Chemical modification of Nitzschia panduriformis's frustules for protein and viral nanoparticle adsorption

The frustule of diatoms, through appropriate chemical modification, can be developed for a high adsorption level of recombinant proteins and viral nanoparticles. Field emission scanning electron microscopy (FE-SEM) analysis of clean frustules revealed a 3D loculate areolae structure (valvar phase porous pattern of the siliceous cell wall). Isocyanatopropyl triethoxysilane (IPS) and iminodiacetic acid (IDA) were used to immobilize Cu²⁺ ions (an average Cu²⁺ adsorption capacity about 190 μmol of Cu²⁺/ml of the Cu²⁺-coupled biosilica reached). FE-SEM, energy dispersion X-ray spectroscopy (EDS) and Fourier transform infrared (FT-IR) were used to confirm the chemical modification of the Cu²⁺-coupled biosilica. Protein adsorption was confirmed with the detection of a recombinant (His)₆-tagged green fluorescent protein binding using fluorescent microscopy. Infectious bursal disease virus VP2-441 subviral particles (SVPs) were found to bind to the Cu²⁺-coupled biosilica (approximately 3 × 10^?9 mol of VP2-441 SVPs/ml of modified frustules), a level higher than the previously obtained 9 × 10^?10 mol/ml for SVP binding using a commercial Ni–NTA resin. These give diatom frustules the potential to be developed into a material useful in viral nanoparticle purification systems or as a biosensor for the detection of viruses.     

Green biosynthesis of silver nanoparticles from Annona squamosa leaf extract and its in vitro cytotoxic effect on MCF-7 cells

The biological method for the synthesis of silver nanoparticles (AgNPs) using Annona squamosa leaf extract and its cytotoxicity against MCF-7 cells are reported. The synthesized AgNPs using A. squamosa leaf extract was determined by UV–visible spectroscopy and it was further characterized by FT-IR, X-ray diffraction (XRD), Transmission electron microscopy (TEM), Zeta potential and energy dispersive spectrometric (EDS) analysis. The UV–visible spectrum showed an absorption peak at 444 nm which reflects surface plasmon resonance (SPR) of AgNPs. TEM photography showed biosynthesized AgNPs were predominantly spherical in shape with an average size ranging from 20 to 100 nm. The Zeta potential value of ?37 mV revealed the stability of biosynthesized AgNPs. Furthermore, the green synthesized AgNPs exhibited a dose-dependent cytotoxicity against human breast cancer cell (MCF-7) and normal breast epithelial cells (HBL-100) and the inhibitory concentration (IC₅₀) were found to be 50 μg/mL, 30 μg/mL, and 80 μg/mL, 60 μg/ml for AgNPs against MCF-7 and normal HBL-100 cells at 24 h and 48 h incubation respectively. An induction of apoptosis was evidenced by (AO/EtBr) and DAPI staining. Application of such eco-friendly nanoparticles makes this method potentially exciting for the large scale synthesis of nanoparticles.     

Superparamagnetic iron oxide nanoparticles may affect endothelial progenitor cell migration ability and adhesion capacity

Background aimsCell labeling with superparamagnetic iron oxide (SPIO) nanoparticles enables non-invasive tracking of transplanted cells. The aim of this study was to investigate whether SPIO nanoparticles have an effect on endothelial progenitor cell (EPC) functional activity and the feasibility of a protocol for labeling swine- and rat-origin EPC using SPIO nanoparticles at an optimized low dosage.MethodsEPC were isolated from the peripheral blood of swine and bone marrow of rat and characterized. After ex vivo cultivation, EPC were labeled with SPIO nanoparticles (to make a series of final concentrations, 50, 100, 200 and 400 μg/mL) or vehicle control. We also investigated the long-term effects of 200 μg/mL SPIO nanoparticles on EPC (4, 8, 12 and 16 days after labeling). The labeling efficiency was tested through Prussian blue (PB) staining and the intracellular iron uptake was also measured quantitatively and confirmed. EPC proliferation and migration were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and transwell chamber assay, respectively. An EPC adhesion assay was performed by replating the cells on fibronectin-coated dishes and then counting the adherent cells. EPC apoptosis was evaluated using an Annexin V–FITC apoptosis kit.ResultsSPIO nanoparticles impaired EPC migration and promoted EPC adhesion. EPC proliferation and apoptosis were not affected. SPIO nanoparticles could label EPC efficiently at 200 μg/mL overnight without significantly affecting EPC functional activity.ConclusionsSPIO nanoparticles impaired the EPC migration ability and promoted the EPC adhesion capacity. EPC could be labeled efficiently at an appropriate concentration (200 μg/mL) without significantly affecting their functional activity.     

Activity of three cytotoxic isoflavonoids from Erythrina excelsa and Erythrina senegalensis (neobavaisoflavone,sigmoidin H and isoneorautenol) toward multi-factorial drug resistant cancer cells

IntroductionResistance of cancer cells to chemotherapy has become a worldwide concern. Naturally occuring isoflavonoids possess a variety of biological activities including anti-cancer effects. The present study was aimed at investigating the cytotoxicity and the modes of action of three naturally occuring isoflavonoids, neobavaisoflavone (1), sigmoidin H (2) and a pterocarpan that is a special type of isoflavonoid, isoneorautenol (3) against a panel of nine cancer cell lines, including various sensitive and drug-resistant phenotypes.MethodsThe cytotoxicity of the compounds was determined using a resazurin reduction assay, whereas the caspase-Glo assay was used to detect the activation of caspases 3/7, caspase 8 and caspase 9 in cells treated with compounds 3. Flow cytometry was used for cell cycle analysis and detection of apoptotic cells, analysis of mitochondrial membrane potential (MMP) as well as measurement of reactive oxygen species (ROS).ResultsCompounds 3 showed significant cytotoxicity toward sensitive and drug-resistant cancer cell lines. Compounds 1 and 2 were selectively active, and IC50 values below 115 μM were obtained on 6/9 and 4/9 cell lines respectively with values ranging from 42.93 μM (toward CCRF-CEM cells) to 114.64 μM [against HCT116 (p53^+/+) cells] for 1 and 25.59 μM (toward U87MG) to 110.51 μM [against HCT116 (p53^+/+) cells] for 2. IC50 values ranging from 2.67 μM (against MDA-MB 237BCRP cells) to 21.84 (toward U87MG) were measured for compound 3 and between 0.20 μM (toward CCRF-CEM cells) and 195.12 μM (toward CEM/ADR5000 cells) for doxorubicin as control drug. BCRP-transfected MDA-MB-231 cells, HCT116 (p53^+/+) and U87MG.ΔEGFR cells were hypersensitive (collateral sensitive) to compound 3 as compared to their counterpart cell lines. Compound 3 induced apoptosis in CCRF-CEM cells via activation of caspases 3/7, 8 and 9 as well as the loss of MMP and increased ROS production.ConclusionsThe cytotoxicity of the studied isoflavonoids and especially the pterocarpan 3 deserve more detailed exploration in the future to develop novel anticancer drugs against sensitive and otherwise drug-resistant phenotypes.     

Folic acid-capped PEGylated magnetic nanoparticles enter cancer cells mostly via clathrin-dependent endocytosis

BackgroundThis work is focused on mechanisms of uptake in cancer cells of rationally designed, covalently assembled nanoparticles, made of superparamagnetic iron oxide nanoparticles (SPIONs), fluorophores (doxorubicin or Nile Blue), polyethylene glycol (PEG) and folic acid (FA), referred hereinafter as SFP-FA.MethodsSFP-FA were characterized by DLS, zetametry and fluorescence spectroscopy. The SFP-FA uptake in cancer cells was monitored using fluorescence-based methods like fluorescence-assisted cell sorting, CLSM with single-photon and two-photon excitation. The SFP-FA endocytosis was also analyzed with electron microscopy approaches: TEM, HAADF-STEM and EELS.ResultsThe SFP-FA have zeta potential below − 6 mW and stable hydrodynamic diameter close to 100 nm in aqueous suspensions of pH range from 5 to 8. They contain ca. 109 PEG-FA, 480 PEG-OCH₃ and 22–27 fluorophore molecules per SPION. The fluorophores protected under the PEG shell allows a reliable detection of intracellular NPs. SFP-FA readily enter into all the cancer cell lines studied and accumulate in lysosomes, mostly via clathrin-dependent endocytosis, whatever the FR status on the cells.ConclusionsThe present study highlights the advantages of rational design of nanosystems as well as the possible involvement of direct molecular interactions of PEG and FA with cellular membranes, not limited to FA-FR recognition, in the mechanisms of their endocytosis.General significanceComposition, magnetic and optical properties of the SFP-FA as well their ability to enter cancer cells are promising for their applications in cancer theranosis. Combination of complementary analytical approaches is relevant to understand the nanoparticles behavior in suspension and in contact with cells.     

Enhanced cytotoxic activity of doxorubicin through the inhibition of autophagy in triple negative breast cancer cell line

BackgroundThe outcome of triple negative breast cancer is still poor and requires improvement with better therapy options. Autophagy has recently been shown to play a role in anticancer drug resistance. Therefore, we investigated if the effectiveness of doxorubicin was augmented by the inhibition of autophagy.MethodsMDA-MB-231 was used as a model cell line for triple negative breast cancer and 3-methyladenine was used as an inhibitor of autophagy. Cells were treated with 0.46–1.84 μM doxorubicin and 2.5–10 μM 3-methyladenine for 48 h. Cell death mode was examined with M30 and M65 ELISA assays. ROS level and LDH activity was examined and the cellular acidic compartment of cells was monitored by acridine orange staining. The expression of various autophagy and apoptosis related proteins/genes were evaluated with Western blotting and RT-qPCR respectively.ResultsSynergism was observed between the compounds (CI value < 1.0). RT-qPCR analysis revealed that the combination resulted in a down-regulation of autophagy-related genes. Moreover, the combination resulted in a different cell death modality, upregulating necroptosis-related genes. This suggests that the mode of cell death may switch from apoptosis to necroptosis, which is a more severe form of cell death, when autophagy is inhibited. These results were further confirmed at protein level by Western blotting.ConclusionInhibition of autophagy seems to sensitize triple negative breast cancer cells to doxorubicin, warranting further in vivo studies for the proof of this concept.General significanceAutophagy has a key role in drug resistance in MDA-MB-231 cells. Therefore combinatorial approaches may effectively overcome resistance.     

Protection effect of cerium oxide nanoparticles against radiation-induced acute lung injuries in rats

IntroductionRadiation therapy is one of the most common tools for treating cancer. The aim is to deliver adequate doses of radiation to kill cancer cells and the most challenging part during this procedure is to protect normal cells from radiation. One strategy is to use a radioprotector to spare normal tissues from ionizing radiation effects. Researchers have pursued cerium oxide nanoparticles as a therapeutic agent, due to its diverse characteristics, which include antioxidant properties, making it a potential radioprotector.Materials and methodsOne hundred rats were divided into five groups of A) control group, intraperitoneal (IP) saline injection was done twice a week; B) bi-weekly IP injection of 14.5 nM (0.00001 mg/kg) CNP for two weeks; C) a single whole thorax radiation dose of 18 Gy; D) a single whole thorax radiation dose of 18 Gy + bi-weekly injection of 14.5 nM CNP for two weeks after radiation; E) bi-weekly IP injection of 14.5 nM CNP for two weeks prior to radiation + a single whole thorax radiation dose of 18 Gy. Thirty days after irradiation, 7 rats from each group were anesthetized and their lungs extracted for histopathological examination.ResultsStatistical analyses revealed that CNP significantly decreased the incidence of tissue collapse and neutrophile aggregation in rats receiving CNP before radiation in comparison with the radiation group.ConclusionThe results suggested the possibility of using CNP as a future radioprotector due to its ability to protect normal cells against radiation-induced damage.     

Cytotoxicity and modes of action of three naturally occurring xanthones (8-hydroxycudraxanthone G,morusignin I and cudraxanthone I) against sensitive and multidrug-resistant cancer cell lines

BackgroundResistance of cancer to chemotherapy remains a challenging issue for scientists as well as physicians. Naturally occurring xanthones possess a variety of biological activities such as anti-inflammatory, anti-bacterial, and anti-cancer effects. The present study was aimed at investigating the cytotoxicity and the modes of action of three naturally occurring xanthones namely, morusignin I (1), 8-hydroxycudraxanthone G (2) and cudraxanthone I (3) against a panel of nine cancer cell lines, including various sensitive and drug-resistant phenotypes.MethodsThe cytotoxicity of the compounds was determined using a resazurin reduction assay, whereas the caspase-Glo assay was used to detect the activation of caspases 3/7, caspase 8 and caspase 9 in cells treated with compounds 3. Flow cytometry was used for cell cycle analysis and detection of apoptotic cells, analysis of mitochondrial membrane potential (MMP) as well as measurement of reactive oxygen species (ROS).ResultsCompounds 1 and 3 inhibited the proliferation of all tested cancer cell lines including sensitive and drug-resistant phenotypes. Compound 2 was active on 8/9 cell lines with the IC₅₀ values ranging from 16.65 μM (against leukemia CCRF-CEM cells) to 70.38 μM (against hepatocarcinoma HepG2 cells). The IC₅₀ value ranged from 7.15 μM (against CCRF-CEM cells) to 53.85 μM [against human glioblastoma U87MG.ΔEGFR cells] for compound 1, and from 2.78 μM (against breast cancer MDA-MB231 BCRP cells) to 22.49 μM (against U87MG cells) for compound 3. P-glycoprotein expressing CEM/ADR5000 cells were cross-resistant to compounds 1 and 2 (4.21- to 610-fold) while no cross-resistance or even collateral cross-sensitivity were observed in other drug-resistant cell lines to the three compounds. Normal AML12 liver cells were more resistant to the three compounds than HepG2 liver cancer cells. Compounds 3 arrested the cell cycle between G0/G1 and S phases, strongly induced apoptosis via caspases 3/7, caspase 8, caspase 9 activation and disrupted the MMP in CCRF-CEM cells.ConclusionsThe cytotoxicity of the studied xanthones and especially compound 3 deserve more detailed exploration in the future to develop novel anticancer drugs against sensitive and otherwise drug-resistant phenotypes.     

Vitamin A (retinol) downregulates the receptor for advanced glycation endproducts (RAGE) by oxidant-dependent activation of p38 MAPK and NF-kB in human lung cancer A549 cells

As an essential component of the diet, retinol supplementation is often considered harmless and its application is poorly controlled. However, recent works demonstrated that retinol may induce a wide array of deleterious effects, especially when doses used are elevated. Controlled clinical trials have demonstrated that retinol supplementation increased the incidence of lung cancer and mortality in smokers. Experimental works in cell cultures and animal models showed that retinol may induce free radical production, oxidative stress and extensive biomolecular damage. Here, we evaluated the effect of retinol on the regulation of the receptor for advanced glycation end-products (RAGE) in the human lung cancer cell line A549. RAGE is constitutively expressed in lungs and was observed to be down-regulated in lung cancer patients. A549 cells were treated with retinol doses reported as physiologic (2 μM) or therapeutic (5, 10 or 20 μM). Retinol at 10 and 20 μM increased free radical production, oxidative damage and antioxidant enzyme activity in A549 cells. These doses also downregulated RAGE expression. Antioxidant co-treatment with Trolox®, a hydrophilic analog of α-tocopherol, reversed the effects of retinol on oxidative parameters and RAGE downregulation. The effect of retinol on RAGE was mediated by p38 MAPK activation, as blockade of p38 with PD169316 (10 μM), SB203580 (10 μM) or siRNA to either p38α (MAPK14) or p38β (MAPK11) reversed the effect of retinol on RAGE. Trolox also inhibited p38 phosphorylation, indicating that retinol induced a redox-dependent activation of this MAPK. Besides, we observed that NF-kB acted as a downstream effector of p38 in RAGE downregulation by retinol, as NF-kB inhibition by SN50 (100 μg/mL) and siRNA to p65 blocked the effect of retinol on RAGE, and p38 inhibitors reversed NF-kB activation. Taken together, our results indicate a pro-oxidant effect of retinol on A549 cells, and suggest that modulation of RAGE expression by retinol is mediated by the redox-dependent activation of p38/NF-kB signaling pathway.     

High levels of plasma selenium are associated with metabolic syndrome and elevated fasting plasma glucose in a Chinese population: A case-control study

BackgroundSelenium is important for human health and involved in various metabolic processes. Deficiency of selenium associates with increased risk for cancer and cardiovascular diseases. There has been an increase use of selenium supplements for the treatment of autoimmune thyroid conditions. However, the potential biological effects of selenium overload arouse the public concern. The aim of this study was to investigate the associations of plasma selenium concentrations of adults with metabolic syndrome (MS) in Chinese population.MethodsA matched case-control study including 204 metabolic syndrome patients and 204 healthy controls was conducted in 2012. The MS cases were defined according to the criteria of Chinese Diabetes Society (CDS). Healthy controls without abnormality of metabolic components were matched with cases in age, gender and region. Plasma concentrations of selenium were determined by graphite furnace atomic absorption spectrometry (GFAAS). Fasting plasma glucose (FPG), total cholesterol (TC), triglycerides (TG), high density lipoprotein cholesterol (HDL), and low density lipoprotein cholesterol (LDL) were detected by automatic biochemical analyzer.ResultsThe median levels of plasma selenium in MS group were 146.3 (107.3–199.4) μg/L, which were significantly higher than that in the control group (127.4: 95.7–176.0) μg/L; Plasma levels of selenium were related to the risk of MS in dose-response manner. Risk of MS was significantly higher in subjects with plasma selenium in the highest tertile (T3: ≥176.0 μg/L) compared to those in the lowest tertile (T1: <95.7 μg/L) [odds ratio (OR) = 2.416 (95% CI: 1.289–4.526)]. The plasma levels of selenium were positively correlated with fasting plasma glucose (FPG) (r_s = 0.268, P < 0.001). Plasma selenium at the median (T2: 95.7–176.0 μg/L) or upper tertile (T3: ≥176.0 μg/L) was associated with increased risk of elevated FPG (defined by FPG ≥ 6.1 mmol/L) as compared with the lowest tertile (T1: ≤95.7 μg/L) [T2 vs. T1, OR = 3.487 (1.738–6.996); T3 vs. T1, OR = 6.245 (3.005–12.981)].ConclusionsHigher levels of plasma selenium might increase the risk of metabolic syndrome and elevated fasting plasma glucose. Selenium supplements should be used with prudence for CVD and cancer prevention.     

6 Iodo-δ-lactone: A derivative of arachidonic acid with antitumor effects in HT-29 colon cancer cells

BackgroundIL-δ (5-hydroxy-6 iodo-8,11,14-eicosatrienoic delta lactone) an iodinated arachidonic acid (AA) derivative, is one of the iodolipids biosynthesized by the thyroid. Although IL-δ regulates several thyroid parameters such as cell proliferation and goiter growth it was found that this iodolipid inhibits the growth of other non thyroid cell lines.ObjectivesTo study the effect of IL-δ on cell proliferation and apoptosis in the colon cancer cell line HT-29.ResultsTreatment with IL-δ reduced cell viability in a concentration-dependent manner: 1 μM 20%, 5 μM 25%, 10 μM 31%, 50 μM 47% and caused a significant decrease of PCNA expression (25%). IL-δ had pro-apoptotic effects, evidenced by morphological features of programmed cell death such as pyknosis, karyorrhexis, cell shrinkage and cell blebbing observed by fluorescence microscopy, and an increase in caspase-3 activity and in Bax/Bcl-2 ratio (2.5 after 3 h of treatment). Furthermore, IL-δ increased ROS production (30%) and lipid peroxidation levels (19%), suggesting that apoptosis could be a result of increased oxidative stress. A maximum increase in c-fos and c-jun protein expression in response to IL-δ was observed 1 h after initiation of the treatment. IL-δ also induced a tumour growth delay of 70% compared to the control group in NIH nude mice implanted with HT-29 cells.ConclusionOur study shows that IL-δ inhibits cell growth and induces apoptosis in the colon cancer cell line, HT-29 and opens the possibility that IL-δ could be a potential useful chemotherapy agent.     

Extreme effects of Seabuckthorn extracts on influenza viruses and human cancer cells and correlation between flavonol glycosides and biological activities of extracts

Seabuckthorn is a medicinal plant that is used to prevent cold. It was tested for its metabolic content followed by activity against cancer and virus. The metabolic distribution of different polarity solvent extractions from the leaves was analyzed by LC–MS/MS. Flavonol glycoside contents in EA and Bu extracts were higher than MeOH and DW was observed. MeOH and EA extracts recorded high activity against influenza A/PR virus with IC₅₀ of 7.2 μg/mL and 10.3 μg/mL compared with known drug Oseltamivir of 60.3 μg/mL. A similar trend showed in influenza A/Victoria virus. In case of influenza B viruses such as B/Lee and B/Maryland, EA extract (2.87 μg/mL and 4.5 μg/mL of IC₅₀) emerged strongest among other extracts and Oseltamivir (103.73 μg/mL and 71.6 μg/mL). Each extract showed potent anticancer activities. Interestingly, Bu extract showed stronger anticancer activity against human cancer cells such as NCL-H1299, HeLa, SKOV and Caski (8.2 μg/mL, 8.6 μg/mL, 18.2 μg/mL and 9.2 μg/mL of IC₅₀) respectively. Correlation study reveals that aglycones and flavonol mono-glycosides highly correlated with anti-influenza activities but not correlated with anticancer activities. Reversely, di-glycosides and tri-glycosides have a high correlation with cytotoxic effect with both normal and cancer cells. Therefore, this study provides significant information concerning Seabuckthorn for further medicinal drug development.     

The human antimicrobial peptide LL-37 and its fragments possess both antimicrobial and antibiofilm activities against multidrug-resistant Acinetobacter baumannii

Acinetobacter baumannii infections are difficult to treat due to multidrug resistance. Biofilm formation by A. baumannii is an additional factor in its ability to resist antimicrobial therapy. The antibacterial and antibiofilm activities of the human antimicrobial peptide LL-37 and its fragments KS-30, KR-20 and KR-12 against clinical isolates of multidrug-resistant (MDR) A. baumannii were evaluated. The minimal inhibitory concentration (MIC) of LL-37 against MDR A. baumannii isolates ranged from 16 to 32 μg/mL. The MIC of KS-30 fragment varied from 8.0 to16 μg/mL and the KR-20 fragment MIC ranged from 16 to 64 μg/mL. LL-37 and KS-30 fragment exhibited 100% bactericidal activity against five A. baumannii strains, including four MDR clinical isolates, within 30 min at concentrations of 0.25–1 μg/mL. By 0.5 h, the fragments KR-20 and KR-12 eliminated all tested strains at 8 and 64 μg/mL respectively. LL-37 and its fragments displayed anti-adherence activities between 32-128 μg/mL. A minimum biofilm eradication concentration (MBEC) biofilm assay demonstrated that LL-37 inhibited and dispersed A. baumannii biofilms at 32 μg/mL respectively. Truncated fragments of LL-37 inhibited biofilms at concentrations of 64–128 μg/mL. KS-30, the truncated variant of LL-37, effectively dispersed biofilms at 64 μg/mL. At 24 h, no detectable toxicity was observed at the efficacious doses when cytotoxicity assays were performed. Thus, LL-37, KS-30 and KR-20 exhibit significant antimicrobial activity against MDR A. baumannii. The prevention of biofilm formation in vitro by LL-37, KS-30 and KR-20 adds significance to their efficacy. These peptides can be potential therapeutics against MDR A. baumannii infections.     

Receptor for hyaluronic acid- mediated motility (RHAMM) regulates HT1080 fibrosarcoma cell proliferation via a β-catenin/c-myc signaling axis

BackgroundHigh levels of hyaluronan (HA) synthesis in various cancer tissues, including sarcomas, are correlated with tumorigenesis and malignant transformation. RHAMM (receptor for hyaluronic acid-mediated motility) is overexpressed during tumor development in different malignancies. β-Catenin is a crucial downstream mediator of the Wnt signaling cascade which facilitates carcinogenic events characterized by deregulated cell proliferation.MethodsReal-time PCR, in vitro cell proliferation assay, siRNA transfection, flow cytometry, immunoprecipitation, western blotting and immunofluorescence were utilized.ResultsThe reduction of RHAMM expression was strongly correlated with an inhibition of HT1080 fibrosarcoma cell growth (p ≤ 0.01). LMWHA, in a RHAMM-dependent manner increases cell growth of HT1080 cells ((p ≤ 0.01). Both basal and LMWHA dependent growth of HT1080 cells was attenuated by β-catenin deficiency (p ≤ 0.01). β-Catenin cytoplasmatic deposition is positively regulated by RHAMM (p ≤ 0.01). Immunoflourescence and immunoprecipitation suggest that RHAMM/β-catenin form an intracellular complex. Transfection experiments identified c-myc as candidate downstream mediator of RHAMM/β-catenin effects on HT1080 fibrosarcoma cell proliferation.ConclusionsLMWHA/RHAMM downstream signaling regulates fibrosarcoma cell growth in a β-catenin/c-myc dependent manner.General significanceThe present study suggests that RHAMM is a novel β-catenin intracellular binding partner, protecting β-catenin from degradation and supporting the nuclear translocation of this key cellular mediator, which results in c-myc activation and enhanced fibrosarcoma cell growth.     

Blood lead level and its relationship to essential elements in preschool children from Nanning,China

ObjectiveOur study aimed to assess the distribution of blood lead level and its relationship to essential elements in preschool children in an urban area of China.Design and methodsA total of 6741 children aged 0- to 6-year-old were recruited. Levels of lead, zinc, copper, iron, calcium, and magnesium in whole blood samples were determined using atomic absorption spectrometry.ResultsThe mean blood lead level (BLL) and the prevalence of BLL ≥ 10 μg/dl (5.26 ± 4.08 μg/dl and 6.84%, respectively) increased with age gradually, and there was a gender-difference for blood lead, copper, zinc and iron levels. Compared with the group of children who had BLLs < 5 μg/dl, the groups of 5 ≤ BLLs < 10 μg/dl and 10 ≤ BLLs < 15 μg/dl showed higher blood zinc, iron and magnesium levels, and a lower blood calcium level. A positive correlation of lead with zinc, iron and magnesium, and a negative correlation of lead with calcium were found in the group of children with BLL < 5 μg/dl.ConclusionAge- and gender-differences were found when assessing the BLL and intoxication prevalence in preschool children. Metabolic disorder of essential elements was found even with a low level of lead exposure.     

A therapeutic trial of human melanomas with combined small interfering RNAs targeting adaptor molecules p130Cas and paxillin activated under expression of ganglioside GD3

We previously demonstrated that focal adhesion kinase (FAK), p130Cas and paxillin are crucially involved in the enhanced malignant properties under expression of ganglioside GD3 in melanoma cells. Therefore, molecules existing in the GD3-mediated signaling pathway could be considered as suitable targets for therapeutic intervention in malignant melanoma. The aim of this study was to determine whether blockade of p130Cas and/or paxillin by RNAi suppresses melanoma growth. We found a suitable dose (40 μM siRNA, 25 μl/tumor) of the siRNA to suppress p130Cas in the xenografts generated in nu/nu mice. Based on these results, we performed intratumoral (i.t.) treatment with anti-p130Cas and/or anti-paxillin siRNAs mixed with atelocollagen as a drug delivery system in a xenograft tumor of a human melanoma cell line, SK-MEL-28. Mixture of atelocollagen (1.75%) and an siRNA (500 or 1000 pmol/tumor) was injected into the tumors every 3 days after the first injection. An siRNA against human p130Cas markedly suppressed tumor growth of the xenograft in a dose-dependent manner, whereas siRNA against human paxillin slightly inhibited the tumor growth. A control siRNA against firefly luciferase showed no effect. To our surprise, siRNA against human p130Cas (500 or 1000 pmol/tumor) combined with siRNA against human paxillin dramatically suppressed tumor growth. In agreement with the tumor suppression effects of the anti-p130Cas siRNA, reduction in Ki-67 positive cell number as well as in p130Cas expression was demonstrated by immunohistostaining. These results suggested that blockade of GD3-mediated growth signaling pathways by siRNAs might be a novel and promising therapeutic strategy against malignant melanomas, provided signaling molecules such as p130Cas and paxillin are significantly expressed in individual cases. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.     

The effect of PI3 kinase inhibitor LY294002 on voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells

AimsWe examined the effect of LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, on voltage-dependent K⁺ (Kv) channels.Main methodsElectrophysiological recordings were performed in freshly isolated rabbit coronary arterial smooth muscle cells.Key findingsThe Kv current amplitude was inhibited by LY294002 in a dose-dependent manner, with a K_d value of 1.48 μM. Without alteration of the kinetics of activation, LY294002 accelerated the decay rate of Kv channel inactivation. The rate constants of association and dissociation for LY294002 were 1.83 ± 0.01 μM^? 1 s^? 1 and 2.59 ± 0.14 s^? 1, respectively. Application of LY294002 had no significant impact on the steady-state activation or inactivation curves. In the presence of LY294002, the recovery time constant from inactivation was increased, and Kv channel inhibition increased under train pulses (1 or 2 Hz). This indicates that LY294002-induced Kv channel inhibition is use-dependent. Furthermore, pretreatment with another PI3K inhibitor, wortmannin (10 μM), did not affect the Kv current, and did not change the inhibitory effect of LY294002.SignificanceBased on these results, we suggest that LY294002 directly blocks Kv current irrespective of PI3K inhibition.     

Transgenic human programmed cell death 5 expression in mice suppresses skin cancer development by enhancing apoptosis

AimsWe sought to probe the role of human programmed cell death 5 (PDCD5) in vivo and to understand its mechanisms.Main methodsA transgenic mouse model of human PDCD5 was generated by pronuclear microinjection. Apoptosis in tissues of three independent transgenic mouse lines was quantified by terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling (TUNEL) and compared to wild type littermates. Their lifespan was compared. 8-Week PDCD5 mice and wild type mice (at a group of 5) were treated with carcinogen 3-methylcholanthrene (3-MC) at 5 μg per week to induce skin cancer. Cancer development was measured by examining hematoxylin and eosin (H&E) stained skin sections after 5 weeks and 10 weeks treatment. Protein expression was determined by Western blot and apoptosis of skin cells was quantified by TUNEL.Key findingsStarting from 5 months after birth, significant autonomous apoptosis was observed in multiple tissues of transgenic mice including skin, liver, spleen, adrenal gland and thyroid gland comparing to their wild type littermates. The average lifespan of PDCD5 mice was reduced to 9.75 months (normally 24–30 months). Moreover, carcinogen 3-MC induced skin cancer development was attenuated in the lesion of PDCD5 transgenic mice by enhancing apoptosis. Pro-apoptotic protein Bax expression was up-regulated in the 3-MC treated skin of transgenic mice.SignificanceThese results suggest PDCD5 plays an antitumor role by enhancing apoptosis in animal physiological settings. Therefore, PDCD5 is a potential target for cancer therapy.