PDF Publication Title:
Text from PDF Page: 014
In conclusion, our results suggest a so-called “Trojan-horse” mechanism [67], in which AgNPs-EPSaer internalized within the cells access the mitochondria and nuclei where release Ag ions, which determine oxidative stress and cell death. The crosstalk between autophagy, redox signaling and mitochondrial dysfunction is not well understood and further efforts are necessary to analyze the oxidative stress-ER stress-mitochondria connectivity and apoptosis/autophagy. These interesting results encourage further studies to highlight the in vivo effects on breast cancer cells. MATERIALS AND METHODS Culture conditions production of two AgNPs- EPS Cells of K. oxytoca DSM 29614 strain were stored in cryovials at –80°C in 25% glycerol solution until they were revitalized in Nutrient Broth (Difco), When the strain was grown under anaerobic and aerobic conditions the NAC medium, buffered at pH 7.8, was used (2.5 g/L NaHCO3, 1.5 g/L ammonium acetate, 1.5 g/L MgSO4.7H2O, 0.6 g/L NaH2PO4, 0.1 g/L potassium acetate, and 14.7 g.l-1 sodium citrate) [22]. The anaerobic medium was cooled under N2 flux and then was sealed. An overnight pre inoculum of cells was prepared and inoculated both into this oxygen- free medium and that aerated medium with 10 ml of DSM 29614 culture (OD600nm = 1.0) and incubated at 30°C. After 3 days incubation for aerobic medium and after 6 days for anaerobic medium, 50 mg of Ag+, such as AgNO3, was added in both cultures. The growth of the strain was followed by determining total proteins according to Bradford micro-method recommended by Bio-Rad reagent. After 2 days from Ag+ additions both precipitates were recovered by a gently centrifugation (1000 g x 20 min). The precipitated cells were removed by further centrifugations. The polysaccharide fraction was washed twice with milliQ water and the polysaccharide was precipitated with 70% ethanol solution overnight at 4°C. Both ethanol precipitates were dried out under vacuum and grinded in a mortar. Thereafter the two biogenerated silver nanoparticles were designed as AgNPs-EPSaer and of AgNPs-EPSanaer. Cell culture and treatments SKBR3, 8701-BC, HT29 and HCT116, were cultured in RPMI 1640 media (Gibco, Paisley, UK), while CaCo2 cells were cultured in DMEM medium, supplemented with 10% heat-inactivated fetal bovine serum and 100U/mL penicillin and 100 μg/mL streptomycin, as already described [33, 68–71]. The human mammary epithelial cell line HB2, already characterized for proteomic profile [70], was grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin, hydrocortisone (5 μg/mL) and insulin (10 μg/mL). All culture cells were maintained at 37°C and 5% CO2. Cells were seeded at cell density of 2 × 104/cm2, and to 70% confluence they were treated with different concentrations of AgNPs- EPSanaer, AgNPs-EPSaer and metal-free EPS (produced in absence of metal), for 24 h for all experiments. Where not expressed, the cells were treated with the corresponding IC50 values. Cell viability assay The cytotoxic activity of both NPs-EPS was determined using MTT assay (Promega, Madison, WI, USA), as previously described [69, 72]. Briefly, cells were plated at 5 × 103 cells/well in 96-well plates. NPs- EPS, diluted to the desiderated concentrations in culture medium, were added to the wells with respective vehicle control (H2O). Doxorubicin hydrochloride (Sigma, st. Louis, MO) was used as reference drug. After 24 h of incubation 20 μL of the Cell titer 96®AQueous reagent was added to each well after three washes with phosphate buffer saline (PBS) and incubated for 1-4 h at 37 °C in a CO2 incubator. The absorbance was recorded at 490 nm using a 96-well plate reader (Spark® 20M, Tecan Trading AG, Switzerland). The percentage of cell viability was calculated with respect to untreated control cells for each treatment after subtraction of the blank. The concentration necessary for 50% of growth inhibition (IC50) was calculated using a dose–response model, which was obtained from sigmoidal fitting of response curves of percent inhibition versus logarithmic concentration of DOSs using Graph Pad Prism software. Each result was the mean value of three different experiments performed in triplicate. Colony formation assay This assay identifies the cell populations that survive following a cytotoxic treatment. Cells were seeded at a density of 200, 400 and 800 cells in 6-well plates. After 2 h, the cells were treated for 1 h and for 24 h with 5 and 50 μg/L of AgNPs-EPS. At the end of the incubation period, medium was replaced with fresh medium and cells were incubated for 10 days. Colonies were fixed and stained with a mixture of 6.0% glutaraldehyde and 0.5% crystal violet and then counted microscopically using 10× high power fields. Clonogenic index was then calculated as the ratio of plating efficiency of treated cells on wells divided by the number of cells in the control wells. Cell migration Cell migration was studied by using an in vitro scratch assay. SKBR3 cells were seeded on 24-well tissue www.impactjournals.com/oncotarget 9698 OncotargetPDF Image | Anticancer activity of biogenerated silver nanoparticles
PDF Search Title:
Anticancer activity of biogenerated silver nanoparticlesOriginal File Name Searched:
oncotarget-09-9685.pdfDIY PDF Search: Google It | Yahoo | Bing
Turbine and System Plans CAD CAM: Special for this month, any plans are $10,000 for complete Cad/Cam blueprints. License is for one build. Try before you buy a production license. More Info
Waste Heat Power Technology: Organic Rankine Cycle uses waste heat to make electricity, shaft horsepower and cooling. More Info
All Turbine and System Products: Infinity Turbine ORD systems, turbine generator sets, build plans and more to use your waste heat from 30C to 100C. More Info
CO2 Phase Change Demonstrator: CO2 goes supercritical at 30 C. This is a experimental platform which you can use to demonstrate phase change with low heat. Includes integration area for small CO2 turbine, static generator, and more. This can also be used for a GTL Gas to Liquids experimental platform. More Info
Introducing the Infinity Turbine Products Infinity Turbine develops and builds systems for making power from waste heat. It also is working on innovative strategies for storing, making, and deploying energy. More Info
Need Strategy? Use our Consulting and analyst services Infinity Turbine LLC is pleased to announce its consulting and analyst services. We have worked in the renewable energy industry as a researcher, developing sales and markets, along with may inventions and innovations. More Info
Made in USA with Global Energy Millennial Web Engine These pages were made with the Global Energy Web PDF Engine using Filemaker (Claris) software.
Infinity Turbine Developing Spinning Disc Reactor SDR or Spinning Disc Reactors reduce processing time for liquid production of Silver Nanoparticles.
| CONTACT TEL: 608-238-6001 Email: greg@infinityturbine.com | RSS | AMP |