bactericidal effect of NO: kinetics and inhibition by nitroxides

NO plays diverse roles in physiological and pathological processes, occasionally resulting in opposing effects, particularly in cells subjected to oxidative stress. NO mostly protects eukaryotes against oxidative injury, but was demonstrated to kill prokaryotes synergistically with H2O2. This could be a promising therapeutic avenue. However, recent conflicting findings were reported describing dramatic protective activity of NO. The previous studies of NO effects on prokaryotes applied a transient oxidative stress while arbitrarily checking the residual bacterial viability after 30 or 60min and ignoring the process kinetics. If NO-induced synergy and the oxidative stress are time-dependent, the elucidation of the cell killing kinetics is essential, particularly for survival curves exhibiting a "shoulder" sometimes reflecting sublethal damage as in the linear-quadratic survival models. We studied the kinetics of NO synergic effects on H2O2-induced killing of microbial pathogens. A synergic pro-oxidative activity toward gram-negative and gram-positive cells is demonstrated even at sub-μM/min flux of NO. For certain strains, the synergic effect progressively increased with the duration of cell exposure, and the linear-quadratic survival model best fit the observed survival data. In contrast to the failure of SOD to affect the bactericidal process, nitroxide SOD mimics abrogated the pro-oxidative synergy of NO/H2O2. These cell-permeative antioxidants, which hardly react with diamagnetic species and react neither with NO nor with H2O2, can detoxify redox-active transition metals and catalytically remove intracellular superoxide and nitrogen-derived reactive species such as (•)NO2 or peroxynitrite. The possible mechanism underlying the bactericidal NO synergy under oxidative stress and the potential therapeutic gain are discussed.