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Sample records for propidium iodide-stained cells

  1. NEW METHOD TO DETERMINE 'GIARDIA' CYST VIABILITY: CORRELATION OF FLUORESCEIN DIACETATE AND PROPIDIUM IODIDE STAINING WITH ANIMAL INFECTIVITY

    EPA Science Inventory

    The viability of Giardia muris cysts was studied using the fluorogenic dyes, fluorescein diacetate (FDA) and propidium iodide (PI). Using the mouse model for giardiasis, FDA or PI stained cysts were inoculated into neonatal mice. Feces were examined at days 3, 5, 8, and 11 post-i...

  2. Measuring Cell Death by Propidium Iodide Uptake and Flow Cytometry.

    PubMed

    Crowley, Lisa C; Scott, Adrian P; Marfell, Brooke J; Boughaba, Jeanne A; Chojnowski, Grace; Waterhouse, Nigel J

    2016-01-01

    Propidium iodide (PI) is a small fluorescent molecule that binds to DNA but cannot passively traverse into cells that possess an intact plasma membrane. PI uptake versus exclusion can be used to discriminate dead cells, in which plasma membranes become permeable regardless of the mechanism of death, from live cells with intact membranes. PI is excited by wavelengths between 400 and 600 nm and emits light between 600 and 700 nm, and is therefore compatible with lasers and photodetectors commonly available in flow cytometers. This protocol for PI staining can be used to quantitate cell death in most modern research facilities and universities. PMID:27371595

  3. Copper iodide staining and determination of proteins adsorbed to microtiter plates.

    PubMed

    Root, D D; Reisler, E

    1990-04-01

    Copper iodide staining and determination of proteins adsorbed to polystyrene microtiter plates are described. The minimum amount of copper iodide-stained protein detected in densitometric measurements is approximately 20 pg/mm2. Enzyme immunoassay readers may also be used for the determination of copper iodide-stained proteins, but are less sensitive than densitometers. The densitometric readings of copper iodide-stained proteins vary linearly with the amount of protein present as verified by enzymatic and radioactive probes. Staining is complete in 2-3 min and may be removed by a 30-min treatment with EDTA without loss of adsorbed protein or immunoreactivity. The exact amount of protein adsorbed to microtiter plate wells can be measured by using protein bound and stained on nitrocellulose as a calibration curve. Copper iodide staining is a rapid, convenient, and inexpensive alternative to radioactive measurements of similar parameters. PMID:1694063

  4. [The application of eosin and propidium iodide in evaluation of vitality of human spermatozoa].

    PubMed

    Ploskonos, М В

    2014-11-01

    The article analyzes comparative assessment of vitality of spermatozoa by condition of permeability of membranes for eosin and propidium iodide and comparison of results acquired using technique of light and fluorescent microscopy. The comparison of data of light microscopy with eosin staining with data of fluorescent microscopy with propidium iodide staining demonstrated that percentage of content of spermatozoa separated from ejaculates of 28 fertile males and stained with eosin was reliably higher (34.8 ± 3.2) than percentage of content of spermatozoa with stained with propidium iodide (2.1 ± 4.0). After incubation of spermatozoa under room temperature during 24 hours percentage of unviable cells with stained eosin also was higher than in case of propidium iodide staining correspondingly (44.5 ± 3.3% and 34.7 ± 3.6%). The analysis of vitality of spermatozoa under damaging effect of oxidative stress on cell membrane developed by 4 hours incubation with 200 mkM of hydrogen peroxide (H2O2) demonstrated that under staining of spermatozoa with propidium iodide significantly higher percentage of damaged cells is detected. In such cases, eosin staining is less suitable for detection of vitality of spermatozoa (73.6 ± 5.8% against 51.7 ± 6.4%). The carried out experiment demonstrates that in case of detected effects on spermatozoa (for example, effect of oxidative stress) the light microscopy insufficiently adequate reflects degree of damage of membranes of spermatozoa. The fluorescent microscopy detects a higher percentage of spermatozoa with damaged membrane. PMID:25850240

  5. Cell Cycle Analysis of CML Stem Cells Using Hoechst 33342 and Propidium Iodide.

    PubMed

    DeSouza, Ngoc; Zhou, Megan; Shan, Yi

    2016-01-01

    Chronic myeloid leukemia (CML) is a myeloproliferative disease with an expansion of white blood cells. The current treatments for CML are shown not to be long-term effective because of CML stem cells' insensitivity to tyrosine kinase inhibitors. Therefore, studying more about CML stem cells is essential to understand the pathways of CML stem cell development and proliferation and finally lead to effective treatments to eliminate CML stem cells and eradicate CML. This chapter describes two methods to analyze cell cycle of CML stem cells. The rare population of CML stem cells can be identified by staining with cell surface markers, and then DNA-binding dyes Hoechst 33342 and propidium iodide (PI) are added to stain the DNA content which is changed when cells go through different phases of the cell cycle. Samples are run through the flow cytometer to be analyzed based on different absorbance and emission wavelengths of different florescent colors. PMID:27581138

  6. Flow cytometric lifetime-based cell viability assay using propidium iodide

    NASA Astrophysics Data System (ADS)

    Steinkamp, John A.; Lehnert, Bruce E.; Lehnert, Nancy M.

    1999-05-01

    Assays which discriminate and enumerate dying or dead cells are important in various types of cellular studies. In many instances, there is a need to identify dead cells that interfere with fluorescent probes which are used to measure functional and physiological properties in viable cells. For example, dead cells can introduce analytical errors arising from (1) nonspecific uptake of fluorescent probes, leading to erroneous percentages of positive labeled cells, (2) increased autofluorescence, and (3) altered antigen expression. The ability to detect dead cells is also of importance in determining the effectiveness of cytotoxic agents. Propidium iodide (PPI) exclusion, which is analogous to the non- fluorescent trypan blue dye test for viability, is used extensively in flow cytometry assays. However, the use of PI can potentially limit the application of additional fluorescent probes due to spectral overlap of the probe with PI. In this report we present phase-resolved fluorescence studies on rat and murine thymus cells labeled with phycoerythrin-antiThy 1.1 and phycoerythrin/Texas Red-antiThy 1.2 immunofluorescence markers, respectively, and PI. Overlapping emission spectra are resolved based on differences in fluorescence lifetimes of the probes and PI. These studies demonstrate a new lifetime-based viability method for use in analysis of immunofluorescent probes and for assaying the dynamics of cell killing.

  7. Use of propidium monoazide for selective profiling of viable microbial cells during Gouda cheese ripening.

    PubMed

    Erkus, Oylum; de Jager, Victor C L; Geene, Renske T C M; van Alen-Boerrigter, Ingrid; Hazelwood, Lucie; van Hijum, Sacha A F T; Kleerebezem, Michiel; Smid, Eddy J

    2016-07-01

    DNA based microbial community profiling of food samples is confounded by the presence of DNA derived from membrane compromised (dead or injured) cells. Selective amplification of DNA from viable (intact) fraction of the community by propidium monoazide (PMA) treatment could circumvent this problem. Gouda cheese manufacturing is a proper model to evaluate the use of PMA for selective detection of intact cells since large fraction of membrane compromised cells emerges as a background in the cheese matrix during ripening. In this study, the effect of PMA on cheese community profiles was evaluated throughout manufacturing and ripening using quantitative PCR (qPCR). PMA effectively inhibited the amplification of DNA derived from membrane compromised cells and enhanced the analysis of the intact fraction residing in the cheese samples. Furthermore, a two-step protocol, which involves whole genome amplification (WGA) to enrich the DNA not modified with PMA and subsequent sequencing, was developed for the selective metagenome sequencing of viable fraction in the Gouda cheese microbial community. The metagenome profile of PMA treated cheese sample reflected the viable community profile at that time point in the cheese manufacturing. PMID:27077825

  8. Enumeration of Viable Listeria monocytogenes Cells by Real-Time PCR with Propidium Monoazide and Ethidium Monoazide in the Presence of Dead Cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Propidium monoazide (PMA) and ethidium monoazide were used for enumeration of viable Listeria monocytogenes cells in the presence of dead cells. PMA had no antimicrobial effect on L. monocytogenes. Viable cell counts were linearly related to real-time PCR threshold cycle values for PMA-treated cel...

  9. Visualization of cell death in mice with focal cerebral ischemia using fluorescent annexin A5, propidium iodide, and TUNEL staining.

    PubMed

    Bahmani, Peyman; Schellenberger, Eyk; Klohs, Jan; Steinbrink, Jens; Cordell, Ryan; Zille, Marietta; Müller, Jochen; Harhausen, Denise; Hofstra, Leo; Reutelingsperger, Chris; Farr, Tracy Deanne; Dirnagl, Ulrich; Wunder, Andreas

    2011-05-01

    To monitor stroke-induced brain damage and assess neuroprotective therapies, specific imaging of cell death after cerebral ischemia in a noninvasive manner is highly desirable. Annexin A5 has been suggested as a marker for imaging cell death under various disease conditions including stroke. In this study, C57BL6/N mice received middle cerebral artery occlusion (MCAO) and were injected intravenously with either active or inactive Cy5.5-annexin A5 48 hours after reperfusion. Some mice also received propidium iodide (PI), a cell integrity marker. Only in mice receiving active Cy5.5-annexin A5 were fluorescence intensities significantly higher over the hemisphere ipsilateral to MCAO than on the contralateral side. This was detected noninvasively and ex vivo 4 and 8 hours after injection. The majority of cells positive for fluorescent annexin A5 were also positive for PI and fragmented DNA as detected by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL) staining. This study demonstrates the high specificity of annexin A5 for visualization of cell death in a mouse model of stroke. To our knowledge, this is the first study to compare the distribution of injected active and inactive annexin A5, PI, and TUNEL staining. It provides important information on the experimental and potential clinical applications of annexin A5-based imaging agents in stroke. PMID:21245871

  10. Rapid decay of host-specific fecal Bacteroidales cells in seawater as measured by quantitative PCR with propidium monoazide.

    PubMed

    Bae, Sungwoo; Wuertz, Stefan

    2009-11-01

    We investigated the persistence of feces-derived Bacteroidales cells and their DNA in seawater under natural conditions using an optimized chemical method based on co-extraction of nucleic acids with propidium monoazide (PMA), which interferes with PCR amplification of molecular markers from extracellular DNA and dead bacterial cells. The previously validated Bacteroidales assays BacUni-UCD, BacHum-UCD, BacCow-UCD, and BacCan-UCD were utilized to determine concentrations of Bacteroidales genetic markers targeting all warm-blooded animals, humans, cows and dogs, specifically, over a period of 24d. Microcosms containing mixed feces in dialysis tubing were exposed to seawater under flow-through conditions at ambient temperature in the presence and absence of sunlight. Using a two-stage plus linear decay model, the average T(99) (two-log reduction) of host-specific Bacteroidales cells was 28h, whereas that of host-specific Bacteroidales DNA was 177h. Natural sunlight did not affect the survival of uncultivable Bacteroidales cells and their DNA with the exception of the BacCow-UCD marker. Bacteroidales DNA, as measured by quantitative PCR (qPCR) without PMA, persisted for as long as 24d at concentrations close to the limit of detection. Culturable Enterococcus cells were detected for only 70h, whereas Enterococcus cells measured by qPCR with and without PMA persisted for 450h. In conclusion, measuring Bacteroidales DNA without differentiating between intact and dead cells or extracellular DNA may misinform about the extent of recent fecal pollution events, particularly in the case of multiple sources of contamination with variable temporal and spatial scales due to the relatively long persistence of DNA in the environment. In contrast, applying qPCR with and without PMA can provide data on the fate and transport of fecal Bacteroidales in water, and help implement management practices to protect recreational water quality. PMID:19656546

  11. Time-resolved, single-cell analysis of induced and programmed cell death via non-invasive propidium iodide and counterstain perfusion

    PubMed Central

    Krämer, Christina E. M.; Wiechert, Wolfgang; Kohlheyer, Dietrich

    2016-01-01

    Conventional propidium iodide (PI) staining requires the execution of multiple steps prior to analysis, potentially affecting assay results as well as cell vitality. In this study, this multistep analysis method has been transformed into a single-step, non-toxic, real-time method via live-cell imaging during perfusion with 0.1 μM PI inside a microfluidic cultivation device. Dynamic PI staining was an effective live/dead analytical tool and demonstrated consistent results for single-cell death initiated by direct or indirect triggers. Application of this method for the first time revealed the apparent antibiotic tolerance of wild-type Corynebacterium glutamicum cells, as indicated by the conversion of violet fluorogenic calcein acetoxymethyl ester (CvAM). Additional implementation of this method provided insight into the induced cell lysis of Escherichia coli cells expressing a lytic toxin-antitoxin module, providing evidence for non-lytic cell death and cell resistance to toxin production. Finally, our dynamic PI staining method distinguished necrotic-like and apoptotic-like cell death phenotypes in Saccharomyces cerevisiae among predisposed descendants of nutrient-deprived ancestor cells using PO-PRO-1 or green fluorogenic calcein acetoxymethyl ester (CgAM) as counterstains. The combination of single-cell cultivation, fluorescent time-lapse imaging, and PI perfusion facilitates spatiotemporally resolved observations that deliver new insights into the dynamics of cellular behaviour. PMID:27580964

  12. Time-resolved, single-cell analysis of induced and programmed cell death via non-invasive propidium iodide and counterstain perfusion.

    PubMed

    Krämer, Christina E M; Wiechert, Wolfgang; Kohlheyer, Dietrich

    2016-01-01

    Conventional propidium iodide (PI) staining requires the execution of multiple steps prior to analysis, potentially affecting assay results as well as cell vitality. In this study, this multistep analysis method has been transformed into a single-step, non-toxic, real-time method via live-cell imaging during perfusion with 0.1 μM PI inside a microfluidic cultivation device. Dynamic PI staining was an effective live/dead analytical tool and demonstrated consistent results for single-cell death initiated by direct or indirect triggers. Application of this method for the first time revealed the apparent antibiotic tolerance of wild-type Corynebacterium glutamicum cells, as indicated by the conversion of violet fluorogenic calcein acetoxymethyl ester (CvAM). Additional implementation of this method provided insight into the induced cell lysis of Escherichia coli cells expressing a lytic toxin-antitoxin module, providing evidence for non-lytic cell death and cell resistance to toxin production. Finally, our dynamic PI staining method distinguished necrotic-like and apoptotic-like cell death phenotypes in Saccharomyces cerevisiae among predisposed descendants of nutrient-deprived ancestor cells using PO-PRO-1 or green fluorogenic calcein acetoxymethyl ester (CgAM) as counterstains. The combination of single-cell cultivation, fluorescent time-lapse imaging, and PI perfusion facilitates spatiotemporally resolved observations that deliver new insights into the dynamics of cellular behaviour. PMID:27580964

  13. Bioactive actions of pomegranate fruit extracts on leukemia cell lines in vitro hold promise for new therapeutic agents for leukemia.

    PubMed

    Dahlawi, Haytham; Jordan-Mahy, Nicola; Clench, Malcolm R; Le Maitre, Christine L

    2012-01-01

    Studies suggest that pomegranates contain bioactive chemicals with potential for treatment and prevention of cancer. Pomegranate juice extracts (PJE) have been shown to inhibit cellular proliferation and tumor growth and induce cell death via apoptosis in a number of cancer cell lines. However, to date, few studies have investigated the potential of PJE in the treatment of leukemia. We investigated the potential effect of PJE on induction of apoptosis and inhibition of cellular proliferation in 8 leukemia cell lines (4 lymphoid and 4 myeloid) and nontumor hematopoietic stem cells (control cells). Apoptosis was assessed by 2 methods: Annexin V-FITC/propidium iodide staining with flow cytometric analysis and 4'-6-diamidino-2-phenylindole (DAPI) morphological assessment. Cell cycle stage was investigated using propidum iodide staining of DNA content and flow cytometric analysis. Live cell counts were also performed using a trypan exclusion assay. PJE significantly induced apoptosis in all cell lines, including nontumor control cells, although lymphoid cells and 2 of the myeloid cell lines were more sensitive. Furthermore, PJE induced cell cycle arrest. These results were confirmed by DAPI analysis and viable cell counts using trypan blue exclusion assay. Our results provide evidence that PJE contain bioactive compounds that could be used in the treatment of leukemia. PMID:22098126

  14. Apoptotic effects on cultured cells of atmospheric-pressure plasma produced using various gases

    NASA Astrophysics Data System (ADS)

    Tominami, Kanako; Kanetaka, Hiroyasu; Kudo, Tada-aki; Sasaki, Shota; Kaneko, Toshiro

    2016-01-01

    This study investigated the effects of low-temperature atmospheric-pressure plasma on various cells such as rat fibroblastic Rat-1 cell line, rat neuroblastoma-like PC12 cell line, and rat macrophage-like NR8383 cell line. The plasma was irradiated directly to a culture medium containing plated cells for 0-20 s. The applied voltage, excitation frequency, and argon or helium gas flow were, respectively, 3-6 kV, 10 kHz, and 3 L/min. Cell viability and apoptotic activity were evaluated using annexin-V/propidium iodide staining. Results showed that the low-temperature atmospheric-pressure plasma irradiation promoted cell death in a discharge-voltage-dependent and irradiation-time-dependent manner. Furthermore, different effects are produced depending on the cell type. Moreover, entirely different mechanisms might be responsible for the induction of apoptosis in cells by helium and argon plasma.

  15. 1-(2,6-Dihydroxy-4-methoxyphenyl)-2-(4-hydroxyphenyl) Ethanone-Induced Cell Cycle Arrest in G1/G0 in HT-29 Cells Human Colon Adenocarcinoma Cells

    PubMed Central

    Lay, Ma Ma; Karsani, Saiful Anuar; Abd Malek, Sri Nurestri

    2014-01-01

    1-(2,6-Dihydroxy-4-methoxyphenyl)-2-(4-hydroxyphenyl) ethanone (DMHE) was isolated from the ethyl acetate fraction of Phaleria macrocarpa (Scheff.) Boerl fruits and the structure confirmed by GC-MS (gas chromatography-mass spectrometry) and NMR (nuclear magnetic resonance) analysis. This compound was tested on the HT-29 human colon adenocarcinoma cell line using MTT (method of transcriptional and translational) cell proliferation assay. The results of MTT assay showed that DMHE exhibited good cytotoxic effect on HT-29 cells in a dose- and time-dependent manner but no cytotoxic effect on the MRC-5 cell line after 72 h incubation. Morphological features of apoptotic cells upon treatment by DMHE, e.g., cell shrinkage and membrane blebbing, were examined by an inverted and phase microscope. Other features, such as chromatin condension and nuclear fragmentation were studied using acridine orange and propidium iodide staining under the fluorescence microscope. Future evidence of apoptosis/necrosis was provided by result fromannexin V-FITC/PI (fluorescein-isothiocyanate/propidium iodide) staining revealed the percentage of early apoptotic, late apoptotic, necrotic and live cells in a dose- and time-dependent manner using flow cytometry. Cell cycle analysis showed G0/G1 arrest in a time-dependent manner. A western blot analysis indicated that cell death might be associated with the up-regulation of the pro-apoptotic proteins Bax PUMA. However, the anit-apotptic proteins Bcl-2, Bcl-xL, and Mcl-1 were also found to increase in a time-dependent manner. The expression of the pro-apoptotic protein Bak was not observed. PMID:24451128

  16. Method to quantify live and dead cells in multi-species oral biofilm by real-time PCR with propidium monoazide

    PubMed Central

    2013-01-01

    Real-time PCR (qPCR) is a widely used technique in analysing environmental and clinical microbiological samples. However, its main limitation was its inability to discriminate between live and dead cells. Recently, propidium monoazide (PMA) together with qPCR has been used to overcome this problem, with good results for different bacterial species in different types of samples. Our objective was to implement this technique for analysing mortality in multi-species oral biofilms formed in vitro with five oral bacteria: Streptococcus oralis, Streptococcus gordonii, Veillonella parvula, Fusobacterium nucleatum and Prevotella intermedia. We also tested its effectiveness on biofilms treated with an antiseptic solution containing 0.07% w/w cetylpyridinium chloride (CPC). Standardisation of the qPCR-PMA method was performed on pure, heat-killed planktonic cultures of each species, detecting mortality higher than 4 log in S. oralis, S. gordonii and F. nucleatum and higher than 2 for V. parvula and P. intermedia. We obtained similar results for all species when using CPC. When we analysed biofilms with qPCR-PMA, we found that the mortality in the non-CPC treated multi-species biofilms was lower than 1 log for all species. After treatment with CPC, the viability reduction was higher than 4 log in S. oralis and S. gordonii, higher than 3 log in F. nucleatum and P. intermedia and approximately 2 in V. parvula. In short, we standardised the conditions for using qPCR-PMA in 5 oral bacterial species and proved its usefulness for quantification of live and dead cells in multi-species oral biofilms formed in vitro, after use of an antiseptic. PMID:23289803

  17. The effect of lance geometry and carbon coating of silicon lances on propidium iodide uptake in lance array nanoinjection of HeLa 229 cells

    NASA Astrophysics Data System (ADS)

    Sessions, John W.; Lindstrom, Dallin L.; Hanks, Brad W.; Hope, Sandra; Jensen, Brian D.

    2016-04-01

    Connecting technology to biologic discovery is a core focus of non-viral gene therapy biotechnologies. One approach that leverages both the physical and electrical function of microelectromechanical systems (MEMS) in cellular engineering is a technology previously described as lance array nanoinjection (LAN). In brief, LAN consists of a silicon chip measuring 2 cm by 2 cm that has been etched to contain an array of 10 μm tall, solid lances that are spaced every 10 μm in a grid pattern. This array of lances is used to physically penetrate hundreds of thousands of cells simultaneously and to then electrically deliver molecular loads into cells. In this present work, two variables related to the microfabrication of the silicon lances, namely lance geometry and coating, are investigated. The purpose of both experimental variables is to assess these parameters’ effect on propidium iodide (PI), a cell membrane impermeable dye, uptake to injected HeLa 229 cells. For the lance geometry experimentation, three different microfabricated lance geometries were used which include a flat/narrow (FN, 1 μm diameter), flat/wide (FW, 2-2.5 μm diameter), and pointed (P, 1 μm diameter) lance geometries. From these tests, it was shown that the FN lances had a slightly better cell viability rate of 91.73% and that the P lances had the best PI uptake rate of 75.08%. For the lance coating experimentation, two different lances were fabricated, both silicon etched lances with some being carbon coated (CC) in a  <100 nm layer of carbon and the other lances being non-coated (Si). Results from this experiment showed no significant difference between lance types at three different nanoinjection protocols (0V, +1.5V DC, and  +5V Pulsed) for both cell viability and PI uptake rates. One exception to this is the comparison of CC/5V Pul and Si/5V Pul samples, where the CC/5V Pul samples had a cell viability rate 5% higher. Both outcomes were unexpected and reveal how to better

  18. Limitations in the Use of Fluorescein Diacetate/Propidium Iodide (FDA/PI) and Cell Permeable Nucleic Acid Stains for Viability Measurements of Isolated Islets of Langerhans

    PubMed Central

    Boyd, Vinc; Cholewa, Olivia Maria; Papas, Klearchos K.

    2010-01-01

    Background A review of current literature shows that the combined use of the cell permeable esterase-substrate fluorescein diacetate (FDA) and the cell impermeant nucleic acid stain propidium iodide (PI) to be one of the most common fluorescence-based methods to assess the viability of isolated islets of Langerhans, and it is currently used for islet product release prior to transplantation in humans. However, results from this assay do not correlate with islet viability and function or islet transplantation success in animals or humans (Eckhard et al. 2004; Ricordi et al. 2001). This may be in part attributed to considerable differences as well as discrepancies in the use of these reagents on islets. We critically surveyed the literature and evaluated the impact of a number of variables associated with the use of FDA/PI to determine their reliability in assessing islet cell viability. In addition, we evaluated other fluorescent stains, such as SYTO®13, SYTO®24 and SYBR®14 as possible alternatives to FDA. Results We found that the stability of stains in storage and stock solutions, the number of islets stained, concentration of stains, staining incubation time, the buffer/media used, and the method of examining islets were significant in the final scoring of viability. For archival file photos, the exposure time and camera/software settings can also impact interpretation of viability. Although our results show that FDA does detect intracellular esterase activity and staining with PI does assess cell membrane integrity, the results obtained from using these stains did not correlate directly with expected islet function and viability per transplantation into diabetic athymic nude mice (Papas et al. 2007). In addition, the use of two nucleic acid stains, such as SYTO®13 and PI, for live/dead scoring exhibited staining anomalies which limit their accuracy in assessing islet viability. Conclusions From a review of the literature and from our observations on the

  19. Predicting electroporation of cells in an inhomogeneous electric field based on mathematical modeling and experimental CHO-cell permeabilization to propidium iodide determination.

    PubMed

    Dermol, Janja; Miklavčič, Damijan

    2014-12-01

    High voltage electric pulses cause electroporation of the cell membrane. Consequently, flow of the molecules across the membrane increases. In our study we investigated possibility to predict the percentage of the electroporated cells in an inhomogeneous electric field on the basis of the experimental results obtained when cells were exposed to a homogeneous electric field. We compared and evaluated different mathematical models previously suggested by other authors for interpolation of the results (symmetric sigmoid, asymmetric sigmoid, hyperbolic tangent and Gompertz curve). We investigated the density of the cells and observed that it has the most significant effect on the electroporation of the cells while all four of the mathematical models yielded similar results. We were able to predict electroporation of cells exposed to an inhomogeneous electric field based on mathematical modeling and using mathematical formulations of electroporation probability obtained experimentally using exposure to the homogeneous field of the same density of cells. Models describing cell electroporation probability can be useful for development and presentation of treatment planning for electrochemotherapy and non-thermal irreversible electroporation. PMID:24731594

  20. Betulinic Acid Inhibits Growth of Cultured Vascular Smooth Muscle Cells In Vitro by Inducing G1 Arrest and Apoptosis

    PubMed Central

    Vadivelu, Raja Kumar; Yeap, Swee Keong; Ali, Abdul Manaf; Hamid, Muhajir; Alitheen, Noorjahan Banu

    2012-01-01

    Betulinic acid is a widely available plant-derived triterpene which is reported to possess selective cytotoxic activity against cancer cells of neuroectodermal origin and leukemia. However, the potential of betulinic acid as an antiproliferative and cytotoxic agent on vascular smooth muscle (VSMC) is still unclear. This study was carried out to demonstrate the antiproliferative and cytotoxic effect of betulinic acid on VSMCs using 3-[4,5-dimethylthizol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry cell cycle assay, BrdU proliferation assay, acridine orange/propidium iodide staining, and comet assay. Result from MTT and BrdU assays indicated that betulinic acid was able to inhibit the growth and proliferation of VSMCs in a dose-dependent manner with IC50 of 3.8 μg/mL significantly (P < 0.05). Nevertheless, betulinic acid exhibited G1 cell cycle arrest in flow cytometry cell cycle profiling and low level of DNA damage against VSMC in acridine orange/propidium iodide and comet assay after 24 h of treatment. In conclusion, betulinic acid induced G1 cell cycle arrest and dose-dependent DNA damage on VSMC. PMID:23056140

  1. Assessment of the cell viability of cultured Perkinsus marinus (Perkinsea), a parasitic protozoan of the Eastern oyster, Crassostrea virginica, using SYBRgreen-propidium iodide double staining and flow cytometry.

    PubMed

    Soudant, Philippe; Chu, Fu-Lin E; Lund, Eric D

    2005-01-01

    A flow cytometry (FCM) assay using SYBRgreen and propidium iodide double staining was tested to assess viability and morphological parameters of Perkinsus marinus under different cold- and heat-shock treatments and at different growth phases. P. marinus meront cells, cultivated at 28 degrees C, were incubated in triplicate for 30 min at -80 degrees C, -20 degrees C, 5 degrees C, and 20 degrees C for cold-shock treatments and at 32 degrees C, 36 degrees C, 40 degrees C, 44 degrees C, 48 degrees C, 52 degrees C, and 60 degrees C for heat-shock treatments. A slight and significant decrease in percentage of viable cells (PVC), from 93.6% to 92.7%, was observed at -20 degrees C and the lowest PVC was obtained at -80 degrees C (54.0%). After 30 min of heat shocks at 40 degrees C and 44 degrees C, PVC decreased slightly but significantly compared to cells maintained at 28 degrees C. When cells were heat shocked at 48 degrees C, 52 degrees C, and 60 degrees C heavy mortality occurred and PVC decreased to 33.8%, 8.0%, and 3.4%, respectively. No change in cell complexity and size was noted until cells were heat shocked at >or=44 degrees C. High cell mortality was detected at stationary phase of P. marinus cell culture. Cell viability dropped below 40% in 28-day-old cultures and ranged 11-25% in 38 to 47-day-old cultures. Results suggest that FCM could be a useful tool for determining viability of cultured P. marinus cells. PMID:16313441

  2. The Effects of Brazilian Green Propolis against Excessive Light-Induced Cell Damage in Retina and Fibroblast Cells

    PubMed Central

    Murase, Hiromi; Shimazawa, Masamitsu; Kakino, Mamoru; Ichihara, Kenji; Tsuruma, Kazuhiro; Hara, Hideaki

    2013-01-01

    Background. We investigated the effects of Brazilian green propolis and its constituents against white light- or UVA-induced cell damage in mouse retinal cone-cell line 661W or human skin-derived fibroblast cells (NB1-RGB). Methods. Cell damage was induced by 3,000lx white light for 24 h or 4/10 J/cm2 UVA exposure. Cell viability was assessed by Hoechst33342 and propidium iodide staining or by tetrazolium salt (WST-8) cell viability assay. The radical scavenging activity of propolis induced by UVA irradiation in NB1-RGB cells was measured using a reactive-oxygen-species- (ROS-) sensitive probe CM-H2DCFDA. Moreover, the effects of propolis on the UVA-induced activation of p38 and extracellular signal-regulated kinase (ERK) were examined by immunoblotting. Results. Treatment with propolis and two dicaffeoylquinic acids significantly inhibited the decrease in cell viability induced by white light in 661W. Propolis and its constituents inhibited the decrease in cell viability induced by UVA in NB1-RGB. Moreover, propolis suppressed the intracellular ROS production by UVA irradiation. Propolis also inhibited the levels of phosphorylated-p38 and ERK by UVA irradiation. Conclusion. Brazilian green propolis may become a major therapeutic candidate for the treatment of AMD and skin damage induced by UV irradiation. PMID:24416064

  3. Cell cycle progression in denV-transfected murine fibroblasts exposed to ultraviolet radiation.

    PubMed

    Kusewitt, D F; Budge, C L; Nolla, H A; Edwards, B S; Ley, R D

    1992-09-01

    Repair-proficient murine fibroblasts transfected with the denV gene of bacteriophage T4 repaired 70-80% of pyrimidine dimers within 24 h after exposure to 150 J/m2 ultraviolet radiation (UVR) from an FS-40 sunlamp. Under the same conditions, control cells repaired only about 20% of UVR-induced pyrimidine dimers. After UVR exposure, both control and denV-transfected cells exhibited some degree of DNA-synthesis inhibition, as determined by flow cytometric analysis of cell-cycle kinetics in propidium iodide-stained cells. DenV-transfected cells had a longer and more profound S phase arrest than control cells, but both control and denV-transfected cells had largely recovered from UVR effects on cell-cycle kinetics by 48 h after UVR exposure. Inhibition of DNA synthesis by UVR was also measured by determining post-UVR incorporation of bromodeoxyuridine (BrdU). The amount of BrdU incorporated was quantitated by determining with flow cytometry the quenching of Hoechst dye 33342 by BrdU incorporated in cellular DNA. DenV-transfected cells showed more marked inhibition of BrdU incorporation after low fluences of UVR than control cells. Differences between denV-transfected and control cells in cell-cycle kinetics following UVR exposure may be related to differences in mechanisms of repair when excision repair of pyrimidine dimers is initiated by endonuclease V instead of cellular repair enzymes. PMID:1380650

  4. The cytotoxic activities of 7-isopentenyloxycoumarin on 5637 cells via induction of apoptosis and cell cycle arrest in G2/M stage

    PubMed Central

    2014-01-01

    Background Bladder cancer is the second common malignancy of genitourinary tract, and transitional cell carcinomas (TCCs) account for 90% of all bladder cancers. Due to acquired resistance of TCC cells to a wide range of chemotherapeutic agents, there is always a need for search on new compounds for treatment of these cancers. Coumarins represent a group of natural compounds, which some of them have exerted valuable anti-tumor activities. The current study was designed to evaluate anti-tumor properties and mechanism of action of 7-isopentenyloxycoumarin, a prenyloxycoumarin, on 5637 cells (a TCC cell line). Results MTT results revealed that the cytotoxic effects of 7-isopentenyloxycoumarin on 5637 cancerous cells were more prominent in comparison to HDF-1 normal cells. This coumarin increased the amount of chromatin condensation and DNA damage in 5637 cells by 58 and 33%, respectively. The results also indicated that it can induce apoptosis most probably via activation of caspase-3 in these cells. Moreover, propidium iodide staining revealed that 7-isopentenyloxycoumarin induced cell cycle arrest at G2/M stage, after 24 h of treatment. Conclusion Our results indicated that 7-isopentenyloxycoumarin had selective toxic effects on this bladder cancer cell line and promoted its effects by apoptosis induction and cell cycle arrest. This coumarin can be considered for further studies to reveal its exact mechanism of action and also its anti-cancer effects in vivo. PMID:24393601

  5. Mechanisms of Propidium Monoazide Inhibition of Polymerase Chain Reaction and implications for Propidium Monoazide Applications

    NASA Astrophysics Data System (ADS)

    Lee, C. M.; Darrach, H.; Ponce, A.; McFarland, E.; Laymon, C.; Fingland, N. K.

    2015-12-01

    PMA-qPCR is a laboratory technique that can be used to identify viable microbes by employing the use of propidium monoazide (PMA), a DNA-intercalating dye, and quantitative polymerase chain reaction (qPCR). The current model of PMA-qPCR operates under the assumption that PMA is only capable of entering membrane-compromised cells, where it irreversibly cross-links to DNA and makes it unavailable for amplification via qPCR. However, the exact mechanism behind PMA's entry into the cell and its interaction with genetic material is not well understood. To better understand PMA's capabilities, we have examined the effect PMA has on enzyme binding and processivity using endonucleases and exonucleases. Our results suggest that the current model behind PMA-qPCR inhibition is incomplete, in that rather than precipitating the entirety of the DNA, PMA also inhibits enzyme binding and/or processivity in soluble DNA. These results have important implications for studying the viable community of microorganisms in various applications, such as environmental monitoring, planetary protection and bioburden assessment, and biohazard detection.

  6. Altered Antioxidant System Stimulates Dielectric Barrier Discharge Plasma-Induced Cell Death for Solid Tumor Cell Treatment

    PubMed Central

    Park, Daehoon; Choi, Eun H.

    2014-01-01

    This study reports the experimental findings and plasma delivery approach developed at the Plasma Bioscience Research Center, Korea for the assessment of antitumor activity of dielectric barrier discharge (DBD) for cancer treatment. Detailed investigation of biological effects occurring after atmospheric pressure non-thermal (APNT) plasma application during in vitro experiments revealed the role of reactive oxygen species (ROS) in modulation of the antioxidant defense system, cellular metabolic activity, and apoptosis induction in cancer cells. To understand basic cellular mechanisms, we investigated the effects of APNT DBD plasma on antioxidant defense against oxidative stress in various malignant cells as well as normal cells. T98G glioblastoma, SNU80 thyroid carcinoma, KB oral carcinoma and a non-malignant HEK293 embryonic human cell lines were treated with APNT DBD plasma and cellular effects due to reactive oxygen species were observed. Plasma significantly decreased the metabolic viability and clonogenicity of T98G, SNU80, KB and HEK293 cell lines. Enhanced ROS in the cells led to death via alteration of total antioxidant activity, and NADP+/NADPH and GSH/GSSG ratios 24 hours (h) post plasma treatment. This effect was confirmed by annexin V-FITC and propidium iodide staining. These consequences suggested that the failure of antioxidant defense machinery, with compromised redox status, might have led to sensitization of the malignant cells. These findings suggest a promising approach for solid tumor therapy by delivering a lethal dose of APNT plasma to tumor cells while sparing normal healthy tissues. PMID:25068311

  7. Metformin inhibits histone H2B monoubiquitination and downstream gene transcription in human breast cancer cells.

    PubMed

    DU, Yu; Zheng, Haiyan; Wang, Jiang; Ren, Ye; Li, Mi; Gong, Chen; Xu, Fei; Yang, Caihong

    2014-08-01

    Metformin, one of the most widely prescribed antihyperglycemic drugs, has recently received increasing attention for its potential effects with regard to cancer prevention and treatment. However, the mechanisms behind the suppression of cancer cell growth by metformin remain far from completely understood. The aim of the present study was to investigate whether metformin could regulate histone modification and its downstream gene transcription, and its potential function in inhibiting breast cancer cell proliferation. A T47D cell proliferation curve was determined by cell counting following metformin treatment with differing doses or time courses. The cell cycle was analyzed by flow cytometry with propidium iodide staining. Histone H2B monoubiquitination was evaluated by western blotting subsequent to histone extraction. The histone H2B monoubiquitination downstream gene expression level was determined by quantitative PCR. The results showed that metformin changed the cell-cycle check-point and inhibited breast cancer cell proliferation in a dose-dependent manner. AMPK was activated and histone H2B monoubiquitination and downstream gene transcription were inhibited following metformin treatment in the T47D cells. The effect of metformin on T47D cell proliferation was dependent on AMPK activity. It was concluded that metformin can suppress breast cancer cell growth by the activation of AMPK and the inhibition of histone H2B monoubiquitination and downstream gene transcription. This study reveals a novel potential mechanism of cancer cell growth suppression by metformin. PMID:25009658

  8. Damage in Escherichia coli Cells Treated with a Combination of High Hydrostatic Pressure and Subzero Temperature▿

    PubMed Central

    Moussa, Marwen; Perrier-Cornet, Jean-Marie; Gervais, Patrick

    2007-01-01

    The relationship between membrane permeability, changes in ultrastructure, and inactivation in Escherichia coli strain K-12TG1 cells subjected to high hydrostatic pressure treatment at room and subzero temperatures was studied. Propidium iodide staining performed before and after pressure treatment made it possible to distinguish between reversible and irreversible pressure-mediated cell membrane permeabilization. Changes in cell ultrastructure were studied using transmission electron microscopy (TEM), which showed noticeable condensation of nucleoids and aggregation of cytosolic proteins in cells fixed after decompression. A novel technique used to mix fixation reagents with the cell suspension in situ under high hydrostatic pressure (HHP) and subzero-temperature conditions made it possible to show the partial reversibility of pressure-induced nucleoid condensation. However, based on visual examination of TEM micrographs, protein aggregation did not seem to be reversible. Reversible cell membrane permeabilization was noticeable, particularly for HHP treatments at subzero temperature. A correlation between membrane permeabilization and cell inactivation was established, suggesting different mechanisms at room and subzero temperatures. We propose that the inactivation of E. coli cells under combined HHP and subzero temperature occurs mainly during their transiently permeabilized state, whereas HHP inactivation at room temperature is related to a balance of transient and permanent permeabilization. The correlation between TEM results and cell inactivation was not absolute. Further work is required to elucidate the effects of pressure-induced damage on nucleoids and proteins during cell inactivation. PMID:17766454

  9. Damage in Escherichia coli cells treated with a combination of high hydrostatic pressure and subzero temperature.

    PubMed

    Moussa, Marwen; Perrier-Cornet, Jean-Marie; Gervais, Patrick

    2007-10-01

    The relationship between membrane permeability, changes in ultrastructure, and inactivation in Escherichia coli strain K-12TG1 cells subjected to high hydrostatic pressure treatment at room and subzero temperatures was studied. Propidium iodide staining performed before and after pressure treatment made it possible to distinguish between reversible and irreversible pressure-mediated cell membrane permeabilization. Changes in cell ultrastructure were studied using transmission electron microscopy (TEM), which showed noticeable condensation of nucleoids and aggregation of cytosolic proteins in cells fixed after decompression. A novel technique used to mix fixation reagents with the cell suspension in situ under high hydrostatic pressure (HHP) and subzero-temperature conditions made it possible to show the partial reversibility of pressure-induced nucleoid condensation. However, based on visual examination of TEM micrographs, protein aggregation did not seem to be reversible. Reversible cell membrane permeabilization was noticeable, particularly for HHP treatments at subzero temperature. A correlation between membrane permeabilization and cell inactivation was established, suggesting different mechanisms at room and subzero temperatures. We propose that the inactivation of E. coli cells under combined HHP and subzero temperature occurs mainly during their transiently permeabilized state, whereas HHP inactivation at room temperature is related to a balance of transient and permanent permeabilization. The correlation between TEM results and cell inactivation was not absolute. Further work is required to elucidate the effects of pressure-induced damage on nucleoids and proteins during cell inactivation. PMID:17766454

  10. Comparative analysis of the cytotoxic effect of 7-prenyloxycoumarin compounds and herniarin on MCF-7 cell line

    PubMed Central

    Mousavi, Seyed Hadi; Davari, Atiyeh-Sadat; Iranshahi, Mehrdad; Sabouri-Rad, Sarvenaz; Tayarani Najaran, Zahra

    2015-01-01

    Objective: 7-prenyloxycoumarins are a group of secondary metabolites that are found mainly in plants belonging to the Rutaceae and Umbelliferae families. This study was designed to evaluate and compare the cytotoxic and apoptotic activity of 7-prenyloxycoumarin compounds and herniarin on MCF-7, a breast carcinoma cell line. Materials and Methods: Cells were cultured in RPMI medium and incubated with different concentrations of auraptene, herniarin, umbelliferone, and umbelliprenin. Cell viability was quantified by MTT assay. Apoptotic cells were determined using propidium iodide staining of DNA fragmentation by flow cytometry (sub-G1peak). Bax protein expression was detected by western blot to investigate the underlying mechanism. Results: Doses which induced 50% cell growth inhibition (IC50) against MCF-7 cells with auraptene, herniarin, umbelliferone, and umbelliprenin were calculated (59.7, 207.6, 476.3, and 73.4 µM), respectively. Auraptene induced a sub-G1 peak in the flow cytometry histogram of treated cells compared to control cells, and DNA fragmentation suggested the induction of apoptosis. Western blot analysis showed that auraptene significantly up-regulated Bax expression in MCF-7 cells compared to untreated controls. Conclusion: Auraptene exerts cytotoxic and apoptotic effects in breast carcinoma cell line and can be considered for further mechanistic evaluations in human cancer cells. These results candidate auraptene for further studies to evaluate its biosafety and anti-cancer effects. PMID:26693409

  11. Caspase dependent apoptotic inhibition of melanoma and lung cancer cells by tropical Rubus extracts.

    PubMed

    George, Blassan Plackal Adimuriyil; Abrahamse, Heidi; Hemmaragala, Nanjundaswamy M

    2016-05-01

    Rubus fairholmianus Gard. inhibits human melanoma (A375) and lung cancer (A549) cell growth by the caspase dependent apoptotic pathway. Herbal products have a long history of clinical use and acceptance. They are freely available natural compounds that can be safely used to prevent various ailments. The plants and plant derived products became the basis of traditional medicine system throughout the world for thousands of years. The effects of R. fairholmianus root acetone extract (RFRA) on the proliferation of A375 and A549 cells was examined in this study. RFRA led to a decrease in cell viability, proliferation and an increase in cytotoxicity in a dose dependent manner when compared with control and normal skin fibroblast cells (WS1). The morphology of treated cells supported apoptotic cell death. Annexin V/propidium iodide staining indicated that RFRA induced apoptosis in A375 and A549 cells and the percentages of early and late apoptotic populations significantly increased. Moreover, the apoptotic inducing ability of RFRA when analysing effector caspase 3/7 activity, indicated a marked increase in treated cells. In summary, we have shown the anticancer effects of RFRA in A375 and A549 cancer cells via induction of caspase dependent apoptosis in vitro. The extract is more effective against melanoma; which may suggest the usefulness of RFRA-based anticancer therapies. PMID:27133056

  12. Metformin inhibits mitochondrial permeability transition and cell death: a pharmacological in vitro study

    PubMed Central

    2004-01-01

    Metformin, a drug widely used in the treatment of Type II diabetes, has recently received attention owing to new findings regarding its mitochondrial and cellular effects. In the present study, the effects of metformin on respiration, complex 1 activity, mitochondrial permeability transition, cytochrome c release and cell death were investigated in cultured cells from a human carcinoma-derived cell line (KB cells). Metformin significantly decreased respiration both in intact cells and after permeabilization. This was due to a mild and specific inhibition of the respiratory chain complex 1. In addition, metformin prevented to a significant extent mitochondrial permeability transition both in permeabilized cells, as induced by calcium, and in intact cells, as induced by the glutathione-oxidizing agent t-butyl hydroperoxide. This effect was equivalent to that of cyclosporin A, the reference inhibitor. Finally, metformin impaired the t-butyl hydroperoxide-induced cell death, as judged by Trypan Blue exclusion, propidium iodide staining and cytochrome c release. We propose that metformin prevents the permeability transition-related commitment to cell death in relation to its mild inhibitory effect on complex 1, which is responsible for a decreased probability of mitochondrial permeability transition. PMID:15175014

  13. Synthesis, structural characterization, and anticancer activity of a monobenzyltin compound against MCF-7 breast cancer cells

    PubMed Central

    Fani, Somayeh; Kamalidehghan, Behnam; Lo, Kong Mun; Hashim, Najihah Mohd; Chow, Kit May; Ahmadipour, Fatemeh

    2015-01-01

    A new monoorganotin Schiff base compound, [N-(3,5-dichloro-2-oxidobenzylidene)-4-chlorobenzyhydrazidato](o-methylbenzyl)aquatin(IV) chloride, (compound C1), was synthesized, and its structural features were investigated by spectroscopic techniques and single-crystal X-ray diffractometry. Compound C1 was exposed to several human cancer cell lines, including breast adenocarcinoma cell lines MCF-7 and MDA-MB-231, ovarian adenocarcinoma cell lines Skov3 and Caov3, and prostate cancer cell line PC3, in order to examine its cytotoxic effect for different forms of cancer. Human hepatic cell line WRL-68 was used as a normal cell line. We concentrated on the MCF-7 cell line to detect possible underlying mechanism involvement of compound C1. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed the strongest cytotoxicity of compound C1 against MCF-7 cells, with a half maximal inhibitory concentration (IC50) value of 2.5±0.50 μg/mL after 48 hours treatment. The IC50 value was >30 μg/mL in WRL-68 cells. Induced antiproliferative activity of compound C1 for MCF-7 cells was further confirmed by lactate dehydrogenase, reactive oxygen species, acridine orange/propidium iodide staining, and DNA fragmentation assays. A significant increase of lactate dehydrogenase release in treated cells was observed via fluorescence analysis. Luminescent analysis showed significant growth in intracellular reactive oxygen species production after treatment. Morphological changes of necrosis and early and late apoptosis stages were observed in treated cells after staining with acridine orange/propidium iodide. DNA fragmentation was observed as a characteristic of apoptosis in treated cells. Results of the present study obviously reveal potential cytotoxic effects of compound C1 against human breast cancer MCF-7 cells. PMID:26648695

  14. Arctigenin enhances chemosensitivity to cisplatin in human nonsmall lung cancer H460 cells through downregulation of survivin expression.

    PubMed

    Wang, Huan-qin; Jin, Jian-jun; Wang, Jing

    2014-01-01

    Arctigenin, a dibenzylbutyrolactone lignan, enhances cisplatin-mediated cell apoptosis in cancer cells. Here, we sought to investigate the effects of arctigenin on cisplatin-treated non-small-cell lung cancer (NSCLC) H460 cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and annexin-V/propidium iodide staining were performed to analyze the proliferation and apoptosis of H460 cells. Arctigenin dose-dependently suppressed cell proliferation and potentiated cell apoptosis, coupled with increased cleavage of caspase-3 and poly(ADP-ribose) polymerase. Moreover, arctigenin sensitized H460 cells to cisplatin-induced proliferation inhibition and apoptosis. Arctigenin alone or in combination with cisplatin had a significantly lower amount of survivin. Ectopic expression of survivin decreased cell apoptosis induced by arctigenin (P < 0.05) or in combination with cisplatin (P < 0.01). Moreover, arctigenin (P < 0.05) or in combination with cisplatin (P < 0.01) induced G1/G0 cell-cycle arrest. Our data provide evidence that arctigenin has a therapeutic potential in combina-tion with chemotherapeutic agents for NSLC. PMID:24395429

  15. Proliferation and differentiation of oligodendrocyte progenitor cells induced from rat embryonic neural precursor cells followed by flow cytometry.

    PubMed

    Lü, He-Zuo; Wang, Yan-Xia; Li, Ying; Fu, Sai-Li; Hang, Qin; Lu, Pei-Hua

    2008-08-01

    Previous studies have shown that a cell-intrinsic timer might determine when oligodendrocyte progenitor cells (OPCs) isolated from the central nervous system (CNS) stop dividing and initiate differentiation in a defined environment. In this report, the proliferation and differentiation of OPCs induced from neural precursor cells (NPCs) were analyzed by flow cytometry combined with carboxyfluorescein diacetate succinimidyl ester labeling and propidium iodide staining, respectively. When OPCs were cultured in OPC-medium, more than 30% of cells were in S- and G2/M-phases, and continuously self-renewed without differentiation. After exposure to thyroid hormone, there was an obvious decrease in the fraction of cells in both S- and G2/M-phases (<10%). Furthermore, the OPCs no longer proliferated, but differentiated into oligodendrocytes. The dynamic proliferation and differentiation characteristics of OPCs induced from NPCs and analyzed by flow cytometry were similar to those of OPCs isolated from the CNS and analyzed by other methods. These studies indicated that the proliferation and differentiation of OPCs can be followed simply and rapidly by flow cytometry. PMID:18473382

  16. In Vitro Antiproliferative Effect of the Acetone Extract of Rubus fairholmianus Gard. Root on Human Colorectal Cancer Cells

    PubMed Central

    Plackal Adimuriyil George, Blassan; Tynga, Ivan Mfouo

    2015-01-01

    Plants and plant derived products exert chemopreventive effects on various cancer cell lines by the induction of cell death mechanisms. The effects of root acetone extract of Rubus fairholmianus (RFRA) on the proliferation of human colorectal cancer (Caco-2) cells have been investigated in this study. The extract led to a dose dependent decrease in both viability and proliferation and increased cytotoxicity using trypan blue exclusion, adenosine 5′-triphosphate (ATP), and lactate dehydrogenase (LDH) assay. The morphological features of the treated cells were supportive for the antiproliferative activity. The Annexin V/propidium iodide staining indicated that R. fairholmianus induced toxic effects in Caco-2 cells and the percentages of the early and late apoptotic population significantly increased when compared with control cells. Also we studied the apoptosis inducing ability of the extract by analysing caspase 3/7 activity and the induction of cell death via the effector caspases was confirmed; the activity increased in treated cells compared with control. Thus the present findings highlight that the R. fairholmianus root acetone extract exhibits antiproliferative activity on Caco-2 cells by the induction of apoptosis via caspase dependent pathway. PMID:26078938

  17. Flow cytometry-based characterization of label-retaining stem cells following transplacental BrdU labelling.

    PubMed

    Poojan, Shiv; Kumar, Sushil

    2011-02-01

    A method to characterize and culture stem cells from neonate mouse epidermis after transplacental BrdU (bromo-deoxyuridine) administration is described. We have characterized stem cells by their properties viz. to retain BrdU label, adhere rapidly onto collagen-fibronectin substratum and express a specific biomarker beta-1-integrin. BrdU-labelled cells (detected using monoclonal antibody) constituted a sum of 18% of the total number of cells. The ability of freshly isolated keratinocytes [LRCs (label-retaining cells)] to bind to primary BrdU antibody or to pick up PI (propidium iodide) stain was distinguishable. Viable LRCs did not retain PI. Such cells, termed EpSC (epidermis stem cell), were PI negative and BrdU positive. EpSC constituted 6% of the total cell yield. Culture in low Ca2+ medium and susceptibility to differentiation in the presence of high Ca2+ levels further characterized the stem cells. This protocol is useful for studying transplacental carcinogenesis. PMID:21261598

  18. The Acetone Extract of Sclerocarya birrea (Anacardiaceae) Possesses Antiproliferative and Apoptotic Potential against Human Breast Cancer Cell Lines (MCF-7)

    PubMed Central

    Tanih, Nicoline Fri; Ndip, Roland Ndip

    2013-01-01

    Interesting antimicrobial data from the stem bark of Sclerocarya birrea, which support its use in traditional medicine for the treatment of many diseases, have been delineated. The current study was aimed to further study some pharmacological and toxicological properties of the plant to scientifically justify its use. Anticancer activity of water and acetone extracts of S. birrea was evaluated on three different cell lines, HT-29, HeLa, and MCF-7 using the cell titre blue viability assay in 96-well plates. Apoptosis was evaluated using the acridine orange and propidium iodide staining method, while morphological structure of treated cells was examined using SEM. The acetone extract exhibited remarkable antiproliferative activities on MCF-7 cell lines at dose- and time-dependent manners (24 h and 48 h of incubation). The extract also exerted apoptotic programmed cell death in MCF-7 cells with significant effect on the DNA. Morphological examination also displayed apoptotic characteristics in the treated cells, including clumping, condensation, and culminating to budding of the cells to produce membrane-bound fragmentation, as well as formation of apoptotic bodies. The acetone extract of S. birrea possesses antiproliferative and apoptotic potential against MCF-7-treated cells and could be further exploited as a potential lead in anticancer therapy. PMID:23576913

  19. Apoptosis induced by oxysterols in murine lymphoma cells and in normal thymocytes.

    PubMed Central

    Christ, M; Luu, B; Mejia, J E; Moosbrugger, I; Bischoff, P

    1993-01-01

    Oxygenated derivatives of cholesterol (oxysterols), a family of naturally occurring compounds, possess marked anti-proliferative and immunosuppressive activities, in particular they have been shown to inhibit T-cell responses to different stimuli. 25-Hydroxycholesterol (25-OHC) and 7 beta,25-dihydroxycholesterol (7.25-OHC) are able to kill not only RDM4 murine lymphoma in vitro, but also, surprisingly, mouse thymocytes after several hours of incubation. In this study, we report that the death of RDM4 and thymocytes induced by oxysterols exhibits the features of apoptosis. This phenomenon was identified by agarose gel electrophoresis of DNA fragments extracted from the cells and quantified by flow cytometric analysis of the DNA fluorescence of propidium iodide-stained cells. Cycloheximide and actinomycin D were found to decrease the number of apoptotic cells and to increase cell viability, indicating a requirement for the synthesis of macromolecules in oxysterol-induced programmed cell death. The pathway by which 25-OHC and 7.25-OHC are able to induce apoptosis in this type of cell and the possible contribution of these compounds to thymus involution during development are discussed. Images Figure 2 Figure 4 PMID:7682990

  20. Effect of taxol from Pestalotiopsis mangiferae on A549 cells-In vitro study.

    PubMed

    Kathiravan, Govindarajan; Sureban, Sripathi M

    2009-12-01

    Pestalotiopsis mangiferae Coelomycete fungi were used to examine the production of taxol. The taxol isolated from this fungus is biologically active against cancer cell lines were investigated for its antiproliferative activity in human Non Small Cell Lung Cancer A549 cells. The results showed that the methylene chloride extraction of Pestalotiopsis mangiferae inhibited the proliferation of A 549 cells as measured by MTT and Trypan blue assay. Flow cytometric analysis showed that methylene chloride extraction of Pestalotiopsis mangiferae blocked cell cycle progression in G0/G1 phase. In addition fungal taxol induced A549 cell apoptosis as determined by propidium iodide staining. Further the percentage of LDH release was increased at increasing concentrations which is a measure of cell death. The levels of sialic acid levels and DNA, RNA and protein levels were decreased after treatment with methylene chloride extraction of Pestalotiopsis mangiferae. We suggests that methylene chloride extraction of Pestalotiopsis mangiferae might be considered for future therapeutic application with further studies against lung cancer. PMID:25206246

  1. Primula auriculata Extracts Exert Cytotoxic and Apoptotic Effects against HT-29 Human Colon Adenocarcinoma Cells.

    PubMed

    Behzad, Sahar; Ebrahim, Karim; Mosaddegh, Mahmoud; Haeri, Ali

    2016-01-01

    Primula auriculata (Tootia) is one of the most important local medicinal plants in Hamedan district, Iran. To investigate cytotoxicity and apoptosis induction of crude methanolic extract and different fraction of it, we compared several methods on HT-29 human colon Adenocarcinoma cells. Cancer cell proliferation was measured by 3-(4, 5‑dimethylthiazolyl)2, 5‑diphenyl‑tetrazolium bromide (MTT) assay and apoptosis induction was analyzed by fluorescence microscopy (acridin orange/ethidium bromide, annexin V/propidium iodide staining, TUNEL assay and Caspase-3 activity assay). Crude methanolic extract (CM) inhibited the growth of malignant cells in a dose-dependent manner. Among solvent fractions, the dichloromethane fraction (CF) was found to be the most toxic compared to other fractions. With double staining methods, high percentage of 40 µg/mL of (CM) and (CF) treated cells exhibited typical characteristics of apoptotic cells. Apoptosis induction was also revealed by apoptotic fragmentation of nuclear DNA and activation of caspas-3 in treated cells. These findings indicate that crude methanolic extract and dichloromethan fraction of P.auriculata induced apoptosis and inhibited proliferation in colon cancer cells and could be used as a source for new lead structures in drug design to combat colon cancer. PMID:27610172

  2. Primula auriculata Extracts Exert Cytotoxic and Apoptotic Effects against HT-29 Human Colon Adenocarcinoma Cells

    PubMed Central

    Behzad, Sahar; Ebrahim, Karim; Mosaddegh, Mahmoud; Haeri, Ali

    2016-01-01

    Primula auriculata (Tootia) is one of the most important local medicinal plants in Hamedan district, Iran. To investigate cytotoxicity and apoptosis induction of crude methanolic extract and different fraction of it, we compared several methods on HT-29 human colon Adenocarcinoma cells. Cancer cell proliferation was measured by 3-(4, 5‑dimethylthiazolyl)2, 5‑diphenyl‑tetrazolium bromide (MTT) assay and apoptosis induction was analyzed by fluorescence microscopy (acridin orange/ethidium bromide, annexin V/propidium iodide staining, TUNEL assay and Caspase-3 activity assay). Crude methanolic extract (CM) inhibited the growth of malignant cells in a dose-dependent manner. Among solvent fractions, the dichloromethane fraction (CF) was found to be the most toxic compared to other fractions. With double staining methods, high percentage of 40 µg/mL of (CM) and (CF) treated cells exhibited typical characteristics of apoptotic cells. Apoptosis induction was also revealed by apoptotic fragmentation of nuclear DNA and activation of caspas-3 in treated cells. These findings indicate that crude methanolic extract and dichloromethan fraction of P.auriculata induced apoptosis and inhibited proliferation in colon cancer cells and could be used as a source for new lead structures in drug design to combat colon cancer. PMID:27610172

  3. Thrombin Induces Tumor Cell Cycle Activation and Spontaneous Growth by Down-regulation of p27Kip1, in Association with the Up-regulation of Skp2 and MiR-222

    PubMed Central

    Hu, Liang; Ibrahim, Sherif; Liu, Cynthia; Skaar, Jeffrey; Pagano, Michele; Karpatkin, Simon

    2009-01-01

    The effect of thrombin on tumor cell cycle activation and spontaneous growth was examined in synchronized serum-starved tumor cell lines and a model of spontaneous prostate cancer development in TRAMP mice. BrdUrd incorporation and propidium iodide staining of prostate LNCaP cells arrested in G0 and treated with thrombin or serum revealed a 48- and 29-fold increase in S phase cells, respectively, at 8 hours. Similar results were obtained with TRAMP cells and a glioblastoma cell line, T98G. Cell cycle kinases and inhibitors in synchronized tumor cells revealed high levels of p27Kip1 and low levels of Skp2 and cyclins D1 and A. Addition of thrombin, TFLLRN, or serum down-regulated p27Kip1 with concomitant induction of Skp2, Cyclin D1, and Cyclin A with similar kinetics. LNCaP p27Kip1-transfected cells or Skp2 knockdown cells were refractory to thrombin-induced cell cycle activation. MicroRNA 222, an inhibitor of p27Kip1, was robustly up-regulated by thrombin. The in vitro observations were tested in vivo with transgenic TRAMP mice. Repetitive thrombin injection enhanced prostate tumor volume 6- to 8-fold (P < 0.04). Repetitive hirudin, a specific potent antithrombin, decreased tumor volume 13- to 24-fold (P < 0.04). Thus, thrombin stimulates tumor cell growth in vivo by down-regulation of p27Kip1. PMID:19351827

  4. Triticumoside induces apoptosis via caspase-dependent mitochondrial pathway and inhibits migration through downregulation of MMP2/9 in human lung cancer cells.

    PubMed

    Poudel, Barun; Ki, Hyeon-Hui; Luyen, Bui Thi Thuy; Lee, Young-Mi; Kim, Young-Ho; Kim, Dae-Ki

    2016-02-01

    Non-small cell lung cancer (NSCLC) is the major cancer-related death worldwide with only 14% five-year survival rate. Triticumoside, a phenolic compound present in Triticum aestivum sprout extract, has been recognized to have antiobesity and anti-inflammatory effects. However, the effect of triticumoside on cancer cell proliferation and migration has not been studied. In order to elucidate whether triticumoside exhibits an anticancer effect, cells were incubated with different doses of triticumoside, and apoptosis was assessed by observing cell viability, cellular morphological changes, and annexin-V-fluorescein isothiocyanate/propidium iodide staining. Cell cycle analysis, western blotting, wound healing assay, and quantitative-polymerase chain reaction were also performed. Triticumoside exhibited marked cytotoxicity in the cells in dose- and time-dependent manner. Triticumoside caused morphological changes, including cellular rounding, nuclear condensation, and shrinkage. Likewise, triticumoside enhanced the sub-G1 proportion of cells. Additionally, triticumoside regulated expression of apoptosis-associated proteins, such as B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X, and procaspase-3/9. Triticumoside also inhibited migration of the cells through downregulation of matrix metalloproteinase-2/9 (MMP2/9). Collectively, these results suggest that triticumoside induces apoptosis through caspase-dependent mitochondrial pathway and suppresses migration via inhibition of MMP2/9 in NSCLC A549 cells. PMID:26758192

  5. miR-34b inhibits nasopharyngeal carcinoma cell proliferation by targeting ubiquitin-specific peptidase 22

    PubMed Central

    Xiao, Jianyong; Li, Yingying; Zhang, Wenyin; Jiang, Yanni; Du, Biaoyan; Tan, Yuhui

    2016-01-01

    Objectives This study aimed to investigate the precise role of miR-34b in nasopharyngeal carcinoma (NPC). Materials and methods: The expression of miR-34b and transcription of ubiquitin-specific peptidase 22 (USP22) were examined using quantitative reverse transcription-polymerase chain reaction. Western blot analysis was used to measure the protein expression of USP22. A dualluciferase assay was used to investigate the interaction between miR-34b and USP22. Cell viability was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. The cell cycle was analyzed by propidium iodide staining followed by flow cytometry analysis. Results miR-34b was significantly downregulated in NPC tissues and NPC cell lines. Overexpression of miR-34b in NPC SUNE-6-10B cells inhibited cell viability and proliferation. USP22 was highly expressed in NPC cells and promoted cell viability and proliferation. Restoration of USP22 expression could reverse the effect of miR-34b on NPC cell viability and proliferation. Conclusion miR-34b acts as a tumor suppressor in NPC, which is mediated via repression of the oncogene USP22. PMID:27051294

  6. Cytotoxic Effect of Coscinium fenestratum on Human Head and Neck Cancer Cell Line (HN31)

    PubMed Central

    Potikanond, Saranyapin; Khonsung, Parirat

    2015-01-01

    Coscinium fenestratum is widely used as a medicinal plant in many Asian countries. This study aimed to investigate the cytotoxic effect of a crude water extract of C. fenestratum (CF extract) compared to 5-fluorouracil (5-FU) on human HN31 cell line, a metastatic squamous cell carcinoma of the pharynx. The results revealed that cell morphology visualized under inverted light microscopy was changed from flat with a polygonal appearance to round appearance after CF extract application. The cell viability assay (MTT test) showed that the concentration producing 50% growth inhibition (IC50) at 48-hour incubation of CF extract on HN31 was 0.12 mg/mL, while the IC50 of 5-FU was 6.6 mg/mL, indicating that CF extract has a higher potency. However, combining various concentrations of 5-FU and CF extract at IC50 did not show synergistic effect. The CF extract dose dependently increased cell apoptosis determined by Annexin-V and propidium iodide staining. It decreased the phosphorylation of p38 MAPK and pAkt, while it increased the tumor suppressor protein p53. In conclusion, the cytotoxicity of CF extract was associated with the modulation of p38 MAPK, pAkt, and p53 signal molecules, leading to inhibiting cell survival and increasing apoptosis. No synergistic effects of CF extract and 5-FU were observed. PMID:26074999

  7. Cytotoxic Effect of Coscinium fenestratum on Human Head and Neck Cancer Cell Line (HN31).

    PubMed

    Potikanond, Saranyapin; Chiranthanut, Natthakarn; Khonsung, Parirat; Teekachunhatean, Supanimit

    2015-01-01

    Coscinium fenestratum is widely used as a medicinal plant in many Asian countries. This study aimed to investigate the cytotoxic effect of a crude water extract of C. fenestratum (CF extract) compared to 5-fluorouracil (5-FU) on human HN31 cell line, a metastatic squamous cell carcinoma of the pharynx. The results revealed that cell morphology visualized under inverted light microscopy was changed from flat with a polygonal appearance to round appearance after CF extract application. The cell viability assay (MTT test) showed that the concentration producing 50% growth inhibition (IC50) at 48-hour incubation of CF extract on HN31 was 0.12 mg/mL, while the IC50 of 5-FU was 6.6 mg/mL, indicating that CF extract has a higher potency. However, combining various concentrations of 5-FU and CF extract at IC50 did not show synergistic effect. The CF extract dose dependently increased cell apoptosis determined by Annexin-V and propidium iodide staining. It decreased the phosphorylation of p38 MAPK and pAkt, while it increased the tumor suppressor protein p53. In conclusion, the cytotoxicity of CF extract was associated with the modulation of p38 MAPK, pAkt, and p53 signal molecules, leading to inhibiting cell survival and increasing apoptosis. No synergistic effects of CF extract and 5-FU were observed. PMID:26074999

  8. Decrease in hyperosmotic stress–induced corneal epithelial cell apoptosis by L-carnitine

    PubMed Central

    Khandekar, Neeta; Willcox, Mark D.P.; Shih, Sharon; Simmons, Peter; Vehige, Joseph

    2013-01-01

    Purpose To characterize the osmoprotective properties of L-carnitine on human corneal epithelial cell volume and apoptosis during hyperosmotic stress. Methods Human corneal limbal epithelial (HCLE) cells were exposed to culture medium at 300 mOsm (isotonic) or 500 mOsm (hyperosmotic) with or without L-carnitine (10 mM). Induction of apoptosis was detected by quantifying the proteolytic activity of caspase-8, caspase-9, and caspase-3/7 using caspase activity assays, the expression of tumor necrosis factor (TNF)-α with enzyme-linked immunosorbent assay, and annexin V/propidium iodide staining of HCLE cells evaluated with confocal microscopy and flow cytometry. Cell volume changes in response to hyperosmotic stress were analyzed using flow cytometry. Results After the HCLE cells were exposed to hyperosmotic medium (500 mOsm), the percentage of shrunken cells and damaged/dead cells (stained positively for annexin V and/or propidium iodide) was six- and three-fold, respectively, higher than that under isotonic conditions (300 mOsm). This was paralleled by an increase in TNF-α concentration in media and caspase-8, -9, and -3/7 activities (six-, four-, ten-, and twelve-fold, respectively; all showing p<0.001). Addition of L-carnitine during hyperosmotic stress partly restored cell volume and significantly reduced the concentration of TNF-α released (p=0.005) and caspase-9 activity (p=0.0125). Addition of L-carnitine reduced the percentage of hyperosmolarity-induced damaged/dead cells to levels observed under isotonic conditions. Conclusions L-carnitine can regulate human corneal epithelial cell volume under hyperosmotic stress and ameliorate hyperosmotic stress–induced apoptosis. PMID:24068862

  9. Kefir induces apoptosis and inhibits cell proliferation in human acute erythroleukemia.

    PubMed

    Jalali, Fatemeh; Sharifi, Mohammadreza; Salehi, Rasoul

    2016-01-01

    Acute erythroleukemia is an uncommon subtype of acute myeloid leukemia which has been considered to be a subtype of AML with a worse prognosis. Intensive chemotherapy is the first line of treatment. In recent years, the effect of kefir on some malignancies has been experimented. Kefir is a kind of beverage, which obtained by incubation of kefir grains with raw milk. Kefir grains are a symbiotic complex of different kinds of yeasts and bacteria, especially lactic acid bacteria which gather in a mostly carbohydrate matrix, named kefiran. We investigated the effect of kefir on acute erythroleukemia cell line (KG-1) and peripheral blood mononuclear cells (PBMCs). The cell line and PBMCs were treated with different doses of kefir and milk and incubated for three different times. We used Polymixin B to block the lipopolysaccharide and NaOH (1 mol/l) to neutralize the acidic media. Viability was detected by MTT assay. Apoptosis and necrosis were assessed by annexin-propidium iodide staining. Our results showed that kefir induced apoptosis and necrosis in KG-1 cell line. It was revealed that kefir decreased proliferation in erythroleukemia cell line. We did not observe a remarkable effect of kefir on PBMCs. Our study suggested that kefir may have potential to be an effective treatment for erythroleukemia. PMID:26708130

  10. Detergent sclerosants at sub-lytic concentrations induce endothelial cell apoptosis through a caspase dependent pathway.

    PubMed

    Cooley-Andrade, Osvaldo; Cheung, Kelvin; Chew, An-Ning; Connor, David Ewan; Parsi, Kurosh

    2016-07-01

    To investigate the apoptotic effects of detergent sclerosants sodium tetradecylsulphate (STS) and polidocanol (POL) on endothelial cells at sub-lytic concentrations. Human umbilical vein endothelial cells (HUVECs) were isolated and labelled with antibodies to assess for apoptosis and examined with confocal microscopy and flow cytometry. Isolated HUVECs viability was assessed using propidium iodide staining. Early apoptosis was determined by increased phosphatidylserine exposure by lactadherin binding. Caspase 3, 8, 9 and Bax activation as well as inhibitory assays with Pan Caspase (Z-VAD-FMK) and Bax (BI-6C9) were assessed to identify apoptotic pathways. Porimin activation was used to assess cell membrane permeability. Cell lysis reached almost 100 % with STS at 0.3 % and with POL at 0.6 %. Apoptosis was seen with both STS and POL at concentrations ranging from 0.075 to 0.15 %. PS exposure increased with both STS and POL and exhibited a dose-dependent trend. Active Caspase 3, 8 and 9 but not Bax were increased in HUVECs stimulated with low concentrations of both STS and POL. Inhibitory assays demonstrated Caspase 3, 8, 9 inhibition at low concentrations (0.075 to 0.6 %) with both STS and POL. Both agents increased the activation of porimin at all concentrations. Both sclerosants induced endothelial cell (EC) apoptosis at sub-lytic concentrations through a caspase-dependant pathway. Both agents induced EC oncosis. PMID:27225250

  11. Antiproliferation and induction of apoptosis by Moringa oleifera leaf extract on human cancer cells.

    PubMed

    Sreelatha, S; Jeyachitra, A; Padma, P R

    2011-06-01

    Medicinal plants provide an inexhaustible source of anticancer drugs in terms of both variety and mechanism of action. Induction of apoptosis is the key success of plant products as anticancer agents. The present study was designed to determine the antiproliferative and apoptotic events of Moringa oleifera leaf extract (MLE) using human tumor (KB) cell line as a model system. KB cells were cultured in the presence of leaf extracts at various concentrations for 48 h and the percentage of cell viability was evaluated by MTT assay. MLE showed a dose-dependent inhibition of cell proliferation of KB cells. The antiproliferative effect of MLE was also associated with induction of apoptosis as well as morphological changes and DNA fragmentation. The morphology of apoptotic nuclei was quantified using DAPI and propidium iodide staining. The degree of DNA fragmentation was analyzed using agarose gel electrophoresis. In addition, MLE at various concentrations was found to induce ROS production suggesting modulation of redox-sensitive mechanism. Eventually, HPTLC analysis indicated the presence of phenolics such as quercetin and kaempferol. Thus, these findings suggest that the leaf extracts from M. oleifera had strong antiproliferation and potent induction of apoptosis. Thus, it indicates that M. oleifera leaf extracts has potential for cancer chemoprevention and can be claimed as a therapeutic target for cancer. PMID:21385597

  12. Dual role of the caspase enzymes in satellite cells from aged and young subjects.

    PubMed

    Fulle, S; Sancilio, S; Mancinelli, R; Gatta, V; Di Pietro, R

    2013-01-01

    Satellite cell (SC) proliferation and differentiation have critical roles in skeletal muscle recovery after injury and adaptation in response to hypertrophic stimuli. Normal ageing hinders SC proliferation and differentiation, and is associated with increased expression of a number of pro-apoptotic factors in skeletal muscle. In light of previous studies that have demonstrated age-related altered expression of genes involved in SC antioxidant and repair activity, this investigation was aimed at evaluating the incidence of apoptotic features in human SCs. Primary cells were obtained from vastus lateralis of nine young (27.3±2.0 years old) and nine old (71.1±1.8 years old) subjects, and cultured in complete medium for analyses at 4, 24, 48, and 72 h. Apoptosis was assessed using AnnexinV/propidium iodide staining, the terminal deoxynucleotidyl transferase dUTP nick-end labelling technique, RT-PCR, DNA microarrays, flow cytometry, and immunofluorescence analysis. There was an increased rate of apoptotic cells in aged subjects at all of the experimental time points, with no direct correlation between AnnexinV-positive cells and caspase-8 activity. On the other hand, CASP2, CASP6, CASP7, and CASP9 and a number of cell death genes were upregulated in the aged SCs. Altogether, our data show age-related enhanced susceptibility of human SCs to apoptosis, which might be responsible for their reduced response to muscle damage. PMID:24336075

  13. Leptin promotes cell proliferation and survival of trophoblastic cells.

    PubMed

    Magariños, María Paula; Sánchez-Margalet, Víctor; Kotler, Mónica; Calvo, Juan Carlos; Varone, Cecilia L

    2007-02-01

    Leptin, the 16-kDa protein product of the obese gene, was originally considered as an adipocyte-derived signaling molecule for the central control of metabolism. However, leptin has been suggested to be involved in other functions during pregnancy, particularly in placenta. In the present work, we studied a possible effect of leptin on trophoblastic cell proliferation, survival, and apoptosis. Recombinant human leptin added to JEG-3 and BeWo choriocarcinoma cell lines showed a stimulatory effect on cell proliferation up to 3 and 2.4 times, respectively, measured by (3)H-thymidine incorporation and cell counting. These effects were time and dose dependent. Maximal effect was achieved at 250 ng leptin/ml for JEG-3 cells and 50 ng leptin/ml for BeWo cells. Moreover, by inhibiting endogenous leptin expression with 2 microM of an antisense oligonucleotide (AS), cell proliferation was diminished. We analyzed cell population distribution during the different stages of cell cycle by fluorescence-activated cell sorting, and we found that leptin treatment displaced the cells towards a G2/M phase. We also found that leptin upregulated cyclin D1 expression, one of the key cell cycle-signaling proteins. Since proliferation and death processes are intimately related, the effect of leptin on cell apoptosis was investigated. Treatment with 2 microM leptin AS increased the number of apoptotic cells 60 times, as assessed by annexin V-fluorescein isothiocyanate/propidium iodide staining, and the caspase-3 activity was increased more than 2 fold. This effect was prevented by the addition of 100 ng leptin/ml. In conclusion, we provide evidence that suggests that leptin is a trophic and mitogenic factor for trophoblastic cells by virtue of its inhibiting apoptosis and promoting proliferation. PMID:17021346

  14. Borax-induced apoptosis in HepG2 cells involves p53, Bcl-2, and Bax.

    PubMed

    Wei, Y; Yuan, F J; Zhou, W B; Wu, L; Chen, L; Wang, J J; Zhang, Y S

    2016-01-01

    Borax, a boron compound and a salt of boric acid, is known to inhibit the growth of tumor cells. HepG2 cells have been shown to be clearly susceptible to the anti-proliferative effects of borax. However, the specific mechanisms regulating this effect are poorly understood. This study aimed to investigate the pathways underlying the growth inhibition induced by borax in HepG2 cells. The effects of borax on HepG2 cell viability were characterized using MTT. Apoptosis was also verified by annexin V/propidium iodide staining. JC-1 dye and western blotting techniques were used to measure mitochondrial membrane potential and p53, Bax, and Bcl-2 protein expression, respectively. Relevant mRNA levels were measured by qRT-PCR. Borax inhibited the proliferation of HepG2 cells in a time- and dose-dependent manner in vitro. The apoptotic process triggered by borax involved the upregulation of p53 and Bax and the downregulation of Bcl-2, which was confirmed by a change in the mitochondrial membrane potential. These results elucidate a borax-induced apoptotic pathway in HepG2 cells that involves the upregulation of p53 and Bax and the downregulation of Bcl-2. PMID:27420953

  15. Cordycepin induces apoptosis in human liver cancer HepG2 cells through extrinsic and intrinsic signaling pathways

    PubMed Central

    Shao, Le-Wen; Huang, Li-Hua; Yan, Sheng; Jin, Jian-Di; Ren, Shao-Yan

    2016-01-01

    Cordycepin, also termed 3′-deoxyadenosine, is a nucleoside analogue from Cordyceps sinensis and has been reported to demonstrate numerous biological and pharmacological properties. Our previous study illustrated that the anti-tumor effect of cordycepin may be associated with apoptosis. In the present study, the apoptotic effect of cordycepin on HepG2 cells was investigated using 4′,6-diamidino-2-phenylindole, tetraethylbenzimidazolylcarbocyanine iodide and propidium iodide staining analysis and flow cytometry. The results showed that cordycepin exhibited the ability to inhibit HepG2 cells in a time- and dose-dependent manner when cells produced typical apoptotic morphological changes, including chromatin condensation, the accumulation of sub-G1 cells and change mitochondrial permeability. A potential mechanism for cordycepin-induced apoptosis of human liver cancer HepG2 cells may occur through the extrinsic signaling pathway mediated by the transmembrane Fas-associated with death domain protein. Apoptosis was also associated with Bcl-2 family protein regulation, leading to altered mitochondrial membrane permeability and resulting in the release of cytochrome c into the cytosol. The activation of the caspase cascade is responsible for the execution of apoptosis. In conclusion, cordycepin-induced apoptosis in HepG2 cells involved the extrinsic and intrinsic signaling pathway and was primarily regulated by the Bcl-2 family proteins. PMID:27446383

  16. In Vivo therapeutic potential of mesenchymal stem cell-derived extracellular vesicles with optical imaging reporter in tumor mice model.

    PubMed

    Kalimuthu, Senthilkumar; Gangadaran, Prakash; Li, Xiu Juan; Oh, Ji Min; Lee, Ho Won; Jeong, Shin Young; Lee, Sang-Woo; Lee, Jaetae; Ahn, Byeong-Cheol

    2016-01-01

    Mesenchymal stem cells (MSCs) can be used as a therapeutic armor for cancer. Extracellular vesicles (EVs) from MSCs have been evaluated for anticancer effects. In vivo targeting of EVs to the tumor is an essential requirement for successful therapy. Therefore, non-invasive methods of monitoring EVs in animal models are crucial for developing EV-based cancer therapies. The present study to develop bioluminescent EVs using Renilla luciferase (Rluc)-expressing MSCs. The EVs from MSC/Rluc cells (EV-MSC/Rluc) were visualized in a murine lung cancer model. The anticancer effects of EVs on Lewis lung carcinoma (LLC) and other cancer cells were assessed. EV-MSC/Rluc were visualized in vivo in the LLC-efffuc tumor model using optical imaging. The induction of apoptosis was confirmed with Annexin-V and propidium iodide staining. EV-MSC/Rluc and EV-MSCs showed a significant cytotoxic effect against LLC-effluc cells and 4T1; however, no significant effect on CT26, B16F10, TC1 cells. Moreover, EV-MSC/Rluc inhibited LLC tumor growth in vivo. EV-MSC/Rluc-mediated LLC tumor inhibitory mechanism revealed the decreased pERK and increased cleaved caspase 3 and cleaved PARP. We successfully developed luminescent EV-MSC/Rluc that have a therapeutic effect on LLC cells in both in vitro and in vivo. This bioluminescent EV system can be used to optimize EV-based therapy. PMID:27452924

  17. In Vivo therapeutic potential of mesenchymal stem cell-derived extracellular vesicles with optical imaging reporter in tumor mice model

    PubMed Central

    Kalimuthu, Senthilkumar; Gangadaran, Prakash; Li, Xiu Juan; Oh, Ji Min; Lee, Ho Won; Jeong, Shin Young; Lee, Sang-Woo; Lee, Jaetae; Ahn, Byeong-Cheol

    2016-01-01

    Mesenchymal stem cells (MSCs) can be used as a therapeutic armor for cancer. Extracellular vesicles (EVs) from MSCs have been evaluated for anticancer effects. In vivo targeting of EVs to the tumor is an essential requirement for successful therapy. Therefore, non-invasive methods of monitoring EVs in animal models are crucial for developing EV-based cancer therapies. The present study to develop bioluminescent EVs using Renilla luciferase (Rluc)-expressing MSCs. The EVs from MSC/Rluc cells (EV-MSC/Rluc) were visualized in a murine lung cancer model. The anticancer effects of EVs on Lewis lung carcinoma (LLC) and other cancer cells were assessed. EV-MSC/Rluc were visualized in vivo in the LLC-efffuc tumor model using optical imaging. The induction of apoptosis was confirmed with Annexin-V and propidium iodide staining. EV-MSC/Rluc and EV-MSCs showed a significant cytotoxic effect against LLC-effluc cells and 4T1; however, no significant effect on CT26, B16F10, TC1 cells. Moreover, EV-MSC/Rluc inhibited LLC tumor growth in vivo. EV-MSC/Rluc-mediated LLC tumor inhibitory mechanism revealed the decreased pERK and increased cleaved caspase 3 and cleaved PARP. We successfully developed luminescent EV-MSC/Rluc that have a therapeutic effect on LLC cells in both in vitro and in vivo. This bioluminescent EV system can be used to optimize EV-based therapy. PMID:27452924

  18. Methods to isolate a large amount of generative cells, sperm cells and vegetative nuclei from tomato pollen for “omics” analysis

    PubMed Central

    Lu, Yunlong; Wei, Liqin; Wang, Tai

    2015-01-01

    The development of sperm cells (SCs) from microspores involves a set of finely regulated molecular and cellular events and the coordination of these events. The mechanisms underlying these events and their interconnections remain a major challenge. Systems analysis of genome-wide molecular networks and functional modules with high-throughput “omics” approaches is crucial for understanding the mechanisms; however, this study is hindered because of the difficulty in isolating a large amount of cells of different types, especially generative cells (GCs), from the pollen. Here, we optimized the conditions of tomato pollen germination and pollen tube growth to allow for long-term growth of pollen tubes in vitro with SCs generated in the tube. Using this culture system, we developed methods for isolating GCs, SCs and vegetative cell nuclei (VN) from just-germinated tomato pollen grains and growing pollen tubes and their purification by Percoll density gradient centrifugation. The purity and viability of isolated GCs and SCs were confirmed by microscopy examination and fluorescein diacetate staining, respectively, and the integrity of VN was confirmed by propidium iodide staining. We could obtain about 1.5 million GCs and 2.0 million SCs each from 180 mg initiated pollen grains, and 10 million VN from 270 mg initiated pollen grains germinated in vitro in each experiment. These methods provide the necessary preconditions for systematic biology studies of SC development and differentiation in higher plants. PMID:26082789

  19. DNA damage, lysosomal degradation and Bcl-xL deamidation in doxycycline- and minocycline-induced cell death in the K562 leukemic cell line.

    PubMed

    Fares, Mona; Abedi-Valugerdi, Manuchehr; Hassan, Moustapha; Potácová, Zuzana

    2015-07-31

    We investigated mechanisms of cytotoxicity induced by doxycycline (doxy) and minocycline (mino) in the chronic myeloid leukemia K562 cell line. Doxy and mino induced cell death in exposure-dependent manner. While annexin V/propidium iodide staining was consistent with apoptosis, the morphological changes in Giemsa staining were more equivocal. A pancaspase inhibitor Z-VAD-FMK partially reverted cell death morphology, but concurrently completely prevented PARP cleavage. Mitochondrial involvement was detected as dissipation of mitochondrial membrane potential and cytochrome C release. DNA double strand breaks detected with γH2AX antibody and caspase-2 activation were found early after the treatment start, but caspase-3 activation was a late event. Decrement of Bcl-xL protein levels and electrophoretic shift of Bcl-xL molecule were induced by both drugs. Phosphorylation of Bcl-xL at serine 62 was ruled out. Similarly, Bcr/Abl tyrosine kinase levels were decreased. Lysosomal inhibitor chloroquine restored Bcl-xL and Bcr/Abl protein levels and inhibited caspase-3 activation. Thus, the cytotoxicity of doxy and mino in K562 cells is mediated by DNA damage, Bcl-xL deamidation and lysosomal degradation with activation of mitochondrial pathway of apoptosis. PMID:26022120

  20. Artesunate induces G0/G1 cell cycle arrest and iron-mediated mitochondrial apoptosis in A431 human epidermoid carcinoma cells.

    PubMed

    Jiang, Zhongyong; Chai, Jin; Chuang, Henry Hon Fung; Li, Shifeng; Wang, Tianran; Cheng, Yi; Chen, Wensheng; Zhou, Deshan

    2012-07-01

    The anticancer effects of artesunate (ART) have been well documented. However, its potential against skin cancer has not been explored yet. Herein we reported that 60 μmol/l ART effectively inhibited A431 (human epidermoid carcinoma cells) growth but not that of HaCaT (normal human keratinocyte cells). Our results revealed that ART induced cell cycle arrest at G0/G1 phase through the downregulation of cyclin A1, cyclin B, cyclin D1, Cdk2, Cdk4, and Cdk6. This correlated with the upregulation of p21 and p27. The 5-bromodeoxyuridine incorporation assay also indicated that ART treatment reduced DNA synthesis in a time-dependent manner. Furthermore, ART induced mitochondrial apoptosis, as evidenced by annexin V/propidium iodide staining and western blot analysis. Interestingly, ART-induced apoptosis diminished under iron-deficient conditions but intensified under iron-overload conditions. Taken together, these findings demonstrated the potential of ART in treating skin cancer through the induction of G0/G1 cell cycle arrest and iron-mediated mitochondrial apoptosis and supported further investigations in other test systems. PMID:22421370

  1. Statins Prevent Dextrose-Induced Endoplasmic Reticulum Stress and Oxidative Stress in Endothelial and HepG2 Cells.

    PubMed

    Kojanian, Hagop; Szafran-Swietlik, Anna; Onstead-Haas, Luisa M; Haas, Michael J; Mooradian, Arshag D

    2014-05-01

    Statins have favorable effects on endothelial function partly because of their capacity to reduce oxidative stress. However, antioxidant vitamins, unlike statins, are not as cardioprotective, and this paradox has been explained by failure of vitamin antioxidants to ameliorate endoplasmic reticulum (ER) stress. To determine whether statins prevent dextrose-induced ER stress in addition to their antioxidative effects, human umbilical vein endothelial cells and HepG2 hepatocytes were treated with 27.5 mM dextrose in the presence of simvastatin (lipophilic statin that is a prodrug) and pravastatin (water-soluble active drug), and oxidative stress, ER stress, and cell death were measured. Superoxide generation was measured using 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-A]pyrazin-3-one hydrochloride. ER stress was measured using the placental alkaline phosphatase assay and Western blot of glucose-regulated protein 75, c-jun-N-terminal kinase, phospho-JNK, eukaryotic initiating factor 2α and phospho-eIF2α, and X-box binding protein 1 mRNA splicing. Cell viability was measured by propidium iodide staining. Superoxide anion production, ER stress, and cell death induced by 27.5 mM dextrose were inhibited by therapeutic concentrations of simvastatin and pravastatin. The salutary effects of statins on endothelial cells in reducing both ER stress and oxidative stress observed with pravastatin and the prodrug simvastatin suggest that the effects may be independent of cholesterol-lowering activity. PMID:24800792

  2. Emodin induces apoptosis of human breast cancer cells by modulating the expression of apoptosis-related genes

    PubMed Central

    ZU, CONG; ZHANG, MINGDI; XUE, HUI; CAI, XIAOPENG; ZHAO, LEI; HE, ANNING; QIN, GUANGYUAN; YANG, CHUNSHU; ZHENG, XINYU

    2015-01-01

    The aim of this study was to investigate the effects of emodin on the proliferation of human breast cancer cells Bcap-37 and ZR-75-30. Cell viability following emodin treatment was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of emodin on apoptosis were determined by flow cytometry using Annexin V-fluorescein isothiocyanate and propidium iodide staining. Quantitative polymerase chain reaction and western blot analysis were used to determine changes in the expression of apoptotic genes and protein, respectively. The effect of emodin on the invasiveness of breast cancer cells was evaluated by Matrigel invasion assay. Treatment of breast cancer cells Bcap-37 and ZR-75-30 with emodin was observed to inhibit the growth and induced apoptosis in a time- and dose-dependent manner. Emodin reduced the level of Bcl-2 and increased levels of cleaved caspase-3, PARP, p53 and Bax. These findings indicate that emodin induces growth inhibition and apoptosis in human breast cancer cells. Emodin may be a potential therapeutic agent for the treatment of breast cancer. PMID:26722264

  3. Encorafenib (LGX818), a potent BRAF inhibitor, induces senescence accompanied by autophagy in BRAFV600E melanoma cells.

    PubMed

    Li, Zhen; Jiang, Ke; Zhu, Xiaofang; Lin, Guibin; Song, Fei; Zhao, Yongfu; Piao, Yongjun; Liu, Jiwei; Cheng, Wei; Bi, Xiaolin; Gong, Peng; Song, Zhiqi; Meng, Songshu

    2016-01-28

    Encorafenib (LGX818) is a new-generation BRAF inhibitor that is under evaluation in clinical trials. However, the underlying mechanism remains to be elucidated. Here we show that LGX818 potently decreased ERK phosphorylation and inhibited proliferation in BRAFV600E melanoma cell lines. Moreover, LGX818 downregulated CyclinD1 in a glycogen synthase kinase 3β-independent manner and induced cell cycle arrest in the G1 phase, Surprisingly, LGX818 triggered cellular senescence in BRAFV600E melanoma cells, as evidenced by increased β-galactosidase staining, while no appreciable induction of apoptosis was detected, as determined by Annexin V and propidium iodide staining and immunoblot analysis of caspase-3 processing and poly (ADP-ribose) polymerase cleavage. Increased p27KIP1 expression and retinoblastoma protein activation were detected during LGX818-induced senescence. Additionally, inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 1B by AZ191 reversed LGX818-induced CyclinD1 turnover and senescence. Interestingly, autophagy is triggered through inhibition of the mTOR/70S6K pathway during LGX818-induced senescence. Moreover, autophagy inhibition by pharmacological and genetic regulation attenuates LGX818-induced senescence. Notably, combining LGX818 with autophagy modulators has anti-proliferative effect in LGX818-resistant BRAF mutant melanoma cells. Altogether, we uncovered a mechanism by which LGX818 exerts its anti-tumor activity in BRAFV600E melanoma cells. PMID:26586345

  4. Alisertib induces apoptosis and autophagy through targeting the AKT/mTOR/AMPK/p38 pathway in leukemic cells.

    PubMed

    Fu, Yunfeng; Zhang, Yanan; Gao, Meng; Quan, Lingli; Gui, Rong; Liu, Jing

    2016-07-01

    Alisertib, a potent and selective Aurora kinase A inhibitor, has been demonstrated to exert potent anti-cancer effects in pre-clinical and clinical studies. However, mechanisms of action of alisertib, including the molecular pathways involved in alisertib-induced apoptosis and autophagy of leukemic cells, have remained elusive. The aim of the present study was to investigate the effects of alisertib on cell growth, apoptosis and autophagy and to delineate the possible molecular mechanisms in leukemic cells. Acid phosphatase, MTT and Annexin V/propidium iodide staining assays as well as immunostaining for light chain 3B showed that treatment of the REH leukemia cell line with alisertib exerted potent growth inhibitory effects, and induced apoptosis and autophagy in a dose‑dependent manner. Western blot analysis indicated that these effects may be attributed to the suppression of the activity of the Akt/mammalian target of rapamycin/5'-AMP-dependent kinase/p38 mitogen-activated protein kinase signaling pathways in REH cells. The present study confirmed that alisertib may represent a promising autophagy-inducing drug for the treatment of leukemia and shed light on its molecular mechanism of action. PMID:27177156

  5. Raman micro-spectroscopic analysis of cultured HCT116 colon cancer cells in the presence of roscovitine

    NASA Astrophysics Data System (ADS)

    Akyuz, S.; Ozel, A. E.; Balci, K.; Akyuz, T.; Coker, A.; Arisan, E. D.; Palavan-Unsal, N.; Ozalpan, A.

    2011-05-01

    Raman micro-spectroscopic analysis of cultured HCT116 colon cancer cells in the presence of roscovitine, [seliciclib, 2-(1-ethyl-2-hydroxy-ethylamino)-6-benzylamino-9-isopropylpurine], a promising drug candidate in cancer therapy, has been performed for the first time. The aim of this study was to investigate modulations in colon cancer cells induced by roscovitine. Raman spectra of the cultured HCT116 colon cancer cells treated with roscovitine at different concentrations (0, 5, 10, 25 and 50 μM) were recorded in the range 400-1850 cm -1. It was shown that the second derivative profile of the experimental spectrum gives valuable information about the wavenumbers and band widths of the vibrational modes of cell components, and it eliminates the appearance of false peaks arising from incorrect baseline corrections. In samples containing roscovitine, significant spectral changes were observed in the intensities of characteristic protein and DNA bands, which indicate roscovitine-induced apoptosis. Roscovitine-induced apoptosis was also assessed by flow cytometry analysis, and analysis of propidium iodide staining. We observed some modifications in amide I and III bands, which arise from alterations in the secondary structure of cell proteins caused by the presence of roscovitine.

  6. Paroxetine-induced apoptosis in human osteosarcoma cells: Activation of p38 MAP kinase and caspase-3 pathways without involvement of [Ca{sup 2+}]{sub i} elevation

    SciTech Connect

    Chou, C.-T.; He Shiping; Jan, C.-R. . E-mail: crjan@isca.vghks.gov.tw

    2007-02-01

    Selective serotonin reuptake inhibitors (SSRIs), a group of antidepressants, are generally used for treatment of various mood and anxiety disorders. There has been much research showing the anti-tumor and cytotoxic activities of some antidepressants; but the detailed mechanisms were unclear. In cultured human osteosarcoma cells (MG63), paroxetine reduced cell viability in a concentration- and time-dependent manner. Paroxetine caused apoptosis as assessed by propidium iodide-stained cells and increased caspase-3 activation. Although immunoblotting data revealed that paroxetine could activate the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH{sub 2}-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), only SB203580 (a p38 MAPK inhibitor) partially prevented cells from apoptosis. Paroxetine also induced [Ca{sup 2+}]{sub i} increases which involved the mobilization of intracellular Ca{sup 2+} stored in the endoplasmic reticulum and Ca{sup 2+} influx from extracellular medium. However, pretreatment with BAPTA/AM, a Ca{sup 2+} chelator, to prevent paroxetine-induced [Ca{sup 2+}]{sub i} increases did not protect cells from death. The results suggest that in MG63 cells, paroxetine caused Ca{sup 2+}-independent apoptosis via inducing p38 MAPK-associated caspase-3 activation.

  7. Antiproliferative effects of anastrozole on MCF-7 human breast cancer cells in vitro are significantly enhanced by combined treatment with testosterone undecanoate.

    PubMed

    Chen, Rong; Cui, Junwei; Wang, Qinqin; Li, Peng; Liu, Xiaoling; Hu, Hui; Wei, Wei

    2015-07-01

    The present study aimed to assess the effects of aromatase inhibitor anastrozole and testosterone undecanoate, separately and in combination, on proliferation and apoptosis in MCF-7 human breast cancer cells cultured in vitro. The effects of various concentrations of these drugs on the proliferation of MCF-7 cells were evaluated by CCK8 assay, the levels of cell apoptosis were evaluated by flow cytometry with Annexin-V/propidium iodide staining and androgen receptor (AR) protein expression was determined by western blot analysis. The results of the CCK8 assay indicated that greater antiproliferative activity was detected in the MCF-7 cells in the combined treatment groups, compared with those treated with anastrozole or testosterone undecanoate alone. Flow cytometric analysis of apoptosis revealed that treatment with a combination of the two drugs generated a higher percentage of apoptotic cells, particularly when the two drugs were applied for 48 h, compared with single drug treatment. Western blot analysis revealed a significant decrease in AR protein expression in the combined treatment groups compared with MCF7 cells treated with single drugs. The results of the present study provided evidence supporting the potential of a combination of anastrozole and testosterone undecanoate as a novel therapeutic strategy for the treatment of breast cancer. Furthermore, it was demonstrated that the antiproliferative effects of anastrozole were significantly enhanced by combined treatment with testosterone undecanoate via the AR signaling pathway. PMID:25738971

  8. Prion Protein Does Not Confer Resistance to Hippocampus-Derived Zpl Cells against the Toxic Effects of Cu2+, Mn2+, Zn2+ and Co2+ Not Supporting a General Protective Role for PrP in Transition Metal Induced Toxicity

    PubMed Central

    Cingaram, Pradeep Kumar Reddy; Nyeste, Antal; Dondapati, Divya Teja; Fodor, Elfrieda; Welker, Ervin

    2015-01-01

    The interactions of transition metals with the prion protein (PrP) are well-documented and characterized, however, there is no consensus on their role in either the physiology of PrP or PrP-related neurodegenerative disorders. PrP has been reported to protect cells from the toxic stimuli of metals. By employing a cell viability assay, we examined the effects of various concentrations of Cu2+, Zn2+, Mn2+, and Co2+ on Zpl (Prnp-/-) and ZW (Prnp+/+) hippocampus-derived mouse neuronal cells. Prnp-/- Zpl cells were more sensitive to all four metals than PrP-expressing Zw cells. However, when we introduced PrP or only the empty vector into Zpl cells, we could not discern any protective effect associated with the presence of PrP. This observation was further corroborated when assessing the toxic effect of metals by propidium-iodide staining and fluorescence activated cell sorting analysis. Thus, our results on this mouse cell culture model do not seem to support a strong protective role for PrP against transition metal toxicity and also emphasize the necessity of extreme care when comparing cells derived from PrP knock-out and wild type mice. PMID:26426582

  9. Cytotoxic activity of novel palladium-based compounds on leukemia cell lines.

    PubMed

    Antunovic, Maja; Kriznik, Bojana; Ulukaya, Engin; Yilmaz, Veysel T; Mihalic, Katarina C; Madunic, Josip; Marijanovic, Inga

    2015-02-01

    Effective treatment methods for human leukemia are under development, but so far none of them have been found to be completely satisfactory. It was recently reported that palladium complexes have significant anticancer activity as well as lower toxicity compared with some clinically used chemotherapeutics. The anticancer activities of two novel palladium(II) complexes, [Pd(sac)(terpy)](sac)·4H2O and [PdCl(terpy)](sac)·2H2O, were tested against three human leukemia cell lines, Jurkat, MOLT-4, and THP-1, in comparison with cisplatin and adriamycin. The cytotoxic effect of the drugs was determined using the MTT assay. Cell death was assessed using fluorescein isothiocyanate-annexin/propidium iodide staining for flow cytometry. Furthermore, p53 phosphorylation, poly(ADP-ribose) polymerase cleavage, and Bax and Bcl-2 mRNA levels were examined to elucidate the mechanism of cell death induction. Both complexes exhibited a significant dose-dependent antigrowth effect in vitro. The complexes predominately induced apoptosis, but necrosis was also observed. In-vitro results have shown that palladium(II) complexes may be regarded as potential anticancer agents for treating human leukemia. Therefore, further analysis to determine the putative mechanism of action and in-vivo studies on animal models are warranted. PMID:25280061

  10. A novel manganese complex selectively induces malignant glioma cell death by targeting mitochondria

    PubMed Central

    Geng, Ji; Li, Jing; Huang, Tao; Zhao, Kaidi; Chen, Qiuyun; Guo, Wenjie; Gao, Jing

    2016-01-01

    Despite advances in treatment, malignant glioma commonly exhibits recurrence, subsequently leading to a poor prognosis. As manganese (Mn) compounds can be transported by the transferrin-transferrin receptor system, the present study synthesized and examined the potential use of Adpa-Mn as a novel antitumor agent. Adpa-Mn time and dose-dependently inhibited U251 and C6 cell proliferation; however, it had little effect on normal astrocytes. Apoptosis was significantly elevated following treatment with Adpa-Mn, as detected by chromatin condensation, Annexin V/propidium iodide staining, cytochrome c release from mitochondria to the cytoplasm, and the activation of caspases-9, -7 and -3 and poly (ADP-ribose) polymerase. In addition, Adpa-Mn enhanced fluorescence intensity of monodansylcadaverine and elevated the expression levels of the autophagy-related protein microtubule-associated protein 1 light chain 3. Pretreatment with the autophagy inhibitors 3-methyladenine and chloroquine enhanced Adpa-Mn-induced cell inhibition, thus indicating that autophagy has an essential role in this process. Furthermore, evidence of mitochondrial dysfunction was detected in the Adpa-Mn-treated group, including disrupted membrane potential, elevated levels of reactive oxygen species (ROS) and depleted adenosine triphosphate. Conversely, treatment with the mitochondrial permeability transition inhibitor cyclosporin A reversed Adpa-Mn-induced ROS production, mitochondrial damage and cell apoptosis, thus suggesting that Adpa-Mn may target the mitochondria. Taken together, these data suggested that Adpa-Mn may be considered for use as a novel anti-glioma therapeutic option. PMID:27432745

  11. Quinones and halogenated monoterpenes of algal origin show anti-proliferative effects against breast cancer cells in vitro.

    PubMed

    de la Mare, Jo-Anne; Lawson, Jessica C; Chiwakata, Maynard T; Beukes, Denzil R; Edkins, Adrienne L; Blatch, Gregory L

    2012-12-01

    Red and brown algae have been shown to produce a variety of compounds with chemotherapeutic potential. A recent report described the isolation of a range of novel polyhalogenated monoterpene compounds from the red algae Plocamium corallorhiza and Plocamium cornutum collected off the coast of South Africa, together with the previously described tetraprenylquinone, sargaquinoic acid (SQA), from the brown algae Sargassum heterophyllum. In our study, the algal compounds were screened for anti-proliferative activity against metastatic MDA-MB-231 breast cancer cells revealing that a number of compounds displayed anti-cancer activity with IC(50) values in the micromolar range. A subset of the compounds was tested for differential toxicity in the MCF-7/MCF12A system and five of these, including sargaquinoic acid, were found to be at least three times more toxic to the breast cancer than the non-malignant cell line. SQA was further analysed in terms of its mechanism of cytotoxicity in MDA-MB-231 cells. The ability to initiate apoptosis was distinguished from the induction of an inflammatory necrotic response via flow cytometry with propidium iodide and Hoescht staining, confocal microscopy with Annexin V and propidium iodide staining as well as the PARP cleavage assay. We report that SQA induced apoptosis while a polyhalogenated monoterpene RU015 induced necrosis in metastatic breast cancer cells in vitro. Furthermore, we demonstrated that apoptosis induction by SQA occurs via caspase-3, -6, -8, -9 and -13 and was associated with down-regulation of Bcl-2. In addition, cell cycle analyses revealed that the compound causes G(1) arrest in MDA-MB-231 cells. PMID:22249429

  12. Cytotoxicity and morphological effects induced by carvacrol and thymol on the human cell line Caco-2.

    PubMed

    Llana-Ruiz-Cabello, María; Gutiérrez-Praena, Daniel; Pichardo, Silvia; Moreno, F Javier; Bermúdez, José María; Aucejo, Susana; Cameán, Ana María

    2014-02-01

    Essential oils used as additives in the food industry due to its flavour, antimicrobial and antioxidant properties. Therefore, human can be exposed orally to these compounds through the ingestion of foods. In this sense, the present work aims to assess toxicological effects of oregano essential oil on the digestive tract. In concrete, the cytotoxic effects of two components of the oregano essential oils, carvacrol and thymol, and their mixture, on the intestinal cells line Caco-2 after 24 and 48 h of exposure are studied. The basal cytotoxicity endpoints assayed (total protein content, neutral red uptake and the tetrazolium salt reduction) and the annexin/propidium iodide staining indicated that carvacrol and the mixture carvacrol/thymol induced toxic effects. Moreover, a morphological study was performed in order to determine the ultrastructural cellular damages caused by these substances. The main morphological alterations were vacuolated cytoplasm, altered organelles and finally cell death. In addition, although no cytotoxic effects were recorded for thymol at any concentration and time of exposure, ultrastructural changes evidenced cellular damage such as lipid degeneration, mitochondrial damage, nucleolar segregation and apoptosis. PMID:24326232

  13. Cigarette smoke extract induces aberrant cytochrome-c oxidase subunit II methylation and apoptosis in human umbilical vascular endothelial cells.

    PubMed

    Yang, Min; Chen, Ping; Peng, Hong; Zhang, Hongliang; Chen, Yan; Cai, Shan; Lu, Qianjin; Guan, Chaxiang

    2015-03-01

    Cigarette smoke-induced apoptosis of vascular endothelial cells contributes to the pathogenesis of chronic obstructive pulmonary disease. However, the mechanisms responsible for endothelial apoptosis remain poorly understood. We conducted an in vitro study to investigate whether DNA methylation is involved in smoking-induced endothelial apoptosis. Human umbilical vascular endothelial cells (HUVECs) were exposed to cigarette smoke extract (CSE) at a range of concentrations (0-10%). HUVECs were also incubated with a demethylating reagent, 5-aza-2'-deoxycytidinem (AZA), with and without CSE. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and flow cytometry using annexin V-FITC/propidium iodide staining. We found that CSE treatment significantly increased HUVEC apoptosis in a dose- and time-dependent manner. Quantitative real-time RT-PCR and immunoblot revealed that CSE treatment decreased cytochrome-c oxidase subunit II (COX II) mRNA and protein levels and decreased COX activity. Methylation-specific PCR and direct bisulfite sequencing revealed positive COX II gene methylation. AZA administration partly increased mRNA and protein expressions of COX II, and COX activity decreased by CSE and attenuated the toxic effects of CSE. Our results showed that CSE induced aberrant COX II methylation and apoptosis in HUVECs. PMID:25500741

  14. Novel Photosensitizers Trigger Rapid Death of Malignant Human Cells and Rodent Tumor Transplants via Lipid Photodamage and Membrane Permeabilization

    PubMed Central

    Moisenovich, Mikhail M.; Ol'shevskaya, Valentina A.; Rokitskaya, Tatyana I.; Ramonova, Alla A.; Nikitina, Roza G.; Tatarskiy, Victor V.; Kaplan, Mikhail A.; Kalinin, Valery N.; Kotova, Elena A.; Uvarov, Oleg V.; Agapov, Igor I.; Antonenko, Yuri N.; Shtil, Alexander A.

    2010-01-01

    Background Apoptotic cascades may frequently be impaired in tumor cells; therefore, the approaches to circumvent these obstacles emerge as important therapeutic modalities. Methodology/Principal Findings Our novel derivatives of chlorin e6, that is, its amide (compound 2) and boronated amide (compound 5) evoked no dark toxicity and demonstrated a significantly higher photosensitizing efficacy than chlorin e6 against transplanted aggressive tumors such as B16 melanoma and M-1 sarcoma. Compound 5 showed superior therapeutic potency. Illumination with red light of mammalian tumor cells loaded with 0.1 µM of 5 caused rapid (within the initial minutes) necrosis as determined by propidium iodide staining. The laser confocal microscopy-assisted analysis of cell death revealed the following order of events: prior to illumination, 5 accumulated in Golgi cysternae, endoplasmic reticulum and in some (but not all) lysosomes. In response to light, the reactive oxygen species burst was concomitant with the drop of mitochondrial transmembrane electric potential, the dramatic changes of mitochondrial shape and the loss of integrity of mitochondria and lysosomes. Within 3–4 min post illumination, the plasma membrane became permeable for propidium iodide. Compounds 2 and 5 were one order of magnitude more potent than chlorin e6 in photodamage of artificial liposomes monitored in a dye release assay. The latter effect depended on the content of non-saturated lipids; in liposomes consisting of saturated lipids no photodamage was detectable. The increased therapeutic efficacy of 5 compared with 2 was attributed to a striking difference in the ability of these photosensitizers to permeate through hydrophobic membrane interior as evidenced by measurements of voltage jump-induced relaxation of transmembrane current on planar lipid bilayers. Conclusions/Significance The multimembrane photodestruction and cell necrosis induced by photoactivation of 2 and 5 are directly associated with

  15. Solamargine triggers hepatoma cell death through apoptosis

    PubMed Central

    XIE, XIAODONG; ZHU, HAITAO; YANG, HUIJIAN; HUANG, WENSI; WU, YINGYING; WANG, YING; LUO, YANLING; WANG, DONGQING; SHAO, GENBAO

    2015-01-01

    Solamargine (SM), a steroidal alkaloid glycoside extracted from the traditional Chinese herb Solanum incanum, has been evidenced to inhibit the growth and induce apoptosis in a number of human cancer cell lines. In the present study, the anticancer effect of SM and underlying molecular mechanism of SM-induced apoptosis were investigated on the human hepatocellular carcinoma cells, SMMC7721 and HepG2. The proliferation effects of SM on the SMMC7721 and HepG2 cell lines were evaluated using MTT and colony formation assays. In addition, the percentage of apoptosis was measured using an Annexin V/propidium iodide staining method and the cell cycle distribution mediated by SM was analyzed using flow cytometry. The expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), caspase-3, caspase-9, proliferating cell nuclear antigen (pcna) and Ki67 proteins were examined to further demonstrate the proliferate and apoptosis effects of SM on the hepatoma cells. The results indicated that SM effectively inhibited hepatoma cell proliferation and promoted apoptosis. SM resulted in cell cycle arrest at the G2/M phase in the two cell lines. In addition, SM downregulated the levels of proliferation-associated (Ki67 and pcna) and anti-apoptotic (Bcl-2) proteins, and promoted the activity of apoptosis-associated proteins (Bax, caspase-3 and caspase-9). Therefore, the activation of the Bcl-2/Bax and caspase signaling pathways may be involved in the SM-induced apoptosis of hepatoma cells. PMID:26170994

  16. Anticancer effects of crocetin in human esophageal squamous cell carcinoma KYSE-150 cells

    PubMed Central

    LI, SHENG; JIANG, SHENG; JIANG, WEI; ZHOU, YUE; SHEN, XIU-YIN; LUO, TAO; KONG, LING-PING; WANG, HUA-QIAO

    2015-01-01

    Crocetin is the main pharmacologically-active component of saffron and has been considered as a promising candidate for cancer chemoprevention. The purpose of the present study was to investigate the anticancer effects of crocetin and the possible mechanisms of these properties in the esophageal squamous cell carcinoma cell line KYSE-150. The KYSE-150 cells were cultured in Dulbecco’s modified Eagle’s medium and incubated with 0, 12.5, 25, 50, 100 or 200 μmol/l crocetin for 48 h. Cell proliferation was measured using an MTT assay. Hoechst 33258 staining and observation under fluorescent microscopy were used to analyze the proapoptotic effects of crocetin. The migration rate was assessed by a wound-healing assay. The cell cycle distribution was analyzed using flow cytometry analysis subsequent to propidium iodide staining. The expression of B-cell lymphoma-2-associated X protein (Bax) and cleaved caspase 3 was determined by western blot analysis. It was found that treatment of KYSE-150 cells with crocetin for 48 h significantly inhibited the proliferation of the cells in a concentration-dependent manner, and the inhibition of proliferation was associated with S phase arrest. Crocetin was also found to induce morphological changes and cell apoptosis in a dose-dependent manner through increased expression of proapoptotic Bax and activated caspase 3. In addition, crocetin suppressed the migration of KYSE-150 cells. The present study provides evidence that crocetin exerts a prominent chemopreventive effect against esophageal cancer through the inhibition of cell proliferation, migration and induction of apoptosis. These findings reveal that crocetin may be considered to be a promising future chemotherapeutic agent for esophageal cancer therapy. PMID:25663893

  17. Arecoline induced cell cycle arrest, apoptosis, and cytotoxicity to human endothelial cells.

    PubMed

    Tseng, Shuei-Kuen; Chang, Mei-Chi; Su, Cheng-Yao; Chi, Lin-Yang; Chang, Jenny Zwei-Ching; Tseng, Wan-Yu; Yeung, Sin-Yuet; Hsu, Ming-Lun; Jeng, Jiiang-Huei

    2012-08-01

    Betel quid (BQ) chewing is a common oral habit in South Asia and Taiwan. BQ consumption may increase the risk of oral squamous cell carcinoma (OSCC), oral submucous fibrosis (OSF), and periodontitis as well as systemic diseases (atherosclerosis, hypertension, etc.). However, little is known about the toxic effect of BQ components on endothelial cells that play important roles for angiogenesis, carcinogenesis, tissue fibrosis, and cardiovascular diseases. EAhy 926 (EAHY) endothelial cells were exposed to arecoline, a major BQ alkaloid, for various time periods. Cytotoxicity was estimated by 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay. The cell cycle distribution of EAHY cells residing in sub-G0/G1, G0/G1, S-, and G2/M phases was analyzed by propidium iodide staining of cellular DNA content and flow cytometry. Some EAHY cells retracted, became round-shaped in appearance, and even detached from the culture plate after exposure to higher concentrations of arecoline (> 0.4 mM). At concentrations of 0.4 and 0.8 mM, arecoline induced significant cytotoxicity to EAHY cells. At similar concentrations, arecoline induced G2/M cell cycle arrest and increased sub-G0/G1 population, a hallmark of apoptosis. Interestingly, prolonged exposure to arecoline (0.1 mM) for 12 and 21 days significantly suppressed the proliferation of EAHY cells, whereas EAHY cells showed adaptation and survived when exposed to 0.05 mM arecoline. These results suggest that BQ components may contribute to the pathogenesis of OSF and BQ chewing-related cardiovascular diseases via toxicity to oral or systemic endothelial cells, leading to impairment of vascular function. During BQ chewing, endothelial damage may be induced by areca nut components and associate with the pathogenesis of OSF, periodontitis, and cardiovascular diseases. PMID:21847594

  18. Human umbilical cord blood mononuclear cells activate the survival protein Akt in cardiac myocytes and endothelial cells that limits apoptosis and necrosis during hypoxia.

    PubMed

    Henning, Robert J; Dennis, Steve; Sawmiller, Darrell; Hunter, Lorynn; Sanberg, Paul; Miller, Leslie

    2012-06-01

    We have previously reported that human umbilical cord blood mononuclear cells (HUCBC), which contain hematopoietic, mesenchymal, and endothelial stem cells, can significantly reduce acute myocardial infarction size. To determine the mechanism whereby HUCBC increase myocyte and vascular endothelial cell survival, we treated cardiac myocytes and coronary artery endothelial cells in separate experiments with HUCBC plus culture media or culture media alone and subjected the cells to 24 h of hypoxia or normoxia. We then determined in myocytes and endothelial cells activation of the cell survival protein Akt by Western blots. We also determined in these cells apoptosis by annexin V staining and necrosis by propidium iodide staining. Thereafter, we inhibited with API, a specific and sensitive Akt inhibitor, Akt activation in myocytes and endothelial cells cultured with HUCBC during hypoxia and determined cell apoptosis and necrosis. In cells cultured without HUCBC, hypoxia only slightly activated Akt. Moreover, hypoxia increased myocyte apoptosis by ≥ 226% and necrosis by 58% in comparison with myocytes in normoxia. Hypoxic treatment of endothelial cells without HUCBC increased apoptosis by 94% and necrosis by 59%. In contrast, hypoxia did not significantly affect HUCBC. Moreover, in myocyte + HUCBC cultures in hypoxia, HUCBC induced a ≥ 135% increase in myocyte phospho-Akt. Akt activation decreased myocyte apoptosis by 76% and necrosis by 35%. In endothelial cells, HUCBC increased phospho-Akt by 116%. HUCBC also decreased endothelial cell apoptosis by 58% and necrosis by 42%. Inhibition of Akt with API in myocytes and endothelial cells cultured with HUCBC during hypoxia nearly totally prevented the HUCBC-induced decrease in apoptosis and necrosis. We conclude that HUCBC can significantly decrease hypoxia-induced myocyte and endothelial cell apoptosis and necrosis by activating Akt in these cells and in this manner HUCBC can limit myocardial ischemia and injury. PMID

  19. Ultraviolet light-emitting diode irradiation-induced cell death in HL-60 human leukemia cells in vitro

    PubMed Central

    XIE, DONG; SUN, YAN; WANG, LINGZHEN; LI, XIAOLING; ZANG, CHUANNONG; ZHI, YUNLAI; SUN, LIRONG

    2016-01-01

    Ultraviolet (UV) radiation is considered to be a potent cell-damaging agent in various cell lineages; however, the effect of UV light-emitting diode (LED) irradiation on human cells remains unclear. The aim of the present study was to examine the effect of UV LED irradiation emitting at 280 nm on cultured HL-60 human leukemia cells, and to explore the underlying mechanisms. HL-60 cells were irradiated with UV LED (8, 15, 30 and 60 J/m2) and incubated for 2 h after irradiation. The rates of cell proliferation and apoptosis, the cell cycle profiles and the mRNA expression of B-cell lymphoma 2 (Bcl-2) were detected using cell counting kit-8, multicaspase assays, propidium iodide staining and reverse transcription-quantitative polymerase chain reaction, respectively. The results showed that UV LED irradiation (8–60 J/m2) inhibited the proliferation of HL-60 cells in a dose-dependent manner. UV LED at 8–30 J/m2 induced dose-dependent apoptosis and G0/G1 cell cycle arrest, and inhibited the expression of Bcl-2 mRNA, while UV LED at 60 J/m2 induced necrosis. In conclusion, 280 nm UV LED irradiation inhibits proliferation and induces apoptosis and necrosis in cultured HL-60 cells. In addition, the cell cycle arrest at the G0/G1 phase and the downregulation of Bcl-2 mRNA expression were shown to be involved in UV LED-induced apoptosis. PMID:26820261

  20. Does MW Radiation Affect Gene Expression, Apoptotic Level, and Cell Cycle Progression of Human SH-SY5Y Neuroblastoma Cells?

    PubMed

    Kayhan, Handan; Esmekaya, Meric Arda; Saglam, Atiye Seda Yar; Tuysuz, Mehmed Zahid; Canseven, Ayşe Gulnihal; Yagci, Abdullah Munci; Seyhan, Nesrin

    2016-06-01

    Neuroblastoma (NB) is a cancer that occurs in sympathetic nervous system arising from neuroblasts and nerve tissue of the adrenal gland, neck, chest, or spinal cord. It is an embryonal malignancy and affects infants and children. In this study, we investigated the effects of microwave (MW) radiation on apoptotic activity, cell viability, and cell cycle progression in human SH-SY5Y NB cells which can give information about MW radiation effects on neural cells covering the period from the embryonic stages to infants. SH-SY5Y NB cells were exposed to 2.1 GHz W-CDMA modulated MW radiation for 24 h at a specific absorption rate of 0.491 W/kg. Control samples were in the same conditions with MW-exposed samples but they were not exposed to MW radiation. The apoptotic activity of cells was measured by Annexin-V-FITC and propidium iodide staining. Moreover, mRNA levels of proliferative and cell cycle proteins were determined by real-time RT-PCR. The change in cell cycle progression was observed by using CycleTest-Plus DNA reagent. No significant change was observed in apoptotic activity of MW-exposed cells compared to control cells. The mRNA levels of c-myc and cyclin D1 were significantly reduced in MW group (p < 0.05). The percentage of MW-exposed cells in G1 phase was significantly higher than the percentage of control cells in G1 phase. MW radiation caused cell cycle arrest in G1 phase. These results showed that 2.1 GHz W-CDMA modulated MW radiation did not cause apoptotic cell death but changed cell cycle progression. PMID:27260669

  1. Synergy between von Hippel-Lindau and P53 contributes to chemosensitivity of clear cell renal cell carcinoma.

    PubMed

    Zhao, Ziyi; Chen, Changjin; Lin, Junzhi; Zeng, Wentong; Zhao, Juan; Liang, Yindan; Tan, Qinrui; Yang, Chao; Li, Hui

    2016-09-01

    The von Hippel-Lindau tumor suppressor (VHL; E3 ubiquitin ligase gene) is frequently mutated or undetectable in clear cell renal cell carcinoma (CCRCC), and therefore these tumors are highly resistant to chemotherapeutic agents, including adriamycin (ADM) and sunitinib. A mutation in the tumor protein p53 (TP53) also leads to chemoresistance in tumors; however, in CCRCC, TP53 is frequently functional, yet the tumors remain highly insensitive to chemotherapy. This indicates the possibility of a synergistic effect of VHL and P53 in CCRCC. The present study aimed to detect the chemosensitivity of CCRCC. The expression of VHL in the MZ1257 cell line sensitized these cells to ADM and sunitinib, and a knockdown of VHL in the ACHN cells increased their chemoresistance. To confirm that VHL and P53 are both required for chemosensitivity, VHL and P53 were co‑expressed in 786‑O cells. The results of the functional antagonist assay (which assessed the IC50 values, i.e. the half maximal inhibitory concentration) confirmed that VHL and P53 act in synergy to promote chemosensitivity. Cell cycle arrest was measured by propidium iodide staining following treatment with ADM or sunitinib. Further analysis indicated that co‑expression of VHL and P53 inhibited cell proliferation by completely inhibiting the cell cycle at the G0/G1 phase, and promoted apoptosis following treatment with ADM or sunitinib. These findings demonstrated that VHL and P53 act synergistically in the regulation of cell proliferation and apoptosis in CCRCC. Overall, VHL and P53 have important roles in the regulation of cell proliferation and apoptosis in CCRCC. Furthermore, the regulatory role of VHL is dependant on the activation P53. PMID:27485825

  2. Reinjury risk of nano-calcium oxalate monohydrate and calcium oxalate dihydrate crystals on injured renal epithelial cells: aggravation of crystal adhesion and aggregation

    PubMed Central

    Gan, Qiong-Zhi; Sun, Xin-Yuan; Bhadja, Poonam; Yao, Xiu-Qiong; Ouyang, Jian-Ming

    2016-01-01

    Background Renal epithelial cell injury facilitates crystal adhesion to cell surface and serves as a key step in renal stone formation. However, the effects of cell injury on the adhesion of nano-calcium oxalate crystals and the nano-crystal-induced reinjury risk of injured cells remain unclear. Methods African green monkey renal epithelial (Vero) cells were injured with H2O2 to establish a cell injury model. Cell viability, superoxide dismutase (SOD) activity, malonaldehyde (MDA) content, propidium iodide staining, hematoxylin–eosin staining, reactive oxygen species production, and mitochondrial membrane potential (Δψm) were determined to examine cell injury during adhesion. Changes in the surface structure of H2O2-injured cells were assessed through atomic force microscopy. The altered expression of hyaluronan during adhesion was examined through laser scanning confocal microscopy. The adhesion of nano-calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) crystals to Vero cells was observed through scanning electron microscopy. Nano-COM and COD binding was quantitatively determined through inductively coupled plasma emission spectrometry. Results The expression of hyaluronan on the cell surface was increased during wound healing because of Vero cell injury. The structure and function of the cell membrane were also altered by cell injury; thus, nano-crystal adhesion occurred. The ability of nano-COM to adhere to the injured Vero cells was higher than that of nano-COD crystals. The cell viability, SOD activity, and Δψm decreased when nano-crystals attached to the cell surface. By contrast, the MDA content, reactive oxygen species production, and cell death rate increased. Conclusion Cell injury contributes to crystal adhesion to Vero cell surface. The attached nano-COM and COD crystals can aggravate Vero cell injury. As a consequence, crystal adhesion and aggregation are enhanced. These findings provide further insights into kidney stone

  3. Trichostatin A potentiates genistein-induced apoptosis and reverses EMT in HEp2 cells

    PubMed Central

    DU, RUIXIA; LIU, ZHE; HOU, XUEDONG; FU, GONGBI; AN, NING; WANG, LIPING

    2016-01-01

    Genistein and trichostatin A (TSA) are two chemotherapeutic compounds with antitumor effects in different types of cancer cell. However, the effects of genistein and TSA on the HEp-2 laryngeal cancer cell line remain to be fully elucidated. In the present study, it was found that genistein and TSA inhibited cell growth and cell migration, and promoted apoptosis in the HEp-2 laryngeal cancer cell line. The HEp-2 cells were treated with genistein, TSA or the two compounds in combination. Cell proliferation and apoptosis were measured using an MTT assay, Annexin V/propidium iodide staining and a TUNEL assay. Cell invasion was determined using a Matrigel-based Transwell assay. Western blotting was used to examine the activation of the Akt pathway and the expression levels of pro-or anti-apoptotic proteins. Treatment with either genistein or TSA alone mildly inhibited cell viability, growth and invasion, and induced the apoptosis of the laryngeal cancer cells, whereas more marked effects were observed in the cells treated with the combination of the two compounds. In addition, genistein reversed endothelial growth factor-induced epithelial-mesenchymal transition (EMT) in the HEp-2 cells, the effect of which were was further increased by joint application with TSA. Treatment of the HEp-2 cells with genistein and TSA led to a significant reduction in the phosphorylation of Akt and activation of its downstream target, and resulted in peroxisome proliferator-activated receptor-γ cleavage, increased expression of B cell lymphoma-2 (Bcl-2)-associated X protein and reduced the expression of Bcl-2. In conclusion, the present study demonstrated that, with the involvement of TSA, genistein exhibited substantial advantages in inhibiting laryngeal carcinoma cell growth, invasion and EMT, and induced apoptosis, compared with genistein treatment alone, which occurred through the regulation of Akt activation and the apoptotic pathway. PMID:27121018

  4. Gallic acid induced apoptotic events in HCT-15 colon cancer cells

    PubMed Central

    Subramanian, Aruna Priyadharshni; Jaganathan, Saravana Kumar; Mandal, Mahitosh; Supriyanto, Eko; Muhamad, Ida Idayu

    2016-01-01

    AIM: To investigate the inhibitory action of diet-derived phenolic compound gallic acid (GA) against HCT-15 colon cancer cells. METHODS: The antiproliferative effect of GA against colon cancer cells was determined by performing thiazolyl blue tetrazolium bromide (MTT) assay. The colony forming ability of GA treated colon cancer cells was evaluated using the colony forming assay. The cell cycle changes induced by GA in HCT-15 cells were analyzed by propidium iodide staining. Levels of reactive oxygen species (ROS) and mitochondrial membrane potential of HCT-15 exposed to GA was assessed using 2’,7’-dichlorfluorescein-diacetate and rhodamine-123 respectively, with the help of flow cytometry. Morphological changes caused by GA treatment in the colon cancer cells were identified by scanning electron microscope and photomicrograph examination. Apoptosis was confirmed using flow cytometric analysis of GA treated HCT-15 cells after staining with Yo-Pro-1. RESULTS: MTT assay results illustrated that GA has an inhibitory effect on HCT-15 cells with IC50 value of 740 μmol/L. A time-dependent inhibition of colony formation was evident with GA treatment. Cell cycle arrest was evident from the accumulation of GA treated HCT-15 cells at sub-G1 phase (0.98 ± 1.03 vs 58.01 ± 2.05) with increasing exposure time. Flow cytometric analysis of GA treated HCT-15 cells depicted early events associated with apoptosis like lipid layer breakage and fall in mitochondrial membrane potential apart from an increase in the generation of ROS which were in a time dependent manner. SEM and photomicrograph images of the GA-treated cells displayed membrane blebbing and cell shrinking characteristics of apoptosis. Further apoptosis confirmation by Yo-Pro-1 staining also showed the time-dependent increase of apoptotic cells after treatment. CONCLUSION: These results show that GA induced ROS dependent apoptosis and inhibited the growth of colon cancer cells. PMID:27099438

  5. Buformin exhibits anti-proliferative and anti-invasive effects in endometrial cancer cells

    PubMed Central

    Kilgore, Joshua; Jackson, Amanda L; Clark, Leslie H; Guo, Hui; Zhang, Lu; Jones, Hannah M; Gilliam, Timothy P; Gehrig, Paola A; Zhou, Chunxiao; Bae-Jump, Victoria L

    2016-01-01

    Objective: Biguanides are anti-diabetic drugs that are thought to have anti-tumorigenic effects. Most pre-clinical studies have focused on metformin for cancer treatment and prevention; however, buformin may be potentially more potent than metformin. Given this, our goal was to evaluate the effects of buformin on cell growth, adhesion and invasion in endometrial cancer cell lines. Methods: The ECC-1 and Ishikawa endometrial cancer cell lines were used. Cell proliferation was assessed by MTT assay. Apoptosis and cell cycle analysis was performed by FITC Annexin V assay and propidium iodide staining, respectively. Adhesion was analyzed using the laminin adhesion assay. Invasion was assessed using the transwell invasion assay. The effects of buformin on the AMPK/mTOR pathway were determined by Western immunoblotting. Results: Buformin and metformin inhibited cell proliferation in a dose-dependent manner in both endometrial cancer cell lines. IC50s were 1.4-1.6 mM for metformin and 8-150 μM for buformin. Buformin induced cell cycle G1 phase arrest in the ECC-1 cells and G2 phase arrest in the Ishikawa cells. For both ECC-1 and Ishikawa cells, treatment with buformin resulted in induction of apoptosis, reduction in adhesion and invasion, activation of AMPK and inhibition of phosphorylated-S6. Buformin potentiated the anti-proliferative effects of paclitaxel in both cell lines. Conclusion: Buformin has significant anti-proliferative and anti-metastatic effects in endometrial cancer cells through modulation of the AMPK/mTOR pathway. IC50 values were lower for buformin than metformin, suggesting that buformin may be more potent for endometrial cancer treatment and worthy of further investigation. PMID:27398153

  6. Effects of salinomycin and CGP37157 on head and neck squamous cell carcinoma cell lines in vitro.

    PubMed

    Scherzed, Agmal; Hackenberg, Stephan; Froelich, Katrin; Rak, Kristen; Ginzkey, Christian; Hagen, Rudolf; Schendzielorz, Philipp; Kleinsasser, Norbert

    2015-09-01

    Surgery, radiation, chemotherapy or a combinations of these are all accepted modalities for the treatment of head and neck squamous cell carcinoma (HNSCC). Despite this, 40‑60% of patients suffering from HNSCC develop loco‑regional failure and/or distant metastases. Salinomycin has been demonstrated to be >100‑fold more effective than paclitaxel at causing cancer stem cell death, therefore, it may offer an important improvement in cancer therapy. However, the toxicity of salinomycin is of concern. A possible solution may be the administration of additive drugs, which reduce the toxicity. By inhibiting the mitochondrial Na+/Ca2+ exchanger using the benzodiazepine derivate, CGP37157 (CGP), a significant reduction in salinomycin neuronal toxicity has been observed. This raises the question of whether CGP also inhibits the tumor toxicity of salinomycin. In the present study, the FaDu and HLaC79 C1 HNSCC cell lines were treated with salinomycin with or without CGP. Comparative viability assessments were performed using microscopy, a fluorescein diacetate assay, an MTT assay, a clonogenic assay and annexin V‑propidium iodide staining. The expression levels of MDR‑1 were monitored using reverse transcription‑quantitative polymerase chain reaction. Salinomycin alone, and in combination with CGP, achieved a significant attenuation of cell viability and increased apoptosis in a dose‑dependent manner. However, the tumor toxicity of salinomycin was not inhibited by CGP. The HLaC79 C1 cells were more sensitive to salinomycin, compared with the FaDu cells, with this sensitivity being due to high expression levels of MDR‑1 by the HLaC79 C1 cells. In conclusion, CGP did not counteract the tumor toxicity of salinomycin in vitro and may be a promising drug in future anticancer therapy. The results of the present study encourages further investigation of the toxicological aspects of salinomycin, particularly in human cells and animal models. PMID:26099997

  7. H9 induces apoptosis via the intrinsic pathway in non-small-cell lung cancer A549 cells.

    PubMed

    Kwon, Sae-Bom; Kim, Min-Je; Ham, Sun Young; Park, Ga Wan; Choi, Kang-Duk; Jung, Seung Hyun; Yoon, Do-Young

    2015-03-01

    H9 is an ethanol extract prepared from nine traditional/medicinal herbs. This study was focused on the anticancer effect of H9 in non-small-cell lung cancer cells. The effects of H9 on cell viability, apoptosis, mitochondrial membrane potential (MMP; Δφm), and apoptosis-related protein expression were investigated in A549 human lung cancer cells. In this study, H9-induced apoptosis was confirmed by propidium iodide staining, expression levels of mRNA were determined by reverse transcriptase polymerase chain reaction, protein expression levels were checked by western blot analysis, and MMP (Δφm) was measured by JC- 1 staining. Our results indicated that H9 decreased the viability of A549 cells and induced cell morphological changes in a dose-dependent manner. H9 also altered expression levels of molecules involved in the intrinsic signaling pathway. H9 inhibited Bcl-xL expression, whereas Bax expression was enhanced and cytochrome C was released. Furthermore, H9 treatment led to the activation of caspase-3/caspase-9 and proteolytic cleavage of poly(ADPribose) polymerase; the MMP was collapsed by H9. However, the expression levels of extrinsic pathway molecules such as Fas/FasL, TRAIL/TRAIL-R, DR5, and Fas-associated death receptor were downregulated by H9. These results indicated that H9 inhibited proliferation and induced apoptosis by activating intrinsic pathways but not extrinsic pathways in human lung cancer cells. Our results suggest that H9 can be used as an alternative remedy for human non-small-cell lung cancer. PMID:25563417

  8. Murine acute leukemia cell line with megakaryocytic differentiation (MK-8057) induced by whole-body irradiation in C sup 3 H/He mice: Cytological properties and kinetics of its leukemic stem cells

    SciTech Connect

    Hirabayashi, Y.; Inoue, T.; Yoshida, K.; Sasaki, H.; Kubo, S.; Kanisawa, M.; Seki, M. )

    1991-01-01

    Five cases of murine leukemia with megakaryocytic differentiation were observed among the 417 cases of radiation-induced leukemias which developed in 30% of C{sup 3}H/HeMs mice exposed at 8 to 10 weeks to 0.5 to 5 gy total body irradiation. Cells from individual leukemic colonies in the spleen of the irradiated mice, and cells from colonies in methylcellulose (MC) culture in vitro, derived from one of these leukemias, MK-8057, were injected into mice; both types of cells caused the deaths of the recipient mice by inducing the same type of leukemia. MK-8057 can be maintained in Dexter-type liquid culture with a feeder layer of irradiated bone marrow cells. There was a linear reciprocal relationship between the increasing number of MK-8057 cells injected versus the survival of the recipient mice. A reciprocal relationship also was seen between an increasing number of leukemic stem cells, corresponding to the number of MK-8057 cells, and the survival of mice injected with MK-8057. Giant nuclear megakaryocytes developed during the course of colony growth in the spleen as they did in the MC culture. Such megakaryocytes were acetylcholinesterase positive, whereas leukemic cells in the peripheral blood showed no sign of platelet production nor of a positive reaction to acetylcholinesterase. Cells maintained in culture were entirely positive in platelet glycoprotein IIb/IIIa when anti-human antibody was used. The larger cells in a splenic cell suspension derived from a moribund mouse were separated and enriched by velocity sedimentation using centrifugal elutriation (CE), and then subjected to flow cytometry using propidium iodide staining. Cells with up to 32N-DNA content were detected. After separating MK-8057 by counter-flow CE, the larger cell fraction produced more leukemic colonies when injected into irradiated mice than did the small cell fraction.

  9. Real-time direct cell concentration and viability determination using a fully automated microfluidic platform for standalone process monitoring.

    PubMed

    Nunes, P S; Kjaerulff, S; Dufva, M; Mogensen, K B

    2015-06-21

    The industrial production of cells has a large unmet need for greater process monitoring, in addition to the standard temperature, pH and oxygen concentration determination. Monitoring the cell health by a vast range of fluorescence cell-based assays can greatly improve the feedback control and thereby ensure optimal cell production, by prolonging the fermentation cycle and increasing the bioreactor output. In this work, we report on the development of a fully automated microfluidic system capable of extracting samples directly from a bioreactor, diluting the sample, staining the cells, and determining the total cell and dead cells concentrations, within a time frame of 10.3 min. The platform consists of custom made stepper motor actuated peristaltic pumps and valves, fluidic interconnections, sample to waste liquid management and image cytometry-based detection. The total concentration of cells is determined by brightfield microscopy, while fluorescence detection is used to detect propidium iodide stained non-viable cells. This method can be incorporated into facilities with bioreactors to monitor the cell concentration and viability during the cultivation process. Here, we demonstrate the microfluidic system performance by monitoring in real time the cell concentration and viability of yeast extracted directly from an in-house made bioreactor. This is the first demonstration of using the Dean drag force, generated due to the implementation of a curved microchannel geometry in conjunction with high flow rates, to promote passive mixing of cell samples and thus homogenization of the diluted cell plug. The autonomous operation of the fluidics furthermore allows implementation of intelligent protocols for administering air bubbles from the bioreactor in the microfluidic system, so that these will be guided away from the imaging region, thereby significantly improving both the robustness of the system and the quality of the data. PMID:25923294

  10. Analyzing Cytotoxic and Apoptogenic Properties of Scutellaria litwinowii Root Extract on Cancer Cell Lines.

    PubMed

    Tayarani-Najaran, Zahra; Emami, Seyed Ahmad; Asili, Javad; Mirzaei, Alireza; Mousavi, Seyed Hadi

    2011-01-01

    The Scutellaria species (Lamiaceae) is used as a source of flavonoids to treat a variety of diseases in traditional medicine. In spite of many reports about the cytotoxic and antitumor effects of some species of this genus, anticancer researches on one of the Iranian species S. litwinowii have not yet been conducted. The cytotoxic properties of total methanol extract of S. litwinowii and its fractions were investigated on different cancer cell lines including AGS, HeLa, MCF-7, PC12 and NIH 3T3. Meanwhile, the role of apoptosis in this toxicity was explored. The cells were cultured in DMEM medium and incubated with different concentrations of herb plant extracts. Cell viability was quantitated by MTT assay. Apoptotic cells were determined using propidium iodide staining of DNA fragmentation by flow cytometry (sub-G1 peak). Scutellaria litwinowii inhibited the growth of malignant cells in a dose-dependent manner. Among solvent fractions of S. litwinowii, the methylene chloride fraction was found to be more toxic compared to other fractions. The IC(50) values of this fraction against AGS, HeLa, MCF-7 and PC12 cell lines after 24 h were determined, 121.2 ± 3.1, 40.9 ± 2.5, 115.9 ± 3.5 and 64.5 ± 3.4 μg/ml, respectively. Scutellaria litwinowii induced a sub-G1 peak in the flow cytometry histogram of treated cells compared to control cells indicating that apoptotic cell death is involved in S. litwinowii toxicity. Scutellaria litwinowii exerts cytotoxic and proapototic effects in a variety of malignant cell lines and could be considered as a potential chemotherapeutic agent in cancer treatment. PMID:20028719

  11. Differential influence of tacrolimus and sirolimus on mitochondrial-dependent signaling for apoptosis in pancreatic cells.

    PubMed

    Constantinescu, Andrei Alexandru; Abbas, Malak; Kassem, Mohamad; Gleizes, Céline; Kreutter, Guillaume; Schini-Kerth, Valerie; Mitrea, Ioan Liviu; Toti, Florence; Kessler, Laurence

    2016-07-01

    To examine and compare the mitochondria-related cellular mechanisms by which tacrolimus (TAC) or sirolimus (SIR) immunosuppressive drugs alter the pancreatic exocrine and endocrine β-cell fate. Human exocrine PANC-1 and rat endocrine insulin-secreting RIN-m5F cells and isolated rat islets were submitted to 1-100 nM TAC or SIR. In cultures, insulin secretion was measured as endocrine cell function marker. Apoptosis was quantified by annexin 5 and propidium iodide staining. Cleaved caspase-3, Bax apoptosis indicators, and p53, p21 cell cycle regulators were detected by Western blot. Cell cycle and mitochondrial membrane potential (ΔΨm) were analyzed by flow cytometry and SA-beta-galactosidase (SA-β-gal) activity by fluorescence microscopy. Only TAC reduced insulin secretion by RIN-m5F after 24 h. TAC and SIR promoted moderate apoptosis in both PANC-1 and RIN-m5F after 24 h. Apoptosis was associated with up-regulated Bax (threefold) and cleaved caspase-3 (fivefold) but only in PANC-1, while p53 and p21 were up-regulated (twofold) in both cell lines. ΔΨm was impaired only in PANC-1 by TAC and SIR. Only SIR prompted cell cycle arrest in both cell lines. The induction of a premature senescence-like phenotype was confirmed in isolated islets by SA-β-gal activity. TAC and SIR are early inducers of pancreatic cell dysfunction and apoptosis but differentially alter endocrine and exocrine cells via mitochondrial-driven pathways. In rat islets, TAC and SIR prompt a senescence-like phenotype. PMID:27344165

  12. Licochalcone A induces T24 bladder cancer cell apoptosis by increasing intracellular calcium levels.

    PubMed

    Yang, Xinhui; Jiang, Jiangtao; Yang, Xinyan; Han, Jichun; Zheng, Qiusheng

    2016-07-01

    Licochalcone A (LCA) has been reported to significantly inhibit cell proliferation, increase reactive oxygen species (ROS) levels, and induce apoptosis of T24 human bladder cancer cells via mitochondria and endoplasmic reticulum (ER) stress-triggered signaling pathways. Based on these findings, the present study aimed to investigate the mechanisms by which LCA induces apoptosis of T24 cells. Cultured T24 cells were treated with LCA, and cell viability was measured using the sulforhodamine B assay. Apoptosis was detected by flow cytometry with Annexin V/propidium iodide staining, and by fluorescent microscopy with Hoechst 33258 staining. The levels of intracellular free calcium ions were determined using Fluo-3 AM dye marker. Intracellular ROS levels were assessed using the 2',7'-dichlorodihydrofluorescein diacetate probe assay. The mitochondrial membrane potential was measured using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzimidazole carbocyanine iodide. Furthermore, the mRNA expression levels of B‑cell lymphoma (Bcl)‑extra large, Bcl‑2‑associated X protein, Bcl‑2‑interacting mediator of cell death, apoptotic protease activating factor‑1 (Apaf‑1), calpain 2, cysteinyl aspartate specific proteinase (caspase)‑3, caspase‑4 and caspase‑9 were determined using reverse transcription semiquantitative and quantitative polymerase chain reaction analyses. Treatment with LCA inhibited proliferation and induced apoptosis of T24 cells, and increased intracellular Ca2+ levels and ROS production. Furthermore, LCA induced mitochondrial dysfunction, decreased mitochondrial membrane potential, and increased the mRNA expression levels of Apaf‑1, caspase‑9 and caspase‑3. Exposure of T24 cells to LCA also triggered calpain 2 and caspase‑4 activation, resulting in apoptosis. These findings indicated that LCA increased intracellular Ca2+ levels, which may be associated with mitochondrial dysfunction. In addition, the ER stress pathway may be

  13. Monobenzyltin Complex C1 Induces Apoptosis in MCF-7 Breast Cancer Cells through the Intrinsic Signaling Pathway and through the Targeting of MCF-7-Derived Breast Cancer Stem Cells via the Wnt/β-Catenin Signaling Pathway

    PubMed Central

    Fani, Somayeh; Dehghan, Firouzeh; Karimian, Hamed; Mun Lo, Kong; Ebrahimi Nigjeh, Siyamak; Swee Keong, Yeap; Soori, Rahman; May Chow, Kit; Kamalidehghan, Behnam; Mohd Ali, Hapipah; Mohd Hashim, Najihah

    2016-01-01

    Monobenzyltin Schiff base complex, [N-(3,5-dichloro-2-oxidobenzylidene)-4-chlorobenzyhydrazidato](o-methylbenzyl)aquatin(IV) chloride, C1, is an organotin non-platinum metal-based agent. The present study was conducted to investigate its effects on MCF-7 cells with respect to the induction of apoptosis and its inhibitory effect against MCF-7 breast cancer stem cells. As determined in a previous study, compound C1 revealed strong antiproliferative activity on MCF-7 cells with an IC50 value of 2.5 μg/mL. Annexin V/propidium iodide staining coupled with flow cytometry indicated the induction of apoptosis in treated cells. Compound C1 induced apoptosis in MCF-7 cells and was mediated through the intrinsic pathway with a reduction in mitochondrial membrane potential and mitochondrial cytochrome c release to cytosol. Complex C1 activated caspase 9 as a result of cytochrome c release. Subsequently, western blot and real time PCR revealed a significant increase in Bax and Bad expression and a significant decrease in the expression levels of Bcl2 and HSP70. Furthermore, a flow cytometric analysis showed that treatment with compound C1 caused a significant arrest of MCF-7 cells in G0/G1 phase. The inhibitory analysis of compound C1 against derived MCF-7 stem cells showed a significant reduction in the aldehyde dehydrogenase-positive cell population and a significant reduction in the population of MCF-7 cancer stem cells in primary, secondary, and tertiary mammospheres. Moreover, treatment with C1 down-regulated the Wnt/β-catenin self-renewal pathway. These findings indicate that complex C1 is a suppressive agent of MCF-7 cells that functions through the induction of apoptosis, cell cycle arrest, and the targeting of MCF-7-derived cancer stem cells. This work may lead to a better treatment strategy for the reduction of breast cancer recurrence. PMID:27529753

  14. Monobenzyltin Complex C1 Induces Apoptosis in MCF-7 Breast Cancer Cells through the Intrinsic Signaling Pathway and through the Targeting of MCF-7-Derived Breast Cancer Stem Cells via the Wnt/β-Catenin Signaling Pathway.

    PubMed

    Fani, Somayeh; Dehghan, Firouzeh; Karimian, Hamed; Mun Lo, Kong; Ebrahimi Nigjeh, Siyamak; Swee Keong, Yeap; Soori, Rahman; May Chow, Kit; Kamalidehghan, Behnam; Mohd Ali, Hapipah; Mohd Hashim, Najihah

    2016-01-01

    Monobenzyltin Schiff base complex, [N-(3,5-dichloro-2-oxidobenzylidene)-4-chlorobenzyhydrazidato](o-methylbenzyl)aquatin(IV) chloride, C1, is an organotin non-platinum metal-based agent. The present study was conducted to investigate its effects on MCF-7 cells with respect to the induction of apoptosis and its inhibitory effect against MCF-7 breast cancer stem cells. As determined in a previous study, compound C1 revealed strong antiproliferative activity on MCF-7 cells with an IC50 value of 2.5 μg/mL. Annexin V/propidium iodide staining coupled with flow cytometry indicated the induction of apoptosis in treated cells. Compound C1 induced apoptosis in MCF-7 cells and was mediated through the intrinsic pathway with a reduction in mitochondrial membrane potential and mitochondrial cytochrome c release to cytosol. Complex C1 activated caspase 9 as a result of cytochrome c release. Subsequently, western blot and real time PCR revealed a significant increase in Bax and Bad expression and a significant decrease in the expression levels of Bcl2 and HSP70. Furthermore, a flow cytometric analysis showed that treatment with compound C1 caused a significant arrest of MCF-7 cells in G0/G1 phase. The inhibitory analysis of compound C1 against derived MCF-7 stem cells showed a significant reduction in the aldehyde dehydrogenase-positive cell population and a significant reduction in the population of MCF-7 cancer stem cells in primary, secondary, and tertiary mammospheres. Moreover, treatment with C1 down-regulated the Wnt/β-catenin self-renewal pathway. These findings indicate that complex C1 is a suppressive agent of MCF-7 cells that functions through the induction of apoptosis, cell cycle arrest, and the targeting of MCF-7-derived cancer stem cells. This work may lead to a better treatment strategy for the reduction of breast cancer recurrence. PMID:27529753

  15. Induction of apoptosis in MCF‑7 human breast cancer cells by Khz (fusion of Ganoderma lucidum and Polyporus umbellatus mycelium).

    PubMed

    Kim, Tae Hwan; Kim, Ju Sung; Kim, Zoo Haye; Huang, Ren Bin; Chae, Young Lye; Wang, Ren Sheng

    2016-02-01

    Khz (fusion of Ganoderma lucidum and Polyporus umbellatus), isolated from the mycelia of G. lucidum and P. umbellatus, exerts anti‑proliferative effects against malignant cells; however, its activity against human breast cancer cells remains to be elucidated. In the present study, cell proliferation was assessed using a 3-(4,5‑dimethylthiazol‑2‑yl)-2,5‑diphenyltetrazolium bromide assay, and poptosis was examined using annexin V‑propidium iodide staining and flow cytometry. The activation of caspases 7, 8 and 9 were detected in the Khz‑treated cells using western blotting. The results demonstrated that Khz increased the intracellular calcium concentration and induced the production of reactive oxygen species in MCF‑7 breast cancer cells, as determined using flow cytometry. The results also demonstrated that Khz inhibited cell proliferation and induced apoptosis in the MCF‑7 cells. In addition, the mechanism by which Khz induces apoptosis in cancer cells was investigated. Khz induced apoptosis preferentially in transformed cells, with a minimal effect on non‑transformed cells, suggesting its potential as an anticancer therapeutic agent. Oxidative stress is associated with apoptotic and non‑apoptotic cell death, although pro‑oxidative conditions are not a pre‑requisite for apoptosis. Assessment of the activation status of caspases 7, 8 and 9 revealed that the levels of cleaved caspases were significantly increased in the cells treated with Khz. It is widely accepted that calcium signaling is important in apoptosis, and the present study observed an increase in [Ca2+]i in response to Khz treatment. The anti‑proliferative and pro‑apoptotic effects of Khz suggest that this extract may be developed as a potential anticancer agent. PMID:26648109

  16. Scutellaria barbate extract induces apoptosis of hepatoma H22 cells via the mitochondrial pathway involving caspase-3

    PubMed Central

    Dai, Zhi-Jun; Wang, Xi-Jing; Li, Zong-Fang; Ji, Zong-Zheng; Ren, Hong-Tao; Tang, Wei; Liu, Xiao-Xu; Kang, Hua-Feng; Guan, Hai-Tao; Song, Ling-Qin

    2008-01-01

    AIM: To study the growth inhibitory and apoptotic effects of Scutellaria barbata D.Don (S. barbata) and to determine the underlying mechanism of its antitumor activity in mouse liver cancer cell line H22. METHODS: Proliferation of H22 cells was examined by MTT assay. Cellular morphology of PC-2 cells was observed under fluorescence microscope and transmission electron microscope (EM). Mitochondrial transmembrane potential was determined under laser scanning confocal microscope (LSCM) with rhodamine 123 staining. Flow cytometry was performed to analyze the cell cycle of H22 cells with propidium iodide staining. Protein level of cytochrome C and caspase-3 was measured by semi-quantitive RT-PCR and Western blot analysis. Activity of caspase-3 enzyme was measured by spectrofluorometry. RESULTS: MTT assay showed that extracts from S. barbata (ESB) could inhibit the proliferation of H22 cells in a time-dependent manner. Among the various phases of cell cycle, the percentage of cells in S phase was significantly decreased, while the percentage of cells in G1 phase was increased. Flow cytometry assay also showed that ESB had a positive effect on apoptosis. Typical apoptotic morphologies such as condensation and fragmentation of nuclei and blebbing membrane of apoptotic cells could be observed under transmission electron microscope and fluorescence microscope. To further investige the molecular mechanism behind ESB-induced apoptosis, ESB-treated cells rapidly lost their mitochondrial transmembrane potential, released mitochondrial cytochrome C into cytosol, and induced caspase-3 activity in a dose-dependent manner. CONCLUSION: ESB can effectively inhibit the proliferation and induce apoptosis of H22 cells involving loss of mitochondrial transmembrane potential, release of cytochrome C, and activation of caspase-3. PMID:19109865

  17. Infrasound sensitizes human glioblastoma cells to cisplatin-induced apoptosis.

    PubMed

    Rachlin, Kenneth; Moore, Dan H; Yount, Garret

    2013-11-01

    The development of nontoxic agents that can selectively enhance the cytotoxicity of chemotherapy is an important aim in oncology. This study evaluates the ability of infrasound exposure to sensitize glioblastoma cells to cisplatin-induced apoptosis. The infrasound was delivered using a device designed to replicate the unique infrasound emissions measured during external Qigong treatments. Human glioblastoma cell lines harboring wild-type p53 (U87) or mutant p53 (U251, SF210, and SF188) were treated in culture with cisplatin, infrasound emissions, or the combination of the 2 agents. Induction of apoptosis was quantified after 24 hours by flow cytometry following annexin V/propidium iodide staining. Infrasound emissions alone, delivered at moderate levels (~10 mPa) with dynamic frequency content (7-13 Hz), did not induce apoptosis, yet combining infrasound with cisplatin augmented the induction of apoptosis by cisplatin in all the 4 cell lines (P < .05). Increased cellular uptake of the fluorophore calcein associated with infrasound exposure was quantified by fluorescence microscopy as well as flow cytometry, demonstrating increased cell membrane permeability. The 4 cell lines differed in the degree to which infrasound exposure increased calcein uptake, and these differences were predictive of the extent to which infrasound enhanced cisplatin-induced apoptosis. When exposed to specific frequencies, membrane permeabilization also appeared to be differentially responsive for each cell line, suggesting the potential for selective targeting of tissue types using isolated infrasonic frequencies. Additionally, the pressure amplitudes used in this study were several orders of magnitude less than those used in similar studies involving ultrasound and shock waves. The results of this study provide support for using infrasound to enhance the chemotherapeutic effects of cisplatin in a clinical setting. PMID:23165942

  18. Honokiol Inhibits Androgen Receptor Activity in Prostate Cancer Cells

    PubMed Central

    Hahm, Eun-Ryeong; Karlsson, A. Isabella; Bonner, Michael Y.; Arbiser, Jack L.; Singh, Shivendra V.

    2014-01-01

    BACKGROUND We have shown previously that honokiol (HNK), a bioactive component of the medicinal plant Magnolia officinalis, inhibits growth of human prostate cancer cells in vitro and in vivo. However, the effect of HNK on androgen receptor (AR) signaling is not known. METHODS LNCaP, C4-2, and TRAMP-C1 cells were used for various assays. Trypan blue dye exclusion assay or clonogenic assay was performed for determination of cell viability. The effects of HNK and/or its analogs on protein levels of AR and its target gene product prostate specific antigen (PSA) were determined by western blotting. RNA interference of p53 was achieved by transient transfection. Reverse transcription-polymerase chain reaction was performed for mRNA expression of AR. Nuclear translocation of AR was visualized by microscopy. Apoptosis was quantified by DNA fragmentation assay or flow cytometry after Annexin V-propidium iodide staining. RESULTS HNK and its dichloroacetate analog (HDCA) were relatively more effective in suppressing cell viability and AR protein level than honokiol epoxide or biseugenol. Nuclear translocation of AR stimulated by a synthetic androgen (R1881) was markedly suppressed in the presence of HNK. Downregulation of AR protein resulting from HNK exposure was attributable to transcriptional repression as well as proteasomal degradation. HNK-mediated suppression of AR protein was maintained in LNCaP cells after knockdown of p53 protein. HNK-induced apoptosis was not affected by R1881 treatment. CONCLUSIONS The present study demonstrates, for the first time, that HNK inhibits activity of AR in prostate cancer cells regardless of the p53 status. PMID:24338950

  19. NF-Kappa B Modulation Is Involved in Celastrol Induced Human Multiple Myeloma Cell Apoptosis

    PubMed Central

    Ni, Haiwen; Zhao, Wanzhou; Kong, Xiangtu; Li, Haitao; Ouyang, Jian

    2014-01-01

    Celastrol is an active compound extracted from the root bark of the traditional Chinese medicine Tripterygium wilfordii Hook F. To investigate the effect of celastrol on human multiple myeloma cell cycle arrest and apoptosis and explore its molecular mechanism of action. The activity of celastrol on LP-1 cell proliferation was detected by WST-8 assay. The celastrol-induced cell cycle arrest was analyzed by flow cytometry after propidium iodide staining. Nuclear translocation of the nuclear factor kappa B (NF-κB) was observed by fluorescence microscope. Celastrol inhibited cell proliferation of LP-1 myeloma cell in a dose-dependent manner with IC50 values of 0.8817 µM, which was mediated through G1 cell cycle arrest and p27 induction. Celastrol induced apoptosis in LP-1 and RPMI 8226 myeloma cells in a time and dose dependent manner, and it involved Caspase-3 activation and NF-κB pathway. Celastrol down-modulated antiapoptotic proteins including Bcl-2 and survivin expression. The expression of NF-κB and IKKa were decreased after celastrol treatment. Celastrol effectively blocked the nuclear translocation of the p65 subunit and induced human multiple myeloma cell cycle arrest and apoptosis by p27 upregulation and NF-kB modulation. It has been demonstrated that the effect of celastrol on NF-kB was HO-1-independent by using zinc protoporphyrin-9 (ZnPPIX), a selective heme oxygenase inhibitor. From the results, it could be inferred that celastrol may be used as a NF-kB inhibitor to inhibit myeloma cell proliferation. PMID:24755677

  20. Efficient Intracellular Delivery of Molecules with High Cell Viability Using Nanosecond-Pulsed Laser-Activated Carbon Nanoparticles

    PubMed Central

    2015-01-01

    Conventional physical and chemical methods that efficiently deliver molecules into cells are often associated with low cell viability. In this study, we evaluated the cellular effects of carbon nanoparticles believed to emit photoacoustic waves due to nanosecond-pulse laser activation to test the hypothesis that this method could achieve efficient intracellular delivery while maintaining high cell viability. Suspensions of DU145 human prostate carcinoma cells, carbon black (CB) nanoparticles, and calcein were exposed to 5–9 ns long laser pulses of near-infrared (1064 nm wavelength) light and then analyzed by flow cytometry for intracellular uptake of calcein and cell viability by propidium iodide staining. We found that intracellular uptake increased and in some cases saturated at high levels with only small losses in cell viability as a result of increasing laser fluence, laser exposure time, and as a unifying parameter, the total laser energy. Changing interpulse spacing between 0.1 and 10 s intervals showed no significant change in bioeffects, suggesting that the effects of each pulse were independent when spaced by at least 0.1 s intervals. Pretreatment of CB nanoparticles to intense laser exposure followed by mixing with cells also had no significant effect on uptake or viability. Similar uptake and viability were seen when CB nanoparticles were substituted with India ink, when DU145 cells were substituted with H9c2 rat cardiomyoblast cells, and when calcein was substituted with FITC-dextran. The best laser exposure conditions tested led to 88% of cells with intracellular uptake and close to 100% viability, indicating that nanosecond-pulse laser-activated carbon nanoparticles can achieve efficient intracellular delivery while maintaining high cell viability. PMID:24547946

  1. Lactobacillus casei Exerts Anti-Proliferative Effects Accompanied by Apoptotic Cell Death and Up-Regulation of TRAIL in Colon Carcinoma Cells

    PubMed Central

    Santarmaki, Valentina; Aindelis, Georgios; Tompoulidou, Evgenia; Lamprianidou, Eleftheria E.; Saxami, Georgia; Ypsilantis, Petros; Lampri, Evangeli S.; Simopoulos, Constantinos; Kotsianidis, Ioannis; Galanis, Alex; Kourkoutas, Yiannis; Dimitrellou, Dimitra; Chlichlia, Katerina

    2016-01-01

    Probiotic microorganisms such as lactic acid bacteria (LAB) exert a number of strain-specific health-promoting activities attributed to their immunomodulatory, anti-inflammatory and anti-carcinogenic properties. Despite recent attention, our understanding of the biological processes involved in the beneficial effects of LAB strains is still limited. To this end, the present study investigated the growth-inhibitory effects of Lactobacillus casei ATCC 393 against experimental colon cancer. Administration of live Lactobacillus casei (as well as bacterial components thereof) on murine (CT26) and human (HT29) colon carcinoma cell lines raised a significant concentration- and time-dependent anti-proliferative effect, determined by cell viability assays. Specifically, a dramatic decrease in viability of colon cancer cells co-incubated with 109 CFU/mL L. casei for 24 hours was detected (78% for HT29 and 52% for CT26 cells). In addition, live L. casei induced apoptotic cell death in both cell lines as revealed by annexin V and propidium iodide staining. The significance of the in vitro anti-proliferative effects was further confirmed in an experimental tumor model. Oral daily administration of 109 CFU live L. casei for 13 days significantly inhibited in vivo growth of colon carcinoma cells, resulting in approximately 80% reduction in tumor volume of treated mice. Tumor growth inhibition was accompanied by L. casei-driven up-regulation of the TNF-related apoptosis-inducing ligand TRAIL and down-regulation of Survivin. Taken together, these findings provide evidence for beneficial tumor-inhibitory, anti-proliferative and pro-apoptotic effects driven by this probiotic LAB strain. PMID:26849051

  2. Activation of the MAPK/Akt/Nrf2-Egr1/HO-1-GCLc axis protects MG-63 osteosarcoma cells against 15d-PGJ2-mediated cell death.

    PubMed

    Koyani, Chintan N; Kitz, Kerstin; Rossmann, Christine; Bernhart, Eva; Huber, Evelyn; Trummer, Christopher; Windischhofer, Werner; Sattler, Wolfgang; Malle, Ernst

    2016-03-15

    Despite considerable efforts to improve treatment modalities for osteosarcoma (OS), patient survival remains poor mainly due to pro-survival pathways in OS cells. Among others, prostaglandins (PGs) are the potent regulators of bone homoeostasis and OS pathophysiology. Therefore, the present study aimed to elucidate the impact of 15-deoxy-Δ(12,14)-PGJ2 (15d-PGJ2, a stable PGD2 degradation product) on cell death/cell survival pathways in p53-deficient MG-63 OS cells. Our findings show that 15d-PGJ2 induces generation of reactive oxygen species that promote p38 MAPK activation and subsequent Akt phosphorylation. This pathway induced nuclear expression of Nrf2 and Egr1, and increased transcription of haem oxygenase-1 (HO-1) and the catalytic subunit of glutamate cysteine ligase (GCLc), catalysing the first step in GSH synthesis. Silencing of Nrf2, Egr1 and HO-1 significantly elevated 15d-PGJ2-mediated reduction of cellular metabolic activity. Activation of cell survival genes including HO-1 and GCLc inhibited 15d-PGJ2-induced cleavage of pro-caspase-3 and PARP. Annexin V/propidium iodide staining showed an increase in early/late apoptotic cells in response to 15d-PGJ2. The observed 15d-PGJ2-mediated signalling events are independent of PGD2 receptors (DP1 and DP2) and PPARγ. In addition, the electrophilic carbon atom C9 is a prerequisite for the observed activity of 15d-PGJ2. The present data show that the intracellular redox imbalance acted as a node and triggered both death and survival pathways in response to 15d-PGJ2. Pharmacological or genetic interference of the pro-survival pathway, the p38 MAPK/Akt/Nrf2-Egr1/HO-1-GCLc axis, sensitizes MG-63 cells towards 15d-PGJ2-mediated apoptosis. PMID:26801686

  3. Lactobacillus casei Exerts Anti-Proliferative Effects Accompanied by Apoptotic Cell Death and Up-Regulation of TRAIL in Colon Carcinoma Cells.

    PubMed

    Tiptiri-Kourpeti, Angeliki; Spyridopoulou, Katerina; Santarmaki, Valentina; Aindelis, Georgios; Tompoulidou, Evgenia; Lamprianidou, Eleftheria E; Saxami, Georgia; Ypsilantis, Petros; Lampri, Evangeli S; Simopoulos, Constantinos; Kotsianidis, Ioannis; Galanis, Alex; Kourkoutas, Yiannis; Dimitrellou, Dimitra; Chlichlia, Katerina

    2016-01-01

    Probiotic microorganisms such as lactic acid bacteria (LAB) exert a number of strain-specific health-promoting activities attributed to their immunomodulatory, anti-inflammatory and anti-carcinogenic properties. Despite recent attention, our understanding of the biological processes involved in the beneficial effects of LAB strains is still limited. To this end, the present study investigated the growth-inhibitory effects of Lactobacillus casei ATCC 393 against experimental colon cancer. Administration of live Lactobacillus casei (as well as bacterial components thereof) on murine (CT26) and human (HT29) colon carcinoma cell lines raised a significant concentration- and time-dependent anti-proliferative effect, determined by cell viability assays. Specifically, a dramatic decrease in viability of colon cancer cells co-incubated with 10(9) CFU/mL L. casei for 24 hours was detected (78% for HT29 and 52% for CT26 cells). In addition, live L. casei induced apoptotic cell death in both cell lines as revealed by annexin V and propidium iodide staining. The significance of the in vitro anti-proliferative effects was further confirmed in an experimental tumor model. Oral daily administration of 10(9) CFU live L. casei for 13 days significantly inhibited in vivo growth of colon carcinoma cells, resulting in approximately 80% reduction in tumor volume of treated mice. Tumor growth inhibition was accompanied by L. casei-driven up-regulation of the TNF-related apoptosis-inducing ligand TRAIL and down-regulation of Survivin. Taken together, these findings provide evidence for beneficial tumor-inhibitory, anti-proliferative and pro-apoptotic effects driven by this probiotic LAB strain. PMID:26849051

  4. Bortezomib and fenretinide induce synergistic cytotoxicity in mantle cell lymphoma through apoptosis, cell-cycle dysregulation, and IκBα kinase downregulation.

    PubMed

    Cowan, Andrew J; Frayo, Shani L; Press, Oliver W; Palanca-Wessels, Maria C; Pagel, John M; Green, Damian J; Gopal, Ajay K

    2015-10-01

    Mantle cell lymphoma (MCL) remains incurable for most patients, and proteasome inhibitors like bortezomib induce responses in a minority of patients with relapsed disease. Fenretinide is a retinoid that has shown preclinical activity in B-cell lymphomas. We hypothesized that these agents could yield augmented antitumor activity. MCL lines (Granta-519, Jeko-1, and Rec-1) were treated with escalating concentrations of bortezomib and fenretinide singly and in combination. Cytotoxicity was assessed using the MTT assay. Flow cytometric methods were used to assess apoptosis and necrosis, with annexin V-FITC/propidium iodide staining, and G1 and G2 cell-cycle changes were assessed by DAPI staining. Changes in cyclin D1, cyclin B, IκBα, and IKKα expressions were quantified by western blotting. Cytotoxicity was mediated through apoptosis; both agents showed observed versus expected cytotoxicities of 92.2 versus 55.1% in Granta-519, of 87.6 versus 36.3% in Jeko-1, and of 63.2 versus 29.8% in Rec-1. Isobolographic analysis confirmed synergy in Jeko-1 and Rec-1 cell lines. Bortezomib induced G2-phase arrest, with a 1.7-fold increase compared with control, and fenretinide resulted in G1-phase arrest, with an increase of 1.3-fold compared with control. In the combination, G2-phase arrest predominated, with a 1.4-fold increase compared with control, and there was reduced expression of cyclin D1 to 24%, cyclin B to 52 and 64%, cyclin D3 to 25 and 43%, IκBα to 23 and 46%, and IκBα kinase to 34 and 44%. Bortezomib and fenretinide exhibit synergistic cytotoxicity against MCL cell lines. This activity is mediated by IκBα kinase modulation, decreased cyclin expression, cell cycle dysregulation, and apoptotic cell death. PMID:26237500

  5. Activation of the MAPK/Akt/Nrf2-Egr1/HO-1-GCLc axis protects MG-63 osteosarcoma cells against 15d-PGJ2-mediated cell death

    PubMed Central

    Koyani, Chintan N.; Kitz, Kerstin; Rossmann, Christine; Bernhart, Eva; Huber, Evelyn; Trummer, Christopher; Windischhofer, Werner; Sattler, Wolfgang; Malle, Ernst

    2016-01-01

    Despite considerable efforts to improve treatment modalities for osteosarcoma (OS), patient survival remains poor mainly due to pro-survival pathways in OS cells. Among others, prostaglandins (PGs) are the potent regulators of bone homoeostasis and OS pathophysiology. Therefore, the present study aimed to elucidate the impact of 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2, a stable PGD2 degradation product) on cell death/cell survival pathways in p53-deficient MG-63 OS cells. Our findings show that 15d-PGJ2 induces generation of reactive oxygen species that promote p38 MAPK activation and subsequent Akt phosphorylation. This pathway induced nuclear expression of Nrf2 and Egr1, and increased transcription of haem oxygenase-1 (HO-1) and the catalytic subunit of glutamate cysteine ligase (GCLc), catalysing the first step in GSH synthesis. Silencing of Nrf2, Egr1 and HO-1 significantly elevated 15d-PGJ2-mediated reduction of cellular metabolic activity. Activation of cell survival genes including HO-1 and GCLc inhibited 15d-PGJ2-induced cleavage of pro-caspase-3 and PARP. Annexin V/propidium iodide staining showed an increase in early/late apoptotic cells in response to 15d-PGJ2. The observed 15d-PGJ2-mediated signalling events are independent of PGD2 receptors (DP1 and DP2) and PPARγ. In addition, the electrophilic carbon atom C9 is a prerequisite for the observed activity of 15d-PGJ2. The present data show that the intracellular redox imbalance acted as a node and triggered both death and survival pathways in response to 15d-PGJ2. Pharmacological or genetic interference of the pro-survival pathway, the p38 MAPK/Akt/Nrf2-Egr1/HO-1-GCLc axis, sensitizes MG-63 cells towards 15d-PGJ2-mediated apoptosis. PMID:26801686

  6. Cell division in the unicellular microalga Dunaliella viridis depends on phosphorylation of extracellular signal-regulated kinases (ERKs).

    PubMed

    Jiménez, Carlos; Cossío, Belén R; Rivard, Christopher J; Berl, Tomás; Capasso, Juan M

    2007-01-01

    In mammalian cells, MAPKs are involved in both stress response (JNK and p38 pathways) and cell proliferation and differentiation [extracellular signal-regulated kinase (ERK)] through protein kinase cascades. Exposure of Dunaliella viridis cell cultures to PD98059, a very specific inhibitor of the ERK signalling pathway, resulted in a total arrest of cell proliferation and a complete dephosphorylation of ERK. As shown by flow cytometry analysis of propidium iodide-stained cells, PD98059 stopped mitosis at the G(2) phase after the S phase has been completed. Multiple physiological parameters such as cell motility and reducing power generation (NADPH) clearly indicate that the treated cells are wholly viable. Exposure of D. viridis to environmental stresses that impair cell division, such as hyperosmotic shock, nitrogen starvation, or sublethal UV irradiation, caused a marked decrease in the phospho-ERK levels as detected by western blot. Two 400 bp polynucleotides from D. viridis with high homologies to published sequences of ERK1 and ERK2 were cloned, sequenced, and submitted to GenBank. Northern blot analysis revealed two mRNA bands of approximately 1.9 kb, consistent with the expected size of ERK proteins ( approximately 40 kDa). Sequence analysis showed that they contained several mitogen-activated protein kinase (MAPK) conserved domains, including II, III, VIb, VII, and the double phosphorylation motif. Interestingly, in D. viridis, this motif was T*DY* instead of the canonic T*EY*. Based on this finding, ERK plant sequences can be divided into two groups, one termed the T*DY* branch and the other termed the T*EY* branch. The molecular and functional data presented here suggest that ERK is a very ancient signalling pathway and that it was already present in the last common ancestor of all eukaryotic cells. PMID:17220513

  7. Selected novel 5'-amino-2'-hydroxy-1, 3-diaryl-2-propen-1-ones arrest cell cycle of HCT-116 in G0/G1 phase

    PubMed Central

    Simon, Lalitha; Srinivasan, K. K.; Kumar, Nitesh; Reddy, Neetinkumar D.; Biswas, Subhankar; Rao, C. Mallikarjuna; Moorkoth, Sudheer

    2016-01-01

    A series of 5'-amino-2'-hydroxy-1,3-diaryl-2-propen-1-ones (AC1-AC15) were synthesized by Claisen-Schmidt condensation of 5'-acetamido-2'-hydroxy acetophenone with various substituted aromatic aldehydes. The synthesized compounds were characterized by FTIR, 1H NMR and mass spectrometry and evaluated for their selective cytotoxicity using MTT assay on two cancer cell lines namely breast cancer cell line (MCF-7), colon cancer cell line (HCT-116) and one normal kidney epithelial cell line (Vero). Among the tested compounds, AC-10 showed maximum cytotoxic effect on MCF-7 cell line with IC50 value 74.7 ± 3.5 µM. On HCT-116 cells, AC-13 exhibited maximum cytotoxicity with IC50 value 42.1 ± 4.0 µM followed by AC-14 and AC-10 with IC50 values 62 ± 2.3 µM and 95.4 ± 1.7 µM respectively. All tested compounds were found to be safe on Vero cell line with IC50 value more than 200 µM. Based on their highest efficacy on HCT-116, AC-10, AC-13 and AC-14 were selected for mechanistic study on this cell line by evaluating changes nucleomorphological characteristics using acridine orange-ethidium bromide (AOEB) dual stain and by analyzing cell cycle with flow cytometry using propidium iodide stain. In AOEB staining, all three tested compounds showed significant (p < 0.05) increase in percentage apoptotic nuclei compared to control cells, with highest increase in apoptotic nuclei by AC-13 treatment (31 %). Flow cytometric studies showed cell cycle arrest by AC-10 and AC-14 treatment in G0/G1 phase and by AC-13 in G0/G1 and G2/M phase. The study reflected the potential of AC-10, AC-13 and AC-14 to be the lead molecules for further optimization. PMID:27152112

  8. Selected novel 5'-amino-2'-hydroxy-1, 3-diaryl-2-propen-1-ones arrest cell cycle of HCT-116 in G0/G1 phase.

    PubMed

    Simon, Lalitha; Srinivasan, K K; Kumar, Nitesh; Reddy, Neetinkumar D; Biswas, Subhankar; Rao, C Mallikarjuna; Moorkoth, Sudheer

    2016-01-01

    A series of 5'-amino-2'-hydroxy-1,3-diaryl-2-propen-1-ones (AC1-AC15) were synthesized by Claisen-Schmidt condensation of 5'-acetamido-2'-hydroxy acetophenone with various substituted aromatic aldehydes. The synthesized compounds were characterized by FTIR, (1)H NMR and mass spectrometry and evaluated for their selective cytotoxicity using MTT assay on two cancer cell lines namely breast cancer cell line (MCF-7), colon cancer cell line (HCT-116) and one normal kidney epithelial cell line (Vero). Among the tested compounds, AC-10 showed maximum cytotoxic effect on MCF-7 cell line with IC50 value 74.7 ± 3.5 µM. On HCT-116 cells, AC-13 exhibited maximum cytotoxicity with IC50 value 42.1 ± 4.0 µM followed by AC-14 and AC-10 with IC50 values 62 ± 2.3 µM and 95.4 ± 1.7 µM respectively. All tested compounds were found to be safe on Vero cell line with IC50 value more than 200 µM. Based on their highest efficacy on HCT-116, AC-10, AC-13 and AC-14 were selected for mechanistic study on this cell line by evaluating changes nucleomorphological characteristics using acridine orange-ethidium bromide (AOEB) dual stain and by analyzing cell cycle with flow cytometry using propidium iodide stain. In AOEB staining, all three tested compounds showed significant (p < 0.05) increase in percentage apoptotic nuclei compared to control cells, with highest increase in apoptotic nuclei by AC-13 treatment (31 %). Flow cytometric studies showed cell cycle arrest by AC-10 and AC-14 treatment in G0/G1 phase and by AC-13 in G0/G1 and G2/M phase. The study reflected the potential of AC-10, AC-13 and AC-14 to be the lead molecules for further optimization. PMID:27152112

  9. β-catenin knockdown inhibits the proliferation of human glioma cells in vitro and in vivo

    PubMed Central

    WANG, ZHONG; CHEN, QIANXUE

    2016-01-01

    β-catenin is a crucial oncogene that is capable of regulating cancer progression. The aim of the present study was to clarify whether β-catenin was associated with the proliferation and progress of glioma. In order to knockdown the expression of β-catenin in human U251 glioma cells, three pairs of small interfering (si)RNA were designed and synthesized and the most effective siRNA was selected and used for silencing the endogenous β-catenin, which was detected by western blot analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Proliferation was subsequently detected using a methylthiazolyl-tetrazolium bromide assay and the results demonstrated that knockdown of β-catenin significantly inhibited the proliferation of U251 cells in a time- and dose-dependent manner (P<0.01). Cell apoptosis rate was analyzed using flow cytometry and Annexin V-fluorescein isothiocyanate/propidium iodide staining demonstrated that β-catenin siRNA significantly increased the apoptosis of U251 cells (P<0.01). Furthermore, the results of an in vitro scratch assay demonstrated that β-catenin silencing suppressed the proliferation of U251 cells, as compared with the control group (P<0.01). In vivo, β-catenin expression levels in U251 cells were significantly inhibited (P<0.01) following β-catenin short hairpin (sh)RNA lentiviral-vector transfection, as detected by western blot analysis and RT-qPCR. Tumorigenicity experiments demonstrated that β-catenin inhibition significantly increased the survival rate of nude mice. The results of the present study demonstrated that knockdown of β-catenin expression significantly inhibited the progression of human glioma cancer cells, in vitro and in vivo; thus suggesting that β-catenin silencing may be a novel therapy for the treatment of human glioma. PMID:26998037

  10. Magnetic Fe₃O₄ nanoparticles and chemotherapy agents interact synergistically to induce apoptosis in lymphoma cells.

    PubMed

    Jing, Hongmei; Wang, Jing; Yang, Ping; Ke, Xiaoyan; Xia, Guohua; Chen, Baoan

    2010-01-01

    The purpose of this study was to investigate the potential effects of combination therapy using magnetic nanoparticles of Fe₃O₄ (MNP-Fe₃O₄) and chemotherapeutic drugs on lymphoma cells. Proliferation, inhibition, and viability of Raji cells were detected by MTT and trypan blue exclusion. The percentage of cells undergoing apoptosis was detected by flow cytometry using fluorescein isothiocyanate-annexin V and propidium iodide staining. p53 and nuclear factor-κB (NF-κB) protein levels were measured by Western blot. The results showed that proliferation of Raji cells was inhibited by adriamycin or daunorubicin in a dose-and time-dependent manner. Cell sensitivity was improved and the 50% inhibitory concentrations of adriamycin and daunorubicin decreased when combined with a MNP-Fe₃O₄ carrier. Interestingly, increased apoptosis in Raji lymphoma cells was accompanied by upregulation of p53 protein and downregulation of NF-κB protein. Furthermore, the combination of MNP-Fe₃O₄ with adriamycin or daunorubicin increased p53 protein levels and decreased NF-κB protein levels more than adriamycin or daunorubicin alone, indicating that MNP-Fe₃O₄ could enhance the effect of chemotherapeutic drugs on p53 and NF-κB. Similar results for cell apoptosis and protein expression were not observed for the groups treated with dexamethasone ± MNP-Fe₃O₄ (P > 0.05). These findings suggest a potential clinical application for MNP-Fe₃O₄ in combination with daunorubicin or adriamycin in the treatment of lymphoma. PMID:21187919

  11. Resident bacteria on leaves enhance survival of immigrant cells of Salmonella enterica.

    PubMed

    Poza-Carrion, Cesar; Suslow, Trevor; Lindow, Steven

    2013-04-01

    Although Salmonella enterica apparently has comparatively low epiphytic fitness on plants, external factors that would influence its ability to survive on plants after contamination would be of significance in the epidemiology of human diseases caused by this human pathogen. Viable population sizes of S. enterica applied to plants preinoculated with Pseudomonas syringae or either of two Erwinia herbicola strains was ≥10-fold higher than that on control plants that were not precolonized by such indigenous bacteria when assessed 24 to 72 h after the imposition of desiccation stress. The protective effect of P. fluorescens, which exhibited antibiosis toward S. enterica in vitro, was only ≈50% that conferred by other bacterial strains. Although S. enterica could produce small cellular aggregates after incubation on wet leaves for several days, and the cells in such aggregates were less susceptible to death upon acute dehydration than solitary cells (as determined by propidium iodide staining), most Salmonella cells were found as isolated cells when it was applied to leaves previously colonized by other bacterial species. The proportion of solitary cells of S. enterica coincident with aggregates of cells of preexisting epiphytic species that subsequently were judged as nonviable by viability staining on dry leaves was as much as 10-fold less than those that had landed on uncolonized portions of the leaf. Thus, survival of immigrant cells of S. enterica on plants appears to be strongly context dependent, and the presence of common epiphytic bacteria on plants can protect such immigrants from at least one key stress (i.e., desiccation) encountered on leaf surfaces. PMID:23506362

  12. A Novel Peptide to Treat Oral Mucositis Blocks Endothelial and Epithelial Cell Apoptosis

    SciTech Connect

    Wu Xiaoyan; Chen Peili; Sonis, Stephen T.; Lingen, Mark W.; Berger, Ann; Toback, F. Gary

    2012-07-01

    Purpose: No effective agents currently exist to treat oral mucositis (OM) in patients receiving chemoradiation for the treatment of head-and-neck cancer. We identified a novel 21-amino acid peptide derived from antrum mucosal protein-18 that is cytoprotective, mitogenic, and motogenic in tissue culture and animal models of gastrointestinal epithelial cell injury. We examined whether administration of antrum mucosal protein peptide (AMP-p) could protect against and/or speed recovery from OM. Methods and Materials: OM was induced in established hamster models by a single dose of radiation, fractionated radiation, or fractionated radiation together with cisplatin to simulate conventional treatments of head-and-neck cancer. Results: Daily subcutaneous administration of AMP-p reduced the occurrence of ulceration and accelerated mucosal recovery in all three models. A delay in the onset of erythema after irradiation was observed, suggesting that a protective effect exists even before injury to mucosal epithelial cells occurs. To test this hypothesis, the effects of AMP-p on tumor necrosis factor-{alpha}-induced apoptosis were studied in an endothelial cell line (human dermal microvascular endothelial cells) as well as an epithelial cell line (human adult low-calcium, high-temperature keratinocytes; HaCaT) used to model the oral mucosa. AMP-p treatment, either before or after cell monolayers were exposed to tumor necrosis factor-{alpha}, protected against development of apoptosis in both cell types when assessed by annexin V and propidium iodide staining followed by flow cytometry or ligase-mediated polymerase chain reaction. Conclusions: These observations suggest that the ability of AMP-p to attenuate radiation-induced OM could be attributable, at least in part, to its antiapoptotic activity.

  13. Induction of apoptosis by ginger in HEp-2 cell line is mediated by reactive oxygen species.

    PubMed

    Vijaya Padma, Viswanadha; Arul Diana Christie, Swamidurai; Ramkuma, Kunga Mohan

    2007-05-01

    Ginger (Zingiber officinale Roscoe, Zingiberaceae) is a commonly used medicinal herb throughout the world. Although some studies have demonstrated its antitumour activities on cancer cells in vitro and in vivo, the exact mechanism is not fully elucidated. Hence, the present study was designed to examine the in vitro cytotoxic activities of saline extract prepared from ginger extract on HEp-2 cell line. The cytotoxic effect of the drug was confirmed by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and cell counting and estimation of protein, DNA and RNA. Meanwhile, propidium iodide staining and agarose gel electrophoresis were performed for determining the induction of apoptosis. In addition, superoxide radical generation, nitrite formation and glutathione studies show involvement of free radicals. The present results show that the extract exerts dose-dependent suppression of cell proliferation; the IC(50) value was found to be 900 microg/ml. At a dose of 250 microg/ml, marked morphological changes including cell shrinkage and condensation of chromosomes were observed. Agarose gel electrophoresis of DNA from HEp-2 cells treated with 250 microg/ml ginger powder for 24 hr showed marked DNA ladder pattern. The involvement of free radicals was confirmed by increased superoxide production, decreased nitrate formation and depletion of glutathione in ginger-treated cells. Further screening of active components using gas chromatography-mass spectrometry analyses showed the presence of clavatol, geraniol and pinostrobin in the extract. The results of the present study suggest that ginger might be useful as a potential antitumour agent. PMID:17448115

  14. Effects of topical corticosteroids on cell proliferation, cell cycle progression and apoptosis: in vitro comparison on HaCaT.

    PubMed

    Guichard, Alexandre; Humbert, Philippe; Tissot, Marion; Muret, Patrice; Courderot-Masuyer, Carole; Viennet, Céline

    2015-02-20

    Topical-corticosteroids are mainly used for the treatment of inflammatory or hyperproliferative skin diseases. The in vivo assay to rank topical-corticosteroids potency, based on the skin blanching, is not adapted to compare their anti-proliferative efficacy. We have compared the antiproliferative effect of six topical-corticosteroids on a model of hyperproliferant keratinocytes (HaCaT). Betamethasone-dipropionate; clobetasol-propionate; betamethasone-valerate; desonide; hydrocortisone-butyrate and hydrocortisone-base, at different concentrations (10(-8)-10(-4)M) have been compared. HaCaT proliferation has been evaluated by MTT-assay and the mechanism of the death was evaluated by annexin V/propidium iodide staining and cell cycle phases analysis. Topical corticosteroids reduced cell growth in a dose-dependent manner. At 10(-4)M, betamethasone dipropionate was the most antiproliferative compound while hydrocortisone-butyrate was the less. Hydrocortisone-base which is usually considered as the less potent topical-corticosteroids showed a clear cytotoxic effect. Betamethasone-dipropionate and betamethasone-valerate induced more apoptosis than necrosis whereas the reverse has been observed for other topical-corticosteroids. All topical-corticosteroids, except clobetasol-propionate, arrested cell cycle mainly in G2-phase. Clobetasol-propionate arrested cell cycle in S-phase population. At 10(-8)M, topical-corticosteroids induced HaCaT proliferation. In terms of antiproliferative effect at 10(-4)M, we propose to rank topical corticosteroids as follow: betamethasone-dipropionate>desonide≥betamethasone-valerate=hydrocortisone-base=clobetasol-propionate>hydrocortisone-butyrate. This classification differs from the current ranking, based on the vasoconstrictive effect, but is more adapted for hyperproliferative disease treatment. PMID:25556056

  15. Corticosteroid treatment of experimental autoimmune encephalomyelitis in the Lewis rat results in loss of V beta 8.2+ and myelin basic protein-reactive cells from the spinal cord, with increased total T-cell apoptosis but reduced apoptosis of V beta 8.2+ cells.

    PubMed

    McCombe, P A; Nickson, I; Tabi, Z; Pender, M P

    1996-11-01

    We have studied the effects of corticosteroid treatment on the numbers of lymphocytes obtained from the spinal cords of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE) induced by inoculation with myelin basic protein (MBP) and adjuvants. Flow cytometric studies showed that treatment with dexamethasone (4 mg/kg) 8-12 h prior to study on day 14 after inoculation resulted in a reduction in the numbers of CD5+, TCR alpha beta + and V beta 8.2+ cells in the spinal cord. Limiting dilution analysis indicated that dexamethasone treatment 12 h prior to study on day 12 after inoculation reduced the frequencies of MBP-reactive and interleukin-2-responsive lymphocytes in the spinal cord to low levels, but reduced the frequency of concanavalin-A-responsive lymphocytes to a lesser extent. Using propidium iodide staining of nuclear chromatin we also studied lymphocyte apoptosis. Greater numbers of apoptotic cells were found in the cells extracted from the spinal cords of rats, examined on day 14, that had been treated 1-12 h previously with dexamethasone, than in saline-treated controls. This increased level of apoptosis was observed in the CD5+ and TCR alpha beta + cell populations. At 1-4 h after dexamethasone treatment there was a reduction in the selective apoptosis of V beta 8.2+ cells that normally occurs during spontaneous recovery from EAE. Therefore apoptosis of V beta 8.2+ cells cannot explain the reduction in the numbers of V beta 8.2+ cells and MBP-reactive cells in the CNS after dexamethasone treatment. By 8-12 h after dexamethasone treatment the selectivity of the apoptotic process was restored. These studies suggest that a reduction in the number of T-lymphocytes in the central nervous system contributes to the beneficial effects of corticosteroids in EAE. PMID:8898717

  16. Anticancer Activities of Pterostilbene-Isothiocyanate Conjugate in Breast Cancer Cells: Involvement of PPARγ

    PubMed Central

    Nikhil, Kumar; Sharan, Shruti; Singh, Abhimanyu K.; Chakraborty, Ajanta; Roy, Partha

    2014-01-01

    Trans-3,5-dimethoxy-4′-hydroxystilbene (PTER), a natural dimethylated analog of resveratrol, preferentially induces certain cancer cells to undergo apoptosis and could thus have a role in cancer chemoprevention. Peroxisome proliferator-activated receptor γ (PPARγ), a member of the nuclear receptor superfamily, is a ligand-dependent transcription factor whose activation results in growth arrest and/or apoptosis in a variety of cancer cells. Here we investigated the potential of PTER-isothiocyanate (ITC) conjugate, a novel class of hybrid compound (PTER-ITC) synthesized by appending an ITC moiety to the PTER backbone, to induce apoptotic cell death in hormone-dependent (MCF-7) and -independent (MDA-MB-231) breast cancer cell lines and to elucidate PPARγ involvement in PTER-ITC action. Our results showed that when pre-treated with PPARγ antagonists or PPARγ siRNA, both breast cancer cell lines suppressed PTER-ITC-induced apoptosis, as determined by annexin V/propidium iodide staining and cleaved caspase-9 expression. Furthermore, PTER-ITC significantly increased PPARγ mRNA and protein levels in a dose-dependent manner and modulated expression of PPARγ-related genes in both breast cancer cell lines. This increase in PPARγ activity was prevented by a PPARγ-specific inhibitor, in support of our hypothesis that PTER-ITC can act as a PPARγ activator. PTER-ITC-mediated upregulation of PPARγ was counteracted by co-incubation with p38 MAPK or JNK inhibitors, suggesting involvement of these pathways in PTER-ITC action. Molecular docking analysis further suggested that PTER-ITC interacted with 5 polar and 8 non-polar residues within the PPARγ ligand-binding pocket, which are reported to be critical for its activity. Collectively, our observations suggest potential applications for PTER-ITC in breast cancer prevention and treatment through modulation of the PPARγ activation pathway. PMID:25119466

  17. Reversal of P-glycoprotein-mediated multidrug resistance in human hepatoma cells by hedyotiscone A, a compound isolated from Hedyotis corymbosa.

    PubMed

    Yue, Grace Gar-Lee; Kin-Ming Lee, Julia; Cheng, Ling; Chung-Lap Chan, Ben; Jiang, Lei; Fung, Kwok-Pui; Leung, Ping-Chung; Bik-San Lau, Clara

    2012-06-01

    Multidrug resistance is a major problem in hepatocellular carcinoma. Hedyotiscone A, a compound isolated from Chinese herbal medicine Hedyotis corymbosa (HC, family Rubiaceae), was used as the chemical marker to distinguish between HC and an anticancer herb Hedyotis diffusa (HD) in our previous study. The present study aimed to investigate whether HA exhibited antiproliferative activities in multidrug-resistant hepatocellular carcinoma cells R-HepG2 and the parental cells HepG2 using MTT assay and [(3)H]-thymidine incorporation assay. Our results showed that HA could significantly inhibit cell proliferation in R-HepG2 and HepG2 (IC(50) = 43.7 and 56.3 µg/mL, respectively), but not in normal human liver cells WRL-68 (IC(50) > 100 µg/mL) cells, suggesting its selective cytotoxic effects. Besides, HA induced apoptosis in R-HepG2 cells, as confirmed by annexin-V & propidium iodide staining, and DNA fragmentation assay. The caspase cascade was activated as shown by a significant increase of cleaved caspases-3, -7 and -9 in HA-treated R-HepG2 cells. The activities and protein expression of P-glycoprotein as well as mRNA expression of MDR1 were also decreased in HA-treated R-HepG2 cells. Our study demonstrated for the first time the antiproliferative activities of hedyotiscone A in multidrug-resistant R-HepG2 cells. The findings revealed the potential of this compound in treating multidrug-resistant tumor. PMID:22352391

  18. Exogenous Nitric Oxide Protects Human Embryonic Stem Cell-Derived Cardiomyocytes against Ischemia/Reperfusion Injury

    PubMed Central

    Pálóczi, János; Varga, Zoltán V.; Szebényi, Kornélia; Sarkadi, Balázs; Madonna, Rosalinda; De Caterina, Raffaele; Csont, Tamás; Eschenhagen, Thomas; Ferdinandy, Péter; Görbe, Anikó

    2016-01-01

    Background and Aims. Human embryonic stem cell- (hESC-) derived cardiomyocytes are one of the useful screening platforms of potential cardiocytoprotective molecules. However, little is known about the behavior of these cardiomyocytes in simulated ischemia/reperfusion conditions. In this study, we have tested the cytoprotective effect of an NO donor and the brain type natriuretic peptide (BNP) in a screening platform based first on differentiated embryonic bodies (EBs, 6 + 4 days) and then on more differentiated cardiomyocytes (6 + 24 days), both derived from hESCs. Methods. Both types of hESC-derived cells were exposed to 150 min simulated ischemia, followed by 120 min reperfusion. Cell viability was assessed by propidium iodide staining. The following treatments were applied during simulated ischemia in differentiated EBs: the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) (10−7, 10−6, and 10−5 M), BNP (10−9, 10−8, and 10−7 M), and the nonspecific NO synthase inhibitor Nω-nitro-L-arginine (L-NNA, 10−5 M). Results. SNAP (10−6, 10−5 M) significantly attenuated cell death in differentiated EBs. However, simulated ischemia/reperfusion-induced cell death was not affected by BNP or by L-NNA. In separate experiments, SNAP (10−6 M) also protected hESC-derived cardiomyocytes. Conclusions. We conclude that SNAP, but not BNP, protects differentiated EBs or cardiomyocytes derived from hESCs against simulated ischemia/reperfusion injury. The present screening platform is a useful tool for discovery of cardiocytoprotective molecules and their cellular mechanisms. PMID:27403231

  19. Biological activity of ruthenium and osmium arene complexes with modified paullones in human cancer cells.

    PubMed

    Mühlgassner, Gerhard; Bartel, Caroline; Schmid, Wolfgang F; Jakupec, Michael A; Arion, Vladimir B; Keppler, Bernhard K

    2012-11-01

    In an attempt to combine the ability of indolobenzazepines (paullones) to inhibit cyclin-dependent kinases (Cdks) and that of platinum-group metal ions to interact with proteins and DNA, ruthenium(II) and osmium(II) arene complexes with paullones were prepared, expecting synergies and an increase of solubility of paullones. Complexes with the general formula [M(II)Cl(η(6)-p-cymene)L]Cl, where M=Ru (1, 3) or Os (2, 4), and L=L(1) (1, 2) or L(2) (3, 4), L(1)=N-(9-bromo-7,12-dihydroindolo[3,2-d][1]-benzazepin-6(5H)-yliden-N'-(2-hydroxybenzylidene)azine and L(2)=N-(9-bromo-7,12-dihydroindolo[3,2-d][1]benzazepin-6-yl)-N'-[3-hydroxy-5-(hydroxymethyl)-2-methylpyridin-4-yl-methylene]azinium chloride (L(2)(*)HCl), were now investigated regarding cytotoxicity and accumulation in cancer cells, impact on the cell cycle, capacity of inhibiting DNA synthesis and inducing apoptosis as well as their ability to inhibit Cdk activity. The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay yielded IC(50) values in the nanomolar to low micromolar range. In accordance with cytotoxicity data, the BrdU assay showed that 1 is the most and 4 the least effective of these compounds regarding inhibition of DNA synthesis. Effects on the cell cycle are minor, although concentration-dependent inhibition of Cdk2/cyclin E activity was observed in cell-free experiments. Induction of apoptosis is most pronounced for complex 1, accompanied by a low fraction of necrotic cells, as observed by annexin V-fluorescein isothiocyanate/propidium iodide staining and flow cytometric analysis. PMID:23037896

  20. Biological activity of ruthenium and osmium arene complexes with modified paullones in human cancer cells

    PubMed Central

    Mühlgassner, Gerhard; Bartel, Caroline; Schmid, Wolfgang F.; Jakupec, Michael A.; Arion, Vladimir B.; Keppler, Bernhard K.

    2012-01-01

    In an attempt to combine the ability of indolobenzazepines (paullones) to inhibit cyclin-dependent kinases (Cdks) and that of platinum-group metal ions to interact with proteins and DNA, ruthenium(II) and osmium(II) arene complexes with paullones were prepared, expecting synergies and an increase of solubility of paullones. Complexes with the general formula [MIICl(η6-p-cymene)L]Cl, where M = Ru (1, 3) or Os (2, 4), and L = L1 (1, 2) or L2 (3, 4), L1 = N-(9-bromo-7,12-dihydroindolo[3,2-d][1]-benzazepin-6(5H)-yliden-N′-(2-hydroxybenzylidene)azine and L2 = N-(9-bromo-7,12-dihydroindolo[3,2-d][1]benzazepin-6-yl)-N′-[3-hydroxy-5-(hydroxymethyl)-2-methylpyridin-4-yl-methylene]azinium chloride (L2*HCl), were now investigated regarding cytotoxicity and accumulation in cancer cells, impact on the cell cycle, capacity of inhibiting DNA synthesis and inducing apoptosis as well as their ability to inhibit Cdk activity. The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay yielded IC50 values in the nanomolar to low micromolar range. In accordance with cytotoxicity data, the BrdU assay showed that 1 is the most and 4 the least effective of these compounds regarding inhibition of DNA synthesis. Effects on the cell cycle are minor, although concentration-dependent inhibition of Cdk2/cyclin E activity was observed in cell-free experiments. Induction of apoptosis is most pronounced for complex 1, accompanied by a low fraction of necrotic cells, as observed by annexin V–fluorescein isothiocyanate/propidium iodide staining and flow cytometric analysis. PMID:23037896

  1. Pterostilbene carboxaldehyde thiosemicarbazone, a resveratrol derivative inhibits 17β-Estradiol induced cell migration and proliferation in HUVECs.

    PubMed

    Nikhil, Kumar; Sharan, Shruti; Wishard, Rohan; Palla, Srinivasa Rao; Krishna Peddinti, Rama; Roy, Partha

    2016-04-01

    Angiogenesis plays important roles in tumor growth and metastasis, thus development of a novel angiogenesis inhibitor is essential for the improvement of therapeutics against cancer. Thrombospondins-1 (TSP-1) is a potent endogenous inhibitor of angiogenesis that acts through direct effects on endothelial cell migration, proliferation, survival, and activating apoptotic pathways. TSP-1 has been shown to disrupt estrogen-induced endothelial cell proliferation and migration. Here we investigated the potential of pterostilbene carboxaldehyde thiosemicarbazone (PTERC-T), a novel resveratrol (RESV) derivative, to inhibit angiogenesis induced by female sex steroids, particularly 17β-Estradiol (E2), on Human umbilical vein endothelial cells (HUVECs) and to elucidate the involvement of TSP-1 in PTERC-T action. Our results showed that PTERC-T significantly inhibited 17β-E2-stimulated proliferation of HUVECs and induced apoptosis as determined by annexin V/propidium iodide staining and cleaved caspase-3 expression. Furthermore, PTERC-T also inhibited endothelial cell migration, and invasion in chick chorioallantoic membrane (CAM) assay. In contrast, RESV failed to inhibit 17β-E2 induced HUVECs proliferation and invasion at similar dose. PTERC-T was also found to increase TSP-1 protein expression levels in a dose-dependent manner which, however, was counteracted by co-incubation with p38MAPK or JNK inhibitors, suggesting involvement of these pathways in PTERC-T action. These results suggest that the inhibitory effect of PTERC-T on 17β-E2 induced angiogenesis is associated, at least in part, with its induction of endothelial cell apoptosis and inhibition of cell migration through targeting TSP-1. Thus, PTERC-T could be considered as a potential lead compound for developing a class of new drugs targeting angiogenesis-related diseases. PMID:26850466

  2. Troxerutin, a natural flavonoid binds to DNA minor groove and enhances cancer cell killing in response to radiation.

    PubMed

    Panat, Niranjan A; Singh, Beena G; Maurya, Dharmendra K; Sandur, Santosh K; Ghaskadbi, Saroj S

    2016-05-01

    Troxerutin, a flavonoid best known for its radioprotective and antioxidant properties is of considerable interest of study due to its broad pharmacological activities. The present study on troxerutin highlights its abilities to bind DNA and enhance cancer cell killing in response to radiation. Troxerutin showed strong binding with calf thymus DNA in vitro. Troxerutin-DNA interaction was confirmed by CD spectropolarimetry. The mode of binding of troxerutin to DNA was assessed by competing troxerutin with EtBr or DAPI, known DNA intercalator and a minor groove binder, respectively. DAPI fluorescence was drastically reduced with linear increase in troxerutin concentration suggesting possible binding of troxerutin to DNA minor groove. Further, computational studies of docking of troxerutin molecule on mammalian DNA also indicated possible troxerutin-DNA interaction at minor groove of DNA. Troxerutin was found to mainly localize in the nucleus of prostate cancer cells. It induced cytotoxicity in radioresistant (DU145) and sensitive (PC3) prostate cancer cells. When troxerutin pre-treated DU145 and PC3 cells were exposed to γ-radiation, cytotoxicity as estimated by MTT assay, was found to be further enhanced. In addition, the % subG1 population detected by propidium iodide staining also showed similar response when combined with radiation. A similar trend was observed in terms of ROS generation and DNA damage in DU145 cells when troxerutin and radiation were combined. DNA binding at minor groove by troxerutin may have contributed to strand breaks leading to increased radiation induced cell death. PMID:27016192

  3. Microarray analysis of nemorosone-induced cytotoxic effects on pancreatic cancer cells reveals activation of the unfolded protein response (UPR)

    PubMed Central

    Holtrup, Frank; Bauer, Andrea; Fellenberg, Kurt; Hilger, Ralf A; Wink, Michael; Hoheisel, Jörg D

    2011-01-01

    BACKGROUND AND PURPOSE Pancreatic cancer is one of the leading cancer-related causes of death due to high chemo-resistance and fast metastasation. Nemorosone, a polycyclic polyprenylated acylphloroglucinol, has recently been identified as a promising anticancer agent. Here, we examine its growth-inhibitory effects on pancreatic cancer cells. Based on transcription profiling, a molecular mode of action is proposed. EXPERIMENTAL APPROACH Nemorosone cytotoxicity was assessed by the resazurin proliferation assay on pancreatic cancer cells and fibroblasts. Apoptosis was determined by Annexin V/propidium iodide staining as well as cytochrome c and caspase activation assays. Staining with the voltage-dependent dye JC-1 and fluorescence microscopy were used to detect effects on mitochondrial membrane potential. Total RNA was isolated from treated cell lines and subjected to microarray analysis, subsequent pathway identification and modelling. Gene expression data were validated by quantitative polymerase chain reaction and siRNA-mediated gene knock-down. KEY RESULTS Nemorosone significantly inhibited cancer cell growth, induced cytochrome c release and subsequent caspase-dependent apoptosis, rapidly abolished mitochondrial membrane potential and elevated cytosolic calcium levels, while fibroblasts were largely unaffected. Expression profiling revealed 336 genes to be affected by nemorosone. A total of 75 genes were altered in all three cell lines, many of which were within the unfolded protein response (UPR) network. DNA damage inducible transcript 3 was identified as a key regulator in UPR-mediated cell death. CONCLUSIONS AND IMPLICATIONS Nemorosone could be a lead compound for the development of novel anticancer drugs amplifying the already elevated UPR level in solid tumours, thus driving them into apoptosis. This study forms the basis for further investigations identifying nemorosone's direct molecular target(s). PMID:21091652

  4. Neuroprotective effects of dimerumic acid and deferricoprogen from Monascus purpureus NTU 568-fermented rice against 6-hydroxydopamine-induced oxidative stress and apoptosis in differentiated pheochromocytoma PC-12 cells.

    PubMed

    Tseng, Wei-Ting; Hsu, Ya-Wen; Pan, Tzu-Ming

    2016-08-01

    Context Oxidative stress plays a key role in neurodegenerative disorders, including Parkinson's disease (PD). Rice fermented with Monascus purpureus Went (Monascaceae) NTU 568 (red mould rice) was found to contain antioxidants, including dimerumic acid (DMA) and deferricoprogen (DFC). Objective The effects of DMA and DFC on 6-hydroxydopamine (6-OHDA)-induced cytotoxicity and potential protective mechanisms in differentiated PC-12 pheochromocytoma cells were investigated. Materials and methods DMA (0-60 μM) or DFC (0-10 μM) was co-treated with 6-OHDA (200 μM, 24 h exposure) in differentiated PC-12 cells. Cell viability and intercellular reactive oxygen species (ROS) were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assays, respectively. Cell apoptosis was determined by DNA fragmentation analysis and propidium iodide staining by flow cytometry. Western blot analysis was used to measure the levels of cell protein expression. Results DMA and DFC significantly increased cell viability to 72% and 81% in 6-OHDA-induced differentiated PC-12 cell cultures, respectively. Furthermore, DMA and DFC reduced 6-OHDA-induced formation of extracellular and intercellular ROS by 25% and 20%, respectively, and decreased NADPH oxidase-2 expression in differentiated PC-12 cells. DMA and DFC inhibited 6-OHDA-induced apoptosis and decreased activation of caspase-3 via regulation of Bcl-2-associated X protein (Bax) and Bcl-2 protein expression in differentiated PC-12 cells. Conclusion DMA and DFC may protect against 6-OHDA toxicity by inhibiting ROS formation and apoptosis. These results showed that the metabolites from M. purpureus NTU 568 fermentation were potential therapeutic agents for PD induced by oxidative damage and should be encouraged for further research. PMID:26794209

  5. Anticancer Activity of Cobra Venom Polypeptide, Cytotoxin-II, against Human Breast Adenocarcinoma Cell Line (MCF-7) via the Induction of Apoptosis

    PubMed Central

    Shirazi, Farshad H.; Vatanpour, Hosein; zare, Abas; Kobarfard, Farzad; Rabiei, Hadi

    2014-01-01

    Purpose Breast cancer is a significant health problem worldwide, accounting for a quarter of all cancer diagnoses in women. Current strategies for breast cancer treatment are not fully effective, and there is substantial interest in the identification of novel anticancer agents especially from natural products including toxins. Cytotoxins are polypeptides found in the venom of cobras and have various physiological effects. In the present study, the anticancer potential of cytotoxin-II against the human breast adenocarcinoma cell line (MCF-7) was investigated. Methods The cytotoxic effects of cytotoxin-II were determined by morphological analysis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The mode and mechanism of cell death were investigated via acridine orange/ethidium bromide (AO/EtBr) double staining, flow cytometric analysis of cell death, detection of mitochondrial membrane potential, measurement of intracellular reactive oxygen species (ROS), annexin V/propidium iodide staining, and caspase-9 activity assays. Results The half maximal inhibitory concentration (IC50) of cytotoxin-II in MCF-7 cells was 4.18±1.23 µg/mL, while the value for cisplatin was approximately 28.02±1.87 µg/mL. Morphological analysis and AO/EtBr double staining showed typical manifestations of apoptotic cell death (in doses lower than 8 µg/mL). Dose- and time-dependent ROS generation, loss of mitochondrial membrane potential, caspase-9 activation, and cell cycle arrest were observed in their respective tests. Conclusion In conclusion, cytotoxin-II has potent anticancer effects in the MCF-7 cell line, which are induced via the intrinsic pathways of apoptosis. Based on these findings, cytotoxin-II is a suitable choice for breast cancer treatment. PMID:25548578

  6. Hesperidin from Citrus seed induces human hepatocellular carcinoma HepG2 cell apoptosis via both mitochondrial and death receptor pathways.

    PubMed

    Banjerdpongchai, Ratana; Wudtiwai, Benjawan; Khaw-On, Patompong; Rachakhom, Wasitta; Duangnil, Natthachai; Kongtawelert, Prachya

    2016-01-01

    Citrus seeds are full of phenolic compounds, such as flavonoids. The aims of this study were to identify the types of flavonoids in Citrus seed extracts, the cytotoxic effect, mode of cell death, and signaling pathway in human hepatic cancer HepG2 cells. The flavonoids contain anticancer, free radical scavenging, and antioxidant activities. Neohesperidin, hesperidin, and naringin, active flavanone glycosides, were identified in Citrus seed extract. The cytotoxic effect of three compounds was in a dose-dependent manner, and IC50 levels were determined. The sensitivity of human HepG2 cells was as follows: hesperidin > naringin > neohesperidin > naringenin. Hesperidin induced HepG2 cells to undergo apoptosis in a dose-dependent manner as evidenced by the externalization of phosphatidylserine and determined by annexin V-fluorescein isothiocyanate and propidium iodide staining using flow cytometry. Hesperidin did not induce the generation of reactive oxygen species, which was determined by using 2',7'-dichlorohydrofluorescein diacetate and flow cytometry method. The number of hesperidin-treated HepG2 cells with the loss of mitochondrial transmembrane potential increased concentration dependently, using 3,3'-dihexyloxacarbocyanine iodide employing flow cytometry. Caspase-9, -8, and -3 activities were activated and increased in hesperidin-treated HepG2 cells. Bcl-xL protein was downregulated whereas Bax, Bak, and tBid protein levels were upregulated after treatment with hesperidin in a dose-dependent manner. In conclusion, the bioflavanone from Citrus seeds, hesperidin, induced human HepG2 cell apoptosis via mitochondrial pathway and death receptor pathway. Citrus seed flavonoids are beneficial and can be developed as anticancer drug or food supplement, which still needs further in vivo investigation in animals and human beings. PMID:26194866

  7. Anticancer Activity of Certain Herbs and Spices on the Cervical Epithelial Carcinoma (HeLa) Cell Line

    PubMed Central

    Berrington, Danielle; Lall, Namrita

    2012-01-01

    Acetone extracts of selected plant species were evaluated for their in vitro cytotoxicity against a noncancerous African green monkey kidney (Vero) cell line and an adenocarcinoma cervical cancer (HeLa) cell line. The plants studied were Origanum vulgare L. (Oregano), Rosmarinus officinalis L. (Upright and ground cove rosemary), Lavandula spica L. (Lavender), Laurus nobilis L. (Bay leaf), Thymus vulgaris L. (Thyme), Lavandula x intermedia L. (Margaret Roberts Lavender), Petroselinum crispum Mill. (Curly leaved parsley), Foeniculum vulgare Mill. (Fennel), and Capsicum annuum L. (Paprika). Antioxidant activity was determined using a quantitative DPPH (1,1-diphenyl-2-picryl hydrazyl) assay. The rosemary species exhibited effective radical scavenging capacity with 50% inhibitory concentration (IC50) of 3.48 ± 0.218 μg/mL and 10.84 ± 0.125 μg/mL and vitamin C equivalents of 0.351 g and 1.09 g for McConnell's Blue and Tuscan Blue, respectively. Cytotoxicity was measured using XTT (Sodium 3′-[1-(phenyl amino-carbonyl)-3,4-tetrazolium]-bis-[4-methoxy-6-nitro] benzene sulfonic acid hydrate) colorimetric assay. Only L. nobilis and O. vulgare exhibited pronounced effects on the HeLa cell line. Dose-dependent studies revealed IC50 of 34.46 ± 0.48 μg/mL and 126.3 ± 1.00 μg/mL on the HeLa cells and on the Vero cells 124.1 μg/mL ± 18.26 and 163.8 μg/mL ± 2.95 for L. nobilis and O. vulgare, respectively. Light (eosin and haematoxylin staining) and confocal microscopy (Hoechst 33342, acridine orange, and propidium iodide staining) were used to evaluate the cytotoxic mechanism of action for L. nobilis and O. vulgare. PMID:22649474

  8. A plasmacytoid dendritic cell (CD123+/CD11c-) based assay system to predict contact allergenicity of chemicals

    PubMed Central

    Ayehunie, Seyoum; Snell, Maureen; Child, Matthew; Klausner, Mitchell

    2009-01-01

    A predictive allergenicity test system for assessing the contact allergenicity of chemicals is needed by the cosmetic and pharmaceutical industry to monitor product safety in the marketplace. Development of such non-animal alternative assay systems for skin sensitization and hazard identification has been pursued by policy makers and regulatory agencies. We investigated whether phenotypic and functional changes to a subset of dendritic cells (DC), plasmacytoid DC (pDC), could be used to identify contact allergens. To achieve this goal, normal human DC were generated from CD34+ progenitor cells and cryopreserved. Frozen DC were thawed and the pDC fraction (CD123+/CD11c-) was harvested using FACS sorting. The pDC were cultured, expanded, and exposed to chemical allergens (N=26) or non-allergens (N=22). Concentrations of each chemical that resulted in >50% viability was determined using FACS analysis of propidium iodide stained cells using pDC from 2-5 donors. Expression of the surface marker, CD86, which has been implicated in dendritic cell maturation, was used as a marker of allergenicity. CD86 expression increased (≥ 1.5 fold) for 25 of 26 allergens (sensitivity = 96%) but did not increase for 19 of 22 non-allergens (specificity = 86%). In a direct comparison to historical data for the regulatory approved, mouse local lymph node assay (LLNA) for 23 allergens and 22 non-allergens, the pDC method had sensitivity and specificity of 96% and 86%, respectively, while the sensitivity and specificity of the LLNA assay was 83% and 82%, respectively. In conclusion, CD86 expression in pDC appears to be a sensitive and specific indicator to identify contact allergenicity. Such an assay method utilizing normal human cells will be useful for high throughput screening of chemicals for allergenicity. PMID:19665512

  9. Anticancer Activity of Certain Herbs and Spices on the Cervical Epithelial Carcinoma (HeLa) Cell Line.

    PubMed

    Berrington, Danielle; Lall, Namrita

    2012-01-01

    Acetone extracts of selected plant species were evaluated for their in vitro cytotoxicity against a noncancerous African green monkey kidney (Vero) cell line and an adenocarcinoma cervical cancer (HeLa) cell line. The plants studied were Origanum vulgare L. (Oregano), Rosmarinus officinalis L. (Upright and ground cove rosemary), Lavandula spica L. (Lavender), Laurus nobilis L. (Bay leaf), Thymus vulgaris L. (Thyme), Lavandula x intermedia L. (Margaret Roberts Lavender), Petroselinum crispum Mill. (Curly leaved parsley), Foeniculum vulgare Mill. (Fennel), and Capsicum annuum L. (Paprika). Antioxidant activity was determined using a quantitative DPPH (1,1-diphenyl-2-picryl hydrazyl) assay. The rosemary species exhibited effective radical scavenging capacity with 50% inhibitory concentration (IC(50)) of 3.48 ± 0.218 μg/mL and 10.84 ± 0.125 μg/mL and vitamin C equivalents of 0.351 g and 1.09 g for McConnell's Blue and Tuscan Blue, respectively. Cytotoxicity was measured using XTT (Sodium 3'-[1-(phenyl amino-carbonyl)-3,4-tetrazolium]-bis-[4-methoxy-6-nitro] benzene sulfonic acid hydrate) colorimetric assay. Only L. nobilis and O. vulgare exhibited pronounced effects on the HeLa cell line. Dose-dependent studies revealed IC(50) of 34.46 ± 0.48 μg/mL and 126.3 ± 1.00 μg/mL on the HeLa cells and on the Vero cells 124.1 μg/mL ± 18.26 and 163.8 μg/mL ± 2.95 for L. nobilis and O. vulgare, respectively. Light (eosin and haematoxylin staining) and confocal microscopy (Hoechst 33342, acridine orange, and propidium iodide staining) were used to evaluate the cytotoxic mechanism of action for L. nobilis and O. vulgare. PMID:22649474

  10. Effect of methoxychlor on Ca²⁺ homeostasis and apoptosis in HA59T human hepatoma cells.

    PubMed

    Horng, Chi-Ting; Chou, Chiang-Ting; Tseng, Hui-Wen; Cheng, Jin-Shiung; Chang, Hong-Tai; Chang, Po-Min; Chen, I-Li; Hung, Ming-Chi; Tsai, Yi-Jen; Tsai, Peng-Chih; Liang, Wei-Zhe; Kuo, Chun-Chi; Kuo, Daih-Huang; Ho, Chin-Man; Lin, Jia-Rong; Shieh, Pochuen; Jan, Chung-Ren

    2015-02-28

    Methoxychlor, an organochlorine pesticide, is thought to be an endocrine disrupter that affects Ca²⁺ homeostasis and cell viability in different cell models. This study explored the action of methoxychlor on cytosolic free Ca²⁺ concentrations ([Ca²⁺]i) and apoptosis in HA59T human hepatoma cells. Fura-2, a Ca²⁺-sensitive fluorescent dye, was applied to measure [Ca²⁺]i. Methoxychlor at concentrations of 0.1-1 μM caused a [Ca²⁺]i rise in a concentration-dependent manner. Removal of external Ca²⁺ abolished methoxychlor's effect. Methoxychlor-induced Ca²⁺ influx was confirmed by Mn²⁺-induced quench of fura-2 fluorescence. Methoxychlor-induced Ca²⁺ entry was inhibited by nifedipine, econazole, SK&F96365, and protein kinase C modulators. Methoxychlor killed cells at concentrations of 10-130 μM in a concentration-dependent fashion. Chelation of cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid/AM (BAPTA/AM) did not prevent methoxychlor's cytotoxicity. Methoxychlor (10 and 50 μM) induced apoptosis concentration-dependently as determined by using Annexin V/propidium iodide staining. Together, in HA59T cells, methoxychlor induced a [Ca²⁺]i rise by inducing Ca²⁺ entry via protein kinase C-sensitive Ca²⁺-permeable channels, without causing Ca²⁺ release from stores. Methoxychlor also induced apoptosis that was independent of [Ca²⁺]i rises. PMID:25687486

  11. Targeting of RUNX3 by miR-130a and miR-495 cooperatively increases cell proliferation and tumor angiogenesis in gastric cancer cells

    PubMed Central

    Lee, Sun Hee; Jung, Yuk Dong; Choi, Young Sun; Lee, You Mie

    2015-01-01

    Mature microRNAs (miRNAs) are 21 to 23 nucleotide noncoding RNA molecules that can downregulate multiple gene expression by mRNA degradation or translational repression. miRNAs are considered to play important roles in cell proliferation, apoptosis, and differentiation during mammalian development. The Runt-related transcription factor 3 (RUNX3) expression and activity are frequently downregulated by various mechanisms in gastric cancer. We have reported that RUNX3 inactivation is crucial for early tumorigenesis. In this study, we investigated the role of miRNAs targeting RUNX3 in early tumorigenesis. miR-130a and miR-495 upregulated under hypoxic conditions that bind to the RUNX3 3′-untranslated region (3′-UTR) were identified in gastric cancer cells by using microarray analysis and bioinformatics programs. Combination of miR-130a and miR-495 inhibited RUNX3 expression at the protein level, but not at the mRNA level. miR-130a and miR-495 significantly inhibited the RUNX3–3′UTR-luciferase activity. Combination of miR-130a and miR-495 significantly decreased apoptosis determined by Annexin V-FITC/propidium iodide staining and flow cytometric analysis, and the expression of Bim in SNU484 gastric cancer cells. In addition, p21 and Bim, RUNX3 target genes, were completely downregulated by the combination of miR-130a and miR-495. Using matrigel plug assay, we found that antagomiRs specific for miR-130a and miR-495 significantly reduced angiogenesis in vivo. In conclusion, targeting miR-130a and miR-495 could be a potential therapeutics to recover RUNX3 expression under hypoxic conditions and in early tumorigenic progression. PMID:26375442

  12. Evaluation of the effect of 2,4-dichlorophenol on oxidative parameters and viability of human blood mononuclear cells (in vitro).

    PubMed

    Bukowska, B; Wieteska, P; Kwiatkowska, M; Sicińska, P; Michalowicz, J

    2016-07-01

    2,4-Dichlorophenol (2,4-DCP) is formed in drinking water as a result of its chlorination, and it is created in the environment during transformation of various xenobiotics such as triclosan or herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The molecular mechanism depicting the action of phenolic compounds on nucleated blood cells has been insufficiently studied, and therefore, we have assessed the effect of 2,4-DCP on the structure and viability of human peripheral blood mononuclear cells (PBMCs). We have evaluated necrotic, apoptotic, and morphological changes (alterations in the size and granulation) in PBMCs incubated with 2,4-DCP in the concentration ranging from 10 to 500 µg mL(-1) for 4 h at 37°C. Moreover, we have estimated changes in reactive oxygen species (ROS) formation, lipid peroxidation, and protein carbonylation in the incubated cells. We have noted that 2,4-DCP increased ROS formation and lipid peroxidation (from 10 µg mL(-1)) and oxidized proteins (from 50 µg mL(-1)) in PBMCs. The compound studied also provoked apoptotic (from 50 µg mL(-1)), necrotic (from 100 µg mL(-1)) and alterations in the size and granulation (from 50 µg mL(-1)) in the incubated cells. The analysis of quinolinium 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethyl-ammonio)-propyl]-diiodide/propidium iodide staining revealed that 2,4-DCP (50-250 µg mL(-1)) more strongly increased the number of apoptotic than necrotic cells, which suggests that this cell death type is mainly provoked by this compound in PBMCs. The observed changes were caused by relatively high concentrations of 2,4-DCP, which cannot influence human organism during environmental exposure and thus may only occur as a result of acute or subacute poisoning with this compound. PMID:26391574

  13. A novel manganese complex selectively induces malignant glioma cell death by targeting mitochondria.

    PubMed

    Geng, Ji; Li, Jing; Huang, Tao; Zhao, Kaidi; Chen, Qiuyun; Guo, Wenjie; Gao, Jing

    2016-09-01

    Despite advances in treatment, malignant glioma commonly exhibits recurrence, subsequently leading to a poor prognosis. As manganese (Mn) compounds can be transported by the transferrin‑transferrin receptor system, the present study synthesized and examined the potential use of Adpa‑Mn as a novel antitumor agent. Adpa‑Mn time and dose‑dependently inhibited U251 and C6 cell proliferation; however, it had little effect on normal astrocytes. Apoptosis was significantly elevated following treatment with Adpa‑Mn, as detected by chromatin condensation, Annexin V/propidium iodide staining, cytochrome c release from mitochondria to the cytoplasm, and the activation of caspases‑9, ‑7 and ‑3 and poly (ADP‑ribose) polymerase. In addition, Adpa‑Mn enhanced fluorescence intensity of monodansylcadaverine and elevated the expression levels of the autophagy‑related protein microtubule‑associated protein 1 light chain 3. Pretreatment with the autophagy inhibitors 3‑methyladenine and chloroquine enhanced Adpa‑Mn‑induced cell inhibition, thus indicating that autophagy has an essential role in this process. Furthermore, evidence of mitochondrial dysfunction was detected in the Adpa‑Mn‑treated group, including disrupted membrane potential, elevated levels of reactive oxygen species (ROS) and depleted adenosine triphosphate. Conversely, treatment with the mitochondrial permeability transition inhibitor cyclosporin A reversed Adpa‑Mn‑induced ROS production, mitochondrial damage and cell apoptosis, thus suggesting that Adpa‑Mn may target the mitochondria. Taken together, these data suggested that Adpa‑Mn may be considered for use as a novel anti‑glioma therapeutic option. PMID:27432745

  14. The Mechanism of Safrole-Induced [Ca²⁺]i Rises and Non-Ca²⁺-Triggered Cell Death in SCM1 Human Gastric Cancer Cells.

    PubMed

    Hung, Tzu-Yi; Chou, Chiang-Ting; Sun, Te-Kung; Liang, Wei-Zhe; Cheng, Jin-Shiung; Fang, Yi-Chien; Li, Yih-Do; Shieh, Pochuen; Ho, Chin-Man; Kuo, Chun-Chi; Lin, Jia-Rong; Kuo, Daih-Huang; Jan, Chung-Ren

    2015-10-31

    Safrole is a carcinogen found in plants. The effect of safrole on cytosolic free Ca²⁺ concentrations ([Ca²⁺](i)) and viability in SCM1 human gastric cancer cells was explored. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺](i). Safrole at concentrations of 150-450 μM induced a [Ca²⁺](i) rise in a concentration-dependent manner. The response was reduced by 60% by removing extracellular Ca²⁺. Safrole-evoked Ca²⁺ entry was not altered by nifedipine, econazole, SKF96365, and protein kinase C activator or inhibitor. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished safrole-evoked [Ca²⁺](i) rises. Conversely, treatment with safrole abolished thapsigargin or BHQ-evoked [Ca²⁺](i) rises. Inhibition of phospholipase C (PLC) with U73122 abolished safrole-induced [Ca²⁺](i) rises. At 250-550 μM, safrole decreased cell viability concentration-dependently, which was not reversed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/propidium iodide staining data suggest that safrole (350-550 μM) induced apoptosis concentration-dependently. These studies suggest that in SCM1 human gastric cancer cells, safrole induced [Ca²⁺](i) rises by inducing PLC-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via non-store-operated Ca²⁺ entry pathways. Safrole-induced cell death may involve apoptosis. PMID:26387654

  15. SDF-1/CXCR4 axis induces apoptosis of human degenerative nucleus pulposus cells via the NF-κB pathway

    PubMed Central

    LIU, ZONGCHAO; MA, CHUAN; SHEN, JIELIANG; WANG, DAWU; HAO, JIE; HU, ZHENMING

    2016-01-01

    Intervertebral disc degeneration (IVDD) is a major cause of lower back pain, and increased cell apoptosis is a key characteristic of IVDD. The present study aimed to investigate the effects and mechanism of the stromal cell-derived factor-1 (SDF-1)/C-X-C motif chemokine receptor 4 (CXCR4) axis on apoptosis in human degenerative nucleus pulposus cells (NPCs). The expression levels of SDF-1 and CXCR4 in human intervertebral discs (IVD) were determined using immunohistochemistry and western blot analysis. Apoptosis of primary cultured NPCs was quantified by Annexin V/propidium iodide staining following stimulation with SDF-1 and knockdown of CXCR4 using small interfering RNA (siRNA). The association with the nuclear factor-κB (NF-κB) signaling pathway was investigated using CXCR4-siRNA and NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), treatment. The results demonstrated that SDF-1 and its receptor, CXCR4, were upregulated in degenerative IVD samples compared with normal samples. Stimulation with SDF-1 increased the level of apoptosis in cultured NPCs, and conversely, the apoptosis level was suppressed post-transfection with CXCR4 siRNA compared with SDF-1 stimulation alone. Furthermore, SDF-1 treatment increased the level of phosphorylated NF-κB subunit P65, which was downregulated following CXCR4 siRNA and PDTC treatment. In addition, CXCR4 siRNA and PDTC inhibited the nuclear translocation of P65, which was induced by SDF-1. Taken together, SDF-1-mediated apoptosis was suppressed by NF-κB inhibition using PDTC. In conclusion, the SDF-1/CXCR4 axis promoted cell apoptosis in human degenerative NPCs via the NF-κB pathway, thus suggesting that SDF-1/CXCR signaling may be a therapeutic target for the treatment of degenerative IVD diseases. PMID:27220474

  16. Crystal Structure of Crataeva tapia Bark Protein (CrataBL) and Its Effect in Human Prostate Cancer Cell Lines

    PubMed Central

    Ferreira, Joana Gasperazzo; Silva, Mariana Cristina Cabral; Silva-Lucca, Rosemeire Aparecida; Mentele, Reinhard; Paredes-Gamero, Edgar Julian; Bertolin, Thiago Carlos; dos Santos Correia, Maria Tereza; Paiva, Patrícia Maria Guedes; Gustchina, Alla; Wlodawer, Alexander; Oliva, Maria Luiza Vilela

    2013-01-01

    A protein isolated from the bark of Crataeva tapia (CrataBL) is both a Kunitz-type plant protease inhibitor and a lectin. We have determined the amino acid sequence and three-dimensional structure of CrataBL, as well as characterized its selected biochemical and biological properties. We found two different isoforms of CrataBL isolated from the original source, differing in positions 31 (Pro/Leu); 92 (Ser/Leu); 93 (Ile/Thr); 95 (Arg/Gly) and 97 (Leu/Ser). CrataBL showed relatively weak inhibitory activity against trypsin (Kiapp = 43 µM) and was more potent against Factor Xa (Kiapp = 8.6 µM), but was not active against a number of other proteases. We have confirmed that CrataBL contains two glycosylation sites and forms a dimer at high concentration. The high-resolution crystal structures of two different crystal forms of isoform II verified the β-trefoil fold of CrataBL and have shown the presence of dimers consisting of two almost identical molecules making extensive contacts (∼645 Å2). The structure differs from those of the most closely related proteins by the lack of the N-terminal β-hairpin. In experiments aimed at investigating the biological properties of CrataBL, we have shown that addition of 40 µM of the protein for 48 h caused maximum growth inhibition in MTT assay (47% of DU145 cells and 43% of PC3 cells). The apoptosis of DU145 and PC3 cell lines was confirmed by flow cytometry using Annexin V/FITC and propidium iodide staining. Treatment with CrataBL resulted in the release of mitochondrial cytochrome c and in the activation of caspase-3 in DU145 and PC3 cells. PMID:23823708

  17. The small molecule curcumin analog FLLL32 induces apoptosis in melanoma cells via STAT3 inhibition and retains the cellular response to cytokines with anti-tumor activity

    PubMed Central

    2010-01-01

    Background We characterized the biologic effects of a novel small molecule STAT3 pathway inhibitor that is derived from the natural product curcumin. We hypothesized this lead compound would specifically inhibit the STAT3 signaling pathway to induce apoptosis in melanoma cells. Results FLLL32 specifically reduced STAT3 phosphorylation at Tyr705 (pSTAT3) and induced apoptosis at micromolar amounts in human melanoma cell lines and primary melanoma cultures as determined by annexin V/propidium iodide staining and immunoblot analysis. FLLL32 treatment reduced expression of STAT3-target genes, induced caspase-dependent apoptosis, and reduced mitochondrial membrane potential. FLLL32 displayed specificity for STAT3 over other homologous STAT proteins. In contrast to other STAT3 pathway inhibitors (WP1066, JSI-124, Stattic), FLLL32 did not abrogate IFN-γ-induced pSTAT1 or downstream STAT1-mediated gene expression as determined by Real Time PCR. In addition, FLLL32 did not adversely affect the function or viability of immune cells from normal donors. In peripheral blood mononuclear cells (PBMCs), FLLL32 inhibited IL-6-induced pSTAT3 but did not reduce signaling in response to immunostimulatory cytokines (IFN-γ, IL 2). Treatment of PBMCs or natural killer (NK) cells with FLLL32 also did not decrease viability or granzyme b and IFN-γ production when cultured with K562 targets as compared to vehicle (DMSO). Conclusions These data suggest that FLLL32 represents a lead compound that could serve as a platform for further optimization to develop improved STAT3 specific inhibitors for melanoma therapy. PMID:20576164

  18. Apoptosis in MCF-7 breast cancer cells induced by S-alkenylmercaptocysteine (CySSR) species derived from Allium tissues in combination with sodium selenite.

    PubMed

    Zhang, Wei; Xiao, Hang; Parkin, Kirk L

    2014-06-01

    S-Allylmercaptocysteine (CySSA) from garlic is known to exhibit anti-cancer effects. Apoptosis induction by CySSA was contrasted with S-1-propenylmercaptocysteine (CySSPe) (the major onion analog) in the presence of Na2SeO3 (Se) in breast cancer cells MCF-7. The dose of CySSA or CySSPe alone required to reduce viable cells by 50% was >400μM, and this was reduced to 62μM and 91μM for CySSA+Se and CySSPe+Se, respectively, at molar ratios of 39:1. Synergism of the mixtures was confirmed by isobologram analysis and the treatments evoked enhanced thiol efflux from MCF-7 cells. Apoptosis was confirmed by Annexin-V and propidium iodide staining. Cell cycle arrest occurred at the G2/M and sub-G1 interphases. Both CySSR+Se mixtures reduced the levels of Akt. CySSPe+Se elevated GSK-3 protein levels, whereas CySSA+Se did not. CySSR+Se mixtures enhanced phospho-c-Jun levels, with CySSA+Se more potent than CySSPe+Se. Corresponding increases in phospho-p53, Bax and Bad levels were observed, indicating apoptosis occurred via the mitochondrial pathway. Lack of caspases 6/7 activation implicated a caspase-independent pathway for apoptosis. Reduction of imported CySSR and export of thiols by MCF-7 cells facilitates the reduction of selenite to yield H2Se, a cytotoxic agent. This appears to be the first report of an anti-cancer effect of CySSPe. PMID:24614136

  19. SDF‑1/CXCR4 axis induces apoptosis of human degenerative nucleus pulposus cells via the NF‑κB pathway.

    PubMed

    Liu, Zongchao; Ma, Chuan; Shen, Jieliang; Wang, Dawu; Hao, Jie; Hu, Zhenming

    2016-07-01

    Intervertebral disc degeneration (IVDD) is a major cause of lower back pain, and increased cell apoptosis is a key characteristic of IVDD. The present study aimed to investigate the effects and mechanism of the stromal cell‑derived factor‑1 (SDF‑1)/C‑X‑C motif chemokine receptor 4 (CXCR4) axis on apoptosis in human degenerative nucleus pulposus cells (NPCs). The expression levels of SDF‑1 and CXCR4 in human intervertebral discs (IVD) were determined using immunohistochemistry and western blot analysis. Apoptosis of primary cultured NPCs was quantified by Annexin V/propidium iodide staining following stimulation with SDF‑1 and knockdown of CXCR4 using small interfering RNA (siRNA). The association with the nuclear factor‑κB (NF‑κB) signaling pathway was investigated using CXCR4‑siRNA and NF‑κB inhibitor, pyrrolidine dithiocarbamate (PDTC), treatment. The results demonstrated that SDF‑1 and its receptor, CXCR4, were upregulated in degenerative IVD samples compared with normal samples. Stimulation with SDF‑1 increased the level of apoptosis in cultured NPCs, and conversely, the apoptosis level was suppressed post‑transfection with CXCR4 siRNA compared with SDF‑1 stimulation alone. Furthermore, SDF‑1 treatment increased the level of phosphorylated NF‑κB subunit P65, which was downregulated following CXCR4 siRNA and PDTC treatment. In addition, CXCR4 siRNA and PDTC inhibited the nuclear translocation of P65, which was induced by SDF‑1. Taken together, SDF‑1‑mediated apoptosis was suppressed by NF‑κB inhibition using PDTC. In conclusion, the SDF‑1/CXCR4 axis promoted cell apoptosis in human degenerative NPCs via the NF‑κB pathway, thus suggesting that SDF‑1/CXCR signaling may be a therapeutic target for the treatment of degenerative IVD diseases. PMID:27220474

  20. Apoptosis induced by lipid-associated membrane proteins from Mycoplasma hyopneumoniae in a porcine lung epithelial cell line with the involvement of caspase 3 and the MAPK pathway.

    PubMed

    Ni, B; Bai, F F; Wei, Y; Liu, M J; Feng, Z X; Xiong, Q Y; Hua, L Z; Shao, G Q

    2015-01-01

    Lipid-associated membrane proteins (LAMPs) are important in the pathogenicity of the Mycoplasma genus of bacteria. We investigated whether Mycoplasma hyopneumoniae LAMPs have pathogenic potential by inducing apoptosis in a St. Jude porcine lung epithelial cell line (SJPL). LAMPs from a pathogenic strain of M. hyopneumoniae (strain 232) were used in the research. Our investigation made use of diamidino-phenylindole (DAPI) and acridine orange/ethidium bromide (AO/EB) staining, terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) analysis, and Annexin-V-propidium iodide staining. After LAMP treatment for 24 h, typical changes were induced, chromosomes were concentrated, apoptotic bodies were observed, the 3'-OH groups of cleaved genomes were exposed, and the percentage of apoptotic cells reached 36.5 ± 11.66%. Caspase 3 and caspase 8 were activated and cytochrome c (cyt c) was released from the mitochondria into the cytoplasm; poly ADP ribose polymerase (PARP) was digested into two fragments; p38 mitogen-activated protein kinase (MAPK) was phosphorylated; and the expression of pro-apoptosis protein Bax increased while the anti-apoptosis protein Bcl-2 decreased. LAMPs also stimulated SJPL cells to produce nitric oxide (NO) and superoxide. This study demonstrated that LAMPs from M. hyopneumoniae can induce apoptosis in SJPL cells through the activation of caspase 3, caspase 8, cyt c, Bax, and p38 MAPK, thereby contributing to our understanding of the pathogenesis of M. hyopneumoniae, which should improve the treatment of M. hyopneumoniae infections. PMID:26436384

  1. Effects of nanosecond pulsed electrical fields (nsPEFs) on the cell cycle of CHO and Jurkat cells

    NASA Astrophysics Data System (ADS)

    Mahlke, Megan A.; Navara, Christopher; Ibey, Bennett L.

    2014-03-01

    Exposure to nano-second pulsed electrical fields (nsPEFs) can cause poration of external and internal cell membranes, DNA damage, and disassociation of cytoskeletal components, all of which are capable of disrupting a cell's ability to replicate. Variations between cell lines in membrane and cytoskeletal structure as well as in survival of nsPEF exposure should correspond to unique line-dependent cell cycle effects. Additionally, phase of cell cycle during exposure may be linked to differential sensitivities to nsPEFs across cell lines, as DNA structure, membrane elasticity, and cytoskeletal structure change dramatically during the cell cycle. Populations of Jurkat and Chinese Hamster Ovary (CHO) cells were examined post-exposure (10 ns pulse trains at 150kV/cm) by analysis of DNA content via propidium iodide staining and flow cytometric analysis at various time points (1, 6, and 12h post-exposure) to determine population distribution in cell cycle phases. Additionally, CHO and Jurkat cells were synchronized in G1/S and G2/M phases, pulsed, and analyzed to evaluate role of cell cycle phase in survival of nsPEFs. CHO populations recovered similarly to sham populations postnsPEF exposure and did not exhibit a phase-specific change in response. Jurkat cells exhibited considerable apoptosis/necrosis in response to nsPEF exposure and were unable to recover and proliferate in a manner similar to sham exposed cells. Additionally, Jurkat cells appear to be more sensitive to nsPEFs in G2/M phases than in G1/S phases. Recovery of CHO populations suggests that nsPEFs do not inhibit proliferation in CHO cells; however, inhibition of Jurkat cells post-nsPEF exposure coupled with preferential cell death in G2/M phases suggest that cell cycle phase during exposure may be an important factor in determining nsPEF toxicity in certain cell lines. Interestingly, CHO cells have a more robust and rigid cytoskeleton than Jurkat cells which is thought to contribute to their ability to

  2. The Effect of Ursolic Acid on Leishmania (Leishmania) amazonensis Is Related to Programed Cell Death and Presents Therapeutic Potential in Experimental Cutaneous Leishmaniasis

    PubMed Central

    Yamamoto, Eduardo S.; Campos, Bruno L. S.; Jesus, Jéssica A.; Laurenti, Márcia D.; Ribeiro, Susan P.; Kallás, Esper G.; Rafael-Fernandes, Mariana; Santos-Gomes, Gabriela; Silva, Marcelo S.; Sessa, Deborah P.; Lago, João H. G.; Levy, Débora; Passero, Luiz F. D.

    2015-01-01

    Among neglected tropical diseases, leishmaniasis is one of the most important ones, affecting more than 12 million people worldwide. The available treatments are not well tolerated, and present diverse side effects, justifying the search for new therapeutic compounds. In the present study, the activity of ursolic acid (UA) and oleanolic acid (OA) were assayed in experimental cutaneous leishmaniasis (in vitro and in vivo). Promastigote forms of L. amazonensis were incubated with OA and UA for 24h, and effective concentration 50% (EC50) was estimated. Ultraestructural alterations in Leishmania amazonensis promastigotes after UA treatment were evaluated by transmission electron microscopy, and the possible mode of action was assayed through Annexin V and propidium iodide staining, caspase 3/7 activity, DNA fragmentation and transmembrane mitochondrial potential. The UA potential was evaluated in intracellular amastigotes, and its therapeutic potential was evaluated in L. amazonensis infected BALB/c mice. UA eliminated L. amazonensis promastigotes with an EC50 of 6.4 μg/mL, comparable with miltefosine, while OA presented only a marginal effect on promastigote forms at 100 μg/mL. The possible mechanism by which promastigotes were eliminated by UA was programmed cell death, independent of caspase 3/7, but it was highly dependent on mitochondria activity. UA was not toxic for peritoneal macrophages from BALB/c mice, and it was able to eliminate intracellular amastigotes, associated with nitric oxide (NO) production. OA did not eliminate amastigotes nor trigger NO. L. amazonensis infected BALB/c mice submitted to UA treatment presented lesser lesion size and parasitism compared to control. This study showed, for the first time, that UA eliminate promastigote forms through a mechanism associated with programed cell death, and importantly, was effective in vivo. Therefore, UA can be considered an interesting candidate for future tests as a prototype drug for the treatment

  3. Heterogeneities in inflammatory and cytotoxic responses of RAW 264.7 macrophage cell line to urban air coarse, fine, and ultrafine particles from six European sampling campaigns

    SciTech Connect

    Jalava, P.I.; Salonen, R.O.; Pennanen, A.S.; Sillanpaa, M.; Halinen, A.I.; Happo, M.S.; Hillamo, R.; Brunekreef, B.; Katsouyanni, K.; Sunyer, J.; Hirvonen, M.R.

    2007-03-15

    We investigated the cytotoxic and inflammatory activities of size-segregated particulate samples (particulate matter, PM) from contrasting air pollution situations in Europe. Coarse (PM10-2.5), fine (PM2.5-0.2), and ultrafine (PM0.2) particulate samples were collected with a modified Harvard high-volume cascade impactor (HVCI). Mouse RAW 264.7 macrophages were exposed to the samples for 24 h. Selected inflammatory mediators, nitric oxide (NO) and cytokines (tumor necrosis factor alpha (TNF alpha), interleukin 6 (IL-6), macrophage inflammatory protein-2 (MIP-2)), were measured together with cytotoxicity (MTT test), and analysis of apoptosis and cell cycle (propidium iodide staining). The PM10-2.5 samples had a much higher inflammatory activity than the PM2.5-0.2 and PM0.2 samples, but the PM2.5-0.2 samples showed the largest differences in inflammatory activity, and the PM0.2 samples in cytotoxicity, between the sampling campaigns. The PM2.5-0.2 samples from traffic environments in springtime Barcelona and summertime Athens had the highest inflammatory activities, which may be related to the high photochemical activity in the atmosphere during the sampling campaigns. The PM0.2 sample from wintertime Prague with proven impacts from local coal and biomass combustion had very high cytotoxic and apoptotic activities and caused a distinct cell cycle arrest. Thus, particulate size, sources, and atmospheric transformation processes affect the toxicity profile of urban air particulate matter. These factors may explain some of the heterogeneity observed in particulate exposure-response relationships of human health effects in epidemiological studies.

  4. Differential nanoreprotoxicity of silver nanoparticles in male somatic cells and spermatogonial stem cells

    PubMed Central

    Zhang, Xi-Feng; Choi, Yun-Jung; Han, Jae Woong; Kim, Eunsu; Park, Jung Hyun; Gurunathan, Sangiliyandi; Kim, Jin-Hoi

    2015-01-01

    Background Silver nanoparticles (AgNPs) possess unique physical, chemical, and biological properties. AgNPs have been increasingly used as anticancer, antiangiogenic, and antibacterial agents for the treatment of bacterial infections in open wounds as well as in ointments, bandages, and wound dressings. The present study aimed to investigate the effects of two different sizes of AgNPs (10 nm and 20 nm) in male somatic Leydig (TM3) and Sertoli (TM4) cells and spermatogonial stem cells (SSCs). Methods Here, we demonstrate a green and simple method for the synthesis of AgNPs using Bacillus cereus culture supernatants. The synthesized AgNPs were characterized using ultraviolet and visible absorption spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and transmission electron microscopy (TEM). The toxicity of the synthesized AgNPs was evaluated by the effects on cell viability, metabolic activity, oxidative stress, apoptosis, and expression of genes encoding steroidogenic and tight junction proteins. Results AgNPs inhibited the viability and proliferation of TM3 and TM4 cells in a dose- and size-dependent manner by damaging cell membranes and inducing the generation of reactive oxygen species, which in turn affected SSC growth on TM3 and TM4 as feeder cells. Small AgNPs (10 nm) were more cytotoxic than medium-sized nanoparticles (20 nm). TEM revealed the presence of AgNPs in the cell cytoplasm and nucleus, and detected mitochondrial damage and enhanced formation of autosomes and autolysosomes in the AgNP-treated cells. Flow cytometry analysis using Annexin V/propidium iodide staining showed massive cell death by apoptosis or necrosis. Real-time polymerase chain reaction and western blot analyses indicated that in TM3 and TM4 cells, AgNPs activated the p53, p38, and pErk1/2 signaling pathways and significantly downregulated the expression of genes related to testosterone synthesis (TM3) and tight junctions (TM4). Furthermore, the exposure of TM3

  5. Selenoprotein S Is Highly Expressed in the Blood Vessels and Prevents Vascular Smooth Muscle Cells From Apoptosis.

    PubMed

    Ye, Yali; Fu, Fen; Li, Xiaoming; Yang, Jie; Liu, Hongmei

    2016-01-01

    Atherosclerosis and related cardiovascular diseases (CVD) represent one of the greatest threats to human health worldwide. The protection of vascular smooth muscle cells (VSMCs) from apoptosis in the plaque has become an important therapeutic target for atherosclerotic plaque stabilization. A significant association of selenoprotein S (SelS) gene polymorphism with atherosclerotic CVD has been reported in epidemiologic studies, but the underlying mechanism remains unknown. In this paper, SelS expression in the thoracic aorta and its role in the protection of VSMCs from apoptosis have been studied. Western blot analysis showed that SelS was highly expressed in rat thoracic aorta. SelS gene silence by small interference RNA (siRNA) rendered VSMCs more sensitive to hydrogen peroxide- or tunicamycin- induced injury and apoptosis, as determined by MTT assay, Hoechst staining, and annexin V/propidium iodide staining. SelS silence aggravated hydrogen peroxide-induced oxidative stress and phosphorylation of p38 MAPK and c-Jun N-terminal kinase (JNK) in VSMCs. Furthermore, SelS silence enhanced endoplasmic reticulum (ER) stress induced by hydrogen peroxide or tunicamycin, as showed by the increased protein levels of ER chaperone 78 kDa glucose-regulated protein (GRP78), ER stress transducer phosphorylated protein kinase RNA like ER kinase (PERK), and the proapoptotic transcription factor C/EBP homologous protein (CHOP). In conclusion, the present study suggested that SelS highly expressed in the blood vessel might protect VSMCs from apoptosis by inhibiting oxidative stress and ER stress. Our finding provided mechanistic insights for the potential preventive role of SelS in atherosclerotic CVD. PMID:26058460

  6. Detecting inactivated endospores in fluorescence microscopy using propidium monoazide

    NASA Astrophysics Data System (ADS)

    Probst, Alexander; Mahnert, Alexander; Weber, Christina; Haberer, Klaus; Moissl-Eichinger, Christine

    2012-04-01

    The differentiation between living and dead bacterial endospores is crucial in many research areas of microbiology. The identification of inactivated, non-pathogenic Bacillus anthracis spores is one reason why improvement of decontamination protocols is so desirable. Another field interested in spore viability is planetary protection, a sub-discipline of astrobiology that estimates the bioburden of spacecraft prior to launch in order to avoid interplanetary cross-contamination. We developed a dedicated, rapid and cost-effective method for identifying bacterial endospores that have been inactivated and consequently show a compromised spore wall. This novel protocol is culture-independent and is based on fluorescence microscopy and propidium monoazide (PMA) as a fluorescent marker, which is suggested to bind to DNA of spores with compromised spore coat, cortex and membranes based on our results. Inactivated preparations (treated with wet heat, irradiation, ultracentrifugation) showed a significant increase in spores that were PMA stained in their core; moreover, Bacillus atrophaeus, Bacillus safensis and Geobacillus stearothermophilus seemed to be best suited for this technique, as the spore cores of all these endospores could be positively stained after inactivation. Lastly, we describe an additional counter-staining protocol and provide an example of the application of the coupled staining methods for planetary protection purposes. The introduction of this novel protocol is expected to provide an initial insight into the various possible future applications of PMA as a non-viability marker for spores in, for example, B. anthracis-related studies, food microbiology and astrobiology.

  7. Molecular Mechanism of the Cell Death Induced by the Histone Deacetylase Pan Inhibitor LBH589 (Panobinostat) in Wilms Tumor Cells

    PubMed Central

    Fang, Fang; Jun, Lu; Gang, Li; Lan, Cao; Na-Na, Wang; Xiao-Juan, Du; Li-Chao, Sun; Wen-Li, Zhao; Pei-Fang, Xiao; He, Zhao; Guang-Hao, Su; Yan-Hong, Li; Yi-Ping, Li; Yun-Yun, Xu; Hui-Ting, Zhou; Yi, Wu; Mei-Fang, Jin; Lin, Liu; Jian, Ni; Shao-Yan, Hu; Xue-Ming, Zhu; Xing, Feng; Jian, Wang; Jian, Pan

    2015-01-01

    Background Wilms tumor (WT) is an embryonic kidney cancer, for which histone acetylation might be a therapeutic target. LBH589, a novel targeted agent, suppresses histone deacetylases in many tumors. This study investigated the antitumor activity of LBH589 in SK-NEP-1 and G401 cells. Methods SK-NEP-1 and G401 cell growth was assessed by CCK-8 and in nude mice experiments. Annexin V/propidium iodide staining followed by flow cytometry detected apoptosis in cell culture. Gene expressions of LBH589-treated tumor cells were analyzed using an Arraystar Human LncRNA Array. The Multi Experiment View cluster software analyzed the expression data. Differentially expressed genes from the cluster analyses were imported into the Ingenuity Pathway Analysis tool. Results LBH589 inhibited cell proliferation of SK-NEP-1 and G401 cells in a dose-dependent manner. Annexin V, TUNEL and Hochest 33342 staining analysis showed that LBH589-treated cells showed more apoptotic features compared with the control. LBH589 treatment inhibited the growth of SK-NEP-1 xenograft tumors in nude mice. Arraystar Human LncRNA Array analysis of genes and lncRNAs regulated by LBH589 identified 6653 mRNAs and 8135 lncRNAs in LBH589-treated SK-NEP-1 cells. The most enriched gene ontology terms were those involved in nucleosome assembly. KEGG pathway analysis identified cell cycle proteins, including CCNA2, CCNB2, CCND1, CCND2, CDK4, CDKN1B and HDAC2, etc. Ingenuity Pathway Analysis identified important upstream molecules: HIST2H3C, HIST1H4A, HIST1A, HIST1C, HIST1D, histone H1, histone H3, RPRM, HSP70 and MYC. Conclusions LBH589 treatment caused apoptosis and inhibition of cell proliferation of SK-NEP-1and G401 cells. LBH589 had a significant effect and few side effects on SK-NEP-1 xenograft tumors. Expression profiling, and GO, KEGG and IPA analyses identified new targets and a new “network” of genes responding to LBH589 treatment in SK-NEP-1 cells. RPRM, HSP70 and MYC may be important regulators

  8. Aspirin inhibits cell viability and mTOR downstream signaling in gastroenteropancreatic and bronchopulmonary neuroendocrine tumor cells

    PubMed Central

    Spampatti, Matilde; Vlotides, George; Spöttl, Gerald; Maurer, Julian; Göke, Burkhard; Auernhammer, Christoph J

    2014-01-01

    AIM: To investigate the effect of aspirin on neuroendocrine tumor (NET) cell growth and signaling in vitro. METHODS: Human pancreatic BON1, bronchopulmonary NCI-H727 and midgut GOT1 neuroendocrine tumor cells were treated with different concentrations of aspirin (from 0.001 to 5 mmol/L), and the resulting effects on metabolic activity/cell proliferation were measured using cell proliferation assays and SYBR-DNA-labeling after 72, 144 and 216 h of incubation. The effects of aspirin on the expression and phosphorylation of several critical proteins that are involved in the most common intracellular growth factor signaling pathways (especially Akt protein kinase B) and mammalian target of rapamycin (mTOR) were determined by Western blot analyses. Propidium iodide staining and flow cytometry were used to evaluate changes in cell cycle distribution and apoptosis. Statistical analysis was performed using a 2-tailed Student’s t-test to evaluate the proliferation assays and cell cycle analyses. The results are expressed as the mean ± SD of 3 or 4 independently performed experiments. Statistical significance was set at P < 0.05. RESULTS: Treatment with aspirin suppressed the viability/proliferation of BON1, NCI-H727 and GOT1 cells in a time- and dose-dependent manner. Significant effects were observed at starting doses of 0.5-1 mmol/L and peaked at 5 mmol/L. For instance, after treatment with 1 mmol/L aspirin for 144 h, the viability of pancreatic BON1 cells decreased to 66% ± 13% (P < 0.05), the viability of bronchopulmonary NCI-H727 cells decreased to 53% ± 8% (P < 0.01) and the viability of midgut GOT1 cells decreased to 89% ± 6% (P < 0.01). These effects were associated with a decreased entry into the S phase, the induction of the cyclin-dependent kinase inhibitor p21 and reduced expression of cyclin-dependent kinase 4 and cyclin D3. Aspirin suppressed mTOR downstream signaling, evidenced by the reduced phosphorylation of the mTOR substrates 4E binding protein 1

  9. Sodium formate induces autophagy and apoptosis via the JNK signaling pathway of photoreceptor cells

    PubMed Central

    WANG, YING; XU, SHAO-LIN; XU, WEN-JING; YANG, HAI-YAN; HU, PING; LI, YU-XIN

    2016-01-01

    Incidents associated with methanol intoxication resulting from the consumption of fake wine occur not infrequently worldwide. Certain individuals are made blind due to methanol poisoning. The present study aimed to investigate the effects of sodium formate exposure on photoreceptor cells (661W cells). The 661W cells were exposed to sodium formate for 6–24 h and cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Subsequently, the 661W cells were exposed to 15 or 30 mM sodium formate for 24 h. The level of apoptosis was determined using Hoechst 33342/propidium iodide staining, visualizing the cells under a fluorescence microscope, and annexin V-fluorescein isothiocyanate staining, using flow cytometric analysis. Intracellular reactive oxygen species (ROS) were measured using 2′,7′-dichlorofluorescein diacetate (DCFH-DA) staining, followed by flow cytometric analysis. Autophagy of the 661W cells was measured by monodansylcadaverine staining. The activation of phosphorylated c-Jun N-terminal kinase (p-JNK), B-cell lymphoma (Bcl-2), Bcl-2-associated X protein, cleaved caspase-3, cleaved caspase-9 and microtubule-associated protein 1A/1B-light chain 3 (LC3) was assessed by western blotting. The effects of Z-VAD-fmk (a pan-caspase inhibitor) and SP600125 (a JNK inhibitor) on the viability of the sodium formate-induced 661W cells were determined using an MTT assay. Sodium formate treatment induced a decrease in the viability of the 661W cells in a time- and a dose-dependent manner. In addition, sodium formate at concentrations of 15 or 30 mM markedly increased the level of apoptosis and the ROS levels, as measured by DCFH-DA staining of the 661W cells. Additionally, 661W cells exposed to sodium formate for 24 h exhibited increased levels of p-JNK, Bax, cleaved caspase-3, cleaved caspase-9 and LC3II (the phosphatidylethanolamine-modified form of LC3), although the level of Bcl-2 was decreased

  10. Sodium formate induces autophagy and apoptosis via the JNK signaling pathway of photoreceptor cells.

    PubMed

    Wang, Ying; Xu, Shao-Lin; Xu, Wen-Jing; Yang, Hai-Yan; Hu, Ping; Li, Yu-Xin

    2016-02-01

    Incidents associated with methanol intoxication resulting from the consumption of fake wine occur not infrequently worldwide. Certain individuals are made blind due to methanol poisoning. The present study aimed to investigate the effects of sodium formate exposure on photoreceptor cells (661W cells). The 661W cells were exposed to sodium formate for 6‑24 h and cell viability was determined using a 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyl‑2H‑tetrazolium bromide (MTT) assay. Subsequently, the 661W cells were exposed to 15 or 30 mM sodium formate for 24 h. The level of apoptosis was determined using Hoechst 33342/propidium iodide staining, visualizing the cells under a fluorescence microscope, and annexin V‑fluorescein isothiocyanate staining, using flow cytometric analysis. Intracellular reactive oxygen species (ROS) were measured using 2',7'‑dichlorofluorescein diacetate (DCFH‑DA) staining, followed by flow cytometric analysis. Autophagy of the 661W cells was measured by monodansylcadaverine staining. The activation of phosphorylated c‑Jun N‑terminal kinase (p‑JNK), B‑cell lymphoma (Bcl‑2), Bcl‑2‑associated X protein, cleaved caspase‑3, cleaved caspase‑9 and microtubule‑associated protein 1A/1B‑light chain 3 (LC3) was assessed by western blotting. The effects of Z‑VAD‑fmk (a pan‑caspase inhibitor) and SP600125 (a JNK inhibitor) on the viability of the sodium formate‑induced 661W cells were determined using an MTT assay. Sodium formate treatment induced a decrease in the viability of the 661W cells in a time‑ and a dose‑dependent manner. In addition, sodium formate at concentrations of 15 or 30 mM markedly increased the level of apoptosis and the ROS levels, as measured by DCFH‑DA staining of the 661W cells. Additionally, 661W cells exposed to sodium formate for 24 h exhibited increased levels of p‑JNK, Bax, cleaved caspase‑3, cleaved caspase‑9 and LC3II (the phosphatidylethanolamine‑modified form

  11. One-step purification of functional human and rat pancreatic alpha cells.

    PubMed

    Köhler, Martin; Daré, Elisabetta; Ali, Muhammed Yusuf; Rajasekaran, Subu Surendran; Moede, Tilo; Leibiger, Barbara; Leibiger, Ingo B; Tibell, Annika; Juntti-Berggren, Lisa; Berggren, Per-Olof

    2012-02-01

    Pancreatic alpha cells contribute to glucose homeostasis by the regulated secretion of glucagon, which increases glycogenolysis and hepatic gluconeogenesis in response to hypoglycemia. Alterations of glucagon secretion are observed in diabetic patients and exacerbate the disease. The restricted availability of purified primary alpha cells has limited our understanding of their function in health and disease. This study was designed to establish convenient protocols for the purification of viable alpha cells from rat and human pancreatic islets by FACS, using intrinsic cellular properties. Islets were isolated from the pancreata of Wistar rats or deceased human organ donors. Dispersed islet cells were separated by FACS based on light scatter and autofluorescence. Purity of sorted cells was evaluated by immunocytochemistry using hormone specific antibodies. Relative hormone expression was further determined by quantitative RT-PCR. Viability was determined by Annexin V and propidium iodide staining and function was assessed by monitoring cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) using Fura-2/AM. We developed species-specific FACS gating strategies that resulted in populations consisting mainly of alpha cells (96.6 ± 1.4%, n = 3 for rat; 95.4 ± 1.7%, n = 4 for human, mean ± SEM). These cell fractions showed ~5-fold and ~4-fold enrichment (rat and human, respectively) of glucagon mRNA expression compared to total ungated islet cells. Most of the sorted cells were viable and functional, as they responded with an increase in [Ca(2+)](i) upon stimulation with L-arginine (10 mM). The majority of the sorted human alpha cells responded also to stimulation with kainate (100 μM), whereas this response was infrequent in rat alpha cells. Using the same sample preparation, but a different gating strategy, we were also able to sort rat and human populations enriched in beta cells. In conclusion, we have simplified and optimized a method for the purification of rat

  12. Enumeration of viable non-culturable Vibrio cholerae using propidium monoazide combined with quantitative PCR.

    PubMed

    Wu, Bin; Liang, Weili; Kan, Biao

    2015-08-01

    The well-known human pathogenic bacterium, Vibrio cholerae, can enter a physiologically viable but non-culturable (VBNC) state under stress conditions. The differentiation of VBNC cells and nonviable cells is essential for both disease prevention and basic research. Among all the methods for detecting viability, propidium monoazide (PMA) combined with real-time PCR is popular because of its specificity, sensitivity, and speed. However, the effect of PMA treatment is not consistent and varies among different species and conditions. In this study, with an initial cell concentration of 1×10(8) CFU/ml, time and dose-effect relationships of different PMA treatments were evaluated via quantitative real-time PCR using live cell suspensions, dead cell suspensions and VBNC cell suspensions of V. cholerae O1 El Tor strain C6706. The results suggested that a PMA treatment of 20 μM PMA for 20 min was optimal under our conditions. This treatment maximized the suppression of the PCR signal from membrane-compromised dead cells but had little effect on the signal from membrane-intact live cells. In addition to the characteristics of PMA treatment itself, the initial concentration of the targeted bacteria showed a significant negative influence on the stability of PMA-PCR assay in this study. We developed a strategy that mimicked a 1×10(8) CFU/ml cell concentration with dead bacteria of a different bacterial species, the DNA of which cannot be amplified using the real time PCR primers. With this strategy, our optimal approach successfully overcame the impact of low cell density and generated stable and reliable results for counting viable cells of V. cholerae in the VBNC state. PMID:26001818

  13. Pulsed electromagnetic field affects intrinsic and endoplasmatic reticulum apoptosis induction pathways in MonoMac6 cell line culture.

    PubMed

    Kaszuba-Zwoinska, J; Chorobik, P; Juszczak, K; Zaraska, W; Thor, P J

    2012-10-01

    Current studies were aimed to elucidate influence of pulsed electromagnetic field stimulation on cell viability and apoptosis induction pathways. For the experimental model we have chosen monocytic cell line MonoMac6 and several apoptosis inducers with different mechanism of death induction like puromycin, colchicine, cyclophosphamide, minocycline and hydrogen peroxide. MonoMac6 cell line was grown at density 1x10(5) cells/well in 96-well culture plates. To induce cell death cell cultures were treated with different apoptosis inducers like puromycin, colchicine, cyclophosphamide, minocycline, hydrogen peroxide and at the same time with pulsed electromagnetic field 50 Hz, 45±5 mT (PEMF) for 4 hour per each stimulation, three times, in 24 hours intervals. Afterwards, cells were harvested for flow cytometry analysis of cell viability measured by annexin V-APC labeled and propidium iodide staining. Expression of apoptosis related genes was evaluated by semi quantitative reverse transcription (RT)-PCR assay. NuPAGE Novex Western blot analysis was carried out for apoptosis inducing factor (AIF) abundance in cytosolic and nuclear extracts of MonoMac6 cells. Puromycin, colchicine and minocycline activated cells and simultaneously treated with PEMF have shown out diminished percentage of annexinV positive (AnV+) cells comparing to controls without PEMF stimulation. MonaMac6 cells puromycin/colchicyne and PEMF treated were to a higher extent double stained (AnV+,PI+), which means increased late apoptotic as well as necrotic (PI+) cells, than non-stimulated controls. On the other hand, minocycline activated cells prior to PEMF treatment showed diminished amount of apoptotic and necrotic (annexin V, annexin V and propidium iodide, propidium iodide positive staining) cells. The opposite effect of PEMF on the percentage of annexin V positively stained cells has been achieved after treatment of MonoMac6 culture with cyclophoshamide and hydrogen peroxide. PEMF enhanced early

  14. Integrin β1-mediated acquired gefitinib resistance in non-small cell lung cancer cells occurs via the phosphoinositide 3-kinase-dependent pathway

    PubMed Central

    DENG, QIN-FANG; SU, BO; ZHAO, YIN-MIN; TANG, LIANG; ZHANG, JIE; ZHOU, CAI-CUN

    2016-01-01

    The present study aimed to explore the role of integrin β1 and the relevant signaling pathways in acquired gefitinib resistance in non-small cell lung cancer (NSCLC). The inhibitory effects of gefitinib, with or without LY294002, on cellular proliferation were evaluated by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide assay. Cell cycle progression and apoptosis were analyzed by flow cytometry, while western blotting was used to evaluate the expression of EGFR, phosphorylated (phospho)-EGFR, protein kinase B (Akt), phospho-Akt, extracellular signal-regulated kinase (Erk) and phospho-Erk. The gene expression profiles of PC9 and PC9/G cells were determined by DNA microarray. Integrin β1 was knocked down in PC9/G cells by transiently transfected short interfering RNA (siRNA). A scrambled siRNA sequence was used as a control. Apoptosis of transfected cells was determined by Annexin V-phycoerythrin-Cy5/propidium iodide staining. Sequencing products were amplified by nested PCR. The resistant index of PC9/G cells to gefitinib was ~138- to 256-fold higher than that of PC9 cells, and this resistance was accompanied by significant increase in integrin β1 expression in PC9/G cells. Knockdown of integrin β1 with short hairpin RNA in PC9/G cells markedly inhibited proliferation and enhanced apoptosis in response to gefitinib, restoring the sensitivity of PC9/G cells gefitinib. Phosphoinositide 3-kinase (PI3K)/Akt activation was observed in PC9/G cells in the presence of gefitinib and the sensitivity of PC9/G cells to gefitinib was also able to be restored by PI3K/Akt pathway inhibitor LY294002. Finally, knockdown of integrin β1 significantly reduced the levels of phospho-Akt. These findings suggest that integrin β1 signaling via the PI3K/Akt pathway may be a significant mechanism underlying gefitinib resistance, and may potentially present an alternative therapeutic target for the treatment of NSCLC unresponsive to EGFR inhibitors. PMID:26870244

  15. Discerning Viable from Nonviable Yersinia pestis pgm- and Bacillus anthracis Sterne using Propidium Monoazide in the Presence of White Powders

    SciTech Connect

    Hess, Becky M.; Kaiser, Brooke LD; Sydor, Michael A.; Wunschel, David S.; Bruckner-Lea, Cindy J.; Hutchison, Janine R.

    2015-12-23

    ABSTRACT Aims To develop and optimize an assay to determine viability status of Bacillus anthracis Sterne and Yersinia pestis pgm- strains in the presence of white powders by coupling propidium monoazide (PMA) treatment with real-time PCR (qPCR) analysis. Methods and Results PMA selectively enters nonviable cells and binds DNA, thereby increasing qPCR assay cycle threshold (CT) values compared to untreated samples. Dye concentration, cell number and fitness, incubation time, inactivation methods, and assay buffer were optimized for B. anthracis Sterne and Y. pestis pgm-. Differences in CT values in nonviable cells compared to untreated samples were consistently > 9 for both B. anthracis Sterne vegetative cells and Y. pestis pgm- in the presence and absence of three different white powders. Our method eliminates the need for a DNA extraction step prior to detection by qPCR. Conclusions The developed assay enables simultaneous identification and viability assessment for B. anthracis Sterne and Y. pestis pgm- under laboratory conditions, even in the presence of white powders. Eliminating the DNA extraction step that is typically used reduces total assay time and labor requirements for sample analysis. Significance and Impact of the Study The method developed for simultaneous detection and viability assessment for B. anthracis and Y. pestis can be employed in forming decisions about the severity of a biothreat event or the safety of food. Keywords Bacillus anthracis, Yersinia pestis, Propidium Monoazide, qPCR, White Powders, Rapid Viability Detection

  16. Laccase purified from Cerrena unicolor exerts antitumor activity against leukemic cells

    PubMed Central

    MATUSZEWSKA, ANNA; KARP, MARTA; JASZEK, MAGDALENA; JANUSZ, GRZEGORZ; OSIŃSKA-JAROSZUK, MONIKA; SULEJ, JUSTYNA; STEFANIUK, DAWID; TOMCZAK, WALDEMAR; GIANNOPOULOS, KRZYSZTOF

    2016-01-01

    Chronic lymphocytic leukemia (CLL) is the most commonly observed adult hematological malignancy in Western countries. Despite the fact that recent improvements in CLL treatment have led to an increased percentage of complete remissions, CLL remains an incurable disease. Cerrena unicolor is a novel fungal source of highly active extracellular laccase (ex-LAC) that is currently used in industry. However, to the best of our knowledge, no reports regarding its anti-leukemic activity have been published thus far. In the present study, it was hypothesized that C. unicolor ex-LAC may possess cytotoxic activity against leukemic cell lines and CLL primary cells. C. unicolor ex-LAC was separated using anion exchange chromatography on diethylaminoethyl cellulose-Sepharose and Sephadex G-50 columns. The cytotoxic effects of ex-LAC upon 24- and 48-h treatment on HL-60, Jurkat, RPMI 8226 and K562 cell lines, as well as CLL primary cells of nine patients with CLL, were evaluated using 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay. Annexin V/propidium iodide staining of Jurkat cells treated with ex-LAC was used to investigate apoptosis via flow cytometry. Ex-LAC induced changes in Jurkat and RPMI 8226 cells, as visualized by fluorescence and scanning electron microscopy (SEM). The XTT assay revealed high cytotoxic rates following treatment with various concentrations of ex-LAC on all the cell lines and CLL primary cells analyzed, with a half maximal inhibitory concentration ranging from 0.4 to 1.1 µg/ml. Fluorescence microscopy and SEM observations additionally revealed apoptotic changes in Jurkat and RPMI 8226 cells treated with ex-LAC, compared with control cells. These results were in agreement with the apoptosis analysis of Jurkat cells on flow cytometry. In conclusion, C. unicolor ex-LAC was able to significantly induce cell apoptosis, and may represent a novel therapeutic agent for the treatment of various hematological neoplasms. PMID

  17. Pro-apoptotic effects of tectorigenin on human hepatocellular carcinoma HepG2 cells

    PubMed Central

    Jiang, Chun-Ping; Ding, Hui; Shi, Da-Hua; Wang, Yu-Rong; Li, Er-Guang; Wu, Jun-Hua

    2012-01-01

    AIM: To investigate the effects of tectorigenin on human hepatocellular carcinoma (HCC) HepG2 cells. METHODS: Tectorigenin, one of the main components of rhizome of Iris tectorum, was prepared by simple methods, such as extraction, filtration, concentration, precipitation and recrystallization. HepG2 cells were incubated with tectorigenin at different concentrations, and their viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was detected by morphological observation of nuclear change, agarose gel electrophoresis of DNA ladder, and flow cytometry with Hoechst 33342, Annexin V-EGFP and propidium iodide staining. Generation of reactive oxygen species was quantified using DCFH-DA. Intracellular Ca2+ was monitored by Fura 2-AM. Mitochondrial membrane potential was monitored using Rhodamine 123. Release of cytochrome c from mitochondria to cytosol was detected by Western blotting. Activities of caspase-3, -8 and -9 were investigated by Caspase Activity Assay Kit. RESULTS: The viability of HepG2 cells treated by tectorigenin decreased in a concentration- and time-dependent manner. The concentration that reduced the number of viable HepG2 cells by 50% (IC50) after 12, 24 and 48 h of incubation was 35.72 mg/L, 21.19 mg/L and 11.06 mg/L, respectively. However, treatment with tectorigenin at 20 mg/L resulted in a very slight cytotoxicity to L02 cells after incubation for 12, 24 or 48 h. Tectorigenin at a concentration of 20 mg/L greatly inhibited the viability of HepG2 cells and induced the condensation of chromatin and fragmentation of nuclei. Tectorigenin induced apoptosis of HepG2 cells in a time- and dose-dependent manner. Compared with the viability rate, induction of apoptosis was the main mechanism of the anti-proliferation effect of tectorigenin in HepG2 cells. Furthermore, tectorigenin-induced apoptosis of HepG2 cells was associated with the generation of reactive oxygen species, increased intracellular [Ca2+]i

  18. Biological and molecular mechanisms of sulfur mustard analogue-induced toxicity in JB6 and HaCaT cells: possible role of ataxia telangiectasia-mutated/ataxia telangiectasia-Rad3-related cell cycle checkpoint pathway.

    PubMed

    Tewari-Singh, Neera; Gu, Mallikarjuna; Agarwal, Chapla; White, Carl W; Agarwal, Rajesh

    2010-06-21

    Effective medical treatment and preventive measures for chemical warfare agent sulfur mustard (HD)-caused incapacitating skin toxicity are lacking, because of limited knowledge of its mechanism of action. The proliferating basal epidermal cells are primary major sites of attack during HD-caused skin injury. Therefore, employing mouse JB6 and human HaCaT epidermal cells, here, we investigated the molecular mechanism of HD analogue 2-chloroethyl ethyl sulfide (CEES)-induced skin cytotoxicity. As compared to the control, up to 1 mM CEES treatment of these cells for 2, 4, and 24 h caused dose-dependent decreases in cell viability and proliferation as measured by DNA synthesis, together with S and G2-M phase arrest in cell cycle progression. Mechanistic studies showed phosphorylation of DNA damage sensors and checkpoint kinases, ataxia telangiectasia-mutated (ATM) at ser1981 and ataxia telangiectasia-Rad3-related (ATR) at ser428 within 30 min of CEES exposure, and modulation of S and G2-M phase-associated cell cycle regulatory proteins, which are downstream targets of ATM and ATR kinases. Hoechst-propidium iodide staining demonstrated that CEES-induced cell death was both necrotic and apoptotic in nature, and the latter was induced at 4 and 24 h of CEES treatment in HaCaT and JB6 cells, respectively. An increase in caspase-3 activity and both caspase-3 and poly(ADP-ribose)polymerase (PARP) cleavage coinciding with CEES-caused apoptosis in both cell lines suggested the involvement of the caspase pathway. Together, our findings suggest a DNA-damaging effect of CEES that activates ATM/ATR cell cycle checkpoint signaling as well as caspase-PARP pathways, leading to cell cycle arrest and apoptosis/necrosis in both JB6 and HaCaT cells. The identified molecular targets, quantitative biomarkers, and epidermal cell models in this study have the potential and usefulness in rapid development of effective prophylactic and therapeutic interventions against HD-induced skin toxicity

  19. Downregulation of HIF-1α inhibits the proliferation and invasion of non-small cell lung cancer NCI-H157 cells

    PubMed Central

    QIAN, JIALIN; BAI, HAO; GAO, ZHIQIANG; DONG, YU; PEI, JUN; MA, MEILI; HAN, BAOHUI

    2016-01-01

    Lung cancer, specifically non-small cell lung cancer (NSCLC), is the leading cause of cancer-associated mortality in the world. In previous years, almost no significant advancements have been made towards the molecular characterization of NSCLC, which highlights the requirement for novel target genes. Hypoxia inducible factor-1α (HIF-1α) is known to be essential in tumorigenesis, as it regulates the expression of numerous factors that are involved in angiogenesis, cellular proliferation and apoptosis. However, no direct association between HIF-1α and NSCLC treatment has previously been established. The aim of the present study was to characterize the effect of HIF-1α on NSCLC and to explore the possible mechanism. Additionally, HIF-1α small interfering (si)RNA and diamminedichloroplatinum (DDP) were used in combination to explore the combined effects on NSCLC cells. Lung carcinoma NCI-H157 cells were treated with HIF-1α small interfering (si)RNA, 5 µg/ml DDP or a combination of the two, and the proliferation, apoptosis and invasion ability of the cells were detected using a cell counting kit-8 assay, Annexin V/propidium iodide staining and a Transwell assay, respectively. In addition, the protein levels of caspase-3/9, anti-apoptotic protein B-cell lymphoma-2 (Bcl-2), vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), phosphoinositide 3-kinase (PI3K), phosphorylated (p-)PI3K, protein kinase B (AKT), p-AKT, extracellular signal-regulated kinase (ERK) and p-ERK were detected using western blot analysis. Similar to DPP treatment, HIF-1α siRNA treatment may reduce cell proliferation and the invasiveness of tumor cells while promoting apoptosis. Additionally, HIF-1α siRNA may increase the levels of the apoptotic proteins caspases 3 and 9 and inhibit the expression of Bcl-2. These anti-tumor effects may be acting through the VEGF/PEDF, PI3K/AKT and Raf/mitogen-activated protein kinase kinase/ERK signaling pathways. The effects

  20. Establishment and characterization of gemcitabine-resistant human cholangiocarcinoma cell lines with multidrug resistance and enhanced invasiveness.

    PubMed

    Wattanawongdon, Wareeporn; Hahnvajanawong, Chariya; Namwat, Nisana; Kanchanawat, Sirimas; Boonmars, Thidarut; Jearanaikoon, Patcharee; Leelayuwat, Chanwit; Techasen, Anchalee; Seubwai, Wunchana

    2015-07-01

    To establish and characterize the gemcitabine-resistant cholangiocarcinoma (CCA) cell lines, CCA KKU‑M139 and KKU‑M214 cell lines were exposed stepwisely to increasing gemcitabine (GEM). The resultant drug-resistant cell lines, KKU‑M139/GEM and KKU‑M214/GEM, retained the resistant phenotype in drug-free medium at least for 2 months. Sulforhodamine B assay demonstrated that KKU‑M139/GEM and KKU‑M214/GEM were 25.88- and 62.31-fold more resistant to gemcitabine than their parental cells. Both gemcitabine-resistant cell lines were cross-resistant to 5-fluorouracil (5-FU), doxorubicin and paclitaxel indicating their multidrug-resistant nature. Using reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR and western blot analyses, gemcitabine-resistant cells showed upregulation of RRM1 and downregulation of hENT1 and dCK. In relation to multidrug resistance, these cell lines showed upregulation of multidrug resistance protein 1 (MRP1) leading to an increase of drug efflux. Using cell adhesion and Boyden chamber transwell assays, these cell lines also showed higher cell adhesion, migration and invasion capabilities via the activations of protein kinase C (PKC), focal adhesion kinase (FAK), extracellular signal-regulated kinase-1/2 (ERK1/2) and nuclear factor-κB (NF-κB). Higher activity of matrix metalloproteinase-9 (MMP-9) and urokinase plasminogen activator (uPA) was also observed by a gelatin zymography assay and a casein-plasminogen zymography assay. Flow cytometry analysis indicated the G2/M arrest regulated by downregulation of cyclin B1 and cyclin-dependent kinase 1 (Cdk1) resulted in an extended population doubling time. Using Annexin V/propidium iodide staining, evasion of apoptosis via an intrinsic pathway was observed in both cell lines in association with upregulation of Bcl-2 and downregulation of Bax. Interestingly, Fas was additionally downregulated in KKU‑M214/GEM supporting the view of its higher GEM resistant

  1. Propidium monoazide treatment to distinguish between live and dead methanogens in pure cultures and environmental samples.

    PubMed

    Heise, Janine; Nega, Marcella; Alawi, Mashal; Wagner, Dirk

    2016-02-01

    In clinical trials investigating human health and in the analysis of microbial communities in cultures and natural environments, it is a substantial challenge to differentiate between living, potentially active communities and dead cells. The DNA-intercalating dye propidium monoazide (PMA) enables the selective masking of DNA from dead, membrane-compromised cells immediately before DNA extraction. In the present study, we evaluated for the first time a PMA treatment for methanogenic archaea in cultures and particle-rich environmental samples. Using microscopic analyses, we confirmed the applicability of the LIVE/DEAD(®) BacLight™ kit to methanogenic archaea and demonstrated the maintenance of intact cell membranes of methanogens in the presence of PMA. Although strain-specific differences in the efficiency of PMA treatment to methanogenic archaea were observed, we developed an optimal procedure using 130 μM PMA and 5min of photo-activation with blue LED light. The results showed that the effectiveness of the PMA treatment strongly depends on the texture of the sediment/soil: silt and clay-rich sediments represent a challenge at all concentrations, whereas successful suppression of DNA from dead cells with compromised membranes was possible for low particle loads of sandy soil (total suspended solids (TSS)≤200 mg mL(-1)). Conclusively, we present two strategies to overcome the problem of insufficient light activation of PMA caused by the turbidity effect (shielding) in particle-rich environmental samples by (i) dilution of the particle-rich sample and (ii) detachment of the cells and the free DNA from the sediment prior to a PMA treatment. Both strategies promise to be usable options for distinguishing living cells and free DNA in complex environmental samples. PMID:26656002

  2. Porphyromonas gingivalis Differentially Modulates Cell Death Profile in Ox-LDL and TNF-α Pre-Treated Endothelial Cells

    PubMed Central

    Bugueno, Isaac Maximiliano; Khelif, Yacine; Seelam, Narendra; Morand, David-Nicolas; Tenenbaum, Henri; Davideau, Jean-Luc; Huck, Olivier

    2016-01-01

    Objective Clinical studies demonstrated a potential link between atherosclerosis and periodontitis. Porphyromonas gingivalis (Pg), one of the main periodontal pathogen, has been associated to atheromatous plaque worsening. However, synergism between infection and other endothelial stressors such as oxidized-LDL or TNF-α especially on endothelial cell (EC) death has not been investigated. This study aims to assess the role of Pg on EC death in an inflammatory context and to determine potential molecular pathways involved. Methods Human umbilical vein ECs (HUVECs) were infected with Pg (MOI 100) or stimulated by its lipopolysaccharide (Pg-LPS) (1μg/ml) for 24 to 48 hours. Cell viability was measured with AlamarBlue test, type of cell death induced was assessed using Annexin V/propidium iodide staining. mRNA expression regarding caspase-1, -3, -9, Bcl-2, Bax-1 and Apaf-1 has been evaluated with RT-qPCR. Caspases enzymatic activity and concentration of APAF-1 protein were evaluated to confirm mRNA results. Results Pg infection and Pg-LPS stimulation induced EC death. A cumulative effect has been observed in Ox-LDL pre-treated ECs infected or stimulated. This effect was not observed in TNF-α pre-treated cells. Pg infection promotes EC necrosis, however, in infected Ox-LDL pre-treated ECs, apoptosis was promoted. This effect was not observed in TNF-α pre-treated cells highlighting specificity of molecular pathways activated. Regarding mRNA expression, Pg increased expression of pro-apoptotic genes including caspases-1,-3,-9, Bax-1 and decreased expression of anti-apoptotic Bcl-2. In Ox-LDL pre-treated ECs, Pg increased significantly the expression of Apaf-1. These results were confirmed at the protein level. Conclusion This study contributes to demonstrate that Pg and its Pg-LPS could exacerbate Ox-LDL and TNF-α induced endothelial injury through increase of EC death. Interestingly, molecular pathways are differentially modulated by the infection in function of the

  3. Effects of combined treatment with rapamycin and cotylenin A, a novel differentiation-inducing agent, on human breast carcinoma MCF-7 cells and xenografts

    PubMed Central

    Kasukabe, Takashi; Okabe-Kado, Junko; Kato, Nobuo; Sassa, Takeshi; Honma, Yoshio

    2005-01-01

    Introduction Rapamycin, an inhibitor of the serine/threonine kinase target of rapamycin, induces G1 arrest and/or apoptosis. Although rapamycin and its analogues are attractive candidates for cancer therapy, their sensitivities with respect to growth inhibition differ markedly among various cancer cells. Using human breast carcinoma cell line MCF-7 as an experimental model system, we examined the growth-inhibitory effects of combinations of various agents and rapamycin to find the agent that most potently enhances the growth-inhibitory effect of rapamycin. Method We evaluated the growth-inhibitory effect of rapamycin plus various agents, including cotylenin A (a novel inducer of differentiation of myeloid leukaemia cells) to MCF-7 cells, using either MTT assay or trypan blue dye exclusion test. The cell cycle was analyzed using propidium iodide-stained nuclei. Expressions of several genes in MCF-7 cells with rapamycin plus cotylenin A were studied using cDNA microarray analysis and RT-PCR. The in vitro results of MCF-7 cells treated with rapamycin plus cotylenin A were further confirmed in vivo in a mouse xenograft model. Results We found that the sensitivity of rapamycin to MCF-7 cells was markedly affected by cotylenin A. This treatment induced growth arrest of the cells at the G1 phase, rather than apoptosis, and induced senescence-associated β-galactosidase activity. We examined the gene expression profiles associated with exposure to rapamycin and cotylenin A using cDNA microarrays. We found that expressions of cyclin G2, transforming growth factor-β-induced 68 kDa protein, BCL2-interacting killer, and growth factor receptor-bound 7 were markedly induced in MCF-7 cells treated with rapamycin plus cotylenin A. Furthermore, combined treatment with rapamycin and cotylenin A significantly inhibited the growth of MCF-7 cells as xenografts, without apparent adverse effects. Conclusion Rapamycin and cotylenin A cooperatively induced growth arrest in breast

  4. Positive allosteric modulation of alpha-7 nicotinic receptors promotes cell death by inducing Ca(2+) release from the endoplasmic reticulum.

    PubMed

    Guerra-Álvarez, María; Moreno-Ortega, Ana J; Navarro, Elisa; Fernández-Morales, José Carlos; Egea, Javier; López, Manuela G; Cano-Abad, María F

    2015-05-01

    Positive allosteric modulation of α7 isoform of nicotinic acetylcholine receptors (α7-nAChRs) is emerging as a promising therapeutic approach for central nervous system disorders such as schizophrenia or Alzheimer's disease. However, its effect on Ca(2+) signaling and cell viability remains controversial. This study focuses on how the type II positive allosteric modulator (PAM II) PNU120596 affects intracellular Ca(2+) signaling and cell viability. We used human SH-SY5Y neuroblastoma cells overexpressing α7-nAChRs (α7-SH) and their control (C-SH). We monitored cytoplasmic and endoplasmic reticulum (ER) Ca(2+) with Fura-2 and the genetically encoded cameleon targeting the ER, respectively. Nicotinic inward currents were measured using patch-clamp techniques. Viability was assessed using methylthiazolyl blue tetrazolium bromide or propidium iodide staining. We observed that in the presence of a nicotinic agonist, PNU120596 (i) reduced viability of α7-SH but not of C-SH cells; (ii) significantly increased inward nicotinic currents and cytosolic Ca(2+) concentration; (iii) released Ca(2+) from the ER by a Ca(2+) -induced Ca(2+) release mechanism only in α7-SH cells; (iv) was cytotoxic in rat organotypic hippocampal slice cultures; and, lastly, all these effects were prevented by selective blockade of α7-nAChRs, ryanodine receptors, or IP3 receptors. In conclusion, positive allosteric modulation of α7-nAChRs with the PAM II PNU120596 can lead to dysregulation of ER Ca(2+) , overloading of intracellular Ca(2+) , and neuronal cell death. This study focuses on how the type II positive allosteric modulator PNU120596 (PAM II PNU12) affects intracellular Ca(2+) signaling and cell viability. Using SH-SY5Y neuroblastoma cells overexpressing α7-nAChRs (α7-SH) and their control (C-SH), we find that PAM of α7-nAChRs with PNU120596: (i) increases inward calcium current (ICa ) and cytosolic Ca(2+) concentration ([Ca(2+) ]cyt ); (ii) releases Ca(2+) from the ER ([Ca(2

  5. Inhibitory effect of schisandrin B on free fatty acid-induced steatosis in L-02 cells

    PubMed Central

    Chu, Jian-Hong; Wang, Hui; Ye, Yan; Chan, Ping-Kei; Pan, Si-Yuan; Fong, Wang-Fun; Yu, Zhi-Ling

    2011-01-01

    AIM: To investigate the effects of schisandrin B (Sch B) on free fatty acid (FFA)-induced steatosis in L-02 cells. METHODS: Cellular steatosis was induced by incubating L-02 cells with a FFA mixture (oleate and palmitate at the ratio of 2:1) for 24 h. Cytotoxicity and apoptosis were evaluated by 3-(4, 5-dmethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay and Annexin V/propidium iodide staining, respectively. Cellular total lipid was determined using a photocolorimetric method after Nile red staining, and triglyceride content was measured using an enzymatic kit. To study the effects of Sch B on steatosis, L-02 cells were treated with Sch B (1-100 μmol/L) in the absence or presence of 1 mmol/L FFA for 24 h, and cellular total lipid and triglyceride levels were measured. To explore the mechanisms of action of Sch B in the steatotic L-02 cells, mRNA levels of several regulators of hepatic lipid metabolism including adipose differentiation related protein (ADRP), sterol regulatory element binding protein 1 (SREBP-1), peroxisome proliferator-activated receptor (PPAR)-α and PPAR-γ were measured by quantitative real-time polymerase chain reaction (PCR), and protein levels of ADRP and SREBP-1 were measured by immunoblotting. RESULTS: Treatment with 1 mmol/L FFA for 24 h induced intracellular lipid accumulation in L-02 cells comparable to that in human steatotic livers without causing apparent apoptosis and cytotoxicity. Sch B mitigated cellular total lipid and triglyceride accumulations in the steatotic L-02 cells in a dose-dependent manner. Quantitative real-time PCR and Western blot analyses revealed that treatment of L-02 cells with 100 μmol/L Sch B reverted the FFA-stimulated up-regulation of ADRP and SREBP-1. CONCLUSION: Sch B inhibits FFA-induced steatosis in L-02 cells by, at least in part, reversing the up-regulation of ADRP and SREBP-1. PMID:21633637

  6. Effects of cordycepin on HepG2 and EA.hy926 cells: Potential antiproliferative, antimetastatic and anti-angiogenic effects on hepatocellular carcinoma

    PubMed Central

    LU, HAISHENG; LI, XITING; ZHANG, JIANYING; SHI, HUI; ZHU, XIAOFENG; HE, XIAOSHUN

    2014-01-01

    Hepatocellular carcinoma (HCC) is a hypervascular tumor and accumulating evidence suggests that angiogenesis plays an important role in HCC development. Cordycepin, also known as 3′-deoxyadenosine, is a derivative of adenosine, and numerous cellular enzymes cannot differentiate the two. The aim of the present study was to determine whether cordycepin regulates proliferation, migration and angiogenesis in a human umbilical vein endothelial cell line (EA.hy926) and in a hepatocellular carcinoma cell line (HepG2). MTT was used to assess cell proliferation. Apoptosis was analyzed by flow cytometry (propidium iodide staining). Transwell and wound healing assays were used to analyze the migration and invasion of HepG2 and EA.hy926 cells. Angiogenesis in EA.hy926 cells was assessed using a tube formation assay. Cordycepin strongly suppressed HepG2 and EA.hy926 cell proliferation in a dose- and time-dependent manner. Cordycepin induced EA.hy926 cell apoptosis in a dose-dependent manner (2,000 μg/ml: 50.20±1.55% vs. 0 μg/ml: 2.62±0.19%; P<0.01). Cordycepin inhibited EA.hy926 cell migration (percentage of wound healing area, 2,000 μg/ml: 3.45±0.29% vs. 0 μg/ml: 85.48±0.84%; P<0.05), as well as tube formation (total length of tubular structure, 1,000 μg/ml: 107±39 μm vs. 0 μg/ml: 936±56 μm; P<0.05). Cordycepin also efficiently inhibited HepG2 cell invasion and migration. High-performance liquid chromatography analysis of the cytosol from EA.hy926 cells showed that cordycepin was stable for 3 h. In conclusion, cordycepin not only inhibited human HepG2 cell proliferation and invasion, but also induced apoptosis and inhibited migration and angiogenesis in vascular endothelial cells, suggesting that cordycepin may be used as a novel anti-angiogenic therapy in HCC. PMID:24765175

  7. Sodium Butyrate Induces Endoplasmic Reticulum Stress and Autophagy in Colorectal Cells: Implications for Apoptosis

    PubMed Central

    Zhang, Jintao; Yi, Man; Zha, Longying; Chen, Siqiang; Li, Zhijia; Li, Cheng; Gong, Mingxing; Deng, Hong; Chu, Xinwei; Chen, Jiehua; Zhang, Zheqing; Mao, Limei; Sun, Suxia

    2016-01-01

    Purpose Butyrate, a short-chain fatty acid derived from dietary fiber, inhibits proliferation and induces cell death in colorectal cancer cells. However, clinical trials have shown mixed results regarding the anti-tumor activities of butyrate. We have previously shown that sodium butyrate increases endoplasmic reticulum stress by altering intracellular calcium levels, a well-known autophagy trigger. Here, we investigated whether sodium butyrate-induced endoplasmic reticulum stress mediated autophagy, and whether there was crosstalk between autophagy and the sodium butyrate-induced apoptotic response in human colorectal cancer cells. Methods Human colorectal cancer cell lines (HCT-116 and HT-29) were treated with sodium butyrate at concentrations ranging from 0.5–5mM. Cell proliferation was assessed using MTT tetrazolium salt formation. Autophagy induction was confirmed through a combination of Western blotting for associated proteins, acridine orange staining for acidic vesicles, detection of autolysosomes (MDC staining), and electron microscopy. Apoptosis was quantified by flow cytometry using standard annexinV/propidium iodide staining and by assessing PARP-1 cleavage by Western blot. Results Sodium butyrate suppressed colorectal cancer cell proliferation, induced autophagy, and resulted in apoptotic cell death. The induction of autophagy was supported by the accumulation of acidic vesicular organelles and autolysosomes, and the expression of autophagy-associated proteins, including microtubule-associated protein II light chain 3 (LC3-II), beclin-1, and autophagocytosis-associated protein (Atg)3. The autophagy inhibitors 3-methyladenine (3-MA) and chloroquine inhibited sodium butyrate induced autophagy. Furthermore, sodium butyrate treatment markedly enhanced the expression of endoplasmic reticulum stress-associated proteins, including BIP, CHOP, PDI, and IRE-1a. When endoplasmic reticulum stress was inhibited by pharmacological (cycloheximide and mithramycin

  8. Liposome-mediated transfection of wild-type P53 DNA into human prostate cancer cells is improved by low-frequency ultrasound combined with microbubbles

    PubMed Central

    BAI, WEN-KUN; ZHANG, WEI; HU, BING; YING, TAO

    2016-01-01

    Prostate cancer is a common type of cancer in elderly men. The aim of the present study was to evaluate the effects of ultrasound exposure in combination with SonoVue microbubbles on liposome-mediated transfection of wild-type P53 genes into human prostate cancer cells. PC-3 human prostate cancer cells were exposed to ultrasound; duty cycle was controlled at 20% (2 sec on, 8 sec off) for 5 min with and without SonoVue microbubble echo-contrast agent using a digital sonifier (frequency, 21 kHz; intensity, 46 mW/cm2). The cells were divided into eight groups, as follows: Group A (SonoVue + wild-type P53), group B (ultrasound + wild-type P53), group C (SonoVue + ultrasound + wild-type P53), group D (liposome + wild-type P53), group E (liposome + SonoVue + wild-type P53), group F (liposome + wild-type P53 + ultrasound), group G (liposome + wild-type P53 + ultrasound + SonoVue) and the control group (wild-type P53). Following treatment, a hemocytometer was used to measure cell lysis, reverse transcription-quantitative polymerase chain reaction and western blotting were performed to detect P53 gene transfection efficiency, Cell Counting Kit-8 was employed to reveal cell proliferation and Annexin V/propidium iodide staining was used to determine cell apoptosis. Cell lysis was minimal in each group. Wild-type P53 gene and protein expression were significantly increased in the PC-3 cells in group G compared with the control and all other groups (P<0.01). Cell proliferation was significantly suppressed in group G compared with the control group and all other groups (P<0.01). Cell apoptosis levels in group G were significantly improved compared with the control group and all other groups (P<0.01). Thus, the results of the present study indicate that the use of low-frequency and low-energy ultrasound in combination with SonoVue microbubbles may be a potent physical method for increasing liposome gene delivery efficiency. PMID:27313702

  9. Diffuse, non-polar electropermeabilization and reduced propidium uptake distinguish the effect of nanosecond electric pulses.

    PubMed

    Semenov, Iurii; Zemlin, Christian; Pakhomova, Olga N; Xiao, Shu; Pakhomov, Andrei G

    2015-10-01

    Ca2+ activation and membrane electroporation by 10-ns and 4-ms electric pulses (nsEP and msEP) were compared in rat embryonic cardiomyocytes. The lowest electric field which triggered Ca2+ transients was expectedly higher for nsEP (36 kV/cm) than for msEP (0.09 kV/cm) but the respective doses were similar (190 and 460 mJ/g). At higher intensities, both stimuli triggered prolonged firing in quiescent cells. An increase of basal Ca2+ level by >10 nM in cells with blocked voltage-gated Ca2+ channels and depleted Ca2+ depot occurred at 63 kV/cm (nsEP) or 0.14 kV/cm (msEP) and was regarded as electroporation threshold. These electric field values were at 150-230% of stimulation thresholds for both msEP and nsEP, notwithstanding a 400,000-fold difference in pulse duration. For comparable levels of electroporative Ca2+ uptake, msEP caused at least 10-fold greater uptake of propidium than nsEP, suggesting increased yield of larger pores. Electroporation by msEP started Ca2+ entry abruptly and locally at the electrode-facing poles of cell, followed by a slow diffusion to the center. In a stark contrast, nsEP evoked a "supra-electroporation" pattern of slower but spatially uniform Ca2+ entry. Thus nsEP and msEP had comparable dose efficiency, but differed profoundly in the size and localization of electropores. PMID:26112464

  10. Identification of Novel Gene Expression Targets for the Ras Association Domain Family 1 (RASSF1A) Tumor Suppressor Gene in Non-Small Cell Lung Cancer and Neuroblastoma1

    PubMed Central

    Agathanggelou, Angelo; Bièche, Ivan; Ahmed-Choudhury, Jalal; Nicke, Barbara; Dammann, Reinhard; Baksh, Shairaz; Gao, Boning; Minna, John D.; Downward, Julian; Maher, Eamonn R.; Latif, Farida

    2012-01-01

    RASSF1A is a recently identified 3p21.3 tumor suppressor gene. The high frequency of epigenetic inactivation of this gene in a wide range of human sporadic cancers including non-small cell lung cancer (NSCLC) and neuroblastoma suggests that RASSF1A inactivation is important for tumor development. Although little is known about the function of RASSF1A, preliminary data suggests that it may have multiple functions. To gain insight into RASSF1A functions in an unbiased manner, we have characterized the expression profile of a lung cancer cell line (A549) transfected with RASSF1A. Initially we demonstrated that transient expression of RASSF1A into the NSCLC cell line A549 induced G1 cell cycle arrest, as measured by propidium iodide staining. Furthermore, an-nexin-V staining showed that RASSF1A-expressing cells had an increased sensitivity to staurosporine-induced apoptosis. We then screened a cDNA microarray containing more than 6000 probes to identify genes differentially regulated by RASSF1A. Sixty-six genes showed at least a 2-fold change in expression. Among these were many genes with relevance to tumorigenesis involved in transcription, cytoskeleton, signaling, cell cycle, cell adhesion, and apoptosis. For 22 genes we confirmed the microarray results by real-time RT-PCR and/or Northern blotting. In silico, we were able to confirm the majority of these genes in other NSCLC cell lines using published data on gene expression profiles. Furthermore, we confirmed 10 genes at the RNA level in two neuroblastoma cell lines, indicating that these RASSF1A target genes have relevance in non-lung cell backgrounds. Protein analysis of six genes (ETS2, Cyclin D3, CDH2, DAPK1, TXN, and CTSL) showed that the changes induced by RASSF1A at the RNA level correlated with changes in protein expression in both non-small cell lung cancer and neuroblastoma cell lines. Finally, we have used a transient assay to demonstrate the induction of CDH2 and TGM2 by RASSF1A in NSCLC cell lines. We

  11. Protocol for Apoptosis Assay by Flow Cytometry Using Annexin V Staining Method

    PubMed Central

    Lakshmanan, Imayavaramban; Batra, Surinder K

    2016-01-01

    This assay is used to count the number of cells that have undergone apoptosis. Apoptosis will be detected by initially staining the cells with Annexin V and propidium Iodide solution followed by flow cytometry analysis. It is based on the principle that normal cells are hydrophobic in nature as they express phosphatidyl serine in the inner membrane (side facing the cytoplasm) and when the cells undergo apoptosis, the inner membrane flips to become the outer membrane, thus exposing phosphatidyl serine. The exposed phosphatidyl serine is detected by Annexin V, and propidium iodide stains the necrotic cells, which have leaky DNA content that help to differentiate the apoptotic and necrotic cells.

  12. Assessment of Probiotic Viability during Cheddar Cheese Manufacture and Ripening Using Propidium Monoazide-PCR Quantification.

    PubMed

    Desfossés-Foucault, Emilie; Dussault-Lepage, Véronique; Le Boucher, Clémentine; Savard, Patricia; Lapointe, Gisèle; Roy, Denis

    2012-01-01

    The use of a suitable food carrier such as cheese could significantly enhance probiotic viability during storage. The main goal of this study was to assess viability of commercial probiotic strains during Cheddar cheesemaking and ripening (4-6 months) by comparing the efficiency of microbiological and molecular approaches. Molecular methods such as quantitative PCR (qPCR) allow bacterial quantification, and DNA-blocking molecules such as propidium monoazide (PMA) select only the living cells' DNA. Cheese samples were manufactured with a lactococci starter and with one of three probiotic strains (Bifidobacterium animalis subsp. lactis BB-12, Lactobacillus rhamnosus RO011, or Lactobacillus helveticus RO052) or a mixed culture containing B. animalis subsp. lactis BB-12 and L. helveticus RO052 (MC1), both lactobacilli strains (MC2), or all three strains (MC3). DNA extractions were then carried out on PMA-treated and non-treated cell pellets in order to assess PMA treatment efficiency, followed by quantification using the 16S rRNA gene, the elongation factor Tu gene (tuf) or the transaldolase gene (tal). Results with intact/dead ratios of bacteria showed that PMA-treated cheese samples had a significantly lower bacterial count than non-treated DNA samples (P < 0.005), confirming that PMA did eliminate dead bacteria from PCR quantification. For both quantification methods, the addition of probiotic strains seemed to accelerate the loss of lactococci viability in comparison to control cheese samples, especially when L. helveticus RO052 was added. Viability of all three probiotic strains was also significantly reduced in mixed culture cheese samples (P < 0.0001), B. animalis subsp. lactis BB-12 being the most sensitive to the presence of other strains. However, all probiotic strains did retain their viability (log 9 cfu/g of cheese) throughout ripening. This study was successful in monitoring living probiotic species in Cheddar cheese samples through PMA

  13. Involvement of NF-κB and HSP70 signaling pathways in the apoptosis of MDA-MB-231 cells induced by a prenylated xanthone compound, α-mangostin, from Cratoxylum arborescens

    PubMed Central

    Ibrahim, Mohamed Yousif; Hashim, Najihah Mohd; Mohan, Syam; Abdulla, Mahmood Ameen; Abdelwahab, Siddig Ibrahim; Kamalidehghan, Behnam; Ghaderian, Mostafa; Dehghan, Firouzeh; Ali, Landa Zeenelabdin; Karimian, Hamed; Yahayu, Maizatulakmal; Ee, Gwendoline Cheng Lian; Farjam, Abdoreza Soleimani; Ali, Hapipah Mohd

    2014-01-01

    Background Cratoxylum arborescens has been used traditionally in Malaysia for the treatment of various ailments. Methods α-Mangostin (AM) was isolated from C. arborescens and its cell death mechanism was investigated. AM-induced cytotoxicity was observed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Acridine orange/propidium iodide staining and annexin V were used to detect cells in early phases of apoptosis. High-content screening was used to observe the nuclear condensation, cell permeability, mitochondrial membrane potential, and cytochrome c release. The role of caspases-3/7, -8, and -9, reactive oxygen species, Bcl-2 and Bax expression, and cell cycle arrest were also investigated. To determine the role of the central apoptosis-related proteins, a protein array followed by immunoblot analysis was conducted. Moreover, the involvement of nuclear factor-kappa B (NF-κB) was also analyzed. Results Apoptosis was confirmed by the apoptotic cells stained with annexin V and increase in chromatin condensation in nucleus. Treatment of cells with AM promoted cell death-transducing signals that reduced MMP by downregulation of Bcl-2 and upregulation of Bax, triggering cytochrome c release from the mitochondria to the cytosol. The released cytochrome c triggered the activation of caspase-9 followed by the executioner caspase-3/7 and then cleaved the PARP protein. Increase of caspase-8 showed the involvement of extrinsic pathway. AM treatment significantly arrested the cells at the S phase (P<0.05) concomitant with an increase in reactive oxygen species. The protein array and Western blotting demonstrated the expression of HSP70. Moreover, AM significantly blocked the induced translocation of NF-κB from cytoplasm to nucleus. Conclusion Together, the results demonstrate that the AM isolated from C. arborescens inhibited the proliferation of MDA-MB-231 cells, leading to cell cycle arrest and programmed cell death, which was suggested to

  14. Synergistic anticancer effects of a bioactive subfraction of Strobilanthes crispus and tamoxifen on MCF-7 and MDA-MB-231 human breast cancer cell lines

    PubMed Central

    2014-01-01

    Background Development of tumour resistance to chemotherapeutic drugs and concerns over their toxic effects has led to the increased use of medicinal herbs or natural products by cancer patients. Strobilanthes crispus is a traditional remedy for many ailments including cancer. Its purported anticancer effects have led to the commercialization of the plant leaves as medicinal herbal tea, although the scientific basis for its use has not been established. We previously reported that a bioactive subfraction of Strobilanthes crispus leaves (SCS) exhibit potent cytotoxic activity against human breast cancer cell lines. The current study investigates the effect of this subfraction on cell death activities induced by the antiestrogen drug, tamoxifen, in estrogen receptor-responsive and nonresponsive breast cancer cells. Methods Cytotoxic activity of SCS and tamoxifen in MCF-7 and MDA-MB-231 human breast cancer cells was determined using lactate dehydrogenase release assay and synergism was evaluated using the CalcuSyn software. Apoptosis was quantified by flow cytometry following Annexin V and propidium iodide staining. Cells were also stained with JC-1 dye to determine changes in the mitochondrial membrane potential. Fluorescence imaging using FAM-FLICA assay detects caspase-8 and caspase-9 activities. DNA damage in the non-malignant breast epithelial cell line, MCF-10A, was evaluated using Comet assay. Results The combined SCS and tamoxifen treatment displayed strong synergistic inhibition of MCF-7 and MDA-MB-231 cell growth at low doses of the antiestrogen. SCS further promoted the tamoxifen-induced apoptosis that was associated with modulation of mitochondrial membrane potential and activation of caspase-8 and caspase-9, suggesting the involvement of intrinsic and extrinsic signaling pathways. Interestingly, the non-malignant MCF-10A cells displayed no cytotoxicity or DNA damage when treated with either SCS or SCS-tamoxifen combination. Conclusions The combined use of

  15. Inhibition on Apoptosis Induced by Elevated Hydrostatic Pressure in Retinal Ganglion Cell-5 via Laminin Upregulating β1-integrin/Focal Adhesion Kinase/Protein Kinase B Signaling Pathway

    PubMed Central

    Li, Yi; Chen, Yan-Ming; Sun, Ming-Ming; Guo, Xiao-Dan; Wang, Ya-Chen; Zhang, Zhong-Zhi

    2016-01-01

    Background: Glaucoma is a progressive optic neuropathy characterized by degeneration of neurons due to loss of retinal ganglion cells (RGCs). High intraocular pressure (HIOP), the main risk factor, causes the optic nerve damage. However, the precise mechanism of HIOP-induced RGC death is not yet completely understood. This study was conducted to determine apoptosis of RGC-5 cells induced by elevated hydrostatic pressures, explore whether laminin is associated with apoptosis under pressure, whether laminin can protect RGCs from apoptosis and affirm the mechanism that regulates the process of RGCs survival. Methods: RGC-5 cells were exposed to 0, 20, 40, and 60 mmHg in a pressurized incubator for 6, 12, and 24 h, respectively. The effect of elevated hydrostatic pressure on RGC-5 cells was measured by Annexin V-fluorescein isothiocyanate/propidium iodide staining, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and Western blotting of cleaved caspase-3 protein. Location and expression of laminin were detected by immunofluorescence. The expression of β1-integrin, phosphorylation of focal adhesion kinase (FAK) and protein kinase B (PKB, or AKT) were investigated with real-time polymerase chain reaction and Western blotting analysis. Results: Elevated hydrostatic pressure induced apoptosis in cultured RGC-5 cells. Pressure with 40 mmHg for 24 h induced a maximum apoptosis. Laminin was declined in RGC-5 cells after exposing to 40 mmHg for 24 h. After pretreating with laminin, RGC-5 cells survived from elevated pressure. Furthermore, β1-integrin and phosphorylation of FAK and AKT were increased compared to 40 mmHg group. Conclusions: The data show apoptosis tendency of RGC-5 cells with elevated hydrostatic pressure. Laminin can protect RGC-5 cells against high pressure via β1-integrin/FAK/AKT signaling pathway. These results suggest that the decreased laminin of RGC-5 cells might be responsible for apoptosis induced by elevated hydrostatic pressure

  16. Quantitative Real-Time PCR Analysis of Total Propidium Monazide -Resistant Fecal Indicator Bacteria in Wastewater

    EPA Science Inventory

    A real-time quantitative PCR (qPCR) method and a modification of this method incorporating pretreatment of samples with propidium monoazide (PMA) were evaluated for respective analyses of total and presumptively viable Enterococcus and Bacteroidales fecal indicator bacteria. Thes...

  17. Assessment of Probiotic Viability during Cheddar Cheese Manufacture and Ripening Using Propidium Monoazide-PCR Quantification

    PubMed Central

    Desfossés-Foucault, Émilie; Dussault-Lepage, Véronique; Le Boucher, Clémentine; Savard, Patricia; LaPointe, Gisèle; Roy, Denis

    2012-01-01

    The use of a suitable food carrier such as cheese could significantly enhance probiotic viability during storage. The main goal of this study was to assess viability of commercial probiotic strains during Cheddar cheesemaking and ripening (4–6 months) by comparing the efficiency of microbiological and molecular approaches. Molecular methods such as quantitative PCR (qPCR) allow bacterial quantification, and DNA-blocking molecules such as propidium monoazide (PMA) select only the living cells’ DNA. Cheese samples were manufactured with a lactococci starter and with one of three probiotic strains (Bifidobacterium animalis subsp. lactis BB-12, Lactobacillus rhamnosus RO011, or Lactobacillus helveticus RO052) or a mixed culture containing B. animalis subsp. lactis BB-12 and L. helveticus RO052 (MC1), both lactobacilli strains (MC2), or all three strains (MC3). DNA extractions were then carried out on PMA-treated and non-treated cell pellets in order to assess PMA treatment efficiency, followed by quantification using the 16S rRNA gene, the elongation factor Tu gene (tuf) or the transaldolase gene (tal). Results with intact/dead ratios of bacteria showed that PMA-treated cheese samples had a significantly lower bacterial count than non-treated DNA samples (P < 0.005), confirming that PMA did eliminate dead bacteria from PCR quantification. For both quantification methods, the addition of probiotic strains seemed to accelerate the loss of lactococci viability in comparison to control cheese samples, especially when L. helveticus RO052 was added. Viability of all three probiotic strains was also significantly reduced in mixed culture cheese samples (P < 0.0001), B. animalis subsp. lactis BB-12 being the most sensitive to the presence of other strains. However, all probiotic strains did retain their viability (log 9 cfu/g of cheese) throughout ripening. This study was successful in monitoring living probiotic species in Cheddar cheese samples through PMA

  18. Evaluation of the cytotoxic effects of Cyperus longus extract, fractions and its essential oil on the PC3 and MCF7 cancer cell lines

    PubMed Central

    MEMARIANI, TOKTAM; HOSSEINI, TOKTAM; KAMALI, HOSSEIN; MOHAMMADI, AMENEH; GHORBANI, MARYAM; SHAKERI, ABDOREZA; SPANDIDOS, DEMETRIOS A.; TSATSAKIS, ARISTIDIS M.; SHAHSAVAND, SHABNAM

    2016-01-01

    Cyperus longus is one of the Iranian endemic species. However, to date, and to the best of our knowledge, there are no availale academic reports on the cytotoxicity of this plant. Thus, this study was carried out to examine the in vitro anti-proliferative and anti-apoptotic effects of Cyperus longus extract, fractions and essential oil (EO) on MCF7 and PC3 cell lines. The chemical constituents of EO were identified using gas chromatography (GC)-mass spectrometry (MS) analysis. The cells were cultured in RPMI-1640 medium and incubated with various concentrations of the plant extract and fractions. Cell viability was quantified by MTT assay following 24, 48 and 72 h of exposure to (12.5–200 µg/ml) of the methanol extract, the dichloromethane (CH2Cl2), ethyl acetate (EtOAc) and water fractions, as well as the EO of the plant. The percentage of apoptotic cells was determined using propidium iodide staining of DNA fragments by flow cytometry (sub-G1 peak). The most effective fraction in the MCF7 cell line was the CH2Cl2 fraction (IC50 after 48 h, 25.34±2.01). The EtOAc fraction (IC50 after 48 h, 35.2±2.69) and the methanol extract (IC50 after 48 h, 64.64±1.64) were also found to be effective. The IC50 values obtained for the PC3 cell line were 37.97±3.87, 51.57±3.87 and 70.33±2.36 for the CH2Cl2 fraction, the EtOAc fraction and the methanol extract, respectively. Based on these data and due to the partial polarity of the most effective fraction (the CH2Cl2 fraction), we also examined the cytotoxicity of the plant EO. The IC50 values after 48 h were 22.25±4.25 and 12.55±3.65 in the PC3 and MCF7 cell lines, respectively. DNA fragmentation assay also confirmed these data. Performing GC-MS analysis for the plant EO revealed that β-himachalene (10.81%), α-caryophyllene oxide (7.6%), irisone (4.78%), β-caryophyllene oxide (4.36%), humulene oxide (12%), viridiflorol (4.73%), aristolone (6.39%) and longiverbenone (6.04%) were the main constituents. Our results

  19. Thymoquinone from Nigella sativa Seeds Promotes the Antitumor Activity of Noncytotoxic Doses of Topotecan in Human Colorectal Cancer Cells in Vitro.

    PubMed

    Khalife, Rana; Hodroj, Mohammad Hassan; Fakhoury, Rajaa; Rizk, Sandra

    2016-03-01

    Topotecan, a topoisomerase I inhibitor, is an anticancer drug widely used in the therapy of lung, ovarian, colorectal, and breast adenocarcinoma. Due to the primary dose-limiting toxicity of topotecan, which is myelosuppressive, it is necessary to identify other chemotherapeutic agents that can work synergistically with topotecan to increase its efficacy and limit its toxicity. Many studies have shown synergism upon the combination of topotecan with other chemotherapeutic agents such as gemcitabine. Other studies have demonstrated that pre-exposing cells to naturally occurring compounds such as thymoquinone, followed by gemcitabine or oxaliplatin, resulted in higher growth inhibition compared to treatment with gemcitabine or oxaliplatin alone. Our aim was to elucidate the underlying mechanism of action of topotecan in the survival and apoptotic pathways in human colon cancer cell lines in comparison to thymoquinone, to study the proapoptotic and antiproliferative effects of thymoquinone on the effectiveness of the chemotherapeutic agent topotecan, and to investigate the potential synergistic effect of thymoquinone with topotecan. Cells were incubated with different topotecan and thymoquinone concentrations for 24 and 48 hours in order to determine the IC50 for each drug. Combined therapy was then tested with ± 2 values for the IC50 of each drug. The reduction in proliferation was significantly dose- and time-dependent. After determining the best combination (40 µM thymoquinone and 0.6 µM topotecan), cell proteins were extracted after treatment, and the expression levels of B-cell lymphoma 2 and of its associated X protein, proteins p53 and p21, and caspase-9, caspase-3, and caspase-8 were studied by Western blot. In addition, cell cycle analysis and annexin/propidium iodide staining were performed. Both drugs induced apoptosis through a p53-independent mechanism, whereas the expression of p21 was only seen in thymoquinone treatment. Cell cycle arrest in

  20. Sanguinarine inhibits angiotensin II-induced apoptosis in H9c2 cardiac cells via restoring reactive oxygen species-mediated decreases in the mitochondrial membrane potential

    PubMed Central

    LIU, YUAN; JIAO, RONG; MA, ZHEN-GUO; LIU, WEI; WU, QING-QING; YANG, ZHENG; LI, FANG-FANG; YUAN, YUAN; BIAN, ZHOU-YAN; TANG, QI-ZHU

    2015-01-01

    Cell apoptosis induced by Angiotensin II (Ang II) has a critical role in the development of cardiovascular diseases. The aim of the present study was to investigate whether sanguinarine (SAN), a drug which was proved to have anti-oxidant, anti-proliferative and immune enhancing effects, can abolish cell apoptosis induced by Ang II. In the present study, H9c2 cardiac cells were stimulated with 10 µM Ang II with or without SAN. The level of intracellular reactive oxygen species (ROS) generation was assessed using dichlorodihydrofluorescein diacetate, and changes of the mitochondrial membrane potential (MMP) were assessed using JC-1 staining. Furthermore, mRNA expression of NOX2 was determined by reverse transcription quantitative polymerase chain reaction, and apoptosis was detected by Annexin V/propidium iodide staining and flow cytometry. The expression of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) as well as cleaved (c)-caspase 3 and -9 were detected by western blot analysis, and the activity of caspase 3 and -9 was detected using an ELISA. The results of the present study showed that NOX2 expression and ROS generation induced by Ang II were inhibited by SAN, and the Ang 2-induced MMP loss was also ameliorated. Furthermore, Ang II-induced H9c2 cardiac cell apoptosis as well as c-caspase 3 and -9 levels were significantly reduced by SAN. Investigation of the possible pathway involved in the anti-apoptotic effect of SAN showed that the expression of Bcl-2 was decreased, while that of Bax was increased following stimulation with Ang II, which was reversed following treatment with SAN. In addition, Ang II enhanced the activity of caspase 9 and cleaved downstream caspases such as caspase-3, initiating the caspase cascade, while pre-treatment of H9c2 cardiac cells with SAN blocked these effects. In conclusion, the findings of the present study indicated that SAN inhibits the apoptosis of H9c2 cardiac cells induced by Ang II, most likely via restoring

  1. ROS-Dependent Mitochondria Molecular Mechanisms Underlying Antitumor Activity of Pleurotus abalonus Acidic Polysaccharides in Human Breast Cancer MCF-7 Cells

    PubMed Central

    Shi, Xiaolong; Zhao, Yan; Jiao, Yadong; Shi, Tengrui; Yang, Xingbin

    2013-01-01

    Background A greater reduction in cancer risk associated with mushroom diet rich in fungus polysaccharides is generally accepted. Meanwhile, edible Pleurotus abalonus as a member of Abalone mushroom family is a popular nutritional supplement that purportedly prevents cancer occurrence. However, these anecdotal claims are supported by limited studies describing tumor-inhibitory responses to the promising polysaccharides, and the molecular mechanisms underlying these properties have not yet been elucidated. Methodology/Principal Findings We here fractionated the crude polysaccharide preparation from the fruiting bodies of P. abalonus into three fractions, namely PAP-1, PAP-2 and PAP-3, and tested these fractions for antiproliferative activity in human breast cancer MCF-7 cells. The largest PAP-3, an acidic polysaccharide fraction with a molecular mass of 3.68×105 Da, was the most active in inhibiting MCF-7 cancer cells with an IC50 of 193 µg/mL. The changes in cell normal morphology were observed by DAPI staining and the PAP-3-induced apoptosis was confirmed by annexin V/propidium iodide staining. The apoptosis was involved in mitochondria-mediated pathway including the loss of mitochondrial membrane potential (Δψm), the increase of Bax/Bcl-2 ratio, caspase-9/3 activation, and poly(ADP-ribose) polymerase (PARP) degradation, as well as intracellular ROS production. PAP-3 also induced up-regulation of p53, and cell cycle arrest at the S phase. The incubation of MCF-7 cells with antioxidant superoxide dismutase (SOD) and N-acetylcysteine (NAC) significantly attenuated the ROS generation and apoptosis caused by PAP-3, indicating that intracellular ROS plays a pivotal role in cell death. Conclusions/Significance These findings suggest that the polysaccharides, especially acidic PAP-3, are very important nutritional ingredients responsible for, at least in part, the anticancer health benefits of P. abalonus via ROS-mediated mitochondrial apoptotic pathway. It is a

  2. Biological assays on the effects of Acra3 peptide from Turkish scorpion Androctonus crassicauda venom on a mouse brain tumor cell line (BC3H1) and production of specific monoclonal antibodies.

    PubMed

    Caliskan, Figen; Ergene, Emel; Sogut, Ibrahim; Hatipoglu, Ibrahim; Basalp, Aynur; Sivas, Hulya; Kanbak, Gungor

    2013-12-15

    Constitutes of the venom scorpion are a rich source of low molecular mass peptides which are toxic to various organisms, including man. Androctonus crassicauda is one of the scorpions from the Southeastern Anatolia of Turkey with public health importance. This work is focused on the investigation of biological effects of Acra3 peptide from Androctonus crassicauda. For this purpose, Acra3 isolated from crude venoms was tested for its cytotoxicity on BC3H1 mouse brain tumor cells using tetrazolium salt cleavage and lactate dehydrogenase activity assays. To determine whether the cytotoxic effects of Acra3 was related to the induction of apoptosis, the morphology of the cells and the nuclear fragmentation was examined by using Acridin Orange staining and DNA fragmentation assay, respectively. Caspase 3 and caspase 9 activities were measured spectrophotometrically and flow cytometric assay was performed using Annexin-V FITC and Propidium Iodide staining. Furthermore toxic peptide Acra3 was used as an antigen for immunological studies. Results showed that Acra3 exerted very strong cytotoxic effect on BC3H1 cells with an IC50 value of 5 μg/ml. Exposure of the cells to 0.1 and 0.5 μg/ml was resulted in very strong appearance of the apoptotic morphology in a dose dependent manner. On the other side, not any DNA fragmentation was observed after treatment of the cells. Caspase 3 and 9 activities were slightly decreased with Acra3. Results from flow cytometry and lactate dehydrogenase activity assays indicate that Acra3 exerts its effects by inducing a stronger necrosis than apoptosis in BC3H1 cells. To evaluate its immunogenicity, monoclonal antibody (MAb) specific for Acra3 antigen (5B9) was developed by hybridoma technology using spleen and lymph nodes of mice and immunoglobulin type of antibody was found to be IgM. We suggest that Acra3 may exert its effects by inducing both necrotic and apoptotic pathway in some way on mouse brain tumor cells. These findings will be

  3. Hepatitis C Virus Core Protein Down-Regulates p21Waf1/Cip1 and Inhibits Curcumin-Induced Apoptosis through MicroRNA-345 Targeting in Human Hepatoma Cells

    PubMed Central

    Shiu, Tzu-Yue; Huang, Shih-Ming; Shih, Yu-Lueng; Chu, Heng-Cheng; Chang, Wei-Kuo; Hsieh, Tsai-Yuan

    2013-01-01

    Background Hepatitis C virus (HCV) has been reported to regulate cellular microRNAs. The HCV core protein is considered to be a potential oncoprotein in HCV-related hepatocellular carcinoma, but HCV core-modulated cellular microRNAs are unknown. The HCV core protein regulates p21Waf1/Cip1 expression. However, the mechanism of HCV core-associated p21Waf1/Cip1 regulation remains to be further clarified. Therefore, we attempted to determine whether HCV core-modulated cellular microRNAs play an important role in regulating p21Waf1/Cip1 expression in human hepatoma cells. Methods Cellular microRNA profiling was investigated in core-overexpressing hepatoma cells using TaqMan low density array. Array data were further confirmed by TaqMan real-time qPCR for single microRNA in core-overexpressing and full-length HCV replicon-expressing cells. The target gene of microRNA was examined by reporter assay. The gene expression was determined by real-time qPCR and Western blotting. Apoptosis was examined by annexin V-FITC apoptosis assay. Cell cycle analysis was performed by propidium iodide staining. Cell proliferation was analyzed by MTT assay. Results HCV core protein up- or down-regulated some cellular microRNAs in Huh7 cells. HCV core-induced microRNA-345 suppressed p21Waf1/Cip1 gene expression through targeting its 3′ untranslated region in human hepatoma cells. Moreover, the core protein inhibited curcumin-induced apoptosis through p21Waf1/Cip1-targeting microRNA-345 in Huh7 cells. Conclusion and Significance HCV core protein enhances the expression of microRNA-345 which then down-regulates p21Waf1/Cip1 expression. It is the first time that HCV core protein has ever been shown to suppress p21Waf1/Cip1 gene expression through miR-345 targeting. PMID:23577194

  4. Quantifying fungal viability in air and water samples using quantitative PCR after treatment with propidium monoazide (PMA)

    SciTech Connect

    Vesper, Stephen; McKinstry, Craig A.; Hartmann, Chris; Neace, Michelle; Yoder, Stephanie; Vesper, Alex

    2007-11-28

    A method is described to discriminate between live and dead cells of the infectious fungi Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Mucor racemosus, Rhizopus stolonifer and Paecilomyces variotii. To test the method, conidial suspensions were heat inactivated at 85 °C or held at 5 °C (controls) for 1 h. Polycarbonate filters (25 mm diameter, 0.8 μm pore size) were placed on "welled" slides (14 mm diameter) and the filters treated with either PBS or PMA. Propidium monoazide (PMA), which enters dead cells but not live cells, was incubated with cell suspensions, exposed to blue wavelength light-emitting diodes (LED) to inactivate remaining PMA and secure intercalation of PMAwith DNA of dead cells. Treated cells were extracted and the live and dead cells evaluated with quantitative PCR (QPCR). After heat treatment and DNA modification with PMA, all fungal species tested showed an approximate 100- to 1000-fold difference in cell viability estimated by QPCR analysis which was consistent with estimates of viability based on culturing.

  5. Anticancer Effects of 1,3-Dihydroxy-2-Methylanthraquinone and the Ethyl Acetate Fraction of Hedyotis Diffusa Willd against HepG2 Carcinoma Cells Mediated via Apoptosis.

    PubMed

    Li, Yun-Lan; Zhang, Jiali; Min, Dong; Hongyan, Zhou; Lin, Niu; Li, Qing-Shan

    2016-01-01

    Hedyotis Diffusa Willd, used in Traditional Chinese Medicine, is a treatment for various diseases including cancer, owing to its mild effectiveness and low toxicity. The aim of this study was to identify the main anticancer components in Hedyotis Diffusa Willd, and explore mechanisms underlying their activity. Hedyotis Diffusa Willd was extracted and fractionated using ethyl acetate to obtain the H-Ethyl acetate fraction, which showed higher anticancer activity than the other fractions obtained against HepG2 cells with sulforhodamine B assays. The active component of the H-Ethyl acetate fraction was identified to be 1,3-dihydroxy-2-methylanthraquinone (DMQ) with much high inhibitory rate up to 48.9 ± 3.3% and selectivity rate up to 9.4 ± 4.5 folds (p<0.01) at 125 μmol/L. HepG2 cells treated with the fraction and DMQ visualized morphologically using light and fluorescence microscopy. Annexin V--fluorescein isothiocyanate / propidium iodide staining flow cytometry, DNA ladder and cell cycle distribution assays. Mechanistic studies showed up-regulation of caspase-3, -8, and -9 proteases activities (p<0.001), indicating involvement of mitochondrial apoptotic and death receptor pathways. Further studies revealed that reactive oxygen species in DMQ and the fraction treated HepG2 cells increased (p<0.01) while mitochondrial membrane potential reduced significantly (p<0.001) compared to the control by flow cytometry assays. Western blot analysis showed that Bax, p53, Fas, FasL, p21 and cytoplasmic cytochrome C were up-regulated (p<0.01), while Bcl-2, mitochondrial cytochrome C, cyclin E and CDK 2 were down-regulated dose-dependently (p<0.01). The reverse transcriptase-polymerase chain reaction showed that mRNA expressions of p53 and Bax increased (p<0.001) while that of Bcl-2 decreased (p<0.001). Pre-treatment with caspase-8 inhibitor Z-IETD-FMK, or caspase-9 inhibitor Z-LEHD-FMK, attenuated the growth-inhibitory and apoptosis-inducing effects of DMQ and the fraction on

  6. Anticancer Effects of 1,3-Dihydroxy-2-Methylanthraquinone and the Ethyl Acetate Fraction of Hedyotis Diffusa Willd against HepG2 Carcinoma Cells Mediated via Apoptosis

    PubMed Central

    Li, Yun-lan; Zhang, Jiali; Min, Dong; Hongyan, Zhou; Lin, Niu; Li, Qing-shan

    2016-01-01

    Hedyotis Diffusa Willd, used in Traditional Chinese Medicine, is a treatment for various diseases including cancer, owing to its mild effectiveness and low toxicity. The aim of this study was to identify the main anticancer components in Hedyotis Diffusa Willd, and explore mechanisms underlying their activity. Hedyotis Diffusa Willd was extracted and fractionated using ethyl acetate to obtain the H-Ethyl acetate fraction, which showed higher anticancer activity than the other fractions obtained against HepG2 cells with sulforhodamine B assays. The active component of the H-Ethyl acetate fraction was identified to be 1,3-dihydroxy-2-methylanthraquinone (DMQ) with much high inhibitory rate up to 48.9 ± 3.3% and selectivity rate up to 9.4 ± 4.5 folds (p<0.01) at 125 μmol/L. HepG2 cells treated with the fraction and DMQ visualized morphologically using light and fluorescence microscopy. Annexin V—fluorescein isothiocyanate / propidium iodide staining flow cytometry, DNA ladder and cell cycle distribution assays. Mechanistic studies showed up-regulation of caspase-3, -8, and -9 proteases activities (p<0.001), indicating involvement of mitochondrial apoptotic and death receptor pathways. Further studies revealed that reactive oxygen species in DMQ and the fraction treated HepG2 cells increased (p<0.01) while mitochondrial membrane potential reduced significantly (p<0.001) compared to the control by flow cytometry assays. Western blot analysis showed that Bax, p53, Fas, FasL, p21 and cytoplasmic cytochrome C were up-regulated (p<0.01), while Bcl-2, mitochondrial cytochrome C, cyclin E and CDK 2 were down-regulated dose-dependently (p<0.01). The reverse transcriptase-polymerase chain reaction showed that mRNA expressions of p53 and Bax increased (p<0.001) while that of Bcl-2 decreased (p<0.001). Pre-treatment with caspase-8 inhibitor Z-IETD-FMK, or caspase-9 inhibitor Z-LEHD-FMK, attenuated the growth-inhibitory and apoptosis-inducing effects of DMQ and the fraction

  7. Isoeugenol destabilizes IL-8 mRNA expression in THP-1 cells through induction of the negative regulator of mRNA stability tristetraprolin.

    PubMed

    Galbiati, Valentina; Carne, Alice; Mitjans, Montserrat; Galli, Corrado Lodovico; Marinovich, Marina; Corsini, Emanuela

    2012-02-01

    We previously demonstrated in the human promyelocytic cell line THP-1 that all allergens tested, with the exception of the prohapten isoeugenol, induced a dose-related release of interleukin-8 (IL-8). In the present study, we investigated whether this abnormal behavior was regulated by the AU-rich element-binding proteins HuR and tristetraprolin (TTP) or by the downstream molecule suppressor of cytokine signaling (SOCS)-3. The contact allergens isoeugenol, diethylmaleate (DEM), and 2,4-dinitrochlorobenzene (DNCB), and the irritant salicylic acid were used as reference compounds. Chemicals were used at concentrations that induced a 20% decrease in cell viability as assessed by propidium iodide staining, namely 100 μg/ml (0.61 mM) for isoeugenol, 100 μg/ml (0.58 mM) for DEM, 3 μg/ml (14.8 μM) for DNCB, and 250 μg/ml (1.81 mM) for salicylic acid. Time course experiments of IL-8 mRNA expression and assessment of IL-8 mRNA half-life, indicated a decreased IL-8 mRNA stability in isoeugenol-treated cells. We could demonstrate that a combination and regulation of HuR and TTP following exposure to contact allergens resulted in a different modulation of IL-8 mRNA half-life and release. The increased expression of TTP in THP-1 cells treated with isoeugenol results in destabilization of the IL-8 mRNA, which can account for the lack of IL-8 release. In contrast, the strong allergen DNCB failing to up-regulate TTP, while inducing HuR, resulted in longer IL-8 mRNA half-life and protein release. SOCS-3 was induced only in isoeugenol-treated cells; however, its modulation did not rescue the lack of IL-8 release, indicating that it is unlikely to be involved in the lack of IL-8 production. Finally, the destabilization effect of isoeugenol on IL-8 mRNA expression together with SOCS-3 expression resulted in an anti-inflammatory effect, as demonstrated by the ability of isoeugenol to modulate LPS or ionomycin-induced cytokine release. PMID:21969073

  8. Use of ethidium monoazide and propidium monoazide to determine viral infectivity upon inactivation by heat, UV- exposure and chlorine.

    PubMed

    Leifels, Mats; Jurzik, Lars; Wilhelm, Michael; Hamza, Ibrahim Ahmed

    2015-11-01

    Despite the great sensitivity of PCR in monitoring enteric viruses in an aquatic environment, PCR detects viral nucleic acids of both infectious and noninfectious viruses, limiting the conclusions regarding significance for public health. Ethidium monoazide (EMA) and propidium monoazide (PMA) are closely related membrane impermeant dyes that selectively penetrate cells with compromised membranes. Inside the cells, the dye can intercalate into nucleic acids and inhibit PCR amplification. To assess whether EMA and PMA pretreatment is a suitable approach to inhibit DNA amplification from noninfectious viruses upon heat treatment, UV exposure or chlorine treatment, viruses were measured by qPCR, EMA-qPCR, PMA-qPCR and cell culture titration. EMA/PMA-qPCR of UV- and heat-treated viruses did not correlate with the results of the cell culture assay. However, the data from EMA/PMA-qPCR of chlorine-inactivated viruses was consistent with the cell culture infectivity assay. Therefore, a dye treatment approach could be a rapid and inexpensive tool to screen the efficacy of chlorine disinfection, but it is not able to distinguish between infectious and noninfectious viruses inactivated via heat treatment or UV irradiation. Indeed, different viruses may have different trends and mechanisms of inactivation; thus, the assay must be evaluated for each virus separately. PMID:25747544

  9. Nanosecond pulsed electric fields and the cell cycle

    NASA Astrophysics Data System (ADS)

    Mahlke, Megan A.

    Exposure to nanosecond pulsed electrical fields (nsPEFs) can cause poration of external and internal cell membranes, DNA damage, and disassociation of cytoskeletal components, all of which are capable of disrupting a cell's ability to replicate. The phase of the cell cycle at the time of exposure is linked to differential sensitivities to nsPEFs across cell lines, as DNA structure, membrane elasticity, and cytoskeletal structure change dramatically during the cell cycle. Additionally, nsPEFs are capable of activating cell cycle checkpoints, which could lead to apoptosis or slow population growth. NsPEFs are emerging as a method for treating tumors via apoptotic induction; therefore, investigating the relevance of nsPEFs and the cell cycle could translate into improved efficacy in tumor treatment. Populations of Jurkat and Chinese Hamster Ovary (CHO) cells were examined post-exposure (10 ns pulse trains at 150kV/cm) by analysis of DNA content via propidium iodide staining and flow cytometric analysis at various time points (1, 6, and 12h post-exposure) to determine population distribution in cell cycle phases. Additionally, CHO and Jurkat cells were synchronized in G1/S and G2/M phases, pulsed, and analyzed to evaluate the role of cell cycle phase in survival of nsPEFs. CHO populations appeared similar to sham populations post-nsPEFs but exhibited arrest in the G1 phase at 6h after exposure. Jurkat cells exhibited increased cell death after nsPEFs compared to CHO cells but did not exhibit checkpoint arrest at any observed time point. The G1/S phase checkpoint is partially controlled by the action of p53; the lack of an active p53 response in Jurkat cells could contribute to their ability to pass this checkpoint and resist cell cycle arrest. Both cell lines exhibited increased sensitivity to nsPEFs in G2/M phase. Live imaging of CHO cells after nsPEF exposure supports the theory of G1/S phase arrest, as a reduced number of cells undergo mitosis within 24 h when

  10. From red to green: the propidium iodide-permeable membrane of Shewanella decolorationis S12 is repairable

    PubMed Central

    Yang, Yonggang; Xiang, Yinbo; Xu, Meiying

    2015-01-01

    Viability is a common issue of concern in almost all microbial processes. Fluorescence-based assays are extensively used in microbial viability assessment, especially for mixed-species samples or biofilms. Propidium iodide (PI) is the most frequently used fluorescence indicator for cell viability based on the membrane permeability. Our results showed that the accumulation of succinate from fumarate respiration could induce PI-permeability in Shewanella decolorationis biofilm cells. Confocal laser scanning microscope further showed that the PI-permeable membrane could be repaired in situ when the extracellular succinate was eliminated by switching fumarate respiration to electrode respiration. Simultaneously with the membrane repair, the electrode respiring capacity of the originally PI-permeable cells was recovered. Agar-colony counts suggested that a major portion of the repaired cells were viable but nonculturable (VBNC). The results evidenced that S. decolorationis S12 has the capacity to repair PI-permeable membranes which suggests a reevaluation of the fate and function of the PI-permeable bacteria and expanded our knowledge on the flexibility of bacterial survival status in harsh environments. PMID:26687136

  11. Argon mediates protection by interleukin-8 suppression via a TLR2/TLR4/STAT3/NF-κB pathway in a model of apoptosis in neuroblastoma cells in vitro and following ischemia-reperfusion injury in rat retina in vivo.

    PubMed

    Ulbrich, Felix; Lerach, Teresa; Biermann, Julia; Kaufmann, Kai B; Lagreze, Wolf A; Buerkle, Hartmut; Loop, Torsten; Goebel, Ulrich

    2016-09-01

    Argon has recently come into scientific focus as a neuroprotective agent. The underlying neuroprotective mechanism remains unknown although toll-like receptors were recently suggested to play an important role. We hypothesized that TLR-associated downstream transcription factors are responsible for argon's effects, leading to anti-apoptotic and anti-inflammatory properties. Apoptosis was induced in human neuroblastoma cells. Immediately afterwards, argon treatment (75 Vol% for 2 h) was initiated. Cells were analyzed, measuring mitochondrial membrane potential, reactive-oxygen-species, annexin-V/propidium iodide staining, transcription factor phosphorylation and binding activity as well as protein and mRNA expression of interleukins. Argon's in vivo effects were analyzed by quantification of retinal ganglion cell density, mRNA expression, serum cytokine analysis and immunohistochemistry after retinal ischemia reperfusion injury (IRI) in rats. Argon diminished rotenone-induced kappa-light-chain-enhancer' of activated B-cells (NF-κB) and signal transducer and activator of transcription 3 (STAT3) but not STAT5 or cAMP-response element-binding protein (CREB) phosphorylation and DNA-binding activity. Argon treatment attenuated apoptosis by preservation of mitochondrial membrane potential and decline in reactive oxygen species (ROS) generation. NF-κB and STAT3 inhibition, as well as TLR2 and TLR4 inhibition reversed argon's effects on IL-8 mRNA expression. Argon attenuated rotenone-induced IL-8 protein and mRNA expression in vitro. Inhibition of TLR2 and 4 attenuated argon's protective effect in vivo reducing IRI driven retinal IL-8 expression. IL-8 expression was found in the retina in co-localization with Müller cells and retinal ganglion cells. Argon mediates its neuroprotective effects by TLR-mediated regulation of transcription factors NF-κB and STAT3, thus decreasing interleukin-8 expression in vitro and in vivo. These findings may open up new

  12. Limitations of Using Propidium Monoazide with qPCR to Discriminate between Live and Dead Legionella in Biofilm Samples

    PubMed Central

    Taylor, Michael J; Bentham, Richard H; Ross, Kirstin E

    2014-01-01

    Accurately quantifying Legionella for regulatory purposes to protect public health is essential. Real-time PCR (qPCR) has been proposed as a better method for detecting and enumerating Legionella in samples than conventional culture method. However, since qPCR amplifies any target DNA in the sample, the technique’s inability to discriminate between live and dead cells means that counts are generally significantly overestimated. Propidium monoazide (PMA) has been used successfully in qPCR to aid live/dead discrimination. We tested PMA use as a method to count only live Legionella cells in samples collected from a modified chemostat that generates environmentally comparable samples. Counts from PMA-treated samples that were pretreated with either heat or three types of disinfectants (to kill the cells) were highly variable, with the only consistent trend being the relationship between biofilm mass and numbers of Legionella cells. Two possibilities explain this result: 1. PMA treatment worked and the subsequent muted response of Legionella to disinfection treatment is a factor of biofilm/microbiological effects; although this does not account for the relationship between the amount of biofilm sampled and the viable Legionella count as determined by PMA-qPCR; or 2. PMA treatment did not work, and any measured decrease or increase in detectable Legionella is because of other factors affecting the method. This is the most likely explanation for our results, suggesting that higher concentrations of PMA might be needed to compensate for the presence of other compounds in an environmental sample or that lower amounts of biofilm need to be sampled. As PMA becomes increasingly toxic at higher concentrations and is very expensive, augmenting the method to include higher PMA concentrations is both counterproductive and cost prohibitive. Conversely, if smaller volumes of biofilm are used, the reproducibility of the method is reduced. Our results suggest that using PMA is not

  13. Limitations of Using Propidium Monoazide with qPCR to Discriminate between Live and Dead Legionella in Biofilm Samples.

    PubMed

    Taylor, Michael J; Bentham, Richard H; Ross, Kirstin E

    2014-01-01

    Accurately quantifying Legionella for regulatory purposes to protect public health is essential. Real-time PCR (qPCR) has been proposed as a better method for detecting and enumerating Legionella in samples than conventional culture method. However, since qPCR amplifies any target DNA in the sample, the technique's inability to discriminate between live and dead cells means that counts are generally significantly overestimated. Propidium monoazide (PMA) has been used successfully in qPCR to aid live/dead discrimination. We tested PMA use as a method to count only live Legionella cells in samples collected from a modified chemostat that generates environmentally comparable samples. Counts from PMA-treated samples that were pretreated with either heat or three types of disinfectants (to kill the cells) were highly variable, with the only consistent trend being the relationship between biofilm mass and numbers of Legionella cells. Two possibilities explain this result: 1. PMA treatment worked and the subsequent muted response of Legionella to disinfection treatment is a factor of biofilm/microbiological effects; although this does not account for the relationship between the amount of biofilm sampled and the viable Legionella count as determined by PMA-qPCR; or 2. PMA treatment did not work, and any measured decrease or increase in detectable Legionella is because of other factors affecting the method. This is the most likely explanation for our results, suggesting that higher concentrations of PMA might be needed to compensate for the presence of other compounds in an environmental sample or that lower amounts of biofilm need to be sampled. As PMA becomes increasingly toxic at higher concentrations and is very expensive, augmenting the method to include higher PMA concentrations is both counterproductive and cost prohibitive. Conversely, if smaller volumes of biofilm are used, the reproducibility of the method is reduced. Our results suggest that using PMA is not an

  14. Detection of viable antibiotic-resistant/sensitive Acinetobacter baumannii in indoor air by propidium monoazide quantitative polymerase chain reaction.

    PubMed

    Tseng, C-C; Hsiao, P-K; Chang, K-C; Cheng, C-C; Yiin, L-M; Hsieh, C-J

    2015-10-01

    Acinetobacter baumannii represents a significant cause of nosocomial infections. Therefore, we combined real-time quantitative polymerase chain reaction (PCR) with the propidium monoazide (PMA-qPCR) to assess the feasibility of detecting viable, airborne A. baumannii. The biological collection efficiencies of three samplers for collecting airborne A. baumannii were evaluated by PMA-qPCR in a chamber study. After sampling, the effects of storage in collection fluid on A. baumannii were evaluated. The results showed that the culturable ratio of A. baumannii measured using the culture method was significantly correlated with the viable ratio measured using PMA-qPCR, but was not significantly correlated with the qPCR results. It was indicated that the AGI-30 impinger and the BioSampler were much more effective than the Nuclepore filter sampler for collecting airborne A. baumannii. The storage temperature was critical for aerosol samples, as the loss of viable A. baumannii was minimized when the PMA-bound DNA was stored at -20°C or if the collected cells were stored at 4°C and subsequently processed by PMA-qPCR within 1 month. The PMA-qPCR method was also to distinguish between colistin-sensitive and colistin-resistant A. baumannii, and no colistin-sensitive A. baumannii was detected by PMA-qPCR upon treatment of the BioSampler collection medium with 2 μg/ml colistin for 5 min. PMID:25283547

  15. Quantification of viable Giardia cysts and Cryptosporidium oocysts in wastewater using propidium monoazide quantitative real-time PCR.

    PubMed

    Alonso, José L; Amorós, Inmaculada; Guy, Rebecca A

    2014-07-01

    Real-time PCR (qPCR) is a rapid tool to quantify pathogens in the aquatic environment; however, it quantifies all pathogens, including both viable and nonviable. Propidium monoazide (PMA) is a membrane-impairment dye that penetrates only membrane-damaged cells. Once inside the cell, PMA is covalently cross-linked to DNA through light photoactivation, and PCR amplification is strongly inhibited. The goal of this study was to evaluate PMA-qPCR assays for rapid quantification of viable and heat-treated Giardia cysts and Cryptosporidium oocysts in wastewater. We observed a reduction in detection of heat-treated Giardia duodenalis cysts of 83.2, 89.9, 98.2, or 97% with PMA-qPCR assays amplifying a 75 base-pair (bp) β-giardin target, 77-bp triosephosphate isomerase (tpi), 133-bp glutamate dehydrogenase (GDH), and 143-bp β-giardin gene target, respectively. Thus, the exclusion of dead cysts was more effective when qPCR assays that produced larger amplicons were used. The PMA treatment of Cryptosporidium oocysts plus/minus heat treatment abolished the fluorescent signal for dead oocysts with a PMA-qPCR assay amplifying a Cryptosporidium parvum (150-bp) oocyst wall protein (COWP) gene. The PMA-qPCR 143-bp β-giardin assay for Giardia and the PMA-qPCR 150-bp COWP assay for Cryptosporidium accurately quantified live oo(cysts), and failed to detect dead oo(cysts), when phosphate-buffered saline and tertiary effluent wastewater were spiked with concentrations of 10(3) or 10(2) dead oo(cysts), respectively. Therefore, these assays are suitable for the detection of viable parasites that are typically present in tertiary wastewater effluents at concentrations of <10(3) oo(cysts)/l and can provide rapid risk assessments of environmental water. PMID:24781028

  16. Monochloramine disinfection kinetics of Nitrosomonas europaea by propidium monoazide quantitative PCR and Live/Dead BacLight Methods

    EPA Science Inventory

    Monochloramine disinfection kinetics were determined for the pure culture ammonia-oxidizing bacterium Nitrosomonas europaea (ATCC 19718) by two culture independent methods: (1) LIVE/DEAD® BacLight™ (LD) and (2) propidium monoazide quantitative PCR (PMA-qPCR). Both methods were f...

  17. Distinction between intact and antibiotic-inactivated bacteria by real-time PCR after treatment with propidium monoazide.

    PubMed

    Kobayashi, Hideo; Oethinger, Margret; Tuohy, Marion J; Hall, Gerri S; Bauer, Thomas W

    2010-09-01

    One limitation to the use of the polymerase chain reaction (PCR) to identify orthopedic infections has been apparent false-positive results, possibly due to the detection of dead bacteria. We recently showed that the use of DNA-binding agent propidium monoazide (PMA) could distinguish viable from heat-inactivated bacteria, and, in this study, we investigated whether the same technique can be applied to bacteria killed by two antibiotics with distinctly different mechanisms of action, a test of greater clinical relevance than thermal inactivation. Staphylococcus aureus and S. epidermidis were inactivated by vancomycin and gentamicin and treated with PMA or left untreated before DNA extraction. The threshold cycle difference of antibiotic-treated bacteria with and without PMA pretreatment was investigated with PCR primers for the 16S rDNA and tuf genes. Our results indicated that PMA effectively inhibited detection by PCR of bacteria, which had been inactivated by either vancomycin or gentamicin. The effect was statistically significant at 24 h after treatment (C(t) difference consistently >3; p < 0.05) and after 10 days of treatment (C(t) difference >4; p < 0.01), when compared to viable cells (C(t) difference 1-2). Vancomycin had a stronger effect on the C(t) value than gentamicin, reflecting the different mechanism of action of each antibiotic. Techniques of this type may help reduce clinically false-positive PCR results caused by the detection of dead bacteria, and may be especially useful in patients who have received antibiotics, such as patients undergoing the second stage of a two-stage revision for infected arthroplasty. PMID:20186836

  18. Utility of propidium monoazide viability assay as a biomarker for a tuberculosis disease.

    PubMed

    Nikolayevskyy, Vladyslav; Miotto, Paolo; Pimkina, Edita; Balabanova, Yanina; Kontsevaya, Irina; Ignatyeva, Olga; Ambrosi, Alessandro; Skenders, Girts; Ambrozaitis, Arvydas; Kovalyov, Alexander; Sadykhova, Anna; Simak, Tatiana; Kritsky, Andrey; Mironova, Svetlana; Tikhonova, Olesya; Dubrovskaya, Yulia; Rodionova, Yulia; Cirillo, Daniela; Drobniewski, Francis

    2015-03-01

    Reliable laboratory diagnosis of tuberculosis (TB), including laboratory biomarkers of cure, remains a challenge. In our study we evaluated the performance of a Propidium Monoazide (PMA) assay for the detection of viable TB bacilli in sputum specimens during anti-TB chemotherapy and its potential use as a TB biomarker. The study was conducted at three centres on 1937 sputum specimens from 310 adult bacteriologically confirmed pulmonary TB patients obtained before commencing anti-TB treatment and at regular intervals afterwards. Performance of the PMA assay was assessed using various readout assays with bacteriology culture results and time to positivity on liquid media used as reference standards. Treatment of sputum with N-acetyl-cysteine was found to be fully compatible with the PMA assay. Good sensitivity and specificity (97.5% and 70.7-80.0%) for detection of live TB bacilli was achieved using the Xpert(®) MTB/RIF test as a readout assay. Tentative Ct and ΔCt thresholds for the Xpert(®) MTB/RIF system were proposed. Good correlation (r = 0.61) between Ct values and time to positivity of TB cultures on liquid media was demonstrated. The PMA method has potential in monitoring bacterial load in sputum specimens and so may have a role as a biomarker of cure in TB treatment. PMID:25534168

  19. A multicenter study of viable PCR using propidium monoazide to detect Legionella in water samples.

    PubMed

    Scaturro, Maria; Fontana, Stefano; Dell'eva, Italo; Helfer, Fabrizia; Marchio, Michele; Stefanetti, Maria Vittoria; Cavallaro, Mario; Miglietta, Marilena; Montagna, Maria Teresa; De Giglio, Osvalda; Cuna, Teresa; Chetti, Leonarda; Sabattini, Maria Antonietta Bucci; Carlotti, Michela; Viggiani, Mariagabriella; Stenico, Alberta; Romanin, Elisa; Bonanni, Emma; Ottaviano, Claudio; Franzin, Laura; Avanzini, Claudio; Demarie, Valerio; Corbella, Marta; Cambieri, Patrizia; Marone, Piero; Rota, Maria Cristina; Bella, Antonino; Ricci, Maria Luisa

    2016-07-01

    Legionella quantification in environmental samples is overestimated by qPCR. Combination with a viable dye, such as Propidium monoazide (PMA), could make qPCR (named then vPCR) very reliable. In this multicentre study 717 artificial water samples, spiked with fixed concentrations of Legionella and interfering bacterial flora, were analysed by qPCR, vPCR and culture and data were compared by statistical analysis. A heat-treatment at 55 °C for 10 minutes was also performed to obtain viable and not-viable bacteria. When data of vPCR were compared with those of culture and qPCR, statistical analysis showed significant differences (P < 0.001). However, although the heat-treatment caused an abatement of CFU/mL ≤1 to 1 log10 unit, the comparison between untreated and heat-treated samples analysed by vPCR highlighted non-significant differences (P > 0.05). Overall this study provided a good experimental reproducibility of vPCR but also highlighted limits of PMA in the discriminating capability of dead and live bacteria, making vPCR not completely reliable. PMID:27133308

  20. The use of propidium monoazide in conjunction with qPCR and Illumina sequencing to identify and quantify live yeasts and bacteria.

    PubMed

    Tantikachornkiat, Mansak; Sakakibara, Stacey; Neuner, Marissa; Durall, Daniel M

    2016-10-01

    Culture-independent methods of microbial identification have been developed, which allow for DNA extraction directly from environmental samples without subjecting microbes to growth on nutrient media. These methods often involve next generation DNA sequencing (NGS) for identifying microbes and qPCR for quantifying them. Despite the benefits of extracting all DNA from the sample, results may be compromised by amplifying DNA from dead cells. To address this short-coming, the use of propidium monoazide (PMA) has been used to deactivate DNA in non-viable cells. Nevertheless, its optimization has not been fully explored under a variety of conditions. In this study, we optimized the PMA method for both yeasts and bacteria. Specifically, we explored the effect different PMA concentrations and different cell densities had on DNA amplification (as part of next generation DNA sequencing) from both dead and viable bacterial and yeast cells. We found PMA was effective in eliminating DNA that was associated with dead yeast and bacterial cells for all cell concentrations. Nevertheless, DNA (extracted from viable yeast and bacterial cells) amplified most abundantly when PMA concentration was at 6μM and when yeast densities ranged between 10(6) to 10(7)CFU/mL and bacterial densities were approximately 10(8)CFU/mL. PMID:27371903

  1. Development of a sensitive and specific qPCR assay in conjunction with propidium monoazide for enhanced detection of live Salmonella spp. in food

    PubMed Central

    2013-01-01

    Background Although a variety of methodologies are available for detection of Salmonella, sensitive, specific, and efficient methods are urgently needed for differentiation of live Salmonella cells from dead cells in food and environmental samples. Propidium monoazide (PMA) can preferentially penetrate the compromised membranes of dead cells and inhibit their DNA amplification, however, such inhibition has been reported to be incomplete by some studies. In the present study, we report an efficient qPCR assay targeting a conserved region of the invA gene of Salmonella in conjunction with PMA treatment for detection of DNA from live Salmonella cells in food samples. Results We investigated the relationship between amplicon length and inhibitory effect of PMA treatment to prevent DNA amplification from dead cells while allowing for DNA amplification from live cells, and found that the two factors are well correlated with each other. An amplicon that is 130 bp in length was determined to be optimal for PMA treatment and was selected for further PMA-qPCR assay development. A PMA-qPCR assay was established by utilizing this amplicon and adopting a modified PMA-treatment procedure. The PMA-qPCR assay provided excellent inhibition of DNA amplification from dead cells (a 17-CT-value, or 128,000-fold reduction) while only a slight DNA amplification difference (0.5 CT value) was noted between the PMA-treated and untreated live cells. This assay has been validated through stringent inclusivity and exclusivity studies using a large number of (n = 167) Salmonella, including all strains of SARA and SARB collections, and non-Salmonella strains (n = 36). This PMA-qPCR assay is capable of detecting live Salmonella cells in live/dead cell mixtures, or 30 CFU/g live Salmonella cells from enriched spiked spinach samples as early as 4 h. Conclusions A 130-bp amplicon in invA gene was demonstrated to be optimal for PMA treatment for selective detection of live Salmonella cells

  2. Detection and Quantification of Viable and Nonviable Trypanosoma cruzi Parasites by a Propidium Monoazide Real-Time Polymerase Chain Reaction Assay.

    PubMed

    Cancino-Faure, Beatriz; Fisa, Roser; Alcover, M Magdalena; Jimenez-Marco, Teresa; Riera, Cristina

    2016-06-01

    Molecular techniques based on real-time polymerase chain reaction (qPCR) allow the detection and quantification of DNA but are unable to distinguish between signals from dead or live cells. Because of the lack of simple techniques to differentiate between viable and nonviable cells, the aim of this study was to optimize and evaluate a straightforward test based on propidium monoazide (PMA) dye action combined with a qPCR assay (PMA-qPCR) for the selective quantification of viable/nonviable epimastigotes of Trypanosoma cruzi PMA has the ability to penetrate the plasma membrane of dead cells and covalently cross-link to the DNA during exposure to bright visible light, thereby inhibiting PCR amplification. Different concentrations of PMA (50-200 μM) and epimastigotes of the Maracay strain of T. cruzi (1 × 10(5)-10 parasites/mL) were assayed; viable and nonviable parasites were tested and quantified by qPCR with a TaqMan probe specific for T. cruzi. In the PMA-qPCR assay optimized at 100 μM PMA, a significant qPCR signal reduction was observed in the nonviable versus viable epimastigotes treated with PMA, with a mean signal reduction of 2.5 logarithm units and a percentage of signal reduction > 98%, in all concentrations of parasites assayed. This signal reduction was also observed when PMA-qPCR was applied to a mixture of live/dead parasites, which allowed the detection of live cells, except when the concentration of live parasites was low (10 parasites/mL). The PMA-qPCR developed allows differentiation between viable and nonviable epimastigotes of T. cruzi and could thus be a potential method of parasite viability assessment and quantification. PMID:27139452

  3. Advantageous Direct Quantification of Viable Closely Related Probiotics in Petit-Suisse Cheeses under In Vitro Gastrointestinal Conditions by Propidium Monoazide - qPCR

    PubMed Central

    Villarreal, Martha Lissete Morales; Padilha, Marina; Vieira, Antonio Diogo Silva; Franco, Bernadette Dora Gombossy de Melo; Martinez, Rafael Chacon Ruiz; Saad, Susana Marta Isay

    2013-01-01

    Species-specific Quantitative Real Time PCR (qPCR) alone and combined with the use of propidium monoazide (PMA) were used along with the plate count method to evaluate the survival of the probiotic strains Lactobacillus acidophilus La-5 and Bifidobacterium animalis subsp. lactis Bb-12, and the bacteriocinogenic and potentially probiotic strain Lactobacillus sakei subsp. sakei 2a in synbiotic (F1) and probiotic (F2) petit-suisse cheeses exposed throughout shelf-life to in vitro simulated gastrointestinal tract conditions. The three strains studied showed a reduction in their viability after the 6 h assay. Bb-12 displayed the highest survival capacity, above 72.6 and 74.6% of the initial populations, respectively, by plate count and PMA-qPCR, maintaining population levels in the range or above 6 log CFU/g. The prebiotic mix of inulin and FOS did not offer any additional protection for the strains against the simulated gastrointestinal environment. The microorganisms' populations were comparable among the three methods at the initial time of the assay, confirming the presence of mainly viable and culturable cells. However, with the intensification of the stress induced throughout the various stages of the in vitro test, the differences among the methods increased. The qPCR was not a reliable enumeration method for the quantification of intact bacterial populations, mixed with large numbers of injured and dead bacteria, as confirmed by the scanning electron microscopy results. Furthermore, bacteria plate counts were much lower (P<0.05) than with the PMA-qPCR method, suggesting the accumulation of stressed or dead microorganisms unable to form colonies. The use of PMA overcame the qPCR inability to differentiate between dead and alive cells. The combination of PMA and species-specific qPCR in this study allowed a quick and unequivocal way of enumeration of viable closely related species incorporated into probiotic and synbiotic petit-suisse cheeses and under stress

  4. Propidium monoazide reverse transcription PCR and RT-qPCR for detecting infectious enterovirus and norovirus

    EPA Science Inventory

    Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the publ...

  5. Effects of ionizing radiation on bone cell differentiation in an experimental murine bone cell model

    NASA Astrophysics Data System (ADS)

    Baumstark-Khan, Christa; Lau, Patrick; Hellweg, Christine; Reitz, Guenther

    During long-term space travel astronauts are exposed to a complex mixture of different radiation types under conditions of dramatically reduced weight-bearing activity. It has been validated that astronauts loose a considerable amount of bone mass at a rate up to one to two percent each month in space. Therapeutic doses of ionizing radiation cause bone damage and increase fracture risks after treatment for head-and-neck cancer and in pelvic irradiation. For low radiation doses, the possibility of a disturbed healing potential of bone was described. Radiation induced damage has been discussed to inflict mainly on immature and healing bone. Little is known about radiation effects on bone remodelling and even less on the combined action of microgravity and radiation. Bone remodelling is a life-long process performed by balanced action of cells from the osteoblast and osteoclast lineages. While osteoblasts differentiate either into bone-lining cells or into osteocytes and play a crucial role in bone matrix synthesis, osteoclasts are responsible for bone resorption. We hypothesize that the balance between bone matrix assembly by osteocytes and bone degradation by osteoclasts is modulated by microgravity as well as by ionizing radiation. To address this, a cell model consisting of murine cell lines with the potential to differentiate into bone-forming osteoblasts (OCT-1, MC3T3-E1 S24, and MC3T3-E1 S4) was used for studying radiation response after exposure to simulated components of cosmic radiation. Cells were exposed to graded doses of 150 kV X-rays, α particles (0.525 MeV/u, 160 keV/µm; PTB, Braunschweig, Germany) and accelerated heavy ions (75 MeV/u carbon, 29 keV/µm; 95 MeV/u argon, 230 keV/µm; GANIL, Caen, France). Cell survival was measured as colony forming ability; cell cycle progression was analyzed via fluorescence-activated cell scanning (FACS) by measurement of the content of propidium iodide-stained DNA, DNA damage was visualized by γH2AX

  6. Effects of ionizing radiation on bone cell differentiation in an experimental murine bone cell model

    NASA Astrophysics Data System (ADS)

    Baumstark-Khan, Christa; Lau, Patrick; Hellweg, Christine; Reitz, Guenther

    During long-term space travel astronauts are exposed to a complex mixture of different radiation types under conditions of dramatically reduced weight-bearing activity. It has been validated that astronauts loose a considerable amount of bone mass at a rate up to one to two percent each month in space. Therapeutic doses of ionizing radiation cause bone damage and increase fracture risks after treatment for head-and-neck cancer and in pelvic irradiation. For low radiation doses, the possibility of a disturbed healing potential of bone was described. Radiation induced damage has been discussed to inflict mainly on immature and healing bone. Little is known about radiation effects on bone remodelling and even less on the combined action of microgravity and radiation. Bone remodelling is a life-long process performed by balanced action of cells from the osteoblast and osteoclast lineages. While osteoblasts differentiate either into bone-lining cells or into osteocytes and play a crucial role in bone matrix synthesis, osteoclasts are responsible for bone resorption. We hypothesize that the balance between bone matrix assembly by osteocytes and bone degradation by osteoclasts is modulated by microgravity as well as by ionizing radiation. To address this, a cell model consisting of murine cell lines with the potential to differentiate into bone-forming osteoblasts (OCT-1, MC3T3-E1 S24, and MC3T3-E1 S4) was used for studying radiation response after exposure to simulated components of cosmic radiation. Cells were exposed to graded doses of 150 kV X-rays, α particles (0.525 MeV/u, 160 keV/µm; PTB, Braunschweig, Germany) and accelerated heavy ions (75 MeV/u carbon, 29 keV/µm; 95 MeV/u argon, 230 keV/µm; GANIL, Caen, France). Cell survival was measured as colony forming ability; cell cycle progression was analyzed via fluorescence-activated cell scanning (FACS) by measurement of the content of propidium iodide-stained DNA, DNA damage was visualized by γH2AX

  7. Propidium monoazide reverse transcriptase PCR and RT-qPCR for detecting infectious enterovirus and norovirus.

    PubMed

    Karim, Mohammad R; Fout, G Shay; Johnson, Clifford H; White, Karen M; Parshionikar, Sandhya U

    2015-07-01

    Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the public health significance of positive findings are limited. In this study, PMA RT-PCR and RT-qPCR assays were evaluated for selective detection of infectious poliovirus, murine norovirus (MNV-1), and Norwalk virus. Viruses were inactivated using heat, chlorine, and ultraviolet light (UV). Infectious and non-infectious viruses were treated with PMA before RT-PCR and RT-qPCR. PMA RT-PCR was able to differentiate selectively between infectious and heat and chlorine inactivated poliovirus. PMA RT-PCR was able to differentiate selectively between infectious and noninfectious murine norovirus only when inactivated by chlorine. However, PMA RT-PCR could not differentiate infectious Norwalk virus from virus particles rendered non-infectious by any treatment. PMA RT-PCR assay was not able to differentiate between infectious and UV inactivated viruses suggesting that viral capsid damage may be necessary for PMA to enter and bind to the viral genome. PMA RT-PCR on naked MNV-1 and Norwalk virus RNA suggest that PMA RT-PCR can be used to detect intact, potentially infectious MNV-1 and Norwalk viruses and can be used to exclude the detection of free viral RNA by PCR assay. PMID:25796356

  8. Evaluation of propidium monoazide-quantitative PCR to detect viable Mycobacterium fortuitum after chlorine, ozone, and ultraviolet disinfection.

    PubMed

    Lee, Eun-Sook; Lee, Man-Ho; Kim, Bog-Soon

    2015-10-01

    We evaluated whether propidium monoazide (PMA) combined with real-time quantitative PCR (qPCR) is suitable for detecting viable Mycobacterium fortuitum after chlorine, ozone, and ultraviolet (UV) disinfection. PMA-qPCR was effective in determining the viability of M. fortuitum compared with qPCR based on the membrane integrity. However, with a mild chlorine concentration, PMA-qPCR as an alternative method was not applicable due to a large gap between loss of culturability and membrane integrity damage. In ozonation, PMA-qPCR was able to differentiate between viable and injured mycobacteria, and the results were similar to those obtained by the culture method. Interestingly, PMA-qPCR was successful in monitoring the viability after UV disinfection due to the long UV exposure needed to effectively inactivate M. fortuitum. The findings of the present study suggested that the characteristics of disinfectants and the M. fortuitum resistance to disinfectants play critical roles in determining the suitability of PMA-qPCR for evaluating the efficacy of disinfection methods. PMID:26143168

  9. Anthocyanin Inhibits Propidium Iodide DNA Fluorescence in Euphorbia pulcherrima: Implications for Genome Size Variation and Flow Cytometry

    PubMed Central

    Bennett, Michael D.; Price, H. James; Johnston, J. Spencer

    2008-01-01

    Background Measuring genome size by flow cytometry assumes direct proportionality between nuclear DNA staining and DNA amount. By 1997 it was recognized that secondary metabolites may affect DNA staining, thereby causing inaccuracy. Here experiments are reported with poinsettia (Euphorbia pulcherrima) with green leaves and red bracts rich in phenolics. Methods DNA content was estimated as fluorescence of propidium iodide (PI)-stained nuclei of poinsettia and/or pea (Pisum sativum) using flow cytometry. Tissue was chopped, or two tissues co-chopped, in Galbraith buffer alone or with six concentrations of cyanidin-3-rutinoside (a cyanidin-3-rhamnoglucoside contributing to red coloration in poinsettia). Key Results There were large differences in PI staining (35–70 %) between 2C nuclei from green leaf and red bract tissue in poinsettia. These largely disappeared when pea leaflets were co-chopped with poinsettia tissue as an internal standard. However, smaller (2·8–6·9 %) differences remained, and red bracts gave significantly lower 1C genome size estimates (1·69–1·76 pg) than green leaves (1·81 pg). Chopping pea or poinsettia tissue in buffer with 0–200 µm cyanidin-3-rutinoside showed that the effects of natural inhibitors in red bracts of poinsettia on PI staining were largely reproduced in a dose-dependent way by this anthocyanin. Conclusions Given their near-ubiquitous distribution, many suspected roles and known affects on DNA staining, anthocyanins are a potent, potential cause of significant error variation in genome size estimations for many plant tissues and taxa. This has important implications of wide practical and theoretical significance. When choosing genome size calibration standards it seems prudent to select materials producing little or no anthocyanin. Reviewing the literature identifies clear examples in which claims of intraspecific variation in genome size are probably artefacts caused by natural variation in anthocyanin levels or

  10. Use of Propidium Monoazide in Reverse Transcriptase PCR To Distinguish between Infectious and Noninfectious Enteric Viruses in Water Samples▿

    PubMed Central

    Parshionikar, Sandhya; Laseke, Ian; Fout, G. Shay

    2010-01-01

    Human enteric viruses can be present in untreated and inadequately treated drinking water. Molecular methods, such as the reverse transcriptase PCR (RT-PCR), can detect viral genomes in a few hours, but they cannot distinguish between infectious and noninfectious viruses. Since only infectious viruses are a public health concern, methods that not only are rapid but also provide information on the infectivity of viruses are of interest. The intercalating dye propidium monoazide (PMA) has been used for distinguishing between viable and nonviable bacteria with DNA genomes, but it has not been used to distinguish between infectious and noninfectious enteric viruses with RNA genomes. In this study, PMA in conjunction with RT-PCR (PMA-RT-PCR) was used to determine the infectivity of enteric RNA viruses in water. Coxsackievirus, poliovirus, echovirus, and Norwalk virus were rendered noninfectious or inactivated by treatment with heat (72°C, 37°C, and 19°C) or hypochlorite. Infectious or native and noninfectious or inactivated viruses were treated with PMA. This was followed by RNA extraction and RT-PCR or quantitative RT-PCR (qRT-PCR) analysis. The PMA-RT-PCR results indicated that PMA treatment did not interfere with detection of infectious or native viruses but prevented detection of noninfectious or inactivated viruses that were rendered noninfectious or inactivated by treatment at 72°C and 37°C and by hypochlorite treatment. However, PMA-RT-PCR was unable to prevent detection of enteroviruses that were rendered noninfectious by treatment at 19°C. After PMA treatment poliovirus that was rendered noninfectious by treatment at 37°C was undetectable by qRT-PCR, but PMA treatment did not affect detection of Norwalk virus. PMA-RT-PCR was also shown to be effective for detecting infectious poliovirus in the presence of noninfectious virus and in an environmental matrix. We concluded that PMA can be used to differentiate between potentially infectious and noninfectious

  11. Use of propidium monoazide in reverse transcriptase PCR to distinguish between infectious and noninfectious enteric viruses in water samples.

    PubMed

    Parshionikar, Sandhya; Laseke, Ian; Fout, G Shay

    2010-07-01

    Human enteric viruses can be present in untreated and inadequately treated drinking water. Molecular methods, such as the reverse transcriptase PCR (RT-PCR), can detect viral genomes in a few hours, but they cannot distinguish between infectious and noninfectious viruses. Since only infectious viruses are a public health concern, methods that not only are rapid but also provide information on the infectivity of viruses are of interest. The intercalating dye propidium monoazide (PMA) has been used for distinguishing between viable and nonviable bacteria with DNA genomes, but it has not been used to distinguish between infectious and noninfectious enteric viruses with RNA genomes. In this study, PMA in conjunction with RT-PCR (PMA-RT-PCR) was used to determine the infectivity of enteric RNA viruses in water. Coxsackievirus, poliovirus, echovirus, and Norwalk virus were rendered noninfectious or inactivated by treatment with heat (72 degrees C, 37 degrees C, and 19 degrees C) or hypochlorite. Infectious or native and noninfectious or inactivated viruses were treated with PMA. This was followed by RNA extraction and RT-PCR or quantitative RT-PCR (qRT-PCR) analysis. The PMA-RT-PCR results indicated that PMA treatment did not interfere with detection of infectious or native viruses but prevented detection of noninfectious or inactivated viruses that were rendered noninfectious or inactivated by treatment at 72 degrees C and 37 degrees C and by hypochlorite treatment. However, PMA-RT-PCR was unable to prevent detection of enteroviruses that were rendered noninfectious by treatment at 19 degrees C. After PMA treatment poliovirus that was rendered noninfectious by treatment at 37 degrees C was undetectable by qRT-PCR, but PMA treatment did not affect detection of Norwalk virus. PMA-RT-PCR was also shown to be effective for detecting infectious poliovirus in the presence of noninfectious virus and in an environmental matrix. We concluded that PMA can be used to differentiate

  12. Effect of DNA extraction procedure, repeated extraction and ethidium monoazide (EMA)/propidium monoazide (PMA) treatment on overall DNA yield and impact on microbial fingerprints for bacteria, fungi and archaea in a reference soil

    PubMed Central

    Wagner, Andreas O.; Praeg, Nadine; Reitschuler, Christoph; Illmer, Paul

    2015-01-01

    Different DNA extraction protocols were evaluated on a reference soil. A wide difference was found in the total extractable DNA as derived from different extraction protocols. Concerning the DNA yield phenol–chloroform–isomyl alcohol extraction resulted in high DNA yield but also in a remarkable co-extraction of contaminants making PCR from undiluted DNA extracts impossible. By comparison of two different extraction kits, the Macherey&Nagel SoilExtract II kit resulted in the highest DNA yields when buffer SL1 and the enhancer solution were applied. The enhancer solution not only significantly increased the DNA yield but also the amount of co-extracted contaminates, whereas additional disintegration strategies did not. Although a three times repeated DNA extraction increased the total amount of extracted DNA, microbial fingerprints were merely affected. However, with the 5th extraction this changed. A reduction of total DGGE band numbers was observed for archaea and fungi, whereas for bacteria the diversity increased. The application of ethidium monoazide (EMA) or propidium monoazide (PMA) treatment aiming on the selective removal of soil DNA derived from cells lacking cell wall integrity resulted in a significant reduction of total extracted DNA, however, the hypothesized effect on microbial fingerprints failed to appear indicating the need for further investigations. PMID:26339125

  13. Simplified method for DNA and protein staining of human hematopoietic cell samples. [Cell flow systems

    SciTech Connect

    Crissman, H.A.; Egmond, J.V.; Holdrinet, R.S.; Pennings, A.; Haanen, C.

    1981-01-01

    A rapid reproducible method yielding high resolution analysis of DNA and protein in human hematopoietic cell samples has been developed by modification of the propidium iodide and fluorescein isothiocyanate procedure. Cell staining involves sequential addition of each reagent (RNase, fluorescein isothiocyanate and propidium iodide) to ethanol-fixed cells and requires no centrifugation steps. Stained cells are analyzed in the reagent solutions. Analysis of bone marrow samples from multiple myeloma patients showed mixed normal and aneuploid populations with a major portion of the aneuploid cells having a significantly higher protein content. This approach permitted differential cell cycle analysis of normal and the aneuploid populations.

  14. Synthesis, Characterization, and Biological Affinity of a Near-Infrared-Emitting Conjugated Oligoelectrolyte

    PubMed Central

    2015-01-01

    A near-IR-emitting conjugated oligoelectrolyte (COE), ZCOE, was synthesized, and its photophysical features were characterized. The biological affinity of ZCOE is compared to that of an established lipid-membrane-intercalating COE, DSSN+, which has blue-shifted optical properties making it compatible for tracking preferential sites of accumulation. ZCOE exhibits diffuse staining of E. coli cells, whereas it displays internal staining of select yeast cells which also show propidium iodide staining, indicating ZCOE is a “dead” stain for this organism. Staining of mammalian cells reveals complete internalization of ZCOE through endocytosis, as supported by colocalization with LysoTracker and late endosome markers. In all cases DSSN+ persists in the outer membranes, most likely due to its chemical structure more closely resembling a lipid bilayer. PMID:24575841

  15. Synthesis and in vitro/in vivo anti-cancer evaluation of curcumin-loaded chitosan/poly(butyl cyanoacrylate) nanoparticles.

    PubMed

    Duan, Jinghua; Zhang, Yangde; Han, Shiwei; Chen, Yuxiang; Li, Bo; Liao, Mingmei; Chen, Wei; Deng, Xingming; Zhao, Jinfeng; Huang, Boyun

    2010-11-15

    We have synthesized novel cationic poly(butyl) cyanoacrylate (PBCA) nanoparticles coated with chitosan, formulation of curcumin nanoparticles. The size and zeta potential of prepared curcumin nanoparticles were about 200 nm and +29.11 mV, respectively with 90.04% encapsulation efficiency. The transmission electron microscopy (TEM) study revealed the spherical nature of the prepared nanoparticles along with confirmation of particle size. Curcumin nanoparticles demonstrate comparable in vitro therapeutic efficacy to free curcumin against a panel of human hepatocellular cancer cell lines, as assessed by cell viability (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide assay [MTT assay]) and proapoptotic effects (annexin V/propidium iodide staining). In vivo, curcumin nanoparticles suppressed hepatocellular carcinoma growth in murine xenograft models and inhibited tumor angiogenesis. The curcumin nanoparticles' mechanism of action on hepatocellular carcinoma cells is a mirror that of free curcumin. PMID:20813175

  16. Roles of BN52021 in platelet-activating factor pathway in inflammatory MS1 cells

    PubMed Central

    Xia, Shi-Hai; Xiang, Xiao-Hui; Chen, Kai; Xu, Wei

    2013-01-01

    AIM: To determine the effects of BN52021 on platelet-activating factor receptor (PAFR) signaling molecules under lipopolysaccharide (LPS)-induced inflammatory conditions in MS1 cells. METHODS: MS1 cells (a mouse pancreatic islet endothelial cell line) were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 2 mmol/L glutamine and 100 μg/mL penicillin/streptomycin in 5% CO2 at 37 °C. After growth to confluency in media, the cells were processed for subsequent studies. The MS1 cells received 0, 0.1, 1 and 10 μg/mL LPS in this experiment. The viability/proliferation of the cells induced by LPS was observed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. Apoptosis and necrosis of the cells under the inflammatory condition described previously were observed using Hoechst 33342-propidium iodide staining. Adenylate cyclase (AC), phospholipase A2 (PLA2), phospholipase Cβ (PLCβ), protein tyrosine kinase (PTK), G protein-coupled receptor kinases (GRK) and p38-mitogen-activated protein kinase (p38 MAPK) mRNA in the PAFR signaling pathway were measured by real-time polymerase chain reaction. The protein expression level of phosphorylated AC (p-AC), phosphorylated PLA2 (p-PLA2), phosphorylated PTK (p-PTK), phosphorylated p38 MAPK (p-p38 MAPK), PLCβ and GRK was measured using Western blotting analysis. RESULTS: The activity of MS1 cells incubated with different concentrations of LPS for 6 h decreased significantly in the 1 μg/mL LPS group (0.49 ± 0.10 vs 0.67 ± 0.13, P < 0.05) and 10 μg/mL LPS group (0.44 ± 0.10 vs 0.67 ± 0.13, P < 0.001), but not in 0.1 μg/mL group. When the incubation time was extended to 12 h (0.33 ± 0.05, 0.32 ± 0.03 and 0.25 ± 0.03 vs 0.69 ± 0.01) and 24 h (0.31 ± 0.01, 0.29 ± 0.03 and 0.25 ± 0.01 vs 0.63 ± 0.01), MS1 cell activity decreased in all LPS concentration groups compared with the blank control (P < 0.001). BN52021 significantly improved the cell

  17. Development of propidium iodide as a fluorescence probe for the on-line screening of non-specific DNA-intercalators in Fufang Banbianlian Injection.

    PubMed

    Niu, Yanyan; Li, Sensen; Lin, Zongtao; Liu, Meixian; Wang, Daidong; Wang, Hong; Chen, Shizhong

    2016-09-01

    Fufang Banbianlian Injection (FBI) has been widely used as an anti-inflammatory and anti-tumor prescription. To understand the relationships between its bioactive ingredients and pharmacological efficacies, our previous study has been successfully identified some DNA-binding compounds in FBI using an established on-line screening system, in which 4',6-diamidino-2-phenylindole (DAPI) was developed as a probe. However, DAPI can be only used to screen ATT-specific DNA minor groove binders, leaving the potential active intercalators unknown in FBI. As a continuation of our studies on FBI, here we present a sensitive analytical method for rapid identification and evaluation of DNA-intercalators using propidium iodide (PI) as a fluorescent probe. We have firstly established the technique of high-performance liquid chromatography-diode-array detector-multistage mass spectrometry-deoxyribonucleic acid-propidium iodide-fluorescence detector (HPLC-DAD-MS(n)-DNA-PI-FLD) system. As a result, 38 of 58 previously identified compounds in FBI were DNA-intercalation active. Interestingly, all previously reported DNA-binders also showed intercalative activities, suggesting they are dual-mode DNA-binders. Quantitative study showed that flavonoid glycosides and chlorogenic acids were the main active compounds in FBI, and displayed similar DNA-binding ability using either DAPI or PI. In addition, 13 active compounds were used to establish the structure-activity relationships. In this study, PI was developed into an on-line method for identifying DNA-intercalators for the first time, and thus it will be a useful high-throughput screening technique for other related samples. PMID:27522151

  18. Rapid detection of viable salmonella in produce by coupling propidium monoazide with loop-mediated isothermal amplification (PMA-LAMP).

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recent outbreaks linked to Salmonella-contaminated produce heightened the need to develop simple, rapid, and accurate detection methods; particularly those capable of determining cell viability. In this study, we examined a novel strategy for the rapid detection and quantification of viable Salmonel...

  19. Quantifying Fungal Viability in Air and Water Samples using Quantitative PCR after Treatment with Propidium Monoazide (PMA)

    EPA Science Inventory

    A method is described to discriminate between live and dead cells of the infectious fungi Aspergillus fumigatus, A. flavus, A. terreus, Mucor racemosus, Rhizopus stolonifer and Paecilomyces variotii. To test the method, conidial suspensions were heat inactivated at 85oC or held ...

  20. Comparison of propidium monoazide-quantitative PCR and reverse transcription quantitative PCR for viability detection of fresh Cryptosporidium oocysts following disinfection and after long-term storage in water samples

    EPA Science Inventory

    Purified oocysts of Cryptosporidium parvum were used to evaluate applicability of two quantitative PCR (qPCR) viability detection methods in raw surface water and disinfection treated water. Propidium monoazide-qPCR targeting hsp70 gene was compared to reverse transcription (RT)-...

  1. Determination of the Effects of Medium Composition on the Monochloramine Disinfection Kinetics of Nitrosomonas europaea by the Propidium Monoazide Quantitative PCR and Live/Dead BacLight Methods

    EPA Science Inventory

    Various media compositions (phosphate 1-50 mM; ionic strength 2.8-150 meq/L) significantly affected Nitrosomonas europaea monochloramine disinfection kinetics determined by Live/Dead BacLight (LD) and propidium monoazide quantitative PCR (PMA-qPCR) methods (lag coefficient 37-490...

  2. Repeated cycles of chemical and physical disinfection and their influence on Mycobacterium avium subsp. paratuberculosis viability measured by propidium monoazide F57 quantitative real time PCR.

    PubMed

    Kralik, Petr; Babak, Vladimir; Dziedzinska, Radka

    2014-09-01

    Mycobacterium avium subsp. paratuberculosis (MAP) has a high degree of resistance to chemical and physical procedures frequently used for the elimination of other bacteria. Recently, a method for the determination of viability by exposure of MAP to propidium monoazide (PMA) and subsequent real time quantitative PCR (qPCR) was established and found to be comparable with culture. The aim of this study was to apply the PMA qPCR method to determine the impact of increasing concentration or time and repeated cycles of the application of selected disinfectants on MAP viability. Different MAP isolates responded to the same type of stress in different ways. The laboratory strain CAPM 6381 had the highest tolerance, while the 8819 low-passage field isolate was the most sensitive. Ultraviolet exposure caused only a partial reduction in MAP viability; all MAP isolates were relatively resistant to chlorine. Only the application of peracetic acid led to the total elimination of MAP. Repeated application of the treatments resulted in more significant decreases in MAP viability compared to single increases in the concentration or time of exposure to the disinfectant. PMID:24934261

  3. Cytotoxic evaluation of different fractions of Salvia chorassanica Bunge on MCF-7 and DU 145 cell lines

    PubMed Central

    Golshan, Alireza; Amini, Elaheh; Emami, Seyed Ahmad; Asili, Javad; Jalali, Zahra; Sabouri-Rad, Sarvenaz; Sanjar-Mousavi, Naghmeh; Tayarani-Najaran, Zahra

    2016-01-01

    Because of antimicrobial, antioxidant, and anticancer potential, Salvia chorassanica Bunge (Lamiaceae) has been considered as a popular herb in Iranian traditional medicine. Previous studies have shown remarkable cytotoxic properties of the methanol, n-hexane and dichloromethane extract of S. chorassanica on human cervical cancer cells. To seek the therapeutic potentials of S. chorassanica, this study was undertaken to evaluate the cytotoxic activities of various extracts of this plant on human breast MCF-7 and prostate cancer DU 145 cells. The DU 145 cells were exposed to different concentrations of plant extracts (1-200 μg/ml). Cytotoxic activities were examined using alamarBlue® assay and apoptosis was assessed by acridine orange/propodium iodide double staining and evaluation of DNA fragmentation by flow cytometry. Our findings indicated that n-hexane and dichloromethane extracts had more cytotoxic activities against DU 145 and MCF-7 cell lines compared with other extracts (P<0.05). The acridine orange/propodium iodide staining showed apoptogenic properties of n-hexane and dichloromethane extracts which was consequently confirmed by flow cytometric histogram that exhibited an increase in sub-G1 peak in treated cells as compared with untreated cancer cell lines. Taken together, these observations demonstrated cytotoxic effects of S. chorassanica extracts on MCF-7 and DU 145 cell lines which is most likely exerted via apoptosis cell death. Therefore, further investigations on S. chorassanica extracts as potential chemotherapeutic agents are warranted. PMID:27051435

  4. Shifts of live bacterial community in secondary effluent by chlorine disinfection revealed by Miseq high-throughput sequencing combined with propidium monoazide treatment.

    PubMed

    Pang, Yu-Chen; Xi, Jin-Ying; Xu, Yang; Huo, Zheng-Yang; Hu, Hong-Ying

    2016-07-01

    Chlorine disinfection is a commonly used disinfection process in wastewater treatment, but its effects on the indigenous bacterial community in treated wastewater have not been fully elucidated. In this study, secondary effluent samples collected in four wastewater treatment plants (WWTPs) were selected for chlorine disinfection. Shifts in the bacterial community compositions in secondary effluent samples upon chlorine disinfection, both immediately and after 24 h of storage, were investigated using Illumina MiSeq sequencing combined with propidium monoazide (PMA) treatment. The results showed that the phylum Proteobacteria was sensitive to chlorine, with the relative proportions of Proteobacteria decreased from 39.2 to 75.9 % in secondary effluent samples to 7.5 to 62.2 % immediately after chlorine disinfection. The phylogenetic analysis indicated that the most dominant genera belonging to Proteobacteria were sensitive to chlorine. In contrast, the phyla Firmicutes and Planctomycetes showed a certain resistance to chlorine, with their relative proportions increasing from 5.1 to 23.1 % and 0.8 to 9.3 % to 11.3 to 44.6 % and 1.5 to 13.3 %, respectively. Most dominant genera belonging to Firmicutes showed resistance to chlorine. A significant reduction in the richness and diversity of the bacterial community was observed after 24 h of storage of chlorinated secondary effluent. During the 24-h storage process, the relative proportions of most dominant phyla shifted in reverse from the changes induced by chlorine disinfection. Overall, chlorine disinfection not only changes the bacterial community compositions immediately after the disinfection process but also exerts further impacts over a longer period (24 h). PMID:27005415

  5. Use of an adenosine triphosphate assay, and simultaneous staining with fluorescein diacetate and propidium iodide, to evaluate the effects of cryoprotectants on hard coral (Echinopora spp.) oocytes.

    PubMed

    Tsai, S; Spikings, E; Kuo, F W; Lin, N C; Lin, C

    2010-03-15

    The objective was to examine the effects of cryoprotectants on oocytes of hard corals (Echinopora spp.) to obtain basic knowledge for cryopreservation procedures. Oocytes were exposed to various concentrations of cryoprotectants (0.25 to 5.0M) for 20 min at room temperature (25 degrees C). Two tests were used to assess ovarian follicle viability: fluorescein diacetate (FDA)+propidium iodide (PI) staining, and adenosine triphosphate (ATP) assay. Both FDA+PI staining and ATP assay indicated that cryoprotectant toxicity to oocytes increased in the order methanol, dimethyl sulfoxide (DMSO), propylene glycol (PG), and ethylene glycol (EG). The no observed effect concentrations for Echinopora spp. oocytes were 1.0, 0.5, 0.25, and 0.25 M for methanol, DMSO, PG, and EG, respectively, when assessed with FDA+PI. The ATP assay was more sensitive than FDA+PI staining (P<0.05). Oocyte viability after 1.0M methanol, DMSO, EG, or PG treatment for 20 min at room temperature assessed with FDA+PI tests and ATP assay were 88.9+/-3.1% and 72.2+/-4.4%, 66.2+/-5.0% and 23.2+/-4.9%, 58.9+/-5.4% and 1.1+/-0.7%, and 49.1+/-5.1% and 0.9+/-0.5%, respectively. We inferred that the ATP assay was a valuable measure of cellular injury after cryoprotectant incubation. The results of this study provided a basis for development of protocols to cryopreserve coral oocytes. PMID:20005561

  6. Propidium monoazide RTqPCR assays for the assessment of hepatitis A inactivation and for a better estimation of the health risk of contaminated waters.

    PubMed

    Fuster, Noemí; Pintó, Rosa M; Fuentes, Cristina; Beguiristain, Nerea; Bosch, Albert; Guix, Susana

    2016-09-15

    The waterborne transmission of hepatitis A virus (HAV), the main cause of acute hepatitis, is well documented. Recently, two ISO proposals for sensitive determination of this pathogen by RTqPCR in water and food have been published (ISO/TS 15216-1 and ISO/TS 15216-2), and could enable the formulation of regulatory standards for viruses in the near future. However, since detected viral genomes do not always correlate with virus infectivity, molecular approaches need to be optimized to better predict infectivity of contaminated samples. Two methods involving the use of propidium monoazide (PMA), with or without Triton X-100, prior to RTqPCR amplification were optimized and adapted to infer the performance of infectious viral inactivation upon two different water treatments: free chlorine and high temperature. Significant correlations between the decrease of genome copies and infectivity were found for both inactivation procedures. The best procedure to infer chlorine inactivation was the PMA-RTqPCR assay, in which 1, 2 or 3-log genome copies reductions corresponded to reductions of infectious viruses of 2.61 ± 0.55, 3.76 ± 0.53 and 4.92 ± 0.76 logs, respectively. For heat-inactivated viruses, the best method was the PMA/Triton-RTqPCR assay, with a 1, 2 or 3-log genome reduction corresponding to reductions of infectious viruses of 2.15 ± 1.31, 2.99 ± 0.79 and 3.83 ± 0.70 logs, respectively. Finally, the level of damaged virions was evaluated in distinct types of water naturally contaminated with HAV. While most HAV genomes quantified in sewage corresponded to undamaged capsids, the analysis of a river water sample indicated that more than 98% of viruses were not infectious. Although the PMA/Triton-RTqPCR assay may still overestimate infectivity, it is more reliable than the RTqPCR alone and it seems to be a rapid and cost-effective method that can be applied on different types of water, and that it undeniably provides a more accurate measure of the

  7. The Effects of Intense Submicrosecond Electrical Pulses on Cells

    PubMed Central

    Deng, Jingdong; Schoenbach, Karl H.; Buescher, E. Stephen; Hair, Pamela S.; Fox, Paula M.; Beebe, Stephen J.

    2003-01-01

    A simple electrical model for living cells predicts an increasing probability for electric field interactions with intracellular substructures of both prokaryotic and eukaryotic cells when the electric pulse duration is reduced into the sub-microsecond range. The validity of this hypothesis was verified experimentally by applying electrical pulses (durations 100 μs–60 ns, electric field intensities 3–150 kV/cm) to Jurkat cells suspended in physiologic buffer containing propidium iodide. Effects on Jurkat cells were assessed by means of temporally resolved fluorescence and light microscopy. For the longest applied pulses, immediate uptake of propidium iodide occurred consistent with electroporation as the cause of increased surface membrane permeability. For nanosecond pulses, more delayed propidium iodide uptake occurred with significantly later uptake of propidium iodide occurring after 60 ns pulses compared to 300 ns pulses. Cellular swelling occurred rapidly following 300 ns pulses, but was minimal following 60 ns pulses. These data indicate that submicrosecond pulses achieve temporally distinct effects on living cells compared to microsecond pulses. The longer pulses result in rapid permeability changes in the surface membrane that are relatively homogeneous across the cell population, consistent with electroporation, while shorter pulses cause surface membrane permeability changes that are temporally delayed and heterogeneous in their magnitude. PMID:12668479

  8. Noncontact microsurgery of cell membranes using femtosecond laser pulses for optoinjection of specified substances into cells

    SciTech Connect

    Il'ina, I V; Ovchinnikov, A V; Chefonov, O V; Sitnikov, D S; Agranat, Mikhail B; Mikaelyan, A S

    2013-04-30

    IR femtosecond laser pulses were used for microsurgery of a cell membrane aimed at local and short-duration change in its permeability and injection of specified extracellular substances into the cells. The possibility of noncontact laser delivery of the propidium iodide fluorescent dye and the pEGFP plasmid, encoding the green fluorescent protein, into the cells with preservation of the cell viability was demonstrated. (extreme light fields and their applications)

  9. Protective effect of cannabidiol on hydrogen peroxide‑induced apoptosis, inflammation and oxidative stress in nucleus pulposus cells.

    PubMed

    Chen, Jie; Hou, Chen; Chen, Xin; Wang, Dong; Yang, Pinglin; He, Xijing; Zhou, Jinsong; Li, Haopeng

    2016-09-01

    Cannabidiol, a major component of marijuana, protects nerves, and exerts antispasmodic, anti-inflammatory and anti‑anxiety effects. In the current study, the protective effect of cannabidiol was observed to prevent hydrogen peroxide (H2O2)‑induced apoptosis, inflammation and oxidative stress in nucleus pulposus cells. Nucleus pulposus cells were isolated from rats and cultured in vitro, and H2O2 was used to construct the nucleus pulposus cell model. Cell viability of the nucleus pulposus cells was assessed using a 3‑(4,5-dimethylthiazol-2-yl)-2,5‑diphenyltetrazolium bromide assay. The ratio of apoptotic cells, and caspase‑3 or cyclooxygenase‑2 (COX‑2) mRNA expression was analyzed by annexin V‑fluorescein isothiocyanate/propidium‑iodide staining and reverse transcription‑quantitative polymerase chain reaction, respectively. The quantities of interleukin (IL)‑1β and interleukin‑6 were measured using a series of assay kits. B-cell lymphoma 2 (Bcl‑2) and inducible nitric oxide synthase (iNOS) protein expression levels were analyzed using western blotting. The present study identified that cannabidiol enhanced cell viability and reduced apoptosis in H2O2‑treated nucleus pulposus cells in vitro using a lumbar disc herniation (LDH) model. In addition, cannabidiol reduced caspase‑3 gene expression and augmented the Bcl‑2 protein expression levels in the nucleus pulposus cells following H2O2 exposure. Pre‑treatment with cannabidiol suppressed the promotion of COX‑2, iNOS, IL‑1β and IL‑6 expression in the nucleus pulposus cells following H2O2 exposure. Taken together, these results suggest that cannabidiol potentially exerts its protective effect on LDH via the suppression of anti‑apoptosis, anti‑inflammation and anti‑oxidative activities in nucleus pulposus cells. PMID:27430346

  10. Variable Resistance of RMS to Interferon γ Signaling.

    PubMed

    Simon-Keller, Katja; Mößinger, Katharina; Bohlender, Anna-Lena; Ströbel, Philipp; Marx, Alexander

    2012-01-01

    Aims. Chimeric T cells directed to the γ-subunit of the fetal acetylcholine receptor (fAChR) produce large amounts of interferon-γ (IFNγ) on coculture with fAChR-expressing rhabdomyosarcoma (RMS) cells prior to RMS cell death. The aim of this study was to elucidate whether IFNγ blocks proliferation and survival of RMS cells and modulates expression of genes with relevance for cytotoxicity of chimeric T cells. Methods. Expression levels of IFNγ receptor (IFNGR), AChR, MHCI, MHCII, and CIITA (class II transactivator) by RMS were checked by flow cytometry, qRT-PCR, and western blot. Proliferation and cell survival were investigated by annexin V and propidium iodide staining and MTT (thiazolyl-blue-tetrazolium-bromide) assay. Key phosphorylation and binding sites of IFNGRs were checked by DNA sequencing. Results. IFNγ treatment blocked proliferation in 3 of 6 RMS cell lines, but reduced survival in only one. IFNGR was expressed at levels comparable to controls and binding sites for JAK and STAT1 were intact. Induction of several target genes (e.g., AChR, MHCI, and MHCII) by IFNγ was detected on the RNA level but not protein level. Conclusions. IFNγ does not significantly contribute to the killing of RMS cells by fAChR directed chimeric T cells. Signalling downstream of the IFNR receptor, including the posttranscriptional level, is impaired in most RMS cell lines. PMID:22919516

  11. Variable Resistance of RMS to Interferon γ Signaling

    PubMed Central

    Simon-Keller, Katja; Mößinger, Katharina; Bohlender, Anna-Lena; Ströbel, Philipp; Marx, Alexander

    2012-01-01

    Aims. Chimeric T cells directed to the γ-subunit of the fetal acetylcholine receptor (fAChR) produce large amounts of interferon-γ (IFNγ) on coculture with fAChR-expressing rhabdomyosarcoma (RMS) cells prior to RMS cell death. The aim of this study was to elucidate whether IFNγ blocks proliferation and survival of RMS cells and modulates expression of genes with relevance for cytotoxicity of chimeric T cells. Methods. Expression levels of IFNγ receptor (IFNGR), AChR, MHCI, MHCII, and CIITA (class II transactivator) by RMS were checked by flow cytometry, qRT-PCR, and western blot. Proliferation and cell survival were investigated by annexin V and propidium iodide staining and MTT (thiazolyl-blue-tetrazolium-bromide) assay. Key phosphorylation and binding sites of IFNGRs were checked by DNA sequencing. Results. IFNγ treatment blocked proliferation in 3 of 6 RMS cell lines, but reduced survival in only one. IFNGR was expressed at levels comparable to controls and binding sites for JAK and STAT1 were intact. Induction of several target genes (e.g., AChR, MHCI, and MHCII) by IFNγ was detected on the RNA level but not protein level. Conclusions. IFNγ does not significantly contribute to the killing of RMS cells by fAChR directed chimeric T cells. Signalling downstream of the IFNR receptor, including the posttranscriptional level, is impaired in most RMS cell lines. PMID:22919516

  12. Adiponectin exerts antiproliferative effect on human placenta via modulation of the JNK/c-Jun pathway

    PubMed Central

    Chen, Haitian; Chen, Hanqing; Wu, Yanxin; Liu, Bin; Li, Zhuyu; Wang, Zilian

    2014-01-01

    To determine the effects of adiponectin on human placenta during gestational diabetes mellitus (GDM) and on high glucose (HG)-induced BeWo cell proliferation. We examined the expression levels of adiponectin in control and GDM placenta using quantitative real-time PCR, Western blot, and immunohistochemistry (IHC). Cell proliferation and viability were assessed using a colorimetric assay (cell counting kit-8), PCNA immunocytochemical staining, and Western blot analysis of cyclin D1. Transfection of siRNA against c-jun was performed using Lipofectamine 2000. Cell cycle analysis was performed using propidium iodide staining and flow cytometry. Results show a decreased expression of adiponectin and an increased degree of trophoblast cell proliferation in GDM placenta compared to the normal placenta. Similarly, HG can promote BeWo cell proliferation that is associated with adiponectin down-regulation. This proliferation could be depressed by addition of exogenous adiponectin, i.e. adiponectin exerts antiproliferative effects on HG-induced trophoblast cells. Adiponectin suppresses the HG-induced BeWo cell proliferation by inhibiting the activation of JNK/c-jun. In conclusion, adiponectin inhibits HG-induced proliferation of BeWo cells through down-regulation of JNK/c-jun phosphorylation. PMID:25031708

  13. Effects of X‑ray irradiation in combination with ascorbic acid on tumor control.

    PubMed

    Hosokawa, Yoichiro; Monzen, Satoru; Yoshino, Hironori; Terashima, Shingo; Nakano, Manabu; Toshima, Keisuke; Saga, Ryo; Kashiwakura, Ikuo

    2015-10-01

    Our previous studies demonstrated that the combination of treatment with ascorbic acid (AsA) and X‑ray irradiation results in increased apoptosis in HL60 cells. The present study was performed to investigate the effects of the combined use of AsA and X‑ray irradiation on epithelial cancer and sarcoma cells, and its potential use in future clinical treatment. X‑ray irradiation combined with AsA treatment resulted in increased suppression of cell growth of HT1080, SAS and A549 cells in vitro compared with X‑ray irradiation alone. The combined treatment also suppressed tumor growth in implanted HT‑1080 cells in vivo. Using annexin V/propidium iodide staining and the detection of activated caspase 3, it was found that X‑ray irradiation increased the apoptotic rate of HT1080 cells and resulted in G2/M arrest. However, apoptosis in the HT1080 cells treated with 5 mM AsA remained unchanged, and no changes were observed in the G2/M fraction. By contrast, AsA treatment caused increased suppression of proliferation compared with X‑ray irradiation. These results suggested that 5 mM AsA slowed the cell cycle and reduced tumor growth. Therefore, X‑ray irradiation combined with AsA treatment may be effective against epithelial cancer and sarcoma cells. PMID:26238154

  14. Luciferase-based protein-denaturation assay for quantification of radiofrequency field-induced targeted hyperthermia: developing an intracellular thermometer

    PubMed Central

    Raoof, Mustafa; Zhu, Cihui; Kaluarachchi, Warna D.; Curley, Steven A.

    2013-01-01

    Background Several studies have reported targeted hyperthermia at the cellular level using remote activation of nanoparticles by radiofrequency waves. To date, methods to quantify intracellular thermal dose have not been reported. In this report we study the relationship between radio wave exposure and luciferase denaturation with and without intracellular nanoparticles. The findings are used to devise a strategy to quantify targeted thermal dose in a primary human liver cancer cell line. Methods Water-bath or non-invasive external RF generator (600W, 13.56 MHz) was used for hyperthermia exposures. Luciferase activity was measured using a bioluminescence assay and viability was assessed using Annexin V-FITC and Propidium iodide staining. Heat shock proteins were analyzed using western-blot analysis Results Duration-dependent luciferase denaturation was observed in SNU449 cells exposed to RF field that preceded measurable loss in viability. Loss of luciferase activity was higher in cetuximab-conjugated gold nanoparticle (C225-AuNP) treated cells. Using a standard curve from water-bath experiments, the intracellular thermal dose was calculated. Cells treated with C225-AuNP accumulated 6.07 times higher intracellular thermal dose than the untreated controls over initial 4 minutes of RF exposure. Conclusions Cancer cells when exposed to an external RF field exhibit dose-dependent protein denaturation. Luciferase denaturation assay can be used to quantify thermal dose delivered after RF exposures to cancer cells with and without nanoparticles. PMID:22515341

  15. IL-4 is able to reverse the CD2-mediated negative apoptotic signal to CD4-CD8- alpha beta and/or gamma delta T lymphocytes.

    PubMed

    Spinozzi, F; Nicoletti, I; Agea, E; Belia, S; Moraca, R; Migliorati, G; Riccardi, C; Grignani, F; Bertotto, A

    1995-11-01

    Activation of immature thymocytes or transformed T lymphocytes via T-cell receptor (TCR)/CD3 signalling can induce programmed cell death (apoptosis). Recent data indicate that anti-CD3/TCR monoclonal antibodies (mAb) also trigger apoptosis in activated (but not resting) mature peripheral blood T lymphocytes. Here we report that triggering of resting CD4-CD8-TCR alpha beta+ and/or TCR gamma delta+ via the alternative CD2-dependent activation pathway is able to induce programmed cell death. A pair of mitogenic anti-CD2 mAb provoked a dramatic rise in [Ca2+]i that was almost entirely sustained by extracellular fluxes, and the inhibition of membrane [Ca2+/Mg2+] ATPase. The resulting endonuclease activation was able to induce DNA fragmentation, as revealed by propidium iodide staining and gel electrophoresis. Induction of apoptosis was prevented by the presence of interleukin-4 (IL-4) as well as by endonuclease inactivation with 100 microM ZnCl2, but enhanced by the contemporary block of protein kinase C. Thus it seems that in resting T lymphocytes the strong calcium signal delivered by the alternative CD2 activation pathway may act as a negative apoptotic signal in both alpha beta and gamma delta T cells with low (non-major histocompatibility complex restricted) antigenic affinity, so limiting the extension of polyclonal T-cell growth. PMID:8550074

  16. Effects of Methylmercury on Harbour Seal Peripheral Blood Leucocytes In Vitro Studied by Electron Microscopy.

    PubMed

    Dupont, Aurélie; De Pauw-Gillet, Marie-Claire; Schnitzler, Joseph; Siebert, Ursula; Das, Krishna

    2016-01-01

    Methylmercury (MeHg) is highly immunotoxic and can alter the health status of the harbour seal, Phoca vitulina, from the North Sea. To investigate the mechanism of MeHg-induced toxicity in harbour seal lymphocytes, Concanavalin A (ConA)-stimulated peripheral blood leucocytes were exposed in vitro to sublethal concentrations of MeHgCl (0.2, 1, and 2 µM) for 72 h and then analysed for their viability and ultrastructure. After 72 h of incubation, cells were counted with a propidium iodide staining technique, a metabolic MTS assay was performed, and cells exposed to 1 µM of MeHgCl were observed by transmission electron microscopy (TEM). Alive cell numbers decreased with increased MeHgCl concentrations. In presence of ConA and 1 µM of MeHgCl, TEM images revealed a higher frequency of apoptotic cells. Exposed cells displayed condensation of the chromatin at the nuclear membrane and mitochondrial damages. The results suggest that in vitro MeHgCl-induced apoptosis in harbour seal lymphocytes through a mitochondrial pathway. PMID:26264045

  17. Rigosertib Is a More Effective Radiosensitizer Than Cisplatin in Concurrent Chemoradiation Treatment of Cervical Carcinoma, In Vitro and In Vivo

    SciTech Connect

    Agoni, Lorenzo; Basu, Indranil; Gupta, Seema; Alfieri, Alan; Gambino, Angela; Goldberg, Gary L.; Reddy, E. Premkumar; Guha, Chandan

    2014-04-01

    Purpose: To compare rigosertib versus cisplatin as an effective radiosensitizing agent for cervical malignancies. Methods and Materials: Rigosertib and cisplatin were tested in cervical cancer cell lines, HeLa and C33A. A 24-hour incubation with rigosertib and cisplatin, before irradiation (2-8 Gy), was used for clonogenic survival assays. Cell cycle analysis (propidium iodide staining) and DNA damage (γ-H2AX expression) were evaluated by fluorescence-activated cell sorter cytometry. Rigosertib was also tested in vivo in tumor growth experiments on cervical cancer xenografts. Results: Rigosertib was demonstrated to induce a G{sub 2}/M block in cancer cells. Survival curve comparison revealed a dose modification factor, as index of radiosensitization effect, of 1.1-1.3 for cisplatin and 1.4-2.2 for rigosertib. With 6-Gy irradiation, an increase in DNA damage of 15%-25% was achieved in both HeLa and C33A cells with cisplatin pretreatment, and a 71-108% increase with rigosertib pretreatment. In vivo tumor growth studies demonstrated higher performance of rigosertib when compared with cisplatin, with 53% longer tumor growth delay. Conclusions: Rigosertib was more effective than cisplatin when combined with radiation and caused minimal toxicity. These data support the need for clinical trials with rigosertib in combination therapy for patients with cervical carcinoma.

  18. The Cytotoxicity of Dacarbazine Potentiated by Sea Cucumber Saponin in Resistant B16F10 Melanoma Cells through Apoptosis Induction

    PubMed Central

    Baharara, Javad; Amini, Elaheh; Nikdel, Najme; Salek-Abdollahi, Farzaneh

    2016-01-01

    Background: Malignant melanoma is a highly aggressive malignant melanocytic neoplasm which resists against the most conventional therapies. Sea cucumber as one of marine organisms contains bioactive compounds such as polysaccharide, terpenoid and other metabolites which have anti-cancer, anti-tumor, anti-inflammatory and antioxidant properties. The present study was designed to investigate the anticancer potential of saponin extracted from sea cucumber Holothuria leucospilata alone and in combination with dacarbazine on B16F10 melanoma cell line. Methods: The B16F10 cell line was treated with different concentrations of saponin (0, 4, 8, 12, 16, 20 μg/ml), dacarbazine (0, 1200, 1400, 1600, 18000, 1200, 1400, 1600, 2000 μg/ml) and co-administration of saponin-dacarbazine (1200 da+8 sp, 1200 da+4 sp) for 24 and 48 hr and the cytotoxic effect was examined by MTT, DAPI, acridine orange/propodium iodide, flow cytometry and caspase colorimetric assay. Results: The results exhibited that sea cucumber saponin, dacarbazine, and co-administration of saponin-dacarbazine inhibited the proliferation of melanoma cells in a dose and time dependent manner with IC50 values of 10, 1400 and 4+1200 μg/ml, respectively. Morphological observation of DAPI and acridine orange/propodium iodide staining documented typical characteristics of apoptotic cell death. Flow cytometry assay indicated accumulation of IC50 treated cells in sub-G1 peak. Additionally, saponin extracted induced intrinsic apoptosis via up-regulation of caspase-3 and caspase-9. Conclusion: These results revealed that the saponin extracted from sea cucumber as a natural anti-cancer compound may be a new treatment modality for metastatic melanoma and the application of sea cucumber saponin in combination with dacarbazine demonstrated the strongest anti-cancer activity as compared with the drug alone. PMID:27563423

  19. Antimicrobial Mechanism of Monocaprylate

    PubMed Central

    Hyldgaard, Morten; Sutherland, Duncan S.; Sundh, Maria; Mygind, Tina

    2012-01-01

    Monoglyceride esters of fatty acids occur naturally and encompass a broad spectrum of antimicrobial activity. Monocaprylate is generally regarded as safe (GRAS) and can function both as an emulsifier and as a preservative in food. However, knowledge about its mode of action is lacking. The aim of this study was therefore to elucidate the mechanism behind monocaprylate's antimicrobial effect. The cause of cell death in Escherichia coli, Staphylococcus xylosus, and Zygosaccharomyces bailii was investigated by examining monocaprylate's effect on cell structure, membrane integrity, and its interaction with model membranes. Changes in cell structure were visible by atomic force microscopy (AFM), and propidium iodide staining showed membrane disruption, indicating the membrane as a site of action. This indication was confirmed by measuring calcein leakage from membrane vesicles exposed to monocaprylate. AFM imaging of supported lipid bilayers visualized the integration of monocaprylate into the liquid disordered, and not the solid ordered, phase of the membrane. The integration of monocaprylate was confirmed by quartz crystal microbalance measurements, showing an abrupt increase in mass and hydration of the membrane after exposure to monocaprylate above a threshold concentration. We hypothesize that monocaprylate destabilizes membranes by increasing membrane fluidity and the number of phase boundary defects. The sensitivity of cells to monocaprylate will therefore depend on the lipid composition, fluidity, and curvature of the membrane. PMID:22344642

  20. The Extracellular Matrix Regulates Granuloma Necrosis in Tuberculosis.

    PubMed

    Al Shammari, Basim; Shiomi, Takayuki; Tezera, Liku; Bielecka, Magdalena K; Workman, Victoria; Sathyamoorthy, Tarangini; Mauri, Francesco; Jayasinghe, Suwan N; Robertson, Brian D; D'Armiento, Jeanine; Friedland, Jon S; Elkington, Paul T

    2015-08-01

    A central tenet of tuberculosis pathogenesis is that caseous necrosis leads to extracellular matrix destruction and bacterial transmission. We reconsider the underlying mechanism of tuberculosis pathology and demonstrate that collagen destruction may be a critical initial event, causing caseous necrosis as opposed to resulting from it. In human tuberculosis granulomas, regions of extracellular matrix destruction map to areas of caseous necrosis. In mice, transgenic expression of human matrix metalloproteinase 1 causes caseous necrosis, the pathological hallmark of human tuberculosis. Collagen destruction is the principal pathological difference between humanised mice and wild-type mice with tuberculosis, whereas the release of proinflammatory cytokines does not differ, demonstrating that collagen breakdown may lead to cell death and caseation. To investigate this hypothesis, we developed a 3-dimensional cell culture model of tuberculosis granuloma formation, using bioelectrospray technology. Collagen improved survival of Mycobacterium tuberculosis-infected cells analyzed on the basis of a lactate dehydrogenase release assay, propidium iodide staining, and measurement of the total number of viable cells. Taken together, these findings suggest that collagen destruction is an initial event in tuberculosis immunopathology, leading to caseous necrosis and compromising the immune response, revealing a previously unappreciated role for the extracellular matrix in regulating the host-pathogen interaction. PMID:25676469

  1. Development of a confocal ultrasound device using an inertial cavitation control for transfection in-vitro

    NASA Astrophysics Data System (ADS)

    Mestas, J. L.; Chettab, K.; Roux, S.; Prieur, F.; Lafond, M.; Dumontet, C.; Lafon, C.

    2015-12-01

    Sonoporation using low-frequency high-pressure ultrasound (US) is a non-viral approach for in vitro and in vivo gene delivery. We developed a new sonoporation device designed for spatial and temporal control of ultrasound cavitation. This device was evaluated for the in vitro transfection efficiency of a plasmid coding for Green Fluorescent Protein (peGFP- C1) in adherent and non-adherent cell lines. The frequency spectrum of the signal receive by a hydrophone is used to compute a cavitation index (CI) representative of the inertial cavitation activity. The influence of the CI on transfection efficiency, as well as reproducibility were determined. A real-time feedback loop control on CI was integrated in the process to regulate the cavitation level during sonoporation. In both adherent and non-adherent cell lines, the sonoporation device produced a highly efficient transfection of peGFP-C1 (40-80%), as determined by flow cytometry analysis of GFP expression, along with a low rate of mortality assessed by propidium iodide staining. Moreover, the sonoporation of non-adherent cell lines Jurkat and K562 was found to be equivalent to nucleofection in terms of efficiency and toxicity while these two cell lines were resistant to transfection with lipofection.

  2. Design, synthesis and biological evaluation of 3,5-disubstituted 2-amino thiophene derivatives as a novel class of antitumor agents

    PubMed Central

    Romagnoli, Romeo; Baraldi, Pier Giovanni; Lopez-Cara, Carlota; Salvador, Maria Kimatrai; Preti, Delia; Tabrizi, Mojgan Aghazadeh; Balzarini, Jan; Nussbaumer, Peter; Bassetto, Marcella; Brancale, Andrea; Fu, Xian-Hua; Yang-Gao; Li, Jun; Zhang, Su-Zhan; Hamel, Ernest; Bortolozzi, Roberta; Basso, Giuseppe; Viola, Giampietro

    2014-01-01

    In search of new compounds with strong antiproliferative activity and simple molecular structure, we designed a novel series of agents based on the 2-amino-3-alkoxycarbonyl/cyano-5-arylethylthiophene scaffold. The presence of the ethyl spacer between the 2′,5′-dimethoxyphenyl and the 5-position of the thiophene ring, as well as the number and location of methoxy substitutents on the phenyl ring, played a profound role in affecting the antiproliferative activity. Among the synthesized compounds, we identified the 2-amino-3-cyano-[2-(2,5-dimethoxyphenyl)ethyl] thiophene 2c as the most promising derivative against a wide panel of cancer cell lines (IC50 = 17–130 nM). The antiproliferative activity of this compound appears to correlate well with its ability to inhibit tubulin assembly and the binding of colchicine to tubulin. Moreover 2c, as determined by flow cytometry, strongly induced arrest in the G2/M phase of the cell cycle, and annexin-V and propidium iodide staining indicate that cell death proceeds through an apoptotic mechanism that follows the intrinsic mitochondrial pathway. PMID:24398384

  3. Low-dose γ-irradiation induces dual radio-adaptive responses depending on the post-irradiation time by altering microRNA expression profiles in normal human dermal fibroblasts.

    PubMed

    Bae, Seunghee; Kim, Karam; Cha, Hwa Jun; Choi, Yeongmin; Shin, Shang Hun; An, In-Sook; Lee, Jae Ho; Lee, Su Jae; Kim, Ji Young; Nam, Seon Young; An, Sungkwan

    2015-01-01

    Exposure to high-dose ionizing radiation, including γ-radiation, induces severe skin disorders. However, the biological consequences and molecular mechanisms responsible for the response of human skin to low-dose γ-radiation (LDR) are largely unknown. In the present study, we demonstrate that LDR (0.1 Gy) induces distinct cellular responses in normal human dermal fibroblasts (NHDFs) depending on the post-irradiation time point. A MTT-based cell viability assay and propidium iodide staining-based cell cycle assay revealed that the viability and proportion of the cells in the G2/M phase were differed at 6 and 24 h post-irradiation. Reverse transcription quantitative PCR (RT-qPCR) revealed that LDR significantly upregulated the mRNA expression of collagen type I alpha 1 (COL1A1), but downregulated the mRNA expression of matrix metalloproteinase 1 (MMP1) at 24 h post-irradiation. MicroRNA (miRNA) microarray analysis further demonstrated that LDR induced changes in the expression profiles of specific miRNAs and that some of the deregulated miRNAs were specific to either the early or late radio-adaptive response. Our results suggest that LDR generates dual radio-adaptive responses depending on the post-irradiation time by altering specific miRNA expression profiles in NHDFs. PMID:25384363

  4. Design, synthesis and biological evaluation of 3,5-disubstituted 2-amino thiophene derivatives as a novel class of antitumor agents.

    PubMed

    Romagnoli, Romeo; Baraldi, Pier Giovanni; Lopez-Cara, Carlota; Salvador, Maria Kimatrai; Preti, Delia; Tabrizi, Mojgan Aghazadeh; Balzarini, Jan; Nussbaumer, Peter; Bassetto, Marcella; Brancale, Andrea; Fu, Xian-Hua; Yang-Gao; Li, Jun; Zhang, Su-Zhan; Hamel, Ernest; Bortolozzi, Roberta; Basso, Giuseppe; Viola, Giampietro

    2014-09-15

    In search of new compounds with strong antiproliferative activity and simple molecular structure, we designed a novel series of agents based on the 2-amino-3-alkoxycarbonyl/cyano-5-arylethylthiophene scaffold. The presence of the ethyl spacer between the 2',5'-dimethoxyphenyl and the 5-position of the thiophene ring, as well as the number and location of methoxy substitutents on the phenyl ring, played a profound role in affecting the antiproliferative activity. Among the synthesized compounds, we identified the 2-amino-3-cyano-[2-(2,5-dimethoxyphenyl)ethyl] thiophene 2c as the most promising derivative against a wide panel of cancer cell lines (IC50=17-130 nM). The antiproliferative activity of this compound appears to correlate well with its ability to inhibit tubulin assembly and the binding of colchicine to tubulin. Moreover 2c, as determined by flow cytometry, strongly induced arrest in the G2/M phase of the cell cycle, and annexin-V and propidium iodide staining indicate that cell death proceeds through an apoptotic mechanism that follows the intrinsic mitochondrial pathway. PMID:24398384

  5. Discovery of an algicidal compound from Brevibacterium sp. BS01 and its effect on a harmful algal bloom-causing species, Alexandrium tamarense

    PubMed Central

    An, Xinli; Zhang, Bangzhou; Zhang, Huajun; Li, Yi; Zheng, Wei; Yu, Zhiming; Fu, Lijun; Zheng, Tianling

    2015-01-01

    Blooms of the dinoflagellate Alexandrium tamarense have become worldwide phenomena and have detrimental impacts on aquatic ecosystems and human health. In this study, a culture supernatant of the marine actinomycete BS01 exerted a strong algicidal effect on A. tamarense (ATGD98-006). The target algicide from BS01 was separated by adsorption chromatography and identified by MALDI-TOF-MS and NMR analysis. The results suggested that the purified algicidal component corresponded to a hydrophobic compound (2-isobutoxyphenyl)amine (C10H15NO) with a molecular weight of 165 Da, which exhibited a significant algicidal effect (64.5%) on A. tamarense. After incubation in 5 μg/mL of (2-isobutoxyphenyl)amine for 24 h, the algae lost mobility and sank to the bottom of the flasks, and 56.5% of the algae cells lost vitality at a concentration of 20 μg/mL (p < 0.01) despite having intact cell profiles. Morphological analysis revealed that the cell structure of A. tamarense was altered by (2-isobutoxyphenyl)amine resulting in cytoplasm degradation and the loss of organelle integrity. The images following propidium iodide staining suggested that the algal nucleus was also severely damaged and eventually degraded due to exposure to the algicidal compound. All of the results indicate that (2-isobutoxyphenyl)amine from the actinomycete might be a candidate for the control of bloom-forming A. tamarense. PMID:26594205

  6. Biofilm formation by Escherichia coli in hypertonic sucrose media.

    PubMed

    Kawarai, Taketo; Furukawa, Soichi; Narisawa, Naoki; Hagiwara, Chisato; Ogihara, Hirokazu; Yamasaki, Makari

    2009-06-01

    High osmotic environments produced by NaCl or sucrose have been used as reliable and traditional methods of food preservation. We tested, Escherichia coli as an indicator of food-contaminating bacterium, to determine if it can form biofilm in a hyperosmotic environment. E. coli K-12 IAM1264 did not form biofilm in LB broth that contained 1 M NaCl. However, the bacterium formed biofilm in LB broth that contained 1 M sucrose, although the planktonic growth was greatly suppressed. The biofilm, formed on solid surfaces, such as titer-plate well walls and glass slides, solely around the air-liquid interface. Both biofilm forming cells and planktonic cells in the hypertonic medium adopted a characteristic, fat and filamentous morphology with no FtsZ rings, which are a prerequisite for septum formation. Biofilm forming cells were found to be alive based on propidium iodide staining. The presence of 1 M sucrose in the food environment is not sufficient to prevent biofilm formation by E. coli. PMID:19447340

  7. A Study of Aberrant Glycosylation in Simulated Microgravity Using Laser Induced AutoFluorescence and Flow Cytometry

    NASA Technical Reports Server (NTRS)

    Lawless, B. DeSales

    1999-01-01

    A number of pathologies and cellular dysfunctions including neoplasms have been correlated with autofluorescence. The complications of aging and diabetes have been associated with the accumulation of non-enzymatic glycosylations of tissue macromolecules. These products are known as the Advanced Glycosylated End Products (AGEs). A physical property associated with AGEs is the emission of 570 mn or 630 nm light energy (autofluorescence) following the absorption of 448 mm energy associated with the argon laser. This investigation sought to assess the induction of argon-laser induced autofluorescence in a variety of in vitro culture systems. Different fluorescence intensities distinguished tumor lines from normal cell populations. Laser-stimulated autofluorescence discriminated primary cultures of lymphocytes grown in the presence of excess glucose as opposed to normal glucose concentrations. The effects of deglycosylating agents upon laser-induced autofluorescence were also assessed. The studies included studies of cell cycle analysis using Propidium Iodide stained DNA of cells grown in simulated microgravity using NASA Bioreactor Vessels in media of normal and elevated glucose concentrations.

  8. Discovery of an algicidal compound from Brevibacterium sp. BS01 and its effect on a harmful algal bloom-causing species, Alexandrium tamarense.

    PubMed

    An, Xinli; Zhang, Bangzhou; Zhang, Huajun; Li, Yi; Zheng, Wei; Yu, Zhiming; Fu, Lijun; Zheng, Tianling

    2015-01-01

    Blooms of the dinoflagellate Alexandrium tamarense have become worldwide phenomena and have detrimental impacts on aquatic ecosystems and human health. In this study, a culture supernatant of the marine actinomycete BS01 exerted a strong algicidal effect on A. tamarense (ATGD98-006). The target algicide from BS01 was separated by adsorption chromatography and identified by MALDI-TOF-MS and NMR analysis. The results suggested that the purified algicidal component corresponded to a hydrophobic compound (2-isobutoxyphenyl)amine (C10H15NO) with a molecular weight of 165 Da, which exhibited a significant algicidal effect (64.5%) on A. tamarense. After incubation in 5 μg/mL of (2-isobutoxyphenyl)amine for 24 h, the algae lost mobility and sank to the bottom of the flasks, and 56.5% of the algae cells lost vitality at a concentration of 20 μg/mL (p < 0.01) despite having intact cell profiles. Morphological analysis revealed that the cell structure of A. tamarense was altered by (2-isobutoxyphenyl)amine resulting in cytoplasm degradation and the loss of organelle integrity. The images following propidium iodide staining suggested that the algal nucleus was also severely damaged and eventually degraded due to exposure to the algicidal compound. All of the results indicate that (2-isobutoxyphenyl)amine from the actinomycete might be a candidate for the control of bloom-forming A. tamarense. PMID:26594205

  9. Antitumor activity of jujuboside B and the underlying mechanism via induction of apoptosis and autophagy.

    PubMed

    Xu, Mei-Ying; Lee, So Young; Kang, Sam Sik; Kim, Yeong Shik

    2014-02-28

    Jujuboside B (1) is one of the saponins isolated from the seeds of Zizyphus jujuba var. spinosa, which are used as a well-known traditional medicine for the treatment of insomnia and anxiety in East Asian countries. This is the first study to investigate the antitumor mechanism of 1 in vivo and in vitro. The results showed that 1 induced apoptosis and autophagy in AGS and HCT 116 human cancer cells and also effectively suppressed tumor growth in a nude mouse xenograft model bearing HCT 116 cells. The apoptosis-inducing effect of 1 was characterized by annexin V/propidium iodide staining, sub-G1 phase increase, and caspase-3 activation. Mechanistic studies showed that 1-induced apoptosis is associated with the extrinsic pathway through an increase in FasL and caspase-8 activation. Moreover, 1 activated p38/c-Jun N-terminal kinase (JNK), and the extrinsic pathway-mediated apoptosis was attenuated by both SB202190 (a p38 inhibitor) and SP600125 (a JNK inhibitor). The autophagy-inducing effect was indicated by the formation of cytoplasmic vacuoles and microtubule-associated protein 1 light chain-3 II (LC3-II) conversion. The autophagy inhibitor bafilomycin A1 (BaF) decreased 1-induced cell viability and increased pp38, pJNK, FasL, caspase-8 activation, and caspase-3 activation. Taken together, these results demonstrate that 1 induced protective autophagy to retard extrinsic pathway-mediated apoptosis. PMID:24547878

  10. The green synthesis, characterization, and evaluation of the biological activities of silver nanoparticles synthesized from Leptadenia reticulata leaf extract

    NASA Astrophysics Data System (ADS)

    Kumara Swamy, M.; Sudipta, K. M.; Jayanta, K.; Balasubramanya, S.

    2015-01-01

    Biosynthesis of silver nanoparticles (Ag Nps) was carried out using methanol leaves extract of L. reticulata. Ag Nps were characterized based on the observations of UV-visible spectroscopy, transmission electron microscopy, and X-ray diffraction (XRD) analysis. These Ag Nps were tested for antimicrobial activity by agar well diffusion method against different pathogenic microorganisms and antioxidant activity was performed using DPPH assay. Further, the in vitro cytotoxic effects of Ag Nps were screened against HCT15 cancer cell line and viability of tumor cells was confirmed using MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole)) assay. The nuclear condensation was studied using the propidium iodide-staining method. The color change from green to dark brown and the absorbance peak at about 420 nm indicated the formation of nanoparticles. XRD pattern showed characteristic peaks indexed to the crystalline planes (111), (200) and (220) of face-centered cubic silver. The nanoparticles were of spherical shape with varying sizes ranging from 50 to 70 nm. Biosynthesized Ag Nps showed potent antibacterial activity and effective radical scavenging activity. MTT assay revealed a dose-dependent decrease in cell viability. Microscopic observations showed distinct cellular morphological changes indicating unhealthy cells, whereas the control appeared normal. Increase in the number of propidium iodide positive cells were observed in maximum concentration. Methanolic leaf extract of L. reticulata acts as an excellent capping agent for the formation of silver nanoparticles and demonstrates immense biological activities. Hence, these Ag NPs can be used as antibacterial, antioxidant as well as cytotoxic agent in treating many medical complications.

  11. Comparison of propidium monoazide-quantitative PCR and reverse transcription quantitative PCR for viability detection of fresh Cryptosporidium oocysts following disinfection and after long-term storage in water samples.

    PubMed

    Liang, Zhanbei; Keeley, Ann

    2012-11-15

    Purified oocysts of Cryptosporidium parvum were used to evaluate the applicability of two quantitative PCR (qPCR) viability detection methods in raw surface water and disinfection treated water. Propidium monoazide-qPCR targeting hsp70 gene was compared to reverse transcription (RT)-qPCR heat induced hsp70 mRNA in water samples spiked with oocysts. Changes in viability of flow cytometry sorted fresh and oocysts having undergone various aging periods (up to 48 months at 4 °C) were evaluated by Ct values obtained from the qPCR before and after disinfection scenarios involving ammonia or hydrogen peroxide. Both qPCR methods achieved stability in dose dependent responses by hydrogen peroxide treatment in distilled water that proved their suitability for the viability evaluations. Oocysts exposed to 3% hydrogen peroxide were inactivated at a rate of 0.26 h(-1) and 0.93 h(-1), as measured by the mRNA assay and the PMA-DNA assay, respectively. In contrast, the PMA-DNA assay was not as sensitive as the mRNA assay in detecting viability alterations followed by exposure to ammonia or after a long-term storage in 4 °C in distilled water since no dose response dependency was achieved. Surface water concentrates containing enhanced suspendable solids determined that changes in viability were frequently detected only by the mRNA method. Failure of, or inconsistency in the detection of oocysts viability with the PMA-DNA method, apparently resulted from solids that might have reduced light penetration through the samples, and thus inhibited the cross-linking step of PMA-DNA assay. PMID:22980572

  12. Purely aqueous PLGA nanoparticulate formulations of curcumin exhibit enhanced anticancer activity with dependence on the combination of the carrier.

    PubMed

    Nair, K Lekha; Thulasidasan, Arun Kumar T; Deepa, G; Anto, Ruby John; Kumar, G S Vinod

    2012-04-01

    Curcumin, a yellow pigment present in turmeric, possess potential anti-proliferative and anti-inflammatory activities but poor aqueous solubility limits its applications. In this study we report a novel comparative study of the formulation and characterization of curcumin nanoparticles (nanocurcumin) using two poly (lactide-co-glycolide) (PLGA) combinations, 50:50 and 75:25 having different lactide to glycolide ratios. Nanocurcumin 50:50 showed smaller size with higher encapsulation efficiency. Thermal evaluation suggested the presence of curcumin in molecular dispersion form which supported its sustained release up to a week where nanocurcumin 50:50 showed faster release. Cellular uptake studies in human epithelial cervical cancer cells (HeLa) exhibited enhanced intracellular fluorescence with nanocurcumin when compared to free curcumin, when both given in purely aqueous media. Antiproliferative studies using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, Annexin V/propidium iodide staining, poly (ADP-ribose) polymerase (PARP) cleavage and downregulation of clonogenic potential of HeLa cells proved the better antitumor activity of nanocurcumin 50:50 administered in aqueous media. Superior efficacy of nanocurcumin 50:50 in comparison to free curcumin was further demonstrated by electrophoretic mobility shift assay and immunocytochemical analysis. In conclusion, the enhanced aqueous solubility and higher anticancer efficacy of nanocurcumin administered in aqueous media clearly demonstrates its potential against cancer chemotherapy, with dependence on the combination of PLGA. PMID:22266528

  13. Experimental Procedures for Demonstration of MicroRNA Mediated Enhancement of Functional Neuroprotective Effects of Estrogen Receptor Agonists.

    PubMed

    Chakrabarti, Mrinmay; Ray, Swapan K

    2016-01-01

    Protection of motoneurons is an important therapeutic goal in the treatment of neurological disorders. Recent reports have suggested that specific microRNAs (miRs) could modulate the expression of particular proteins for significant alterations in the pathogenesis of different neurological disorders. Thus, combination of overexpression of a specific neuroprotective miR and treatment with a neuroprotective agent could be a novel strategy for functional protection of motoneurons. The protocols described herein demonstrate that miR-7-1, a neuroprotective miR, can enhance the functional neuroprotective effects of estrogen receptor agonists such as 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT), Way 200070 (WAY), and estrogen (E2) in preventing apoptosis in A23187 calcium ionophore (CI) exposed VSC4.1 motoneurons. This article describes the protocols for the cell viability assay, transfection of VSC4.1 motoneurons with miRs, Annexin V/propidium iodide staining for apoptosis, Western blotting, patch-clamp recording of whole-cell membrane potential, and JC-1 staining for detection of mitochondrial membrane potential. Taken together, these protocols are used to demonstrate that miR-7-1 caused significant enhancement of the efficacy of estrogen receptor agonists for functional neuroprotection in VSC4.1 motoneurons. PMID:26585150

  14. Neuroprotective effects of atorvastatin against glutamate-induced excitotoxicity in primary cortical neurones.

    PubMed

    Bösel, Julian; Gandor, Florin; Harms, Christoph; Synowitz, Michael; Harms, Ulrike; Djoufack, Pierre Chryso; Megow, Dirk; Dirnagl, Ulrich; Hörtnagl, Heide; Fink, Klaus B; Endres, Matthias

    2005-03-01

    Statins [3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors] exert cholesterol-independent pleiotropic effects that include anti-thrombotic, anti-inflammatory, and anti-oxidative properties. Here, we examined direct protective effects of atorvastatin on neurones in different cell damage models in vitro. Primary cortical neurones were pre-treated with atorvastatin and then exposed to (i) glutamate, (ii) oxygen-glucose deprivation or (iii) several apoptosis-inducing compounds. Atorvastatin significantly protected from glutamate-induced excitotoxicity as evidenced by propidium iodide staining, nuclear morphology, release of lactate dehydrogenase, and mitochondrial tetrazolium metabolism, but not from oxygen-glucose deprivation or apoptotic cell death. This anti-excitototoxic effect was evident with 2-4 days pre-treatment but not with daily administration or shorter-term pre-treatment. The protective properties occurred independently of 3-hydroxy-3-methylglutaryl-CoA reductase inhibition because co-treatment with mevalonate or other isoprenoids did not reverse or attenuate neuroprotection. Atorvastatin attenuated the glutamate-induced increase of intracellular calcium, which was associated with a modulation of NMDA receptor function. Taken together, atorvastatin exerts specific anti-excitotoxic effects independent of 3-hydroxy-3-methylglutaryl-CoA reductase inhibition, which has potential therapeutic implications. PMID:15748157

  15. The influence of selected antimicrobial peptides on the physiology of the immune system

    NASA Astrophysics Data System (ADS)

    Golab, Karolina; Mittag, Anja; Pierzchalski, Arkadiusz; Bocsi, Jozsef; Kamysz, Wojciech; Tarnok, Attila

    2011-02-01

    Antimicrobial peptides (AMPs) are an essential part of the innate immune system that serves as a first line of defense against invading pathogens. Recently, immunomodulatory activities of AMPs have begun to be appreciated, implying the usefulness of AMPs in the treatment of infectious disease. The aim of this strategy is the modulation of host immune responses to enhance clearance of infectious agents and reduce tissue damage due to inflammation. Although AMPs could be used as therapeutic agents, a more detailed understanding of how they affect host cells is needed. Hence, several AMPs have been investigated for their potential as a new class of antimicrobial drugs in this study. Synthetic AMPs and AMPs of natural origin were tested on human leukocytes by flow cytometry. Dose- and time-dependent cytotoxic effects could be observed by propidium iodide staining. Different leukocyte subtypes seem to be susceptible to AMP treatment while others were not affected, even in high concentrations. In conclusion, AMPs have an impact on host immune cells. However, their role in stimulation of chemokine production and enhanced leukocyte recruitment remains a crucial aspect and further studies are needed.

  16. Functional expression of IL-12 receptor by human eosinophils: IL-12 promotes eosinophil apoptosis.

    PubMed

    Nutku, E; Zhuang, Q; Soussi-Gounni, A; Aris, F; Mazer, B D; Hamid, Q

    2001-07-15

    In murine models of allergic inflammation, IL-12 has been shown to decrease tissue eosinophilia, but the underlying mechanisms are not known. We evaluated the expression of IL-12R and the effect of IL-12 on eosinophil survival. In situ hybridization demonstrated the presence of mRNA and immunoreactivity for IL-12Rbeta1 and -beta2 subunits in human peripheral blood eosinophils. Surface expression of IL-12Rbeta1 and -beta2 subunits on freshly isolated human eosinophils was optimally expressed after incubation with PMA. To determine the functional significance of IL-12R studies, we studied cell viability and apoptosis. Morphological analysis and propidium iodide staining for cell cycle demonstrated that recombinant human IL-12 increased in vitro human eosinophil apoptosis in a dose-dependent manner. Addition of IL-5 together with IL-12 abrogated eosinophil apoptosis, suggesting that IL-12 and IL-5 have antagonistic effects. Our findings provide evidence for a novel role for IL-12 in regulating eosinophil function by increasing eosinophil apoptosis. PMID:11441113

  17. Analysis of cell-tissue grafts under weightless conditions using confocal fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Volova, L. T.; Milyakova, M. N.; Rossinskaya, V. V.; Boltovskaya, V. V.; Kulagina, L. N.; Kurganskaya, L. V.; Timchenko, P. E.; Timchenko, E. V.; Zherdeva Taskina, Larisa A.

    2015-03-01

    The research results of monitoring of viable cells in a cellular-tissue graft using confocal laser fluorescence microscopy at 488 nm and 561 nm with the use of fluorophore propidium iodide (propidium iodide, PI Sigma Aldrich USA) are presented. The processing of the received images was carried out using the software ANDOR. It is experimentally shown that the method of confocal fluorescence microscopy is one of the informational methods for detecting cells populated in a 3-D bio-carrier with a resolution of at least 400 nm. Analysis of the received micrographs suggests that the cells that were in a bio-carrier for 30 days in a synchronous ground-based experiment retained their viability compared to a similar space-based experiment in which the cells were hardly detected in a bio-carrier.

  18. In vitro immunomodulatory effects of extracts from three plants of the Labiatae family and isolation of the active compound(s).

    PubMed

    Amirghofran, Zahra; Hashemzadeh, Reihaneh; Javidnia, Katayoun; Golmoghaddam, Hossein; Esmaeilbeig, Ahmadreza

    2011-01-01

    Plants may have the ability to modulate immune responses. In the present study, the effects of three plants belonging to Labiatae family, each traditionally used for the treatments of infections and inflammatory diseases, as well as the role of thymol (as one the major components of these plants), were investigated for their potential to affect the activation of lymphocytes. Four organic extracts of Thymus vulgaris and two other plants (i.e., T. daenensis and Zataria multiflora) were prepared. The effect of the extracts on mitogen (PHA)-stimulated peripheral blood lymphocytes was determined using a cell proliferation assay. The hexane extracts obtained from the three plants showed the strongest inhibitory effects on PHA-induced proliferation. Use of preparative thin layer and gas chromatographies in conjunction with the proliferation assay confirmed that thymol was the major component responsible for the observed effects from the three plants. Thymol inhibited inducible lymphocyte proliferation in a concentration-dependent manner, with reductions ranging from 62.8% at 50 µg/ml to 89.8% at 200 µg/ml (> 0.1 µg/ml (p < 0.01). Flow cytometric analysis using propidium iodide staining showed that the inhibitory effect of thymol at 200 µg/ml was due to a cytotoxic activity. In conclusion, the three Labiatae plants studied here each showed immunosuppressive effects against lymphocytes and it was most likely that thymol was the compound in these plants responsible for this effect. PMID:21711089

  19. Whole-mount immunolocalization to study female meiosis in Arabidopsis.

    PubMed

    Escobar-Guzmán, Rocio; Rodríguez-Leal, Daniel; Vielle-Calzada, Jean-Philippe; Ronceret, Arnaud

    2015-10-01

    Here we describe a whole-mount immunolocalization protocol to follow the subcellular localization of proteins during female meiosis in Arabidopsis thaliana, a model species that is used to study sexual reproduction in flowering plants. By using confocal microscopy, the procedure allows one to follow megasporogenesis at all stages before differentiation of the functional megaspore. This in particular includes stages that occur during prophase I, such as the installation of the axial and central elements of the synaptonemal complex along the meiotic chromosomes. In contrast to procedures that require microtome sectioning or enzymatic isolation and smearing to separate female meiocytes from neighboring cells, this 3-day protocol preserves the constitution of the developing primordium and incorporates the architecture of the ovule to provide a temporal and spatial context to meiotic divisions. This opens up the possibility to systematically compare the dynamics of protein localization during female and male meiosis. Steps describe tissue collection and fixation, preparation of slides and polyacrylamide embedding, tissue permeabilization, antibody incubation, propidium iodide staining, and finally image acquisition by confocal microscopy. The procedure adds an essential technique to the toolkit of plant meiotic analysis, and it represents a framework for technical adaptations that could soon allow the analysis of plant reproductive alternatives to sexual reproduction. PMID:26357009

  20. Tackling Obstacles for Gene Therapy Targeting Neurons: Disrupting Perineural Nets with Hyaluronidase Improves Transduction

    PubMed Central

    Wanisch, Klaus; Kovac, Stjepana; Schorge, Stephanie

    2013-01-01

    Gene therapy has been proposed for many diseases in the nervous system. In most cases for successful treatment, therapeutic vectors must be able to transduce mature neurons. However, both in vivo, and in vitro, where preliminary characterisation of viral particles takes place, transduction of neurons is typically inefficient. One possible explanation is that the extracellular matrix (ECM), forming dense perineural nets (PNNs) around neurons, physically blocks access to the cell surface. We asked whether co-administration of lentiviral vectors with an enzyme that disrupts the ECM could improve transduction efficiency. Using hyaluronidase, an enzyme which degrades hyaluronic acid, a high molecular weight molecule of the ECM with mainly a scaffolding function, we show that in vitro in mixed primary cortical cultures, and also in vivo in rat cortex, hyaluronidase co-administration increased the percentage of transduced mature, NeuN-positive neurons. Moreover, hyaluronidase was effective at doses that showed no toxicity in vitro based on propidium iodide staining of treated cultures. Our data suggest that limited efficacy of neuronal transduction is partly due to PNNs surrounding neurons, and further that co-applying hyaluronidase may benefit applications where efficient transduction of neurons in vitro or in vivo is required. PMID:23301052

  1. Cationic surfactants in the form of nanoparticles and micelles elicit different human neutrophil responses: a toxicological study.

    PubMed

    Hwang, Tsong-Long; Sung, Calvin T; Aljuffali, Ibrahim A; Chang, Yuan-Ting; Fang, Jia-You

    2014-02-01

    Cationic surfactants are an ingredient commonly incorporated into nanoparticles for clinical practicability; however, the toxicity of cationic surfactants in nanoparticles is not fully elucidated. We aimed to evaluate the inflammatory responses of cationic nanobubbles and micelles in human neutrophils. Soyaethyl morpholinium ethosulfate (SME) and hexadecyltrimethyl-ammonium bromide (CTAB) are the two cationic surfactants employed in this study. The zeta potential of CTAB nanobubbles was 80 mV, which was the highest among all formulations. Nanobubbles, without cationic surfactants, showed no cytotoxic effects on neutrophils in terms of inflammatory responses. Cationic nanobubbles caused a concentration-dependent cytotoxicity of degranulation (elastase release) and membrane damage (release of lactate dehydrogenase, LDH). Among all nanoparticles and micelles, CTAB-containing nanosystems showed the greatest inflammatory responses. A CTAB nanobubble diluent (1/150) increased the LDH release 80-fold. Propidium iodide staining and scanning electron microscopy (SEM) verified cell death and morphological change of neutrophils treated by CTAB nanobubbles. SME, in a micelle form, strengthened the inflammatory response more than SME-loaded nanobubbles. Membrane interaction and subsequent Ca(2+) influx were the mechanisms that triggered inflammation. The information obtained from this work is beneficial in designing nanoparticulate formulations for balancing clinical activity and toxicity. PMID:24246197

  2. Characterisation of the efficacy of endodontic medications using a three-dimensional fluorescent tooth model: An ex vivo study.

    PubMed

    Chen, Emily W; Carey, Alison J; Ulett, Glen C; George, Roy

    2015-08-01

    The purpose of this study was to establish a three-dimensional fluorescent tooth model to investigate bacterial viability against intra-canal medicaments across the thickness and surface of root dentine. Dental microbial biofilms (Enterococcus faecalis and Streptococcus mutans) were established on the external root surface and bacterial kill was monitored over time against intra-canal medicament (Ca(OH)2 ) using fluorescent microscopy in conjunction with BacLight SYTO9 and propidium iodide stains. An Olympus digital camera fitted to SZX16 fluorescent microscope captured images of bacterial cells in biofilms on the external root surface. Viability of biofilm was measured by calculating the total pixel area of green (viable bacteria) and red (non-viable bacteria) for each image using ImageJ® software. All data generated were assessed for normality and then analysed using a Mann-Whitney t-test. The viability of S. mutans biofilm following Ca(OH)2 treatment showed a significant decline compared with the untreated group (P = 0.0418). No significant difference was seen for E. faecalis biofilm between the Ca(OH)2 and untreated groups indicating Ca(OH)2 medicament is ineffective against E. faecalis biofilm. This novel three-dimensional fluorescent biofilm model provides a new clinically relevant tool for testing of medicaments against dental biofilms. PMID:25583457

  3. The Edible Marine Alga Gracilariopsis chorda Alleviates Hypoxia/Reoxygenation-Induced Oxidative Stress in Cultured Hippocampal Neurons.

    PubMed

    Mohibbullah, Md; Hannan, Md Abdul; Choi, Ji-Young; Bhuiyan, Mohammad Maqueshudul Haque; Hong, Yong-Ki; Choi, Jae-Suk; Choi, In Soon; Moon, Il Soo

    2015-09-01

    Age-related neurological disorders are of growing concern among the elderly, and natural products with neuroprotective properties have been attracting increasing attention as candidates for the prevention or treatment of neurological disorders induced by oxidative stress. In an effort to explore natural resources, we collected some common marine seaweed from the Korean peninsula and Indonesia and screened them for neuroprotective activity against hypoxia/reoxygenation (H/R)-induced oxidative stress. Of the 23 seaweeds examined, the ethanol extract of Gracilariopsis chorda (GCE) provided maximum neuroprotection at an optimum concentration of 15 μg/mL, followed by Undaria pinnatifida. GCE increased cell viability after H/R, decreased the formation of reactive oxygen species (measured by 2',7'-dichlorodihydrofluorescein diacetate [DCF-DA] staining), and inhibited the double-stranded DNA breaks (measured by H2AX immunocytochemistry), apoptosis (measured by Annexin V/propidium iodide staining), internucleosomal DNA fragmentation (measured by DNA laddering), and dissipation of mitochondrial membrane potential (measured by JC-1 staining). Using reverse-phase high-pressure liquid chromatography, we quantitated the arachidonic acid (AA) in GCE, which provides neuroprotection against H/R-induced oxidative stress. This neuroprotective effect of AA was comparable to that of GCE. These findings suggest that the neuroprotective effect of GCE against H/R-induced neuronal death is due, at least in part, to the AA content that suppresses neuronal apoptosis. PMID:26106876

  4. Duramycin-induced calcium release in cancer cells.

    PubMed

    Broughton, Laura J; Crow, Chris; Maraveyas, Anthony; Madden, Leigh A

    2016-03-01

    Duramycin, through binding with phosphatidylethanolamine (PE), has shown potential to be an effective antitumour agent. However, its mode of action in relation to tumour cells is not fully understood. PE expression on the surface of a panel of cancer cell lines was analysed using duramycin and subsequent antibody labelling, and then analysed by flow cytometry. Cell viability was also assessed by flow cytometry using annexin V and propidium iodide. Calcium ion (Ca) release by tumour cells in response to duramycin was determined by spectrofluorometry following incubation with Fluo-3, AM. Confocal microscopy was performed on the cancer cell line AsPC-1 to assess real-time cell response to duramycin treatment. Duramycin could detect cell surface PE expression on all 15 cancer cell lines screened, which was shown to be duramycin concentration dependent. However, higher concentrations induced necrotic cell death. Duramycin induced calcium ion (Ca) release from the cancer cell lines also in a concentration-dependent and time-dependent manner. Confocal microscopy showed an influx of propidium iodide into the cells over time and induced morphological changes. Duramycin induces Ca release from cancer cell lines in a time-dependent and concentration-dependent manner. PMID:26512767

  5. Simplified method for DNA and protein staining of human hematopoietic cell samples

    SciTech Connect

    Crissman, H.A.; Egmond, J.V.; Holdrinet, R.S.; Pennings, A.; Haanen, C.

    1980-01-01

    A rapid reproducible method yielding high resolution analysis of DNA and protein in human hematopoietic cell samples was developed by modification of the propidium iodide (PI) and fluorescein isothiocyanate (FITC) procedure. Cell staining involved sequential addition of each reagent (RNase, FITC, and PI) to ethanol-fixed cells and requires no centrifiguation steps. Stained cells are analyzed in the reagent solutions. Analysis of bone marrow samples from multiple myeloma patients revealed mixed 2C DNA and aneuploid populations with the aneuploid cells having a significantly higher protein content. This approach permitted differential cell cycle kinetic analysis of the 2C DNA and the aneuploid population.

  6. Apoptosis of V beta 8.2+ T lymphocytes in the spinal cord during recovery from experimental autoimmune encephalomyelitis induced in Lewis rats by inoculation with myelin basic protein.

    PubMed

    McCombe, P A; Nickson, I; Tabi, Z; Pender, M P

    1996-07-01

    To study T cell apoptosis during spontaneous recovery from experimental autoimmune encephalomyelitis (EAE), we extracted lymphocytes from the spinal cords of Lewis rats with EAE induced by inoculation with myelin basic protein (MBP) and adjuvants. Using flow cytometry we assessed the numbers of CD5+ and TCR alpha beta + lymphocytes, as well as V beta 8.2+ lymphocytes, which constitute the predominant encephalitogenic MBP-reactive cells in Lewis rats. Rats developed neurological signs of disease 10-12 days after inoculation. The peak of disease was on day 14 after inoculation and was followed by clinical recovery. The numbers of CD5+, TCR alpha beta + and V beta 8.2+ cells obtained from the spinal cord were greatest on day 13. During spontaneous clinical recovery, there was a decline in the numbers of all the cells studied, with a selective loss of V beta 8.2+ cells from the CD5+ and TCR alpha beta + populations. To determine whether the decline in lymphocyte numbers was due to apoptosis, we used simultaneous surface labelling and propidium iodide staining of the DNA of the cells extracted from the spinal cord. From day 14 onwards, there was selective enrichment of V beta 8.2+ cells in the apoptotic population, and the percentage of V beta 8.2+ cells undergoing apoptosis was greater than the percentages of CD5+ and TCR alpha beta + cells undergoing apoptosis. These findings indicate that recovery from acute EAE is associated with the selective apoptosis, in the central nervous system, of these disease-relevant cells. The findings in this study of actively induced EAE are similar to those of our previous study of EAE induced by transfer of encephalitogenic MBP-specific T cells (Z. Tabi et al., Eur. J. Immunol. 24: 2609-2617, 1994) and further support the hypothesis that selective apoptosis of autoreactive T cells in the central nervous system is of primary importance in spontaneous recovery from EAE. PMID:8836965

  7. The in vitro influences of epidermal growth factor and heregulin-β1 on the efficacy of trastuzumab used in Her-2 positive breast adenocarcinoma

    PubMed Central

    2013-01-01

    Background Human epidermal growth factor receptor-2 (Her-2) is over expressed in approximately 25-30% of all primary breast tumors resulting in a distinctive breast cancer subtype associated with a poor prognosis and a decrease in overall survival. Trastuzumab (Herceptin®), an anti-Her-2 monoclonal antibody, has dramatically altered the prognosis of Her-2 positive breast cancer. Trastuzumab is, however, associated with primary and acquired resistance. Aim and methods To investigate the in-vitro effects of trastuzumab on cell viability (tetrazolium conversion assay), cell cycling (propidium iodide staining), apoptosis (executioner caspases and annexin-V) and relative surface Her-2 receptor expression (anti-Her-2 affibody molecule) in Her-2-positive (SK-Br-3) and oestrogen receptor positive (MCF-7) breast adenocarcinoma cells and to determine potential augmentation of these effects by two endogenous ligands, epidermal growth factor (EGF) and heregulin-β1 (HRG- β1). Results Cell viability was decreased in SK-Br-3 cells by exposure to trastuzumab. This was associated with G1 accumulation and decreased relative surface Her-2 receptor density, supporting the cytostatic nature of trastuzumab in vitro. SK-Br-3 cells exposed to EGF and heregulin-β1 produced differential cell responses alone and in combination with trastuzumab, in some instances augmenting cell viability and cell cycling. Relative surface Her-2 receptor density was reduced substantially by trastuzumab, EGF and heregulin-β1. These reductions were amplified when ligands were used in combination with trastuzumab. Conclusion Cell type specific interactions of endogenous ligands appear to be dependent on absolute Her-receptor expression and cross activation of signaling pathways. This supports the notion that receptor density of Her-family members and multiplicity of growth ligands are of mutual importance in breast cancer cell proliferation and therefore also in resistance associated with trastuzumab. PMID

  8. Can Platelet rich plasma stimulate human ACL growth in culture? A preliminary experience

    PubMed Central

    Dhillon, Mandeep Singh; Karna, Saroj Kumar; Dhatt, Sarvdeep Singh; Behera, Prateek; Bhatia, Alka

    2015-01-01

    Summary Introduction Platelet Rich Plasma (PRP) contains numerous growth factors; Platelet poor plasma (PPP) is plasma proteins without platelets, containing growth factors other than platelet derived. We planned to evaluate the effect of both autologous PRP & PPP on human ACL cell growth characteristics in culture conditions to see if one was better than the other. Methods ACL remnants were collected from eleven patients during ACL reconstruction surgery; PPP and PRP were prepared from blood of these patients. Cells were isolated, identified and cultured and were then divided into six groups. Groups A–D had Fetal Bovine Serum (FBS) added to them along with different concentrations of PRP and PPP. Groups E and F had 5% and 10% PRP respectively but lacked FBS. Cell viability was assayed by MTT and Annexin V assay, and DNA content was evaluated by propidium iodide staining and flow cytometry. Results analysis of cultured cells showed that addition of PRP (5 or 10%) increased the viability of ACL cells in 4 out of 11 and promoted cell proliferation in 8 of 11 donor samples; 10% PRP was more effective than 5% PRP. However, the difference in effectiveness of 10% PRP was not significantly better than 5% PRP. 5% PPP had no significant effect on cell viability, but it led to an increase in DNA content in 5 of 11. There was no statistically significant effect of either PRP or PPP in preventing cell death (depicted by apoptosis rate). Conclusion PRP may have an enhancing effect on ACL cell viability and promotion of cell proliferation but the ideal concentration of PRP for these positive effects needs to be determined before it could be used in clinical settings for enhancing primary repair of torn ACL. Also larger, more controlled and better studies are needed to confirm its clinical utility. PMID:26605188

  9. MLN4924, a novel protein neddylation inhibitor, suppresses proliferation and migration of human urothelial carcinoma: In vitro and in vivo studies.

    PubMed

    Kuo, Kuan Lin; Ho, I Lin; Shi, Chung Sheng; Wu, June Tai; Lin, Wei Chou; Tsai, Yu Chieh; Chang, Hong Chiang; Chou, Chien Tso; Hsu, Chen Hsun; Hsieh, Ju Ton; Chang, Shih Chen; Pu, Yeong Shiau; Huang, Kuo How

    2015-07-28

    MLN4924, a small molecule inhibitor of NEDD8 activating enzyme (NAE), has been reported to elicit an anti-tumor effect on various malignancies. In this study, we investigated the anti-tumor effect of MLN4924 in human urothelial carcinoma (UC) in vitro and in vivo by using three human UC cell lines of various grading (T24, NTUB1 and RT4). The impact of MLN4924 on UC cells was determined by measuring viability (MTT), proliferation (BrdU incorporation), cell cycle progression (flow cytometry with propidium iodide staining) and apoptosis (flow cytometry with annexin V-FITC labeling). The cell cycle regulatory molecules, apoptosis-related molecules, and cell stress-related proteins were examined by Western blotting. The influence of tumor cell migration and invasion was analyzed by Transwell and wound healing assays. We also evaluated the effects of MLN4924 on tumor growth by a SCID xenograft mouse model. The data show that MLN4924 induced dose-dependent cytotoxicity, anti-proliferation, anti-migration, anti-invasion and apoptosis in human UC cells, accompanied by activations of Bad, phospho-histone H2A.X, caspase-3, 7 and PARP, decreased level of phospho-Bcl2, and caused cell cycle retardation at the G2M phase. Moreover, MLN4924 activated endoplasmic reticulum stress-related molecules (caspase-4, phospho-eIF2α, ATF-4 and CHOP) and other stress responses (JNK and c-Jun activations). Finally, we confirmed MLN4924 inhibited tumor growth in a UC xenograft mouse model with minimal general toxicity. We concluded that MLN4924 induces apoptosis and cell cycle arrest, as well as activation of cell stress responses in human UC. These findings imply MLN4924 provides a novel strategy for the treatment of UC. PMID:25615422

  10. The Comet Assay to Determine the Mode of Cell Death for the Ultrasonic Delivery of Doxorubicin to Human Leukemia (HL-60 cells) from Pluronic P105 Micelles

    PubMed Central

    Husseini, Ghaleb A.; O'Neill, Kim L.; Pitt, William G.

    2006-01-01

    This notes examines the mode of cell death of HL-60 cells exposed to 70 kHz and 1.3 W/cm2 in the presence of 1% Pluronic P105 and 1.67 μg/ml doxorubicin (Dox). The cells were ultrasonicated for 30, 60 and 120 minutes. They were then lysed, electrophorised, stained using propidium iodide, and their DNA profile captured using a fluorescent microscope. The gradual DNA damage observed and the comet tails captured after 1 and 2 hours of insonation suggest that the mode of cell killing is apoptosis. PMID:16292892

  11. Enhanced Egress of Intracellular Eimeria tenella Sporozoites by Splenic Lymphocytes from Coccidian-Infected Chickens▿

    PubMed Central

    Dong, Xiaojuan; Abdelnabi, Ghada H.; Lee, Sung H.; Li, Guangxing; Jin, Hong; Lillehoj, Hyun S.; Suo, Xun

    2011-01-01

    Egress, which describes the mechanism that some intracellular parasites use to exit from parasitophorous vacuoles and host cells, plays a very important role in the parasite life cycle and is central to Eimeria propagation and pathogenesis. Despite the importance of egress in the intracellular parasite's life cycle, very little information is known on this process compared to other steps, e.g., invasion. The present study was conducted to investigate the interplay between the host adaptive immune system and Eimeria egression. Splenic lymphocytes or soluble immune factors were incubated with parasite-infected host cells for 3 or 5 h, and the percentage of egress was calculated according to an established formula. Viability of egressed parasites and host cells was tested using trypan blue exclusion and annexin V and propidium iodide staining, respectively. We found that premature egression of sporozoites from Eimeria tenella-infected primary chicken kidney cells or from chicken peripheral blood mononuclear cells occurred when the cells were cocultured in vitro with spleen lymphocytes from E. tenella-infected chickens but not when they were cocultured with splenocytes from uninfected chickens. Eimeria-specific antibodies and cytokines (gamma interferon [IFN-γ], interleukin-2 [IL-2], and IL-15), derived from E. tenella-primed B and T lymphocytes, respectively, were capable of promoting premature egress of sporozoites from infected host cells. Both egressed parasites and host cells were viable, although the latter showed reduced reinvasion ability. These results suggest a novel, immune-mediated mechanism that the host exploits to interrupt the normal Eimeria life cycle in vivo and thereby block the release of mature parasites into the environment. PMID:21628515

  12. Synthesis, characterization, and antitumor activity of three ternary dinuclear copper (II) complexes with a reduced Schiff base ligand and diimine coligands in vitro and in vivo.

    PubMed

    Jia, Lei; Xu, Jun; Zhao, Xiaolei; Shen, Shanshan; Zhou, Tao; Xu, Zhouqing; Zhu, Taofeng; Chen, Ruhua; Ma, Tieliang; Xie, Jing; Dong, Kun; Huang, Jiancui

    2016-06-01

    Three ternary copper (II) complexes containing 1,10-phenanthroline (phen, 1), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq, 2) and dipyrido[3,2-a:2',3'-c]phenazine (dppz, 3), with the formulation [Cu2(NCL)2(H4PASP)]·4.5H2O (1-3) (where NCL=the diimine coligand, H4PASP=N,N'-(p-xylylene)di-2-aminosuccinic acid), were isolated and characterized. The binding of these complexes with calf thymus DNA was studied using UV-visible absorption titration, emission, and circular dichroism spectroscopy, among other methods. The changes in physicochemical properties that occurred upon binding of these complexes with DNA indicate that binding occurs primarily through intercalative interactions. Human tumor cell lines HeLa, PC3, and HepG2 were treated with the copper(II) complexes in vitro and cell survival rate was assessed by 3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay and crystal violet survival assay. Flow cytometry was performed on treated cells labeled with AnnexinV/Propidium Iodide staining to determine rates of apoptosis. Western blot was performed to determine the expression levels of the apoptotic markers p53, Bax, and Bcl-2. The complexes reduced cell viability and induced apoptosis in cells of human tumor cell lines in a dose-dependent manner. In addition, using a nude mouse xenograft model, we found that the three ternary copper (II) complexes inhibited human tumor cell growth in vivo. In conclusion, these novel synthetic copper complexes have profound antitumor effects on human tumor cells and are promising therapeutic agents for human tumors. PMID:26974885

  13. Influence of lepidocrocite (gamma-FeOOH) on Escherichia coli cultivability in drinking water.

    PubMed

    Grandjean, D; Jorand, F; Yañez, C; Appenzeller, B M R; Block, J-C

    2005-02-01

    A washed suspension of the bacteria Escherichia coli, pre-grown on a complex culture medium, was stored in sterilized drinking water for 21 days at 25 degrees C in glass flasks in order to assess the effect of iron corrosion products on the persistenceof the bacteria in drinking water. Four conditions were tested: aerobic with 50 mM lepidocrocite (gamma-FeOOH, an insoluble iron corrosion product), anaerobic with 50 mM lepidocrocite, aerobic without lepidocrocite and anaerobic without lepidocrocite. The survival of E. coli was monitored by their cultivability and their membrane integrity (propidium iodide staining). When the samples were not supplemented with the iron oxide, the cultivability and cell integrity of the bacteria were dramatically altered: from the 10(7) initially added, only 10 CFU ml(-1) remained after 21 days; 90% of the cells exhibited membrane alteration after 2 weeks of storage. In contrast, bacteria with lepidocrocite preserved their cultivability and integrity over the 21 days of storage. In the presence of di-oxygen and without iron oxide, the alteration of cell cultivability was more pronounced than that in anaerobic conditions, suggesting that oxidative stress was part of the phenomenon. When the cells were pre-grown in a growth medium supplemented by a large excess of an easily available form of iron (ferric-citrate), the cells stored a higher amount of iron and persisted one week longer in the iron-free drinking water than cells pre-grown in the standard growth medium. Therefore, in an oligotrophic environment like drinking water, E. coli cells can find the ability to survive a long time through the presence of iron corrosion products. The necessity of controlling the corrosiveness of drinking water for sanitary reasons is therefore emphasized by this study. PMID:15791802

  14. Inhibition of Salmonella-induced apoptosis as a marker of the protective efficacy of virulence gene-deleted live attenuated vaccine.

    PubMed

    Kamble, Nitin M; Nandre, Rahul M; Lee, John Hwa

    2016-01-01

    Vaccination is one of the best protection strategies against Salmonella infection in humans and chickens. Salmonella bacteria must induce apoptosis prior to initiating infection, pathogenesis and evasion of host immune responses. In this study, we evaluated the efficacy of vaccinating chickens against Salmonella Enteritidis (SE) using a vaccine candidate strain (JOL919), constructed by deleting the lon and cpxR genes from a wild-type SE using an allelic exchange method. In present study day old chickens were inoculated with 1×10(7)cfu (colony forming unit) of JOL919 per os. We measured cell-mediated immunity, protective efficacy and extent of apoptosis induction in splenocytes. Seven days post-immunization, the number of CD3+CD4+ and CD3+ CD8+ T cells was significantly higher in the immunized group compared to the control group, indicating a significant augmentation of systemic immune response. The internal organs of chickens immunized with JOL919 had a significantly lower challenge-strain recovery, indicating effective protection and clearance of the challenge strain. Post-challenge, the number of apoptotic cells in the immunized group was significantly lower than in the control group. Additionally, AV/PI (Annexin V/propidium iodide) staining was performed to differentiate between apoptotic cells and necrotic cells, which corroborated TUNEL-assay (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling) results. The proportions of AV+/PI- and AV+/PI+ cells, which represent the proportions of early apoptotic and late apoptotic/early necrotic cells present, respectively, were significantly lower in the immunized group. Our findings suggest that the apoptotic splenocytes in immunized chickens significantly decreased in number, which occurred concomitantly with a significant rise in systemic immune response and bacterial clearance. This suggests that inhibition of apoptosis may be a marker of protection efficacy in immunized chickens. PMID

  15. Analysis of cell cycle position in mammalian cells.

    PubMed

    Cecchini, Matthew J; Amiri, Mehdi; Dick, Frederick A

    2012-01-01

    The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell's decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments. DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects

  16. Kinetics of plasma membrane and mitochondrial alterations in cells undergoing apoptosis

    SciTech Connect

    Lizard, G.; Fournel, S.; Genestier, L.; Dhedin, N.

    1995-11-01

    Programmed cell death or apoptosis is characterized by typical morphological alterations. By transmission electron microscopy, apoptotic cells are identified by condensation of the chromatin in tight apposition to the nuclear envelope, alteration of the nuclear envelope and fragmentation of the nucleus, whereas integrity of the plasma membrane and organelles is preserved. Conversely cells undergoing necrosis display and early desintegration of cytoplasmic membrane and swelling of mitochondria. In this study we assessed by flow cytometry the sequential alterations of forward angle light scatter, 90{degrees} light scatter, and fluorescence associated with fluorescein diacetate, rhodamine 123, and propidium iodide in two human B cell lines undergoing apoptosis induced by the topoisomerase II inhibitor VP-16. The kinetics of these modifications were compared to those of cells undergoing necrosis induced by the topoisomerase II inhibitor VP-16. The kinetics of these modifications were compared to those of cells undergoing necrosis induced by sodium azide. At the same time intervals, cells were examined by transmission electron microscopy and by UV microscopy after staining with Hoechst 33342. We report that sequential changes in light scatters and fluorescein diacetate are similar in cells undergoing apoptosis or necrosis, whereas apoptosis is characterized by a slightly delayed decrease of mitochondrial activity as assessed by rhodamine 123 staining. Surprisingly, a part of cells undergoing apoptosis displayed an early uptake of propidium iodide followed by a condensation and then a fragmentation of their nuclei. It is concluded that uptake of propidium iodide is a very early marker of cell death which does not discriminate between necrosis and apoptosis. Along with biochemical criteria, nuclear morphology revealed by staining with Hoechst 33342 would seem to be of the most simple and most discriminative assay of apoptosis. 33 refs., 5 figs., 1 tab.

  17. Antioxidative activity of diarylheptanoids from the bark of black alder (Alnus glutinosa) and their interaction with anticancer drugs.

    PubMed

    Dinić, Jelena; Novaković, Miroslav; Podolski-Renić, Ana; Stojković, Sonja; Mandić, Boris; Tešević, Vele; Vajs, Vlatka; Isaković, Aleksandra; Pešić, Milica

    2014-08-01

    Diarylheptanoids belong to polyphenols, a group of plant secondary metabolites with multiple biological properties. Many of them display antioxidative, cytotoxic, or anticancer actions and are increasingly recognized as potential therapeutic agents. The aim of this study was to evaluate antioxidant and cytoprotective activity of two diarylheptanoids: platyphylloside 5(S)-1,7-di(4-hydroxyphenyl)-3-heptanone-5-O-β-D-glucopyranoside (1) and its newly discovered analog 5(S)-1,7-di(4-hydroxyphenyl)-5-O-β-D-[6-(E-p-coumaroylglucopyranosyl)]heptane-3-one (2), both isolated from the bark of black alder (Alnus glutinosa). To that end, we have employed a cancer cell line (NCI-H460), normal human keratinocytes (HaCaT), and peripheral blood mononuclear cells. The effects on cell growth were assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. Cell death was examined by annexin V/propidium iodide staining on a flow cytometer. Reactive oxygen species production was examined by dihydroethidium staining. Mitochondrial structure and doxorubicin localization were visualized by fluorescent microscopy. Gene expression of manganese superoxide dismutase and hypoxia-inducible factor-1α was determined by reverse transcription polymerase chain reaction. Diarylheptanoids antagonized the effects of either doxorubicin or cisplatin, significantly increasing their IC50 values in normal cells. Diarylheptanoid 1 induced the retention of doxorubicin in cytoplasm and reduced mitochondrial fragmentation associated with doxorubicin application. Diarylheptanoid 2 reduced the reactive oxygen species production induced by cisplatin. Both compounds increased the messenger ribonucleic acid expression of enzymes involved in reactive oxygen species elimination (manganese superoxide dismutase and hypoxia-inducible factor-1α). These results indicate that neutralization of reactive oxygen species is an important mechanism of diarylheptanoid action, although these

  18. Fumaric acid esters promote neuronal survival upon ischemic stress through activation of the Nrf2 but not HIF-1 signaling pathway.

    PubMed

    Lin-Holderer, Jiemeng; Li, Lexiao; Gruneberg, Daniel; Marti, Hugo H; Kunze, Reiner

    2016-06-01

    Oxidative stress is a hallmark of ischemic stroke pathogenesis causing neuronal malfunction and cell death. Up-regulation of anti-oxidative genes through activation of the NF-E2-related transcription factor 2 (Nrf2) is one of the key mechanisms in cellular defense against oxidative stress. Fumaric acid esters (FAEs) represent a class of anti-oxidative and anti-inflammatory molecules that are already in clinical use for multiple sclerosis therapy. Purpose of this study was to investigate whether FAEs promote neuronal survival upon ischemia, and analyze putative underlying molecular mechanisms in neurons. Murine organotypic hippocampal slice cultures, and two neuronal cell lines were treated with dimethyl fumarate (DMF) and monomethyl fumarate (MMF). Ischemic conditions were generated by exposing cells and slice cultures to oxygen-glucose deprivation (OGD), and cell death was determined through propidium iodide staining. Treatment with both DMF and MMF immediately after OGD during reoxygenation strongly reduced cell death in hippocampal cultures ex vivo. Both DMF and MMF promoted neuronal survival in HT-22 and SH-SY5Y cell lines exposed to ischemic stress. DMF but not MMF activated the anti-oxidative Nrf2 pathway in neurons. Accordingly, Nrf2 knockdown in murine neurons abrogated the protective effect of DMF but not MMF. Moreover, FAEs did not activate the hypoxia-inducible factor (HIF) pathway suggesting that this pathway may not significantly contribute to FAE mediated neuroprotection. Our results may provide the basis for a new therapeutic approach to treat ischemic pathologies such as stroke with a drug that already has a broad safety record in humans. PMID:26801077

  19. Cryopreservation method affects DNA fragmentation in trophectoderm and the speed of re-expansion in bovine blastocysts.

    PubMed

    Inaba, Yasushi; Miyashita, Satoshi; Somfai, Tamás; Geshi, Masaya; Matoba, Satoko; Dochi, Osamu; Nagai, Takashi

    2016-04-01

    This study investigated re-expansion dynamics during culture of bovine blastocysts cryopreserved either by slow-freezing or vitrification. Also, the extent and localization of membrane damage and DNA fragmentation in re-expanded embryos were studied. Frozen-thawed embryos showed a significantly lower re-expansion rate during 24 h of post-thawing culture compared to vitrified embryos. Vitrified embryos reached the maximum level of re-expansion rate by 12 h of culture whereas frozen embryos showed a gradual increase in re-expansion rate by 24 h of culture. When assayed by Hoechst/propidium iodide staining there was no difference in the numbers and ratio of membrane damaged cells between re-expanded frozen and vitrified embryos; however, the extent of membrane damage in blastomeres was significantly higher in both groups compared with non-cryopreserved embryos (control). TUNEL assay combined with differential ICM and TE staining revealed a significantly higher number and ratio of TE cells showing DNA-fragmentation in frozen-thawed re-expanded blastocysts compared to vitrified ones; however, vitrification also resulted in an increased extent of DNA fragmentation in TE cells compared with control blastocysts. In frozen-thawed blastocysts increased extent of DNA fragmentation was associated with reduced numbers and proportion of TE cells compared with vitrified and control embryos. The number and ratio of ICM cells and the extent of DNA fragmentation in ICM did not differ among control, frozen and vitrified groups. In conclusion, compared with vitrified embryos, blastocysts preserved by slow-freezing showed a delayed timing of re-expansion which was associated with an increased frequency of DNA fragmentation in TE cells. PMID:26996887

  20. Host cell death due to enteropathogenic Escherichia coli has features of apoptosis.

    PubMed

    Crane, J K; Majumdar, S; Pickhardt, D F

    1999-05-01

    Enteropathogenic Escherichia coli (EPEC) is a cause of prolonged watery diarrhea in children in developing countries. The ability of EPEC to kill host cells was investigated in vitro in assays using two human cultured cell lines, HeLa (cervical) and T84 (colonic). EPEC killed epithelial cells as assessed by permeability to the vital dyes trypan blue and propidium iodide. In addition, EPEC triggered changes in the host cell, suggesting apoptosis as the mode of death; such changes included early expression of phosphatidylserine on the host cell surface and internucleosomal cleavage of host cell DNA. Genistein, an inhibitor of tyrosine kinases, and wortmannin, an inhibitor of host phosphatidylinositol 3-kinase, markedly increased EPEC-induced cell death and enhanced the features of apoptosis. EPEC-induced cell death was contact dependent and required adherence of live bacteria to the host cell. A quantitative assay for EPEC-induced cell death was developed by using the propidium iodide uptake method adapted to a fluorescence plate reader. With EPEC, the rate and extent of host cell death were less that what has been reported for Salmonella, Shigella, and Yersinia, three other genera of enteric bacteria known to cause apoptosis. However, rapid apoptosis of the host cell may not favor the pathogenic strategy of EPEC, a mucosa-adhering, noninvasive pathogen. PMID:10225923

  1. Isolation of a Glucosamine Binding Leguminous Lectin with Mitogenic Activity towards Splenocytes and Anti-Proliferative Activity towards Tumor Cells

    PubMed Central

    Chan, Yau Sang; Wong, Jack Ho; Fang, Evandro Fei; Pan, Wenliang; Ng, Tzi Bun

    2012-01-01

    A dimeric 64-kDa glucosamine-specific lectin was purified from seeds of Phaseolus vulgaris cv. “brown kidney bean.” The simple 2-step purification protocol involved affinity chromatography on Affi-gel blue gel and gel filtration by FPLC on Superdex 75. The lectin was absorbed on Affi-gel blue gel and desorbed using 1M NaCl in the starting buffer. Gel filtration on Superdex 75 yielded a major absorbance peak that gave a single 32-kDa band in SDS-PAGE. Hemagglutinating activity was completely preserved when the ambient temperature was in the range of 20°C–60°C. However, drastic reduction of the activity occurred at temperatures above 65°C. Full hemagglutinating activity of the lectin was observed at an ambient pH of 3 to 12. About 50% activity remained at pH 0–2, and only residual activity was observed at pH 13–14. Hemagglutinating activity of the lectin was inhibited by glucosamine. The brown kidney bean lectin elicited maximum mitogenic activity toward murine splenocytes at 2.5 µM. The mitogenic activity was nearly completely eliminated in the presence of 250 mM glucosamine. The lectin also increased mRNA expression of the cytokines IL-2, TNF-α and IFN-γ. The lectin exhibited antiproliferative activity toward human breast cancer (MCF7) cells, hepatoma (HepG2) cells and nasopharyngeal carcinoma (CNE1 and CNE2) cells with IC50 of 5.12 µM, 32.85 µM, 3.12 µM and 40.12 µM respectively after treatment for 24 hours. Flow cytometry with Annexin V and propidum iodide staining indicated apoptosis of MCF7 cells. Hoechst 33342 staining also indicated formation of apoptotic bodies in MCF7 cells after exposure to brown kidney bean lectin. Western blotting revealed that the lectin-induced apoptosis involved ER stress and unfolded protein response. PMID:22720002

  2. Ley specific antibody with potent anti-tumor activity is internalized and degraded in lysosomes.

    PubMed Central

    Garrigues, J.; Garrigues, U.; Hellström, I.; Hellström, K. E.

    1993-01-01

    BR96 is a monoclonal antibody (MAb) that recognizes many human carcinomas and can kill antigen-positive tumor cells in vitro. Using both gold and radiolabeled MAb, the distribution and cellular processing of BR96 during cytolysis has been determined. After a brief (< 3 minutes) MAb treatment, cells in suspension are stained by the nuclear viability dye propidium iodide. Whole MAb and F(ab')2 fragments are equally cytotoxic; monovalent F(ab) fragments, however, have no effect on dye uptake unless cross-linked with goat anti-mouse IgG. The level of toxicity is dependent on both MAb dose and on cell surface receptor density. Cell contact may regulate receptor expression. BR96 receptors are more abundant on cells migrating into the open areas of a scratch wounded confluent culture than on the adjacent contact-inhibited cells. BR96 can also inhibit the anchorage-independent growth of tumor cells in soft agar showing that its effects on propidium iodide staining are not due to transient changes in membrane permeability. Immunogold electron microscopy reveals that, after a 1-minute treatment, BR96 induces significant infolding of the plasma membrane and that internalized MAb is localized to these structures. Immediately thereafter, large cell surface and intracellular vesicles form, mitochondria are swollen, and membrane integrity is lost. Therefore, BR96 seems to cause morphological changes characteristic of necrosis rather than apoptosis. When bound to adherent carcinoma cells, BR96 is distributed uniformly on the apical surface of cells labeled at 4 C and is enriched at points of cell substratum contact. Upon warming of the cells to 37 C, BR96 localizes in small perinuclear clusters and the cell margin is now devoid of label. Immunogold electron microscopy reveals that BR96 undergoes receptor mediated internalization and is localized within the same coated pits, endosomes, and lysosomes as the transferrin receptor. Quantitative studies using iodinated BR96 show that

  3. Indirect radio-chemo-beta therapy: a targeted approach to increase biological efficiency of x-rays based on energy

    NASA Astrophysics Data System (ADS)

    Oktaria, Sianne; Corde, Stéphanie; Lerch, Michael L. F.; Konstantinov, Konstantin; Rosenfeld, Anatoly B.; Tehei, Moeava

    2015-10-01

    Despite the use of multimodal treatments incorporating surgery, chemotherapy and radiotherapy, local control of gliomas remains a major challenge. The potential of a new treatment approach called indirect radio-chemo-beta therapy using the synergy created by combining methotrexate (MTX) with bromodeoxyuridine (BrUdR) under optimum energy x-ray irradiation is assessed. 9L rat gliosarcoma cells pre-treated with 0.01 μM MTX and/or 10 μM BrUdR were irradiated in vitro with 50 kVp, 125 kVp, 250 kVp, 6 MV and 10 MV x-rays. The cytotoxicity was assessed using clonogenic survival as the radiobiological endpoint. The photon energy with maximum effect was determined using radiation sensitization enhancement factors at 10% clonogenic survival (SER10%). The cell cycle distribution was investigated using flow cytometric analysis with propidium iodide staining. Incorporation of BrUdR in the DNA was detected by the fluorescence of labelled anti-BrUdR antibodies. The radiation sensitization enhancement exhibits energy dependence with a maximum of 2.3 at 125 kVp for the combined drug treated cells. At this energy, the shape of the clonogenic survival curve of the pharmacological agents treated cells changes substantially. This change is interpreted as an increased lethality of the local radiation environment and is attributed to supplemented inhibition of DNA repair. Radiation induced chemo-beta therapy was demonstrated in vitro by the targeted activation of combined pharmacological agents with optimized energy tuning of x-ray beams on 9 L cells. Our results show that this is a highly effective form of chemo-radiation therapy.

  4. Circulating immune complexes of calves with bronchopneumonia modulate the function of peripheral blood leukocytes: In vitro evaluation.

    PubMed

    Buač, Marijana; Mojsilović, Slavko; Mišić, Dušan; Vuković, Dejan; Savić, Olivera; Valčić, Olivera; Marković, Dragana; Gvozdić, Dragan; Ilić, Vesna; Fratrić, Natalija

    2016-06-01

    In this work we studied if circulating immune complexes (CIC) of calves with bronchopneumonia have the capacity to modulate function of peripheral blood leukocytes of healthy cattle. CIC of three month old calves (6 healthy and 6 diseased) were isolated by PEG precipitation. Peripheral blood mononuclear cells (MNCs) and granulocytes from healthy calves and cows were the CIC responder cells in in vitro tests. The most remarkable increase of adhesiveness to polystyrene and ROS synthesis (assessed by NBT test) was detected in cows' granulocytes stimulated with CIC of diseased calves. Results of MTT test showed that CIC of both healthy and diseased calves reduced granulocytes' viability. The strongest effect of inhibition of cows' granulocytes resulted from CIC of diseased calves. CIC only moderately reduced spontaneous viability of calves' MNCs. Again, the strongest effect of CIC isolated from diseased calves was observed. In contrast to the low impact of CIC on non-stimulated cells, their inhibitory effect on viability of mitogen stimulated MNCs was very strong. With CFSE assay we showed that both types of CIC stimulated spontaneous, but inhibited mitogen induced proliferation of calves' MNCs. Propidium iodide staining reviled that CIC increased apoptosis/necrosis of both non-stimulated and mitogen stimulated MNCs. CIC of both healthy and diseased calves modulated the function of peripheral blood MNCs and granulocytes, but a stronger effect of CIC of diseased calves was shown. The age of the donors (calves or cows) of the responder cells, and the activation state of these cells, were also of influence. PMID:27234551

  5. Spatial and Temporal Control of Cavitation Allows High In Vitro Transfection Efficiency in the Absence of Transfection Reagents or Contrast Agents

    PubMed Central

    Chettab, Kamel; Roux, Stéphanie; Mathé, Doriane; Cros-Perrial, Emeline; Lafond, Maxime; Lafon, Cyril; Dumontet, Charles; Mestas, Jean-Louis

    2015-01-01

    Sonoporation using low-frequency high-pressure ultrasound (US) is a non-viral approach for in vitro and in vivo gene delivery. In this study, we developed a new sonoporation device designed for spatial and temporal control of ultrasound cavitation. The regulation system incorporated in the device allowed a real-time control of the cavitation level during sonoporation. This device was evaluated for the in vitro transfection efficiency of a plasmid coding for Green Fluorescent Protein (pEGFP-C1) in adherent and non-adherent cell lines. The transfection efficiency of the device was compared to those observed with lipofection and nucleofection methods. In both adherent and non-adherent cell lines, the sonoporation device allowed high rate of transfection of pEGFP-C1 (40–80%), as determined by flow cytometry analysis of GFP expression, along with a low rate of mortality assessed by propidium iodide staining. The transfection efficiency and toxicity of sonoporation on the non-adherent cell lines Jurkat and K562 were similar to those of nucleofection, while these two cell lines were resistant to transfection by lipofection. Moreover, sonoporation was used to produce three stably transfected human lymphoma and leukemia lines. Significant transfection efficiency was also observed in two fresh samples of human acute myeloid leukemia cells. In conclusion, we developed a user-friendly and cost-effective ultrasound device, well adapted for routine in vitro high-yield transfection experiments and which does not require the use of any transfection reagent or gas micro-bubbles. PMID:26274324

  6. Mitochondrial Hyperpolarization and ATP Depletion in Patients With Systemic Lupus Erythematosus

    PubMed Central

    Gergely, Peter; Grossman, Craig; Niland, Brian; Puskas, Ferenc; Neupane, Hom; Allam, Fatme; Banki, Katalin; Phillips, Paul E.; Perl, Andras

    2014-01-01

    Objective Peripheral blood lymphocytes (PBLs) from systemic lupus erythematosus (SLE) patients exhibit increased spontaneous and diminished activation-induced apoptosis. We tested the hypothesis that key biochemical checkpoints, the mitochondrial transmembrane potential (ΔΨm) and production of reactive oxygen intermediates (ROIs), mediate the imbalance of apoptosis in SLE. Methods We assessed the ΔΨm with potentiometric dyes, measured ROI production with oxidation-sensitive fluorochromes, and monitored cell death by annexin V and propidium iodide staining of lymphocytes, using flow cytometry. Intracellular glutathione levels were measured by high-performance liquid chromatography, while ATP and ADP levels were assessed by the luciferin–luciferase assay. Results Both ΔΨm and ROI production were elevated in the 25 SLE patients compared with the 25 healthy subjects and the 10 rheumatoid arthritis patients. Intracellular glutathione contents were diminished, suggesting increased utilization of reducing equivalents in SLE. H2O2, a precursor of ROIs, increased ΔΨm and caused apoptosis in normal PBLs. In contrast, H2O2-induced apoptosis and ΔΨm elevation were diminished, particularly in T cells, and the rate of necrotic cell death was increased in patients with SLE. The intracellular ATP content and the ATP:ADP ratio were reduced and correlated with the ΔΨm elevation in lupus. CD3:CD28 costimulation led to transient elevation of the ΔΨm, followed by ATP depletion, and sensitization of normal PBLs to H2O2-induced necrosis. Depletion of ATP by oligomycin, an inhibitor of F0F1–ATPase, had similar effects. Conclusion T cell activation and apoptosis are mediated by ΔΨm elevation and increased ROI production. Mitochondrial hyperpolarization and the resultant ATP depletion sensitize T cells for necrosis, which may significantly contribute to inflammation in patients with SLE. PMID:11817589

  7. Protective effect of hydrogen-rich saline against radiation-induced immune dysfunction

    PubMed Central

    Zhao, Sanhu; Yang, Yanyong; Liu, Wen; Xuan, Zhiqiang; Wu, Shouming; Yu, Shunfei; Mei, Ke; Huang, Yijuan; Zhang, Pei; Cai, Jianming; Ni, Jin; Zhao, Yaoxian

    2014-01-01

    Recent studies showed that hydrogen can be used as an effective radioprotective agent through scavenging free radicals. This study was undertaken to evaluate the radioprotective effects of hydrogen on immune system in mice. H2 was dissolved in physiological saline using an apparatus produced by our department. Spleen index and histological analysis were used to evaluate the splenic structural damage. Spleen superoxide dismutase, GSH, MDA were measured to appraise the antioxidant capacity and a DCF assay for the measurement of radical oxygen species. Cell apoptosis was evaluated by an Annexin V-FITC and propidium iodide staining method as well as the apoptotic proteins such as Bcl-2, Bax, caspase-3 and c-caspase-3. CD4+ and CD8+ T cells subtypes were detected by flow cytometry with FITC-labelled antimouse CD4 and PE antimouse CD8 staining. Real-time PCR was utilized to determine the CD4+ T cell subtypes and related cytokines. Our study demonstrated that pre-treatment with H2 could increase the spleen index and attenuate the radiation damage on splenic structure. Radical oxygen species level was also reduced by H2 treatment. H2 also inhibited radiation-induced apoptosis in splenocytes and down-regulated pro-apoptotic proteins in living mice. Radiation-induced imbalance of T cells was attenuated by H2. Finally, we found that H2 could regulate the polarization of CD4+ T cells and the level of related cytokines. This study suggests H2 as an effective radioprotective agent on immune system by scavenging reactive oxygen species. PMID:24618260

  8. Strong anticancer potential of nano-triterpenoid from Phytolacca decandra against A549 adenocarcinoma via a Ca(2+)-dependent mitochondrial apoptotic pathway.

    PubMed

    Das, Jayeeta; Das, Sreemanti; Paul, Avijit; Samadder, Asmita; Khuda-Bukhsh, Anisur Rahman

    2014-06-01

    We isolated a triterpenoid from an ethanolic extract of Phytolacca decandra and nanoencapsulated it with biodegradable nontoxic polymers of poly(lactide-co-glycolide) to examine if the nanoform of this hitherto unexplored betulinic-acid derivative (NdBA) could produce a stronger anticancer effect by rendering better drug bioavailability and targeted delivery than the nonencapsulated betulinic-acid derivative (dBA). The nanoparticles were characterized with the help of physicochemical and morphological studies involving dynamic light scattering and atomic force microscopy. A549 cancer cells were exposed to NdBA and dBA at the IC50 doses of 50 μg/mL and 100 μg/mL, respectively. Mitochondrial dysfunction-mediated apoptosis was determined by examining the changes in the intracellular calcium content, the reactive oxygen species accumulation, the cytochrome c release, the upregulation of Bcl-2-associated-X protein (Bax) and caspase 3, the downregulation of B cell lymphoma 2, and the mitochondrial membrane potential (ΔΨm) depolarization. Apoptosis was also verified by acridine orange staining observed under fluorescence microscopy and annexin V-fluorescein isothiocyanate/propidium iodide staining through flow cytometric studies. The levels of intracellular adenosine triphosphate/adenosine diphosphate ratio decreased, and the ATPase activity increased more strikingly in A549 cells exposed to NdBA than in A549 cells exposed to dBA. Overall results showed that both drugs directly target the mitochondrial oxidative phosphorylation system, with NdBA having a stronger effect, indicating NdBA to be a better candidate for the development of an anticancer drug for use against lung adenocarcinomas. PMID:24929458

  9. Quantification of the DNA content of structurally abnormal X chromosomes and X chromosome aneuploidy using high resolution bivariate flow karyotyping.

    PubMed

    Trask, B; van den Engh, G; Nussbaum, R; Schwartz, C; Gray, J

    1990-01-01

    Quantification of the Hoechst and chromomycin A3 fluorescence intensities of mitotic human chromosomes isolated from karyotypically normal and abnormal cells was performed with a dual beam flow cytometer. The resultant flow karyotypes contain information about the relative DNA content and base composition of chromosomes and their relative frequencies in the mitotic cell sample. The relative copy number of X and Y chromosomes was determined for 38 normal males and females and 6 cell lines with X or Y chromosome aneuploidy. Flow karyotype diagnoses corresponded with conventional cytogenetic results in all cases. We show that chromosome DNA content can be derived from peak position in Hoechst vs. chromomycin flow karyotypes. These values are linearly related to propidium iodide staining intensity as measured with flow cytometry and to the binding of gallocyanin chrome alum to phosphate groups as measured with slide-based scanning photometry. Cell lines with deleted or dicentric X chromosomes ranging in length from 0.53 to 1.95 times normal were analyzed by using flow cytometry. The measured difference in DNA content between a normal X and each of the structurally abnormal chromosomes was linearly correlated to the difference predicted from cytogenetics and/or probe analyses. Deletions of 3-5 Mb, which were at and below the detection limits of conventional cytogenetics, could be quantified by flow karyotyping in individuals with X-linked diseases such as Duchenne muscular dystrophy, choroideremia, and ocular albinism/ichthyosis. The results show that the use of flow karyotyping to quantify the size of restricted regions of the genome can complement conventional cytogenetics and other physical mapping techniques in the study of genetic disorders. PMID:2106419

  10. Allyl alcohol activation of protein kinase C delta leads to cytotoxicity of rat hepatocytes.

    PubMed

    Maddox, Jane F; Roth, Robert A; Ganey, Patricia E

    2003-05-01

    Hepatotoxicity of allyl alcohol involves its bioactivation to acrolein and subsequent protein sulfhydryl loss and lipid peroxidation. However, the links between these events and hepatocellular death are not known. The purpose of these studies was to examine whether specific signal transduction pathways are associated with allyl alcohol toxicity in hepatocytes. Inhibition or augmentation of cyclic AMP and/or protein kinase A (PKA) by Rp-Ado-3N,5N-cyclic monophosphorothioate triethylamine salt or 3-isobutyl-1-methylxanthine had no effect on allyl alcohol-induced cell death. H-7, an inhibitor of PKA, PKC, and PKG, partially inhibited cell killing by allyl alcohol, whereas chelerythrine chloride, a nonselective PKC inhibitor, almost completely abolished allyl alcohol cytotoxicity. Neither 2,2N,3,3N,4,4N-hexahydroxy-1,1N,-biphenyl-6,6N-dimethanol-dimethyl ether, a selective PKC alpha and beta inhibitor, nor bisindolylmaleimide I, an inhibitor of PKC alpha, beta, and epsilon, had any effect on allyl alcohol cytotoxicity. In contrast, rottlerin, a selective PKCdelta inhibitor, blocked hepatocellular killing by allyl alcohol. Cytoprotection by chelerythrine chloride and rottlerin was not the result of inhibition of bioactivation of allyl alcohol because each inhibitor also prevented cell death from acrolein. Western blotting and immunohistochemical techniques revealed that allyl alcohol stimulated phosphorylation and translocation of PKCdelta to hepatocyte membranes (i.e., activation), and this activity was inhibited by rottlerin. Cell death appeared to occur via oncotic necrosis rather than apoptosis based on single-stranded DNA ELISA and propidium iodide staining. Together, these results indicate that activation of PKCdelta is a critical, early event in initiating hepatocyte injury and death from allyl alcohol. PMID:12755590

  11. Development of a single cell electroporation method using a scanning ion conductance microscope with a theta nanopipette

    NASA Astrophysics Data System (ADS)

    Sakurai, Satoshi; Yamazaki, Koji; Ushiki, Tatsuo; Iwata, Futoshi

    2015-08-01

    We developed a novel electroporation method using a scanning ion conductance microscope (SICM) with a theta capillary nanopipette probe that has two apertures at the edge of the pipette. One aperture of the pipette probe was used to control the pipette-surface distance and to apply pulse voltage for electroporation. The other was used to eject material over the cell by local electrophoresis. Using the nanopipette, propidium iodide was successfully introduced into a targeted single Hela cell without influencing the surrounding cells. Furthermore, by scanning the theta nanopipette probe using the SICM, the morphological behaviors of the electroporated cells could be observed.

  12. Clostridium perfringens Delta-Toxin Induces Rapid Cell Necrosis

    PubMed Central

    Seike, Soshi; Miyamoto, Kazuaki; Kobayashi, Keiko; Takehara, Masaya; Nagahama, Masahiro

    2016-01-01

    Clostridium perfringens delta-toxin is a β-pore-forming toxin and a putative pathogenic agent of C. perfringens types B and C. However, the mechanism of cytotoxicity of delta-toxin remains unclear. Here, we investigated the mechanisms of cell death induced by delta-toxin in five cell lines (A549, A431, MDCK, Vero, and Caco-2). All cell lines were susceptible to delta-toxin. The toxin caused rapid ATP depletion and swelling of the cells. Delta-toxin bound and formed oligomers predominantly in plasma membrane lipid rafts. Destruction of the lipid rafts with methyl β-cyclodextrin inhibited delta-toxin-induced cytotoxicity and ATP depletion. Delta-toxin caused the release of carboxyfluorescein from sphingomyelin-cholesterol liposomes and formed oligomers; toxin binding to the liposomes declined with decreasing cholesterol content in the liposomes. Flow cytometric assays with annexin V and propidium iodide revealed that delta-toxin treatment induced an elevation in the population of annexin V-negative and propidium iodide-positive cells. Delta-toxin did not cause the fragmentation of DNA or caspase-3 activation. Furthermore, delta-toxin caused damage to mitochondrial membrane permeability and cytochrome c release. In the present study, we demonstrate that delta-toxin produces cytotoxic activity through necrosis. PMID:26807591

  13. Flow cytometric method for measuring chromatin fragmentation in fixed sperm from yellow perch (Perca flavescens)

    USGS Publications Warehouse

    Jenkins, Jill A.; Draugelis-Dale, Rassa O.; Pinkney, Alfred E.; Iwanowicz, Luke R.; Blazer, Vicki

    2015-01-01

    Declining harvests of yellow perch, Perca flavescens, in urbanized watersheds of Chesapeake Bay have prompted investigations of their reproductive fitness. The purpose of this study was to establish a flow cytometric technique for DNA analysis of fixed samples sent from the field to provide reliable gamete quality measurements. Similar to the sperm chromatin structure assay, measures were made on the susceptibility of nuclear DNA to acid-induced denaturation, but used fixed rather than live or thawed cells. Nuclei were best exposed to the acid treatment for 1 minute at 37 °C followed by the addition of cold (4 °C) propidium iodide staining solution before flow cytometry. The rationale for protocol development is presented graphically through cytograms. Field results collected in 2008 and 2009 revealed DNA fragmentation up to 14.5%. In 2008, DNA fragmentation from the more urbanized watersheds was significantly greater than from reference sites (P = 0.026) and in 2009, higher percentages of haploid testicular cells were noted from the less urbanized watersheds (P = 0.032) indicating better reproductive condition at sites with less urbanization. For both years, total and progressive live sperm motilities by computer-assisted sperm motion analysis ranged from 19.1% to 76.5%, being significantly higher at the less urbanized sites (P < 0.05). This flow cytometric method takes advantage of the propensity of fragmented DNA to be denatured under standard conditions, or 1 minute at 37 °C with 10% buffered formalin–fixed cells. The study of fixed sperm makes possible the restrospective investigation of germplasm fragmentation, spermatogenic ploidy patterns, and chromatin compaction levels from samples translocated over distance and time. The protocol provides an approach that can be modified for other species across taxa.

  14. The Antimicrobial Mechanism of Action of Epsilon-Poly-l-Lysine

    PubMed Central

    Hyldgaard, Morten; Mygind, Tina; Vad, Brian S.; Stenvang, Marcel; Otzen, Daniel E.

    2014-01-01

    Epsilon-poly-l-lysine (ε-PL) is a natural antimicrobial cationic peptide which is generally regarded as safe (GRAS) as a food preservative. Although its antimicrobial activity is well documented, its mechanism of action is only vaguely described. The aim of this study was to clarify ε-PL's mechanism of action using Escherichia coli and Listeria innocua as model organisms. We examined ε-PL's effect on cell morphology and membrane integrity and used an array of E. coli deletion mutants to study how specific outer membrane components affected the action of ε-PL. We furthermore studied its interaction with lipid bilayers using membrane models. In vitro cell studies indicated that divalent cations and the heptose I and II phosphate groups in the lipopolysaccharide layer of E. coli are critical for ε-PL's binding efficiency. ε-PL removed the lipopolysaccharide layer and affected cell morphology of E. coli, while L. innocua underwent minor morphological changes. Propidium iodide staining showed that ε-PL permeabilized the cytoplasmic membrane in both species, indicating the membrane as the site of attack. We compared the interaction with neutral or negatively charged membrane systems and showed that the interaction with ε-PL relied on negative charges on the membrane. Suspended membrane vesicles were disrupted by ε-PL, and a detergent-like disruption of E. coli membrane was confirmed by atomic force microscopy imaging of supported lipid bilayers. We hypothesize that ε-PL destabilizes membranes in a carpet-like mechanism by interacting with negatively charged phospholipid head groups, which displace divalent cations and enforce a negative curvature folding on membranes that leads to formation of vesicles/micelles. PMID:25304506

  15. A new and simple method to evaluate early membrane changes in frozen-thawed boar spermatozoa.

    PubMed

    Peña, F J; Saravia, F; Johannisson, A; Walgren, M; Rodríguez-Martínez, H

    2005-04-01

    Detection of early changes in the sperm plasma membrane during cryopreservation is of utmost importance when designing freezing protocols and has previously been studied in the pig species using annexin-V detection of phosphatidylserine translocation. In the present study we designed a new assay to detect these changes in boar spermatozoa, based on the slight increase of sperm membrane permeability occurring during the early stages of cryoinjury, using the combination of three fluorescent probes, SNARF-1, YO-PRO-1 and ethidium homodimer. Four ejaculates from five different boars were frozen-thawed and flow cytometrically (FC) evaluated as paired samples. One of the samples was assayed using the annexin-V/propidium iodide staining and the other sample was evaluated using the new triple staining. Using this combination of probes, four sperm subpopulations were easily detected: viable, with stable membranes (SNARF-1 positive cells), and three with compromised membranes, one of YO-PRO-1+/Eth- cells, one ethidium homodimer+ spermatozoa and, finally spermatozoa stained both with YO-PRO-1 and ethidium homodimer (YO-PRO-1+/Eth+). The latter three categories corresponded to dead spermatozoa, but with different degree of membrane damage, being YO-PRO+/Eth- an earlier stage of membrane destabilization, (manifested by an increase in membrane permeability, while still maintaining membrane integrity) than YO-PRO+/Eth+. A method agreement analysis between both methods was performed revealing good agreement, although the percentage of live cells was 9.44% larger for the triple stain than the annexin-V assay. The new assay stained all sperm sub-populations present in the sample, making it especially suitable for both fluorescence microscopy and flow cytometry, facilitating the exclusion of debris and egg-yolk particles when using FC. PMID:15811072

  16. The Calpain Inhibitor MDL28170 Induces the Expression of Apoptotic Markers in Leishmania amazonensis Promastigotes

    PubMed Central

    Marinho, Fernanda A.; Gonçalves, Keyla C. S.; Oliveira, Simone S. C.; Gonçalves, Diego S.; Matteoli, Filipe P.; Seabra, Sergio H.; Oliveira, Ana Carolina S.; Bellio, Maria; Oliveira, Selma S.; Souto-Padrón, Thaïs; d'Avila-Levy, Claudia M.; Santos, André L. S.; Branquinha, Marta H.

    2014-01-01

    Background Human cutaneous leishmaniasis is caused by distinct species, including Leishmania amazonensis. Treatment of cutaneous leishmaniasis is far from satisfactory due to increases in drug resistance and relapses, and toxicity of compounds to the host. As a consequence for this situation, the development of new leishmanicidal drugs and the search of new targets in the parasite biology are important goals. Methodology/Principal Findings In this study, we investigated the mechanism of death pathway induced by the calpain inhibitor MDL28170 on Leishmania amazonensis promastigote forms. The combined use of different techniques was applied to contemplate this goal. MDL28170 treatment with IC50 (15 µM) and two times the IC50 doses induced loss of parasite viability, as verified by resazurin assay, as well as depolarization of the mitochondrial membrane, which was quantified by JC-1 staining. Scanning and transmission electron microscopic images revealed drastic alterations on the parasite morphology, some of them resembling apoptotic-like death, including cell shrinking, surface membrane blebs and altered chromatin condensation pattern. The lipid rearrangement of the plasma membrane was detected by Annexin-V labeling. The inhibitor also induced a significant increase in the proportion of cells in the sub-G0/G1 phase, as quantified by propidium iodide staining, as well as genomic DNA fragmentation, detected by TUNEL assay. In cells treated with MDL28170 at two times the IC50 dose, it was also possible to observe an oligonucleossomal DNA fragmentation by agarose gel electrophoresis. Conclusions/Significance The data presented in the current study suggest that MDL28170 induces apoptotic marker expression in promastigotes of L. amazonensis. Altogether, the results described in the present work not only provide a rationale for further exploration of the mechanism of action of calpain inhibitors against trypanosomatids, but may also widen the investigation of the

  17. Comparison of apoptosis between adult worms of Schistosoma japonicum from susceptible (BALB/c mice) and less-susceptible (Wistar rats) hosts.

    PubMed

    Wang, Tao; Guo, Xiaoyong; Hong, Yang; Han, Hongxiao; Cao, Xiaodan; Han, Yanhui; Zhang, Min; Wu, Miaoli; Fu, Zhiqiang; Lu, Ke; Li, Hao; Zhao, Zhixin; Lin, Jiaojiao

    2016-10-30

    Schistosomiasis remains a serious public health concern in China. BALB/c mice are susceptible to Schistosoma japonicum infection, whereas the Wistar rats are less susceptible. Apoptosis phenomenon was observed in 42d adult worms of S. japonicum from both rats and mice at the morphologic, DNA, cellular, and gene levels by transmission electron microscopy (TEM), fluorometric terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis, fluorescein isothiocyanate-annexin-V/propidium iodide staining flow cytometry (FCM) analysis, and real-time PCR. The results showed that the apoptotic state in worms from two different susceptible hosts was diverse. Several classical hallmarks of apoptosis, including cell shrinkage, chromatin condensation and lunate marginalization, splitting of the nucleoli, nuclear shrinkage and apoptotic body formation were observed by TEM. TUNEL analysis showed that there were much more apoptosis spots in adult worms from rats than those from mice. Statistical analysis revealed that the degree of apoptosis and percentage of necrotic cells in adult worms from Wistar rats were significantly greater (P<0.01) than those from BALB/c mice by flow cytometry. A total of 15 apoptosis-associated genes including the major components of an intrinsic cell-death pathway were identified from S. japonicum in this study, suggested that a similar apoptosis pathway might occur in S. japonicum. Real-time PCR analyses revealed that the expression levels of most of the tested apoptosis-associated genes, except CASP7, were significantly higher or at the similar level in adult worms from Wistar rats, as compared to those from BALB/c mice. The results obtained in this study collectively demonstrated that differential development of adult S. japonicum in less-susceptible rats and susceptible mice was significantly associated with apoptosis in the worm, and provided valuable information to guide further investigations of the mechanisms governing

  18. Design, synthesis and biological evaluation of 1,3,6-trisubstituted β-carboline derivatives for cytotoxic and anti-leishmanial potential.

    PubMed

    Lunagariya, Nitin A; Gohil, Vikrantsinh M; Kushwah, Varun; Neelagiri, Soumya; Jain, Sanyog; Singh, Sushma; Bhutani, Kamlesh K

    2016-02-01

    In the present study, 23 derivatives of 1,3,6-trisubstituted β-carboline were synthesized and evaluated for cytotoxic potential against four human cancer cells, namely A-549, HeLa, Hep G2 and MCF-7 as well as anti-leishmanial activity against Leishmania donovani (MHOM/80/IN/Dd8) promastigotes. Among the studied compounds, compounds 13c and 13q showed potent cytotoxic activity better than the parent compound 10. For instance, compound 13c was found to be the most cytotoxic with IC50 of 4.72, 3.59, 3.65 and 4.17 μM against A-549, HeLa, Hep G2 and MCF-7 respectively, while for compound 13q, IC50 were 15.47, 5.30, 6.15 and 13.39 μM against the same cancer cells respectively. Further, these two compounds were found to be apoptotic in A-549 and MCF-7 cells when observed using Annexin V/propidium iodide staining under confocal microscope. All the compounds were also tested for anti-leishmanial potential. In which, compounds 13u and 13c were found to show moderate inhibition with IC50 of 23.5±9.0 and 68.0±0.0 μM respectively, while compound 10 was the most active with IC50 of 9.0±2.8 μM, suggesting the modification at C-6 detrimental for anti-leishmanial activity. Interestingly, amongst all, compound 13c was found to be the most active for cytotoxic and moderately active for anti-leishmanial activity which can be further developed as a lead for these disease areas. PMID:26791014

  19. Tumescent Liposuction without Lidocaine

    PubMed Central

    Goldman, Joshua J.; Fang, Xin-Hua; Williams, Shelley J.; Baynosa, Richard C.

    2016-01-01

    Background: Our previous study demonstrated that lidocaine has a negative impact on adipose-derived stem cell (ASC) survival. Currently for large-volume liposuction, patients often undergo general anesthesia; therefore, lidocaine subcutaneous anesthesia is nonessential. We hypothesized that removing lidocaine from tumescent might improve stromal vascular fraction (SVF) and ASC survival from the standard tumescent with lidocaine. Ropivacaine is also a commonly used local anesthetic. The effect of ropivacaine on ASC survival was examined. Methods: Adults who underwent liposuction on bilateral body areas were included (n = 10). Under general anesthesia, liposuction on 1 area was conducted under standard tumescent with lidocaine. On the contralateral side, liposuction was conducted under the modified tumescent without lidocaine. Five milliliters of lipoaspirate were processed for the isolation of SVF. The adherent ASCs were counted after 24 hours of SVF culture. Apoptosis and necrosis of SVF cells were examined by Annexin/propidium iodide staining and analyzed by flow cytometry. Results: Average percentage of live SVF cells was 68.0% ± 4.0% (28.5% ± 3.8% of apoptosis and 3.4% ± 1.0% of necrosis) in lidocaine group compared with 86.7% ± 3.7% (11.5% ± 3.1% of apoptosis and 1.8% ± 0.7% of necrosis) in no-lidocaine group (P = 0.002). Average number of viable ASC was also significantly lower (367,000 ± 107) in lidocaine group compared with that (500,000 ± 152) in no-lidocaine group (P = 0.04). No significant difference was found between lidocaine and ropivacaine on ASC cytotoxicity. Conclusions: Removing lidocaine from tumescent significantly reduced SVF and ASC apoptosis in the lipoaspirate. We recommend tumescent liposuction without lidocaine, particularly if patient’s lipoaspirate will be used for fat grafting. PMID:27622097

  20. The effects of oncolytic reovirus in canine lymphoma cell lines.

    PubMed

    Hwang, C C; Umeki, S; Igase, M; Coffey, M; Noguchi, S; Okuda, M; Mizuno, T

    2016-08-01

    Reovirus is a potent oncolytic virus in many human neoplasms that has reached phase II and III clinical trials. Our laboratory has previously reported the oncolytic effects of reovirus in canine mast cell tumour (MCT). In order to further explore the potential of reovirus in veterinary oncology, we tested the susceptibility of reovirus in 10 canine lymphoma cell lines. Reovirus-induced cell death, virus replication and infectivity were confirmed in four cell lines with variable levels of susceptibility. The level of Ras activation varied among the cell lines with no correlation with reovirus susceptibility. Reovirus-susceptible cell lines underwent apoptosis as proven by propidium iodide (PI) staining, Annexin V-FITC/PI assay, cleavage of PARP and inhibition of cell death by caspase inhibitor. A single intratumoral injection of reovirus suppressed the growth of canine lymphoma subcutaneous tumour in NOD/SCID mice. Unlike canine MCT, canine lymphoma is less susceptible to reovirus. PMID:25319493

  1. Determination of intracellular reactive oxygen species and high mitochondrial membrane potential in Percoll-treated viable boar sperm using fluorescence-activated flow cytometry.

    PubMed

    Guthrie, H D; Welch, G R

    2006-08-01

    The use of frozen semen in the swine industry is limited by problems with viability and fertility compared with liquid semen. Part of the reduction in sperm motility and fertility associated with cryopreservation may be due to oxidative damage from excessive or inappropriate formation of reactive oxygen species (ROS). Chemiluminescence measurements of ROS are not possible in live cells and are problematic because of poor specificity. An alternative approach, flow cytometry, was developed to identify viable boar sperm containing ROS utilizing the dyes hydroethidine and 2', 7'-dichlorodihydrofluorescein diacetate as oxidizable substrates and impermeant DNA dyes to exclude dead sperm. The percentage of sperm with high mitochondrial transmembrane potential was determined by flow cytometry using the mitochondrial probe 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolylcarbocyanine iodide with propidium iodide staining to exclude nonviable cells. Sperm were incubated with and without ROS generators and free radical scavengers. Basal ROS formation was low (less than 4%) and did not differ (P = 0.26) between viable fresh and frozen-thawed boar sperm. In addition, fresh and frozen-thawed viable sperm were equally susceptible (P = 0.20) to intracellular formation of ROS produced by xanthine/xanthine oxidase (94.4 and 87.9% of sperm, respectively). Menadione increased (P < 0.05) ROS formation, decreased (P < 0.05) JC-1-aggregate fluorescence intensity, and decreased (P < 0.05) motion variables by 25 to 60%. The mechanism of inhibition of motility by ROS formation may be related to a decrease in mitochondrial charge potential below a critical threshold. Catalase and superoxide dismutase treatment in the presence of xanthine/xanthine oxidase indicated that hydrogen peroxide was the primary intracellular ROS measured. Further, catalase, but not superoxide dismutase, was capable of attenuating ROS-induced inhibition of motility. Whereas basal intracellular hydrogen

  2. Celastrol Potentiates Radiotherapy by Impairment of DNA Damage Processing in Human Prostate Cancer

    SciTech Connect

    Dai Yao; DeSano, Jeffrey T.; Meng Yang; J, Qing; Ljungman, Mats; Lawrence, Theodore S.; Xu Liang

    2009-07-15

    Purpose: Celastrol is an active ingredient of traditional herbal medicine and has recently been identified as a potent natural proteasome inhibitor. In the present study, we evaluated the radiosensitizing potential of celastrol in the human prostate cancer PC-3 model. Methods and Materials: Clonogenic assays were performed to determine the radiosensitizing effect of celastrol. Apoptosis was examined by flow cytometry using Annexin V and propidium iodide staining and by a caspase-3 activation assay. DNA damage processing was examined by immunofluorescent staining and Western blot for phosphorylated H2AX ({gamma}H2AX). The PC-3 xenograft model in the athymic nude mouse was used for the determination of the in vivo efficacy of celastrol combined with radiotherapy. The tumor samples were also analyzed for apoptosis and angiogenesis. Results: Celastrol sensitized PC-3 cells to ionizing radiation (IR) in a dose- and schedule-dependent manner, in which pretreatment with celastrol for 1 h followed by IR achieved maximal radiosensitization. Celastrol significantly prolonged the presence of IR-induced {gamma}H2AX and increased IR-induced apoptosis. Celastrol, combined with fractionated radiation, significantly inhibited PC-3 tumor growth in vivo without obvious systemic toxicity. The combination treatment increased {gamma}H2AX levels and apoptosis, induced cleavage of poly(adenosine diphosphate-ribose)polymerase and Mcl-1, and reduced angiogenesis in vivo compared with either treatment alone. Conclusion: Celastrol sensitized PC-3 cells to radiation both in vitro and in vivo by impairing DNA damage processing and augmenting apoptosis. Celastrol might represent a promising new adjuvant regimen for the treatment of hormone-refractory prostate cancer.

  3. Nutrient reserves may allow for genome size increase: evidence from comparison of geophytes and their sister non-geophytic relatives

    PubMed Central

    Veselý, Pavel; Bureš, Petr; Šmarda, Petr

    2013-01-01

    Background and Aims The genome size of an organism is determined by its capacity to tolerate genome expansion, given the species' life strategy and the limits of a particular environment, and the ability for retrotransposon suppression and/or removal. In some giant-genomed bulb geophytes, this tolerance is explained by their ability to pre-divide cells in the dormant stages or by the selective advantage of larger cells in the rapid growth of their fleshy body. In this study, a test shows that the tendency for genome size expansion is a more universal feature of geophytes, and is a subject in need of more general consideration. Methods Differences in monoploid genome sizes were compared using standardized phylogenetically independent contrasts in 47 sister pairs of geophytic and non-geophytic taxa sampled across all the angiosperms. The genome sizes of 96 species were adopted from the literature and 53 species were newly measured using flow cytometry with propidium iodide staining. Key Results The geophytes showed increased genome sizes compared with their non-geophytic relatives, regardless of the storage organ type and regardless of whether or not vernal geophytes, polyploids or annuals were included in the analyses. Conclusions The universal tendency of geophytes to possess a higher genome size suggests the presence of a universal mechanism allowing for genome expansion. It is assumed that this is primarily due to the nutrient and energetic independence of geophytes perhaps allowing continuous synthesis of DNA, which is known to proceed in the extreme cases of vernal geophytes even in dormant stages. This independence may also be assumed as a reason for allowing large genomes in some parasitic plants, as well as the nutrient limitation of small genomes of carnivorous plants. PMID:23960044

  4. Mechanisms of cell death in canine parvovirus-infected cells provide intuitive insights to developing nanotools for medicine.

    PubMed

    Nykky, Jonna; Tuusa, Jenni E; Kirjavainen, Sanna; Vuento, Matti; Gilbert, Leona

    2010-01-01

    Viruses have great potential as nanotools in medicine for gene transfer, targeted gene delivery, and oncolytic cancer virotherapy. Here we have studied cell death mechanisms of canine parvovirus (CPV) to increase the knowledge on the CPV life cycle in order to facilitate the development of better parvovirus vectors. Morphological studies of CPV-infected Norden laboratory feline kidney (NLFK) cells and canine fibroma cells (A72) displayed characteristic apoptotic events. Apoptosis was further confirmed by activation of caspases and cellular DNA damage. However, results from annexin V-propidium iodide (PI) labeling and membrane polarization assays indicated disruption of the plasma membrane uncommon to apoptosis. These results provide evidence that secondary necrosis followed apoptosis. In addition, two human cancer cell lines were found to be infected by CPV. This necrotic event over apoptotic cell death and infection in human cells provide insightful information when developing CPV as a nanotool for cancer treatments. PMID:20957163

  5. Mechanisms of cell death in canine parvovirus-infected cells provide intuitive insights to developing nanotools for medicine

    PubMed Central

    Nykky, Jonna; Tuusa, Jenni E; Kirjavainen, Sanna; Vuento, Matti; Gilbert, Leona

    2010-01-01

    Viruses have great potential as nanotools in medicine for gene transfer, targeted gene delivery, and oncolytic cancer virotherapy. Here we have studied cell death mechanisms of canine parvovirus (CPV) to increase the knowledge on the CPV life cycle in order to facilitate the development of better parvovirus vectors. Morphological studies of CPV-infected Norden laboratory feline kidney (NLFK) cells and canine fibroma cells (A72) displayed characteristic apoptotic events. Apoptosis was further confirmed by activation of caspases and cellular DNA damage. However, results from annexin V-propidium iodide (PI) labeling and membrane polarization assays indicated disruption of the plasma membrane uncommon to apoptosis. These results provide evidence that secondary necrosis followed apoptosis. In addition, two human cancer cell lines were found to be infected by CPV. This necrotic event over apoptotic cell death and infection in human cells provide insightful information when developing CPV as a nanotool for cancer treatments. PMID:20957163

  6. Riccardin C derivatives cause cell leakage in Staphylococcus aureus.

    PubMed

    Morita, Daichi; Sawada, Hiromi; Ogawa, Wakano; Miyachi, Hiroyuki; Kuroda, Teruo

    2015-10-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a major problem in clinical settings, and because it is resistant to most antimicrobial agents, MRSA infections are difficult to treat. We previously reported that synthetic macrocyclic bis(bibenzyl) derivatives, which were originally discovered in liverworts, had anti-MRSA activity. However, the action mechanism responsible was unclear. In the present study, we elucidated the action mechanism of macrocyclic bis(bibenzyl) RC-112 and its partial structure, IDPO-9 (2-phenoxyphenol). Survival experiments demonstrated that RC-112 had a bactericidal effect on MRSA, whereas IDPO-9 had bacteriostatic effects. IDPO-9-resistant mutants exhibited cross-resistance to triclosan, but not to RC-112. The mutation was identified in the fabI, enoyl-acyl carrier protein reductase gene, a target of triclosan. We have not yet isolated the RC-112-resistant mutant. On the other hand, the addition of RC-112, unlike IDPO-9, caused the inflow of ethidium and propidium into S. aureus cells. RC-112-dependent ethidium outflow was observed in ethidium-loaded S. aureus cells. Transmission electron microscopy also revealed that S. aureus cells treated with RC-112 had intracellular lamellar mesosomal-like structures. Intracellular Na+ and K+ concentrations were significantly changed by the RC-112 treatment. These results indicated that RC-112 increased membrane permeability to ethidium, propidium, Na+, and K+, and also that the action mechanism of IDPO-9 was different from those of the other compounds. PMID:26003535

  7. Xanthohumol induces apoptosis and S phase cell cycle arrest in A549 non-small cell lung cancer cells

    PubMed Central

    Yong, Wai Kuan; Ho, Yen Fong; Malek, Sri Nurestri Abd

    2015-01-01

    Background: Xanthohumol, a major prenylated chalcone found in female hop plant, Humulus lupulus, was reported to have various chemopreventive and anti-cancer properties. However, its apoptotic effect on human alveolar adenocarcinoma cell line (A549) of non-small cell lung cancer (NSCLC) was unknown. Objective: This study aimed to investigate the effects of xanthohumol on apoptosis in A549 human NSCLC cells. Materials and Methods: A549 cell proliferation was determined by sulforhodamine B assay. Morphological changes of the cells were studied via phase contrast and fluorescent microscopy. Induction of apoptosis was assessed by Annexin-V fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) staining, DNA fragmentation (TUNEL) assay mitochondrial membrane potential assay, cell cycle analysis, and caspase activity studies. Results: Xanthohumol was found to decrease cell proliferation in A549 cells but had relatively low cytotoxicity on normal human lung fibroblast cell line (MRC-5). Typical cellular and nuclear apoptotic features were also observed in A549 cells treated with xanthohumol. Onset of apoptosis in A549 cells was further confirmed by externalization of phosphatidylserine, changes in mitochondrial membrane potential, and DNA fragmentation in the cells after treatment. Xanthohumol induced accumulation of cells in sub G1 and S phase based on cell cycle analysis and also increased the activities of caspase-3, -8, and -9. Conclusion: This work suggests that xanthohumol as an apoptosis inducer, may be a potent therapeutic compound for NSCLC. PMID:26664015

  8. Increased Susceptibility to Oxidative Death of Lymphocytes from Alzheimer Patients Correlates with Dementia Severity

    PubMed Central

    Ponce, Daniela P.; Salech, Felipe; SanMartin, Carol D.; Silva, Monica; Xiong, Chengjie; Roe, Catherine M.; Henriquez, Mauricio; Quest, Andrew F.; Behrens, Maria I.

    2015-01-01

    We previously reported on enhanced susceptibility to death of lymphocytes from Alzheimer’s disease (AD) patients when exposed to hydrogen peroxide (H2O2)-induced oxidative stress and an increased resistance to death in those of patients with a history of skin cancer. This is consistent with our hypothesis proposing that the cellular machinery controlling cell death is deregulated in opposite directions in Alzheimer’s disease (AD) and cancer, to explain the inverse association observed in epidemiological studies. Here we investigated whether the observed increased susceptibility correlates with the degree of dementia severity. Peripheral lymphocytes from 23 AD patients, classified using the Clinical Dementia Rating (CDR) into severe dementia (CDR 3, n=10) and mild-to-moderate dementia (CDR 1–2, n=13), and 15 healthy controls (HC) (CDR 0), were exposed to H2O2 for 20 hours. Lymphocyte death was determined by flow cytometry and propidium iodide staining. The greatest susceptibility to H2O2-induced death was observed for lymphocytes from severe dementia patients, whereas those with mild-to-moderate dementia exhibited intermediate values, compared to healthy controls. A significant increase in the apoptosis/necrosis ratio was found in AD patients. Poly (ADP-ribosyl) polymerase-1 (PARP-1) inhibition significantly protected from H2O2-induced death of lymphocytes, whereby a lower degree of protection was observed in severe AD patients. Moreover, inhibition of PARP-1 abolished the differences in apoptosis/necrosis ratios observed between the three groups of patients. These results support the notion that AD is a systemic disorder, whereby enhanced susceptibility to H2O2-induced death in peripheral lymphocytes correlates with dementia severity and enhanced death in AD patients is attributable to a PARP-dependent increase in the apoptosis/necrosis ratio. PMID:25274115

  9. Sulphasalazine accelerates apoptosis in neutrophils exposed to immune complex: Role of caspase pathway.

    PubMed

    Bertolotto, M; Dallegri, F; Dapino, P; Quercioli, A; Pende, A; Ottonello, L; Montecucco, Fabrizio

    2009-11-01

    1. Neutrophils release several histotoxic molecules that cause tissue injury. Neutrophil apoptosis is a crucial process that governs the persistence of inflammatory disorders and tissue damage. Thus, in the present study, we investigated whether the anti-inflammatory drug sulphasalazine (SSZ) affects neutrophil apoptosis in the presence of insoluble immune complex (IC). 2. Neutrophils were obtained from healthy donors. Neutrophils were resuspended in incubation medium and incubated for 2-12 h with or without 10, 30 or 100 micromol/L SSZ and 25 microg/mL IC. In some experiments, cells were co-incubated with 20 micromol/L Z-IETD-fmk (a caspase 8 inhibitor) or 20 micromol/L Z-LEHD-fmk (a caspase 9 inhibitor). Apoptosis was evaluated morphologically on cytological preparations stained with May-Grünwald-Giemsa as well as by flow cytometry analysis of annexin V and propidium iodide staining. Caspase 3 activity was determined spectrophotometrically. 3. At 100 micromol/L, SSZ significantly accelerated IC-induced neutrophil apoptosis. Treatment of neutrophils with 20 micromol/L of the caspase 8 or 9 inhibitors Z-IETD-fmk or Z-LEHD-fmk, respectively, demonstrated that the SSZ-induced pro-apoptotic effect was mediated by a caspase 8- but not caspase 9-dependent pathway. The caspase 3 activity assay showed that treatment with 100 micromol/L SSZ increased caspase 3 activation. 4. In conclusion, the results of the present study indicate that it is possible that the molecular mechanism underlying SSZ protection against neutrophil-mediated tissue injury inflammatory disorders, such as rheumatoid arthritis and inflammatory bowel diseases, involves a caspase 8-dependent pathway. PMID:19473188

  10. Sources of variation in flow cytometric analysis of aquatic species sperm: The effect of cryoprotectants on flow cytometry scatter plots and subsequent population gating.

    PubMed

    Daly, Jonathan; Tiersch, Terrence R

    2012-12-11

    The use of fluorescent staining and flow cytometry to assess sperm quality in aquatic species has increased over the past decade, but comparisons among studies are difficult or impossible due to variation in application, analysis, and reporting of protocols and data.The goal of the present study was to determine the effect of exposure to two cryoprotectants commonly used for cryopreservation of sperm from aquatic species on the accuracy of flow cytometric assessment of sperm quality.Membrane integrity of zebrafish (Danio rerio) sperm exposed to 10% and 20%methanol and dimethyl sulfoxide (DMSO)in 300 mOsm kg(-1) Hanks' balanced salt solution (HBSS) or calcium-free HBSSwas determined using SYBR 14/propidium iodide staining. Both cryoprotectants significantly affected forward-scatter and side-scatter characteristics of sperm samples, resulting in significant changes in the number of total and gated events, and in the number and percentage of intact cells. These results indicate that it cannot be assumed that the approach to flow cytometric analysis of fresh sperm will be applicable to cryoprotectant-treated or cryopreserved sperm. In total, we document examples of five potentially interacting factors that produce errors of 5 to 50% each, resulting in underestimates and overestimates of total and intact sperm (actual numbers and percentages) in the presence of the two most commonly used cryoprotectants at the concentrations used most often for cryopreservation of sperm from aquatic species. This study provides methods to reduce or eliminate these errors and recommendations necessary for standardization and reporting. PMID:23175587

  11. Antibiofilm efficacy of silver nanoparticles against biofilm of extended spectrum β-lactamase isolates of Escherichia coli and Klebsiella pneumoniae

    NASA Astrophysics Data System (ADS)

    Ansari, Mohammad Azam; Khan, Haris M.; Khan, Aijaz A.; Cameotra, Swaranjit Singh; Pal, Ruchita

    2014-10-01

    The ability of bacteria to develop antibiotic resistance and colonize abiotic surfaces by forming biofilms is a major cause of medical implant-associated infections and results in prolonged hospitalization periods and patient mortality. Different approaches have been used for preventing biofilm-related infections in health care settings. Many of these methods have their own demerits that include chemical-based complications; emergent antibiotic-resistant strains, and so on. Silver nanoparticles (AgNPs) are renowned for their influential antimicrobial activity. We demonstrate the biofilm formation by extended spectrum β-lactamases-producing Escherichia coli and Klebsiella spp. by direct visualization applying tissue culture plate, tube, and Congo red agar methods. Double fluorescent staining for confocal laser scanning microscopy (CLSM) consisted of propidium iodide staining to detect bacterial cells and concanavalin A-fluorescein isothiocyanate staining to detect the exopolysaccharides matrix were used. Scanning electron microscopy observations clearly indicate that AgNPs reduced the surface coverage by E. coli and Klebsiella spp. thus prevent the biofilm formations. Double-staining technique using CLSM provides the visual evidence that AgNPs arrested the bacterial growth and prevent the exopolysaccharides formation. The AgNPs-coated surfaces effectively restricted biofilm formation of the tested bacteria. In our study, we could demonstrate the complete antibiofilm activity AgNPs at a concentration as low as 50 μg/ml. Our findings suggested that AgNPs can be exploited towards the development of potential antibacterial coatings for various biomedical and environmental applications. These formulations can be used for the treatment of drug-resistant bacterial infections caused by biofilms, at much lower nanosilver loading with higher efficiency.

  12. Chrysophanic Acid Induces Necrosis but not Necroptosis in Human Renal Cell Carcinoma Caki-2 Cells

    PubMed Central

    Choi, Joon-Seok

    2016-01-01

    Background: Chrysophanic acid, also known as chrysophanol, has a number of biological activities. It enhances memory and learning abilities, raises superoxide dismutase activity, and has anti-cancer effects in several model systems. According to previous reports, chrysophanic acid-induced cell death shares features of necrotic cell death. However, the molecular and cellular processes underlying chrysophanic acid-induced cell death remain poorly understood. Methods: Chrysophanic acid-induced cell death was monitored by cell viability assay and Annexin V-propidium iodide (PI) staining of renal cell carcinoma Caki-2 cells. The induction of intracellular reactive oxygen species (ROS) by chrysophanic acid and the suppression of ROS by anti-oxidants were evaluated by 2′,7′-dichlorofluorescin diacetate staining. The expression and phosphorylation of proteins that are involved in apoptosis and necroptosis were detected by immunoblotting. Results: The extent of chrysophanic acid-induced cell death was concentration and time dependent, and dead cells mainly appeared in the PI-positive population, which is a major feature of necrosis, upon fluorescence-activated cell sorting analysis. Chrysophanic acid-induced cell death was associated with the generation of intracellular ROS, and this effect was reversed by pretreatment with N-acetyl cysteine. Chrysophanic acid-induced cell death was not associated with changes in apoptotic or necroptotic marker proteins. Conclusions: The cell death induced by chrysophanic acid resembled neither apoptotic nor necroptotic cell death in human renal cell carcinoma Caki-2 cells. PMID:27390736

  13. Photodynamic hyperthermal therapy with indocyanine green (ICG) induces apoptosis and cell cycle arrest in B16F10 murine melanoma cells.

    PubMed

    Radzi, Rozanaliza; Osaki, Tomohiro; Tsuka, Takeshi; Imagawa, Tomohiro; Minami, Saburo; Nakayama, Yuji; Okamoto, Yoshiharu

    2012-05-01

    We examined the effects of photodynamic hyperthemal therapy (PHT), which is a combination of photodynamic therapy (PDT) and hyperthermia (HT), on the apoptosis and cell cycle progression of murine melanoma B16F10 cells. The percentage of apoptotic cell was determined by flow cytometry using fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide (PI) double staining. The cell cycle analysis was performed by PI staining with flow cytometry. The expression of cyclins and heat shock protein 70 (Hsp70) were examined by a Western blotting analysis. PHT induces death in B16F10 cells, and PHT-mediated apoptosis occurred acutely and persistently in vitro. Our study demonstrated that PHT using indocyanine green (ICG) and near infrared (NIR) light source induces apoptosis and G0/G1 cell cycle arrest in the B16F10 cells. PMID:22146339

  14. Induction of apoptosis in human endothelial cells by nanodiamond particles.

    PubMed

    Solarska, K; Gajewska, A; Bartosz, G; Mitura, K

    2012-06-01

    Carbon nanoparticles are a promising material which finds application in different fields in industry and medicine. For medical applications, biocompatibility of nanoparticles is of critical importance because a lot of medical implants are coated by carbon coating. Our previous results showed that nanoparticles may induce increased production of ROS by the cells so we decided to checked if nanopowders can induce apoptosis. Apoptosis was quantified by double-staining with acridine orange and ethidium bromide. For comparison, we identified apoptotic cells with annexin V-FITC/propidium iodide. Our data demonstrate that treatment of the cells with diamond nanopowders may induce apoptosis and necrosis and this effect is dependent on the time of treatment and concentration of the nanopowders. The highest level of apoptotic cells was observed after incubation with Ultrananocrystalline Detonation Diamond (UDD) suggesting that the size is the main determinant of nanoparticle cytotoxicity. PMID:22905588

  15. Nanopore formation in neuroblastoma cells following ultrashort electric pulse exposure

    NASA Astrophysics Data System (ADS)

    Roth, Caleb C.; Payne, Jason A.; Wilmink, Gerald J.; Ibey, Bennett L.

    2011-03-01

    Ultrashort or nanosecond electrical pulses (USEP) cause repairable damage to the plasma membranes of cells through formation of nanopores. These nanopores are able to pass small ions such as sodium, calcium, and potassium, but remain impermeable to larger molecules like trypan blue and propidium iodide. What remains uncertain is whether generation of nanopores by ultrashort electrical pulses can inhibit action potentials in excitable cells. In this paper, we explored the sensitivity of excitable cells to USEP using Calcium Green AM 1 ester fluorescence to measure calcium uptake indicative of nanopore formation in the plasma membrane. We determined the threshold for nanopore formation in neuroblastoma cells for three pulse parameters (amplitude, pulse width, and pulse number). Measurement of such thresholds will guide future studies to determine if USEP can inhibit action potentials without causing irreversible membrane damage.

  16. Cytotoxic Action of Serratia marcescens Hemolysin on Human Epithelial Cells

    PubMed Central

    Hertle, Ralf; Hilger, Martina; Weingardt-Kocher, Sandra; Walev, Iwan

    1999-01-01

    Incubation of human epithelial cells with nanomolar concentrations of chromatographically purified Serratia marcescens hemolysin (ShlA) caused irreversible vacuolation and subsequent lysis of the cells. Vacuolation differed from vacuole formation by Helicobacter pylori VacA. Sublytic doses of ShlA led to a reversible depletion of intracellular ATP. Restoration to the initial ATP level was presumably due to the repair of the toxin damage and was inhibited by cycloheximide. Pores formed in epithelial cells and fibroblasts without disruption of the plasma membrane, and the pores appeared to be considerably smaller than those observed in artificial lipid membranes and in erythrocytes and did not allow the influx of propidium iodide or trypan blue. All cytotoxic effects induced by isolated recombinant ShlA were also obtained with exponentially growing S. marcescens cells. The previously suggested role of the hemolysin in the pathogenicity of S. marcescens is supported by these data. PMID:9916096

  17. Prediction of clinical toxicity in locally advanced head and neck cancer patients by radio-induced apoptosis in peripheral blood lymphocytes (PBLs)

    PubMed Central

    2010-01-01

    Head and neck cancer is treated mainly by surgery and radiotherapy. Normal tissue toxicity due to x-ray exposure is a limiting factor for treatment success. Many efforts have been employed to develop predictive tests applied to clinical practice. Determination of lymphocyte radio-sensitivity by radio-induced apoptosis arises as a possible method to predict tissue toxicity due to radiotherapy. The aim of the present study was to analyze radio-induced apoptosis of peripheral blood lymphocytes in head and neck cancer patients and to explore their role in predicting radiation induced toxicity. Seventy nine consecutive patients suffering from head and neck cancer, diagnosed and treated in our institution, were included in the study. Toxicity was evaluated using the Radiation Therapy Oncology Group scale. Peripheral blood lymphocytes were isolated and irradiated at 0, 1, 2 and 8 Gy during 24 hours. Apoptosis was measured by flow cytometry using annexin V/propidium iodide. Lymphocytes were marked with CD45 APC-conjugated monoclonal antibody. Radiation-induced apoptosis increased in order to radiation dose and fitted to a semi logarithmic model defined by two constants: α and β. α, as the origin of the curve in the Y axis determining the percentage of spontaneous cell death, and β, as the slope of the curve determining the percentage of cell death induced at a determined radiation dose, were obtained. β value was statistically associated to normal tissue toxicity in terms of severe xerostomia, as higher levels of apoptosis were observed in patients with low toxicity (p = 0.035; Exp(B) 0.224, I.C.95% (0.060-0.904)). These data agree with our previous results and suggest that it is possible to estimate the radiosensitivity of peripheral blood lymphocytes from patients determining the radiation induced apoptosis with annexin V/propidium iodide staining. β values observed define an individual radiosensitivity profile that could predict late toxicity due to radiotherapy

  18. Nanosecond pulsed electric field (nsPEF) enhance cytotoxicity of cisplatin to hepatocellular cells by microdomain disruption on plasma membrane.

    PubMed

    Yin, Shengyong; Chen, Xinhua; Xie, Haiyang; Zhou, Lin; Guo, Danjing; Xu, Yuning; Wu, Liming; Zheng, Shusen

    2016-08-15

    Previous studies showed nanosecond pulsed electric field (nsPEF) can ablate solid tumors including hepatocellular carcinoma (HCC) but its effect on cell membrane is not fully understood. We hypothesized nsPEF disrupt the microdomains on outer-cellular membrane with direct mechanical force and as a result the plasma membrane permeability increases to facilitate the small molecule intake. Three HCC cells were pulsed one pulse per minute, an interval longer than nanopore resealing time. The cationized ferritin was used to mark up the electronegative microdomains, propidium iodide (PI) for membrane permeabilization, energy dispersive X-ray spectroscopy (EDS) for the negative cell surface charge and cisplatin for inner-cellular cytotoxicity. We demonstrated that the ferritin marked-microdomain and negative cell surface charge were disrupted by nsPEF caused-mechanical force. The cell uptake of propidium and cytotoxicity of DNA-targeted cisplatin increased with a dose effect. Cisplatin gains its maximum inner-cellular cytotoxicity when combining with nsPEF stimulation. We conclude that nsPEF disrupt the microdomains on the outer cellular membrane directly and increase the membrane permeabilization for PI and cisplatin. The microdomain disruption and membrane infiltration changes are caused by the mechanical force from the changes of negative cell surface charge. PMID:27375200

  19. Anticancer effect of ethanol Lycium barbarum (Goji berry) extract on human breast cancer T47D cell line.

    PubMed

    Wawruszak, Anna; Czerwonka, Arkadiusz; Okła, Karolina; Rzeski, Wojciech

    2016-09-01

    The anticancer activity of ethanol extract isolated from Goji berry (EEGB) on T47D human breast cancer cell line has been reported. Cell viability and cell proliferation were examined with the use of BrdU, MTT and NR methods. Induction of apoptosis was assessed by propidium iodide and Hoechst 33342 staining. Expression of genes involved in cell proliferation, apoptosis, cell cycle control and regulation of transcription was estimated using Western blotting analysis. EEGB inhibited the proliferation of breast cancer cells in a time-, and dose-dependent manner. The study confirmed the lack of EEGB cytotoxic activity to normal human skin fibroblasts. Western blot analysis demonstrated an increase in pro-apoptotic and a decrease in anti-apoptotic proteins' expression in cells treated with the extract. Anticancer activity and lack of toxicity against normal cells indicate a chemopreventive potential of Goji berries in breast cancer treatment. PMID:26525080

  20. Local electroporation of a single cell using a scanning ion conductance microscope

    NASA Astrophysics Data System (ADS)

    Iwata, Futoshi; Yamazaki, Koji; Ishizaki, Kimihiro; Ushiki, Tatuo

    2014-03-01

    We developed a novel electroporation technique for molecular delivery into a single cell. A nanopipette, a thermally pulled glass capillary, is prepared as to act as a pair of tiny electrodes for single-cell electroporation. An Ag/AgCl wire is inserted into the nanopipette, and the outside edge of the nanopipette is coated by Ag sputtering. Electric pulses are applied between the outside and inside electrodes to form a local electric field at the edge of the nanopipette. To position the pipette edge in the vicinity of the cell membrane, we control the probe-surface distance using a scanning ion conductance microscope (SICM). The SICM technique achieves non-contact approach of the nanopipette edge on the cell membrane, which allows low-invasive electroporation of a single cell. As a demonstration of this technique, a fluorescent molecule of propidium iodide was successfully delivered into a single HeLa cell.

  1. Bax is not involved in the resveratrol-induced apoptosis in human lung adenocarcinoma cells

    NASA Astrophysics Data System (ADS)

    Zhang, Wei-wei; Wang, Zhi-ping; Chen, Tong-sheng

    2010-02-01

    Resveratrol (RV) is a natural plant polyphenol widely present in foods such as grapes, wine, and peanuts. Previous studies indicate that RV has an ability to inhibit various stages of carcinogenesis and eliminate preneoplastic cells in vitro and in vivo. However, little is known about the molecular mechanism of RV-induced apoptosis in human lung adenocarcinoma (ASTC-a-1) cell. In this report, we analyzed whether Bax translocation from cytoplasm to mitochondria during RV-induced apoptosis in single living cell using onfocal microscopey. Cells were transfected with GFP-Bax plasmid. Cell counting kit (CCK-8) assay was used to assess the inhibition of RV on the cells viability. Apoptotic activity of RV was detected by Hoechst 33258 and propidium iodide (PI) staining. Our results showed that RV induced a dose-dependent apoptosis in which Bax did not translocate to mitochondrias.

  2. Anticancer and apoptotic activities of oleanolic acid are mediated through cell cycle arrest and disruption of mitochondrial membrane potential in HepG2 human hepatocellular carcinoma cells.

    PubMed

    Zhu, Yue-Yong; Huang, Hong-Yan; Wu, Yin-Lian

    2015-10-01

    Hepatocellular carcinoma (HCC) is an aggressive form of cancer, with high rates of morbidity and mortality, a poor prognosis and limited therapeutic options. The objective of the present study was to demonstrate the anticancer activity of oleanolic acid in HepG2 human HCC cells. Cell viability was evaluated using an MTT assay, following administration of various doses of oleanolic acid. The effect of oleanolic acid on cell cycle phase distribution and mitochondrial membrane potential was evaluated using flow cytometry with propidium iodide and rhodamine‑123 DNA‑binding cationic fluorescent dyes. Fluorescence microscopy was employed to detect morphological changes in HepG2 cells following oleanolic acid treatment. The results revealed that oleanolic acid induced a dose‑dependent, as well as time‑dependent inhibition in the growth of HepG2 cancer cells. Following acridine orange and ethidium bromide staining, treatment with various doses (0, 5, 25 and 50 µM) of oleanolic acid induced typical morphological changes associated with apoptosis, including cell shrinkage, membrane blebbing, nuclear condensation and apoptotic body formation. Cell cycle analysis revealed that oleanolic acid induced cell cycle arrest in HepG2 cells at the sub‑G1 (apoptotic) phase of the cell cycle, in a dose‑dependent manner. Staining with Annexin V‑fluorescein isothiocyanate and propidium iodide revealed that apoptosis occurred early in these cells. Oleanolic acid treatment also resulted in fragmentation of nuclear DNA in a dose‑dependent manner, producing the typical features of DNA laddering on an agarose gel. The results also demonstrated that oleanolic acid treatment resulted in a potent loss of mitochondrial membrane potential, which also occurred in a dose‑dependent manner. Therefore, oleanolic acid may be used as a therapeutic agent in the treatment of human HCC. PMID:26151733

  3. Lycopene and beta-carotene induce cell-cycle arrest and apoptosis in human breast cancer cell lines.

    PubMed

    Gloria, Nathalie Fonseca; Soares, Nathalia; Brand, Camila; Oliveira, Felipe Leite; Borojevic, Radovan; Teodoro, Anderson Junger

    2014-03-01

    Lycopene and beta-carotene are carotenoids widely distributed in fruits and vegetables, with potential anticancer activity. Epidemiological trials rarely provide evidence for the mechanisms of action of these compounds, and their biological effects at different times of treatment are still unclear. The aim of the present study was to determine the effect of carotenoids on the cell cycle and cell viability in human breast cancer cell lines. Human breast cell lines were treated with carotenoids (0.5-10 μM) for 48 and 96 h. Cell viability was monitored using the MTT method (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; thiazolyl blue). The cell cycle was analyzed by flow cytometry, and apoptotic cells were identified by annexin/propidium iodide (PI) biomarkers. Our data showed a significant decrease in the number of viable breast cancer cells on treatment with carotenoids. Carotenoids also promoted cell-cycle arrest followed by decreased cell viability in the majority of cell lines after 96 h, compared to controls. Furthermore, an increase in apoptosis was observed in cell lines when cells were treated with carotenoids. Our findings show the capacity of lycopene and beta-carotene to inhibit cell proliferation, arrest the cell cycle in different phases, and increase apoptosis. These findings indicate that the effect was cell type-dependent and that carotenoids are potential agents for biological interference with cancer. PMID:24596385

  4. Iodinated contrast media cause direct tubular cell damage, leading to oxidative stress, low nitric oxide, and impairment of tubuloglomerular feedback

    PubMed Central

    Liu, Zhi Zhao; Schmerbach, Kristin; Lu, Yuan; Perlewitz, Andrea; Nikitina, Tatiana; Cantow, Kathleen; Seeliger, Erdmann; Persson, Pontus B.; Liu, Ruisheng; Sendeski, Mauricio M.

    2014-01-01

    Iodinated contrast media (CM) have adverse effects that may result in contrast-induced acute kidney injury. Oxidative stress is believed to play a role in CM-induced kidney injury. We test the hypothesis that oxidative stress and reduced nitric oxide in tubules are consequences of CM-induced direct cell damage and that increased local oxidative stress may increase tubuloglomerular feedback. Rat thick ascending limbs (TAL) were isolated and perfused. Superoxide and nitric oxide were quantified using fluorescence techniques. Cell death rate was estimated using propidium iodide and trypan blue. The function of macula densa and tubuloglomerular feedback responsiveness were measured in isolated, perfused juxtaglomerular apparatuses (JGA) of rabbits. The expression of genes related to oxidative stress and the activity of superoxide dismutase (SOD) were investigated in the renal medulla of rats that received CM. CM increased superoxide concentration and reduced nitric oxide bioavailability in TAL. Propidium iodide fluorescence and trypan blue uptake increased more in CM-perfused TAL than in controls, indicating increased rate of cell death. There were no marked acute changes in the expression of genes related to oxidative stress in medullary segments of Henle's loop. SOD activity did not differ between CM and control groups. The tubuloglomerular feedback in isolated JGA was increased by CM. Tubular cell damage and accompanying oxidative stress in our model are consequences of CM-induced direct cell damage, which also modifies the tubulovascular interaction at the macula densa, and may therefore contribute to disturbances of renal perfusion and filtration. PMID:24431205

  5. Conducting and permeable states of cell membrane submitted to high voltage pulses: mathematical and numerical studies validated by the experiments.

    PubMed

    Leguèbe, M; Silve, A; Mir, L M; Poignard, C

    2014-11-01

    The aim of this paper is to present a new model of in vitro cell electropermeabilization, which describes separately the conducting state and the permeable state of the membrane submitted to high voltage pulses. We first derive the model based on the experimental observations and we present the numerical methods to solve the non-linear partial differential equations. We then present numerical simulations that corroborate qualitatively the experimental data dealing with the uptake of propidium iodide (PI) after millipulses. This tends to justify the validity of our modeling. Forthcoming work will be to calibrate the parameters of the model for quantitative description of the uptake. PMID:25010659

  6. Dendrimer-Based Selective Proteostasis-Inhibition Strategy to Control NSCLC Growth and Progression

    PubMed Central

    Walworth, Kyla; Bodas, Manish; Campbell, Ryan John; Swanson, Doug; Sharma, Ajit; Vij, Neeraj

    2016-01-01

    Elevated valosin containing protein (VCP/p97) levels promote the progression of non-small cell lung carcinoma (NSCLC). Although many VCP inhibitors are available, most of these therapeutic compounds have low specificity for targeted tumor cell delivery. Hence, the primary aim of this study was to evaluate the in vitro efficacy of dendrimer-encapsulated potent VCP-inhibitor drug in controlling non-small cell lung carcinoma (NSCLC) progression. The VCP inhibitor(s) (either in their pure form or encapsulated in generation-4 PAMAM-dendrimer with hydroxyl surface) were tested for their in vitro efficacy in modulating H1299 (NSCLC cells) proliferation, migration, invasion, apoptosis and cell cycle progression. Our results show that VCP inhibition by DBeQ was significantly more potent than NMS-873 as evident by decreased cell proliferation (p<0.0001, MTT-assay) and migration (p<0.05; scratch-assay), and increased apoptosis (p<0.05; caspase-3/7-assay) as compared to untreated control cells. Next, we found that dendrimer-encapsulated DBeQ (DDNDBeQ) treatment increased ubiquitinated-protein accumulation in soluble protein-fraction (immunoblotting) of H1299 cells as compared to DDN-control, implying the effectiveness of DBeQ in proteostasis-inhibition. We verified by immunostaining that DDNDBeQ treatment increases accumulation of ubiquitinated-proteins that co-localizes with an ER-marker, KDEL. We observed that proteostasis-inhibition with DDNDBeQ, significantly decreased cell migration rate (scratch-assay and transwell-invasion) as compared to the control-DDN treatment (p<0.05). Moreover, DDNDBeQ treatment showed a significant decrease in cell proliferation (p<0.01, MTT-assay) and increased caspase-3/7 mediated apoptotic cell death (p<0.05) as compared to DDN-control. This was further verified by cell cycle analysis (propidium-iodide-staining) that demonstrated significant cell cycle arrest in the G2/M-phase (p<0.001) by DDNDBeQ treatment as compared to control-DDN. Moreover

  7. Dendrimer-Based Selective Proteostasis-Inhibition Strategy to Control NSCLC Growth and Progression.

    PubMed

    Walworth, Kyla; Bodas, Manish; Campbell, Ryan John; Swanson, Doug; Sharma, Ajit; Vij, Neeraj

    2016-01-01

    Elevated valosin containing protein (VCP/p97) levels promote the progression of non-small cell lung carcinoma (NSCLC). Although many VCP inhibitors are available, most of these therapeutic compounds have low specificity for targeted tumor cell delivery. Hence, the primary aim of this study was to evaluate the in vitro efficacy of dendrimer-encapsulated potent VCP-inhibitor drug in controlling non-small cell lung carcinoma (NSCLC) progression. The VCP inhibitor(s) (either in their pure form or encapsulated in generation-4 PAMAM-dendrimer with hydroxyl surface) were tested for their in vitro efficacy in modulating H1299 (NSCLC cells) proliferation, migration, invasion, apoptosis and cell cycle progression. Our results show that VCP inhibition by DBeQ was significantly more potent than NMS-873 as evident by decreased cell proliferation (p<0.0001, MTT-assay) and migration (p<0.05; scratch-assay), and increased apoptosis (p<0.05; caspase-3/7-assay) as compared to untreated control cells. Next, we found that dendrimer-encapsulated DBeQ (DDNDBeQ) treatment increased ubiquitinated-protein accumulation in soluble protein-fraction (immunoblotting) of H1299 cells as compared to DDN-control, implying the effectiveness of DBeQ in proteostasis-inhibition. We verified by immunostaining that DDNDBeQ treatment increases accumulation of ubiquitinated-proteins that co-localizes with an ER-marker, KDEL. We observed that proteostasis-inhibition with DDNDBeQ, significantly decreased cell migration rate (scratch-assay and transwell-invasion) as compared to the control-DDN treatment (p<0.05). Moreover, DDNDBeQ treatment showed a significant decrease in cell proliferation (p<0.01, MTT-assay) and increased caspase-3/7 mediated apoptotic cell death (p<0.05) as compared to DDN-control. This was further verified by cell cycle analysis (propidium-iodide-staining) that demonstrated significant cell cycle arrest in the G2/M-phase (p<0.001) by DDNDBeQ treatment as compared to control-DDN. Moreover

  8. Clostridium perfringens Iota-Toxin b Induces Rapid Cell Necrosis▿

    PubMed Central

    Nagahama, Masahiro; Umezaki, Mariko; Oda, Masataka; Kobayashi, Keiko; Tone, Shigenobu; Suda, Taiji; Ishidoh, Kazumi; Sakurai, Jun

    2011-01-01

    Clostridium perfringens iota-toxin is a binary toxin composed of an enzyme component (Ia) and a binding component (Ib). Each component alone lacks toxic activity, but together they produce cytotoxic effects. We examined the cytotoxicity of iota-toxin Ib in eight cell lines. A431 and A549 cells were susceptible to Ib, but MDCK, Vero, CHO, Caco-2, HT-29, and DLD-1 cells were not. Ib bound and formed oligomers in the membranes of A431 and MDCK cells. However, Ib entered MDCK cells but not A431 cells, suggesting that uptake is essential for cellular survival. Ib also induced cell swelling and the rapid depletion of cellular ATP in A431 and A549 cells but not the insensitive cell lines. In A431 cells, Ib binds and oligomerizes mainly in nonlipid rafts in the membranes. Disruption of lipid rafts by methyl-β-cyclodextrin did not impair ATP depletion or cell death caused by Ib. Ib induced permeabilization by propidium iodide without DNA fragmentation in A431 cells. Ultrastructural studies revealed that A431 cells undergo necrosis after treatment with Ib. Ib caused a disruption of mitochondrial permeability and the release of cytochrome c. Staining with active-form-specific antibodies showed that the proapoptotic Bcl-2-family proteins Bax and Bak were activated and colocalized with mitochondria in Ib-treated A431 cells. We demonstrate that Ib by itself produces cytotoxic activity through necrosis. PMID:21911469

  9. Quercetin-Induced Cell Death in Human Papillary Thyroid Cancer (B-CPAP) Cells

    PubMed Central

    Mutlu Altundağ, Ergül; Kasacı, Tolga; Yılmaz, Ayşe Mine; Karademir, Betül; Koçtürk, Semra; Taga, Yavuz; Yalçın, A. Süha

    2016-01-01

    In this study, we have investigated the antiproliferative effect of quercetin on human papillary thyroid cancer cells and determined the apoptotic mechanisms underlying its actions. We have used different concentrations of quercetin to induce apoptosis and measured cell viability. Apoptosis and cell cycle analysis was determined by flow cytometry using Annexin V and propidium iodide. Finally, we have measured changes in caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) protein expression levels as hallmarks of apoptosis and Hsp90 protein expression level as a marker of proteasome activity in treated and control cells. Quercetin treatment of human papillary thyroid cancer cells resulted in decreased cell proliferation and increased rate of apoptosis by caspase activation. Furthermore, it was demonstrated that quercetin induces cancer cell apoptosis by downregulating the levels of Hsp90. In conclusion, we have shown that quercetin induces downregulation of Hsp90 expression that may be involved in the decrease of chymotrypsin-like proteasome activity which, in order, induces inhibition of growth and causes cell death in thyroid cancer cells. Thus, quercetin appears to be a promising candidate drug for Hsp90 downregulation and apoptosis of thyroid cancer cells. PMID:27057371

  10. Spatial Organization of Dual-Species Bacterial Aggregates on Leaf Surfaces

    PubMed Central

    Monier, J.-M.; Lindow, S. E.

    2005-01-01

    The spatial organization of cells within bacterial aggregates on leaf surfaces was determined for pair-wise mixtures of three different bacterial species commonly found on leaves, Pseudomonas syringae, Pantoea agglomerans, and Pseudomonas fluorescens. Cells were coinoculated onto bean plants and allowed to grow under moist conditions, and the resulting aggregates were examined in situ by epifluorescence microscopy. Each bacterial strain could be localized because it expressed either the green or the cyan fluorescent protein constitutively, and the viability of individual cells was assessed by propidium iodide staining. Each pair of bacterial strains that was coinoculated onto leaves formed mixed aggregates. The degree of segregation of cells in mixed aggregates differed between the different coinoculated pairs of strains and was higher in mixtures of P. fluorescens A506 and P. agglomerans 299R and mixtures of P. syringae B728a and P. agglomerans 299R than in mixtures of two isogenic strains of P. agglomerans 299R. The fractions of the total cell population that were dead in mixed and monospecific aggregates of a gfp-marked strain of P. agglomerans 299R and a cfp-marked strain of P. agglomerans 299R, or of P. fluorescens A506 and P. agglomerans 299R, were similar. However, the proportion of dead cells in mixed aggregates of P. syringae B728a and P. agglomerans 299R was significantly higher (13.2% ± 8.2%) than that in monospecific aggregates of these two strains (1.6% ± 0.7%), and it increased over time. While dead cells in such mixed aggregates were preferentially found at the interface between clusters of cells of these strains, cells of these two strains located at the interface did not exhibit equal probabilities of mortality. After 9 days of incubation, about 77% of the P. agglomerans 299R cells located at the interface were dead, while only about 24% of the P. syringae B728a cells were dead. The relevance of our results to understanding bacterial interactions

  11. Effects of air transient spark discharge and helium plasma jet on water, bacteria, cells, and biomolecules.

    PubMed

    Hensel, Karol; Kučerová, Katarína; Tarabová, Barbora; Janda, Mário; Machala, Zdenko; Sano, Kaori; Mihai, Cosmin Teodor; Ciorpac, Mitică; Gorgan, Lucian Dragos; Jijie, Roxana; Pohoata, Valentin; Topala, Ionut

    2015-01-01

    Atmospheric pressure DC-driven self-pulsing transient spark (TS) discharge operated in air and pulse-driven dielectric barrier discharge plasma jet (PJ) operated in helium in contact with water solutions were used for inducing chemical effects in water solutions, and the treatment of bacteria (Escherichia coli), mammalian cells (Vero line normal cells, HeLa line cancerous cells), deoxyribonucleic acid (dsDNA), and protein (bovine serum albumin). Two different methods of water solution supply were used in the TS: water electrode system and water spray system. The effects of both TS systems and the PJ were compared, as well as a direct exposure of the solution to the discharge with an indirect exposure to the discharge activated gas flow. The chemical analysis of water solutions was performed by using colorimetric methods of UV-VIS absorption spectrophotometry. The bactericidal effects of the discharges on bacteria were evaluated by standard microbiological plate count method. Viability, apoptosis and cell cycle were assessed in normal and cancerous cells. Viability of cells was evaluated by trypan blue exclusion test, apoptosis by Annexin V-FITC/propidium iodide assay, and cell cycle progression by propidium iodide/RNase test. The effect of the discharges on deoxyribonucleic acid and protein were evaluated by fluorescence and UV absorption spectroscopy. The results of bacterial and mammalian cell viability, apoptosis, and cell cycle clearly show that cold plasma can inactivate bacteria and selectively target cancerous cells, which is very important for possible future development of new plasma therapeutic strategies in biomedicine. The authors found that all investigated bio-effects were stronger with the air TS discharge than with the He PJ, even in indirect exposure. PMID:25947389

  12. PACAP protects against TNFα-induced cell death in olfactory epithelium and olfactory placodal cell lines

    PubMed Central

    Kanekar, Shami; Gandham, Mahendra; Lucero, Mary T

    2010-01-01

    In mouse olfactory epithelium (OE), pituitary adenylate cyclase activating peptide (PACAP) protects against axotomy-induced apoptosis. We used mouse OE to determine whether PACAP protects neurons during exposure to the inflammatory cytokine TNFα. Live slices of neonatal mouse OE were treated with 40 ng/ml TNFα ± 40 nM PACAP for 6 hours and dying cells were live-labeled with 0.5% propidium iodide. TNFα significantly increased the percentage of dying cells while co-incubation with PACAP prevented cell death. PACAP also prevented TNFα-mediated cell death in the olfactory placodal (OP) cell lines, OP6 and OP27. Although OP cell lines express all three PACAP receptors (PAC1, VPAC1,VPAC2), PACAP’s protection of these cells from TNFα was mimicked by the specific PAC1 receptor agonist maxadilan and abolished by the PAC1 antagonist PACAP6–38. Treatment of OP cell lines with blockers or activators of the PLC and AC/MAPKK pathways revealed that PACAP-mediated protection from TNFα involved both pathways. PACAP may therefore function through PAC1 receptors to protect neurons from cell death during inflammatory cytokine release in vivo as would occur upon viral infection or allergic rhinitis-associated injury. PMID:20654718

  13. The antiproliferative effect of C2-ceramide on lung cancer cells through apoptosis by inhibiting Akt and NFκB

    PubMed Central

    2014-01-01

    The anticancer effects of ceramide have been reported in many types of cancers but less in lung cancer. In this study, we used C2-ceramide to further investigate its possible anticancer effects and mechanisms on non-small cell lung cancer (NSCLC) H1299 cells. The result of cell proliferation in terms of trypan blue assay showed high dose of C2-ceramide inhibited cell survival after 24 h treatment. The flow cytometry-based assays indicated the effect of apoptosis, chromatin condensation, and G1 arrest in terms of Annexin V/propidium iodide (PI), DAPI, and PI stainings, respectively. Moreover, the decreased protein level of p-Akt, p-NFκB, survivin and cyclin A2 were detected by Western blot assay. Taken together, these results indicated the antiproliferative effect of C2-ceramide is majorly responsible for cell apoptosis in lung cancer H1299 cells. PMID:24393431

  14. Histone H2AX phosphorylation as a measure of DNA double-strand breaks and a marker of environmental stress and disease activity in lupus

    PubMed Central

    Namas, Rajaie; Renauer, Paul; Ognenovski, Mikhail; Tsou, Pei-Suen; Sawalha, Amr H

    2016-01-01

    Objective Defective or inefficient DNA double-strand break (DSB) repair results in failure to preserve genomic integrity leading to apoptotic cell death, a hallmark of systemic lupus erythematosus (SLE). Compelling evidence linked environmental factors that increase oxidative stress with SLE risk and the formation of DSBs. In this study, we sought to further explore genotoxic stress sensitivity in SLE by investigating DSB accumulation as a marker linking the effect of environmental stressors and the chromatin microenvironment. Methods DSBs were quantified in peripheral blood mononuclear cell subsets from patients with SLE, healthy controls, and patients with rheumatoid arthritis (RA) by measuring phosphorylated H2AX (phospho-H2AX) levels with flow cytometry. Phospho-H2AX levels were assessed in G0/G1, S and G2 cell-cycle phases using propidium iodide staining, and after oxidative stress using 0.5 µM hydrogen peroxide exposure for 0, 2, 5, 10, 30 and 60 min. Results DSB levels were significantly increased in CD4+ T cells, CD8+ T cells and monocytes in SLE compared with healthy controls (p=2.16×10−4, 1.68×10−3 and 4.74×10−3, respectively) and RA (p=1.05×10−3, 1.78×10−3 and 2.43×10−2, respectively). This increase in DSBs in SLE was independent of the cell-cycle phase, and correlated with disease activity. In CD4+ T cells, CD8+ T cells and monocytes, oxidative stress exposure induced significantly higher DSB accumulation in SLE compared with healthy controls (60 min; p=1.64×10−6, 8.11×10−7 and 2.04×10−3, respectively). Conclusions Our data indicate that SLE T cells and monocytes have increased baseline DSB levels and an increased sensitivity to acquiring DSBs in response to oxidative stress. Although the mechanism underlying DSB sensitivity in SLE requires further investigation, accumulation of DSB may serve a biomarker for disease activity in SLE and help explain increased apoptotic cell accumulation in this disease. PMID:27158526

  15. Glucagonlike Peptide 2 Analogue Teduglutide

    PubMed Central

    Chaturvedi, Lakshmi S.; Basson, Marc D.

    2015-01-01

    IMPORTANCE Short bowel syndrome occurs when a shortened intestine cannot absorb sufficient nutrients or fluids. Teduglutide is a recombinant analogue of human glucagonlike peptide 2 that reduces dependence on parenteral nutrition in patients with short bowel syndrome by promoting enterocytic proliferation, increasing the absorptive surface area. However, enterocyte function depends not only on the number of cells that are present but also on differentiated features that facilitate nutrient absorption and digestion. OBJECTIVE To test the hypothesis that teduglutide impairs human intestinal epithelial differentiation. DESIGN AND SETTING We investigated the effects of teduglutide in the modulation of proliferation and differentiation in human Caco-2 intestinal epithelial cells at a basic science laboratory. This was an in vitro study using Caco-2 cells, a human-derived intestinal epithelial cell line commonly used to model enterocytic biology. EXPOSURE Cells were exposed to teduglutide or vehicle control. MAINOUTCOMESAND MEASURES We analyzed the cell cycle by bromodeoxyuridine incorporation or propidium iodide staining and flow cytometry and measured cell proliferation by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. We used quantitative reverse transcription–polymerase chain reaction to assay the expression of the enterocytic differentiation markers villin, sucrase-isomaltase, glucose transporter 2 (GLUT2), and dipeptidyl peptidase 4 (DPP-4), as well as that of the putative differentiation signals schlafen 12 (SLFN12) and caudal-related homeobox intestine-specific transcription factor (Cdx2). Villin promoter activity was measured by a luciferase-based assay. RESULTS The MTS assay demonstrated that teduglutide increased cell numbers by a mean (SD) of 10% (2%) over untreated controls at a maximal 500nM (n = 6, P < .05). Teduglutide increased bromodeoxyuridine-positive cells vs untreated controls by a mean (SD

  16. Experimental Adaptation of Rotaviruses to Tumor Cell Lines

    PubMed Central

    Guerrero, Carlos A.; Guerrero, Rafael A.; Silva, Elver; Acosta, Orlando; Barreto, Emiliano

    2016-01-01

    A number of viruses show a naturally extended tropism for tumor cells whereas other viruses have been genetically modified or adapted to infect tumor cells. Oncolytic viruses have become a promising tool for treating some cancers by inducing cell lysis or immune response to tumor cells. In the present work, rotavirus strains TRF-41 (G5) (porcine), RRV (G3) (simian), UK (G6-P5) (bovine), Ym (G11-P9) (porcine), ECwt (murine), Wa (G1-P8), Wi61 (G9) and M69 (G8) (human), and five wild-type human rotavirus isolates were passaged multiple times in different human tumor cell lines and then combined in five different ways before additional multiple passages in tumor cell lines. Cell death caused by the tumor cell-adapted isolates was characterized using Hoechst, propidium iodide, 7-AAD, Annexin V, TUNEL, and anti-poly-(ADP ribose) polymerase (PARP) and -phospho-histone H2A.X antibodies. Multiple passages of the combined rotaviruses in tumor cell lines led to a successful infection of these cells, suggesting a gain-of-function by the acquisition of greater infectious capacity as compared with that of the parental rotaviruses. The electropherotype profiles suggest that unique tumor cell-adapted isolates were derived from reassortment of parental rotaviruses. Infection produced by such rotavirus isolates induced chromatin modifications compatible with apoptotic cell death. PMID:26828934

  17. SGTB Promotes the Caspase-Dependent Apoptosis in Chondrocytes of Osteoarthritis.

    PubMed

    Bao, Guofeng; Xu, Libin; Xu, Xinbao; Zhai, Leilei; Duan, Chengwei; Xu, Dawei; Song, Jie; Liu, Zhongbing; Tao, Ran; Cui, Zhiming; Yang, Huilin

    2016-04-01

    The purpose of this study is to investigate the expression of small glutamine-rich tetratricopeptide repeat (TPR)-containing β (SGTB) in articular cartilage of osteoarthritis (OA) and analyze the relationship between SGTB and chondrocyte apoptosis. We established an OA rat model by the meniscal/ligamentous injury (MLI) modeling method and observed the expression of SGTB in articular cartilage by immunohistochemistry and RT-PCR. Human SW1353 chondrosarcoma cells were treated with interleukin-1β (IL-1β) to mimic the OA-like chondrocyte injury in vitro, and Western blot was employed to examine the IL-1β-induced expression of SGTB and active caspase-3. The co-localization of SGTB and active caspase-3 was confirmed by immunofluorescence. We knocked down SGTB expression by RNA interference (RNAi) and overexpressed SGTB by plasmid transfection. Western blot was carried out to detect the knockdown/overexpressing efficiency of SGTB and evaluate its effects on IL-1β-stimulated expression of active caspase-3 in SW1353 cells. Annexin V/propidium iodide staining was used to detect chondrocyte apoptosis. Then, Western blot was carried out to examine the IL-1β-induced expression of Hsp70 and evaluate SGTB effects on IL-1β-stimulated expression of Hsp70 in SW1353 cells. SGTB expression was significantly up-regulated in articular cartilage of OA rat model. IL-1β stimulation increased the expression of SGTB and active caspase-3 in SW1353 cells. SGTB co-localized with active caspase-3 in IL-1β-treated SW1353 cells. SGTB inhibition significantly reduced IL-1β-stimulated expression of active caspase-3 in SW1353 cells. In line with this, overexpressing SGTB via Myc-SGTB transfection increased the active caspase-3 level in IL-1β-stimulated SW1353 cells. Moreover, flow cytometry assay demonstrated that SGTB knockdown alleviated IL-1β-induced apoptosis, but it was increased in SW1353 cells that overexpressed SGTB. Overexpressing SGTB via Myc-SGTB transfection decreased the Hsp

  18. Sapodilla plum (Achras sapota) induces apoptosis in cancer cell lines and inhibits tumor progression in mice.

    PubMed

    Srivastava, Mrinal; Hegde, Mahesh; Chiruvella, Kishore K; Koroth, Jinsha; Bhattacharya, Souvari; Choudhary, Bibha; Raghavan, Sathees C

    2014-01-01

    Intake of fruits rich in antioxidants in daily diet is suggested to be cancer preventive. Sapota is a tropical fruit grown and consumed extensively in several countries including India and Mexico. Here we show that methanolic extracts of Sapota fruit (MESF) induces cytotoxicity in a dose-dependent manner in cancer cell lines. Cell cycle analysis suggested activation of apoptosis, without arresting cell cycle progression. Annexin V-propidium iodide double-staining demonstrated that Sapota fruit extracts potentiate apoptosis rather than necrosis in cancer cells. Loss of mitochondrial membrane potential, upregulation of proapoptotic proteins, activation of MCL-1, PARP-1, and Caspase 9 suggest that MESF treatment leads to activation of mitochondrial pathway of apoptosis. More importantly, we show that MESF treatment leads to significant inhibition of tumor growth and a 3-fold increase in the life span of tumor bearing animals compared to untreated tumor mice. PMID:25142835

  19. Sapodilla Plum (Achras sapota) Induces Apoptosis in Cancer Cell Lines and Inhibits Tumor Progression in Mice

    PubMed Central

    Srivastava, Mrinal; Hegde, Mahesh; Chiruvella, Kishore K.; Koroth, Jinsha; Bhattacharya, Souvari; Choudhary, Bibha; Raghavan, Sathees C.

    2014-01-01

    Intake of fruits rich in antioxidants in daily diet is suggested to be cancer preventive. Sapota is a tropical fruit grown and consumed extensively in several countries including India and Mexico. Here we show that methanolic extracts of Sapota fruit (MESF) induces cytotoxicity in a dose-dependent manner in cancer cell lines. Cell cycle analysis suggested activation of apoptosis, without arresting cell cycle progression. Annexin V-propidium iodide double-staining demonstrated that Sapota fruit extracts potentiate apoptosis rather than necrosis in cancer cells. Loss of mitochondrial membrane potential, upregulation of proapoptotic proteins, activation of MCL-1, PARP-1, and Caspase 9 suggest that MESF treatment leads to activation of mitochondrial pathway of apoptosis. More importantly, we show that MESF treatment leads to significant inhibition of tumor growth and a 3-fold increase in the life span of tumor bearing animals compared to untreated tumor mice. PMID:25142835

  20. Correlated analysis of cellular DNA, membrane antigens and light scatter of human lymphoid cells

    SciTech Connect

    Braylan, R.C.; Benson, N.A.; Nourse, V.; Kruth, H.S.

    1982-03-01

    Flow cytometric correlated analysis of membrane antigens, DNA, and light scatter was performed on human lymphoid cells using fluorescein (FITC)-conjugated antibodies to label B- and T-cell antigens and propidium iodide (PI) to stain DNA after ethanol fixation and RNase treatment. A FACS II flow cytometer was modified to obtain digitized measurements of two color fluorescence and light scatter emissions, simultaneously. Software was written to allow single parameter analysis or correlated analysis of any two of the three parameters acquired. Ethanol fixation preserved FITC surface labeling for at least 15 weeks, but produced marked changes in light scatter. No changes in FITC distributions were observed after RNase treatment and PI staining, and the presence of FITC labeling did not affect DNA distributions. Within heterogeneous cell populations, the DNA distribution of cell subpopulations identified by a membrane antigen was clearly demonstrated.

  1. Comparative susceptibility of peripheral blood leucocytes and related cell lines to killing by T-cell perforin.

    PubMed Central

    Jones, J; Morgan, B P

    1994-01-01

    The comparative susceptibility of lymphocyte subsets, monocytes and polymorphonuclear leucocytes (PMN) to killing by murine perforin was measured using physical separation techniques, cell-surface phenotyping and scatter characteristics to isolate cell types, together with propidium iodide (PI) uptake as a measure of cell death. In the majority of individuals, PMN were more resistant to perforin than other peripheral blood cells including natural killer (NK) cells and CD8+ lymphocytes. Among the lymphocytes, CD4+ cells were the most susceptible subset, followed by CD19+, CD8+ and CD56+ lymphocytes respectively. The human promyelocytic leukaemia cell line, HL-60, and the human histiocytic lymphoma cell line, U937, were readily killed by perforin. When HL-60 were differentiated to either macrophage- or neutrophil-like end cells, and U937 differentiated to macrophage-like end cells, there was no difference between differentiated and undifferentiated cells in their relative susceptibility to perforin. The relative resistance of PMN to perforin may be important in protecting them from damage in in vivo situations where both NK cells and neutrophils are localized in the same inflammatory areas. PMID:7835917

  2. Evaluation of the damage of cell wall and cell membrane for various extracellular polymeric substance extractions of activated sludge.

    PubMed

    Guo, Xuesong; Liu, Junxin; Xiao, Benyi

    2014-10-20

    Extracellular polymeric substances (EPS) are susceptible to contamination by intracellular substances released during the extraction of EPS owing to the damage caused to microbial cell structures. The damage to cell walls and cell membranes in nine EPS extraction processes of activated sludge was evaluated in this study. The extraction of EPS (including proteins, carbohydrates and DNA) was the highest using the NaOH extraction method and the lowest using formaldehyde extraction. All nine EPS extraction methods in this study resulted in cell wall and membrane damage. The damage to cell walls, evaluated by 2-keto-3-deoxyoctonate (KDO) and N-acetylglucosamine content changes in extracted EPS, was the most significant in the NaOH extraction process. Formaldehyde extraction showed a similar extent of damage to cell walls to those detected in the control method (centrifugation), while those in the formaldehyde-NaOH and cation exchange resin extractions were slightly higher than those detected in the control. N-acetylglucosamine was more suitable than KDO for the evaluation of cell wall damage in the EPS extraction of activated sludge. The damage to cell membranes was characterized by two fluorochromes (propidium iodide and FITC Annexin V) with flow cytometry (FCM) measurement. The highest proportion of membrane-damaged cells was detected in NaOH extraction (26.54% of total cells) while membrane-damaged cells comprised 8.19% of total cells in the control. PMID:25173614

  3. Akebia saponin PA induces autophagic and apoptotic cell death in AGS human gastric cancer cells.

    PubMed

    Xu, Mei-Ying; Lee, Dong Hwa; Joo, Eun Ji; Son, Kun Ho; Kim, Yeong Shik

    2013-09-01

    In this study, we investigated the anticancer mechanism of akebia saponin PA (AS), a natural product isolated from Dipsacus asperoides in human gastric cancer cell lines. It was shown that AS-induced cell death is caused by autophagy and apoptosis in AGS cells. The apoptosis-inducing effect of AS was characterized by annexin V/propidium (PI) staining, increase of sub-G1 phase and caspase-3 activation, while the autophagy-inducing effect was indicated by the formation of cytoplasmic vacuoles and microtubule-associated protein 1 light chain-3 II (LC3-II) conversion. The autophagy inhibitor bafilomycin A1 (BaF1) decreased AS-induced cell death and caspase-3 activation, but caspase-3 inhibitor Ac-DEVD-CHO did not affect LC3-II accumulation or AS-induced cell viability, suggesting that AS induces autophagic cell death and autophagy contributes to caspase-3-dependent apoptosis. Furthermore, AS activated p38/c-Jun N-terminal kinase (JNK), which could be inhibited by BaF1, and caspase-3 activation was attenuated by both SB202190 and SP600125, indicating that AS-induced autophagy promotes mitogen-activated protein kinases (MAPKs)-mediated apoptosis. Taken together, these results demonstrate that AS induces autophagic and apoptotic cell death and autophagy plays the main role in akebia saponin PA-induced cell death. PMID:23850994

  4. Caspofungin Kills Candida albicans by Causing both Cellular Apoptosis and Necrosis

    PubMed Central

    Hao, Binghua; Cheng, Shaoji; Nguyen, M. Hong

    2013-01-01

    Caspofungin exerts candidacidal activity by inhibiting cell wall (1,3)-β-d-glucan synthesis. We investigated the physiologic mechanisms of caspofungin-induced Candida albicans cell death. Apoptosis (programmed cell death) and necrosis were studied after C. albicans SC5314 cells were exposed to caspofungin at 0.06, 0.125, and 0.5 μg/ml (0.5×, 1×, and 4× the MIC, respectively) for 3 h. Caspofungin at 0.125 and 0.5 μg/ml reduced cellular viability by >50%, as measured by colony counts and methylene blue exclusion. Apoptosis and necrosis were demonstrated by annexin V and propidium iodide staining for phosphatidylserine externalization and loss of membrane integrity, respectively. At all concentrations of caspofungin, 20 to 25% and 5 to 7% of C. albicans cells exhibited early apoptosis and late apoptosis/necrosis, respectively (P value was not significant [NS]). Necrosis, on the other hand, was significantly greater at 0.125 (43%) and 0.5 (48%) μg/ml than at 0.06 μg/ml (26%) (P values of 0.003 and 0.003, respectively). The induction of apoptosis at concentrations less than or equal to the MIC was corroborated by dihydrorhodamine 123 (DHR-123) and dihydroethidium (DHE) staining (reactive oxygen species production), JC-1 staining (mitochondrial membrane potential dissipation), and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining (DNA damage and nuclear fragmentation). Moreover, electron microscopy of cells exposed to 0.125 μg/ml of caspofungin showed hallmark apoptotic features like chromatin margination and condensation and nuclear blebs. Apoptosis was associated with metacaspase 1 activation, as demonstrated by D2R staining. Caspofungin exerts activity against C. albicans by directly killing cells (resulting in necrosis) and causing others to undergo programmed cell death (apoptosis). Apoptosis is initiated at subinhibitory concentrations, suggesting that strategies to

  5. An RNA Aptamer Provides a Novel Approach for the Induction of Apoptosis by Targeting the HPV16 E7 Oncoprotein

    PubMed Central

    Nicol, Clare; Cesur, Özlem; Forrest, Sophie; Belyaeva, Tamara A.; Bunka, David H. J.; Blair, G. Eric; Stonehouse, Nicola J.

    2013-01-01

    Background Human papillomavirus 16 (HPV16) is a high-risk DNA tumour virus, which is a major causative agent of cervical cancer. Cellular transformation is associated with deregulated expression of the E6 and E7 oncogenes. E7 has been shown to bind a number of cellular proteins, including the cell cycle control protein pRb. In this study, RNA aptamers (small, single-stranded oligonucleotides selected for high-affinity binding) to HPV16 E7 were employed as molecular tools to further investigate these protein-protein interactions. Methodology/Principal Findings This study is focused on one aptamer (termed A2). Transfection of this molecule into HPV16-transformed cells resulted in inhibition of cell proliferation (shown using real-time cell electronic sensing and MTT assays) due to the induction of apoptosis (as demonstrated by Annexin V/propidium iodide staining). GST-pull down and bead binding assays were used to demonstrate that the binding of A2 required N-terminal residues of E7 known to be involved in interaction with the cell cycle control protein, pRb. Using a similar approach, A2 was shown to disrupt the interaction between E7 and pRb in vitro. Furthermore, transfection of HPV16-transformed cells with A2 appeared to result in the loss of E7 and rise in pRb levels, as observed by immunoblotting. Conclusions/Significance This paper includes the first characterisation of the effects of an E7 RNA aptamer in a cell line derived from a cervical carcinoma. Transfection of cells with A2 was correlated with the loss of E7 and the induction of apoptosis. Aptamers specific for a number of cellular and viral proteins have been documented previously; one aptamer (Macugen) is approved for clinical use and several others are in clinical trials. In addition to its role as a molecular tool, A2 could have further applications in the future. PMID:23738000

  6. Discovery of molecular pathways mediating 1,25-dihydroxyvitamin D3 protection against cytokine-induced inflammation and damage of human and male mouse islets of Langerhans.

    PubMed

    Wolden-Kirk, H; Rondas, D; Bugliani, M; Korf, H; Van Lommel, L; Brusgaard, K; Christesen, H T; Schuit, F; Proost, P; Masini, M; Marchetti, P; Eizirik, D L; Overbergh, L; Mathieu, C

    2014-03-01

    Protection against insulitis and diabetes by active vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), in nonobese diabetic mice has until now mainly been attributed to its immunomodulatory effects, but also protective effects of this hormone on inflammation-induced β-cell death have been reported. The aim of this study was to clarify the molecular mechanisms by which 1,25(OH)2D3 contributes to β-cell protection against cytokine-induced β-cell dysfunction and death. Human and mouse islets were exposed to IL-1β and interferon-γ in the presence or absence of 1,25(OH)2D3. Effects on insulin secretion and β-cell survival were analyzed by glucose-stimulated insulin release and electron microscopy or Hoechst/propidium iodide staining, respectively. Gene expression profiles were assessed by Affymetrix microarrays. Nuclear factor-κB activity was tested, whereas effects on secreted chemokines/cytokines were confirmed by ELISA and migration studies. Cytokine exposure caused a significant increase in β-cell apoptosis, which was almost completely prevented by 1,25(OH)2D3. In addition, 1,25(OH)2D3 restored insulin secretion from cytokine-exposed islets. Microarray analysis of murine islets revealed that the expression of approximately 4000 genes was affected by cytokines after 6 and 24 hours (n = 4; >1.3-fold; P < .02), of which nearly 250 genes were modified by 1,25(OH)2D3. These genes belong to functional groups involved in immune response, chemotaxis, cell death, and pancreatic β-cell function/phenotype. In conclusion, these findings demonstrate a direct protective effect of 1,25(OH)2D3 against inflammation-induced β-cell dysfunction and death in human and murine islets, with, in particular, alterations in chemokine production by the islets. These effects may contribute to the beneficial effects of 1,25(OH)2D3 against the induction of autoimmune diabetes. PMID:24424042

  7. Induction of apoptosis in the human Leukemic U937 cell line by Kaempferia parviflora Wall.ex.Baker extract and effects of paclitaxel and camptothecin.

    PubMed

    Banjerdpongchai, Ratana; Chanwikruy, Yupa; Rattanapanone, Viboon; Sripanidkulchai, Bungorn

    2009-01-01

    Kaempferia parviflora Wall.ex.Baker is a Thai medicinal herb that has high antioxidant and anti-inflammatory activities. Apoptotic effects of the herbal extract alone and in combination with chemotherapeutic drugs, paclitaxel and camptothecin, were here studied in the human promonocytic leukemic U937 cell line. K. parviflora extract suppressed cell proliferation and decreased cell viability in a dose- and time-dependent manner as assessed using the trypan blue exclusion assay. Staining of extract-treated cells with propidium iodide and examination under a fluorescence microscope showed condensed nuclei and apoptotic bodies. Mitochondrial transmembrane potential (MTP) decreased after treatment and the number of cells with decreased MTP also increased. Furthermore, activation of caspase-3 was found in herbal extract-treated cells. When the extract was combined with paclitaxel, an additive effect on U937 cell apoptosis was obtained, whereas camptothecin exerted an antagonistic effect. PMID:20192599

  8. A biocompatible microchip and methodology for efficiently trapping and positioning living cells into array based on negative dielectrophoresis

    NASA Astrophysics Data System (ADS)

    Guo, Xiaoliang; Zhu, Rong

    2015-06-01

    We present a microchip and trapping methodology based on negative dielectrophoresis (nDEP), whereby living cells were manipulated and positioned into an array with high trapping efficiency while maintaining good viability. The main factors that ensured good viability of cells were investigated including temperature of medium, extra transmembrane potential on cells, and electrolysis effect in DEP-based trapping. Optimum DEP conditions for the microchip were determined by considering both biocompatibility and trapping efficiency. Experiments demonstrated that under a voltage of 3.6-4 Vpp and at a frequency of 100 kHz, HeLa cells could be trapped and positioned into an array in less than 10 s while maintaining good viability. The normal adherence morphology and fluorescence of the cells, dyed with propidium iodide and Calcein-AM, were observed and verified the biocompatibility of the microchip and trapping methodology.

  9. Bacteroides fragilis induce necrosis on mice peritoneal macrophages: In vitro and in vivo assays

    SciTech Connect

    Vieira, J.M.B.D.; Seabra, S.H.; Vallim, D.C.; Americo, M.A.; Fracallanza, S.E.L.; Vommaro, R.C.; Domingues, R.M.C.P.

    2009-10-02

    Bacteroides fragilis is an anaerobic bacteria component of human intestinal microbiota and agent of infections. In the host B. fragilis interacts with macrophages, which produces toxic radicals like NO. The interaction of activated mice peritoneal macrophages with four strains of B. fragilis was evaluated on this study. Previously was shown that such strains could cause metabolic and morphologic alterations related to macrophage death. In this work propidium iodide staining showed the strains inducing macrophage necrosis in that the labeling was evident. Besides nitroblue tetrazolium test showed that B. fragilis stimulates macrophage to produce oxygen radicals. In vivo assays performed in BalbC mice have results similar to those for in vitro tests as well as scanning electron microscopy, which showed the same surface pore-like structures observed in vitro before. The results revealed that B. fragilis strains studied lead to macrophage death by a process similar to necrosis.

  10. Blue light inhibits proliferation of melanoma cells

    NASA Astrophysics Data System (ADS)

    Becker, Anja; Distler, Elisabeth; Klapczynski, Anna; Arpino, Fabiola; Kuch, Natalia; Simon-Keller, Katja; Sticht, Carsten; van Abeelen, Frank A.; Gretz, Norbert; Oversluizen, Gerrit

    2016-03-01

    Photobiomodulation with blue light is used for several treatment paradigms such as neonatal jaundice, psoriasis and back pain. However, little is known about possible side effects concerning melanoma cells in the skin. The aim of this study was to assess the safety of blue LED irradiation with respect to proliferation of melanoma cells. For that purpose we used the human malignant melanoma cell line SK-MEL28. Cell proliferation was decreased in blue light irradiated cells where the effect size depended on light irradiation dosage. Furthermore, with a repeated irradiation of the melanoma cells on two consecutive days the effect could be intensified. Fluorescence-activated cell sorting with Annexin V and Propidium iodide labeling did not show a higher number of dead cells after blue light irradiation compared to non-irradiated cells. Gene expression analysis revealed down-regulated genes in pathways connected to anti-inflammatory response, like B cell signaling and phagosome. Most prominent pathways with up-regulation of genes were cytochrome P450, steroid hormone biosynthesis. Furthermore, even though cells showed a decrease in proliferation, genes connected to the cell cycle were up-regulated after 24h. This result is concordant with XTT test 48h after irradiation, where irradiated cells showed the same proliferation as the no light negative control. In summary, proliferation of melanoma cells can be decreased using blue light irradiation. Nevertheless, the gene expression analysis has to be further evaluated and more studies, such as in-vivo experiments, are warranted to further assess the safety of blue light treatment.

  11. Gold nanoparticle sensitize radiotherapy of prostate cancer cells by regulation of the cell cycle

    NASA Astrophysics Data System (ADS)

    Roa, Wilson; Zhang, Xiaojing; Guo, Linghong; Shaw, Andrew; Hu, Xiuying; Xiong, Yeping; Gulavita, Sunil; Patel, Samir; Sun, Xuejun; Chen, Jie; Moore, Ronald; Xing, James Z.

    2009-09-01

    Glucose-capped gold nanoparticles (Glu-GNPs) have been used to improve cellular targeting and radio-sensitization. In this study, we explored the mechanism of Glu-GNP enhanced radiation sensitivity in radiation-resistant human prostate cancer cells. Cell survival and proliferation were measured using MTT and clonogenic assay. Flow cytometry with staining by propidium iodide (PI) was performed to study the cell cycle changes induced by Glu-GNPs, and western blotting was used to determine the expression of p53 and cyclin proteins that correlated to cell cycle regulation. With 2 Gy of ortho-voltage irradiation, Glu-GNP showed a 1.5-2.0 fold enhancement in growth inhibition when compared to x-rays alone. Comparing the cell cycle change, Glu-GNPs induced acceleration in the G0/G1 phase and accumulation of cells in the G2/M phase at 29.8% versus 18.4% for controls at 24 h. G2/M arrest was accompanied by decreased expression of p53 and cyclin A, and increased expression of cyclin B1 and cyclin E. In conclusion, Glu-GNPs trigger activation of the CDK kinases leading to cell cycle acceleration in the G0/G1 phase and accumulation in the G2/M phase. This activation is accompanied by a striking sensitization to ionizing radiation, which may have clinical implications.

  12. Human Wharton's jelly stem cells, its conditioned medium and cell-free lysate inhibit the growth of human lymphoma cells.

    PubMed

    Lin, Hao Daniel; Fong, Chui Yee; Biswas, Arijit; Choolani, Mahesh; Bongso, Ariff

    2014-08-01

    Several groups have reported that primitive mesenchymal stem cells from the gelatinous matrix of the Wharton's jelly of the human umbilical cord (hWJSCs) possess tumoricidal properties and inhibit the growth of solid tumours such as human mammary carcinoma, ovarian carcinoma and osteosarcoma. This unique characteristic led to the hypothesis that hWJSCs serve as a natural defence against migrating cancer cells from mother to fetus thus explaining why tumorigenesis in the fetus is rare. However, it is not known whether non-solid malignant hematopoietic cells are also inhibited by hWJSCs and what the exact tumoricidal mechanisms are. We therefore evaluated the influence of hWJSCs and its extracts on Burkitt's lymphoma cells. Cell proliferation (BrdU and Ki67+), viability (MTT) and cell death (Annexin V-Propidium iodide and live/dead) assays showed significant inhibition of lymphoma cell growth after 48 h exposure to hWJSCs or its extracts compared to controls. Increased cell death was observed at sub-G1 and S and decreased proliferation at G2/M phases of the mitotic cycle. Superoxide dismutase and hydrogen peroxide activity were significantly increased and glutathione peroxidase significantly decreased in treated lymphoma cells. Time lapse imaging and confocal z-stack images showed yellow fluorescent in situ hybridization (FISH) signals of lymphoma cell Y chromosomes within the cytoplasm of female red labelled hWJSCs. We hypothesize that the growth of lymphoma cells is inhibited by the molecules secreted by hWJSCs that use oxidative stress pathways to induce cell death followed by engulfment of the apoptotic remains of the lymphoma cells by the hWJSCs. PMID:24789672

  13. Overexpression of AQP3 Modifies the Cell Cycle and the Proliferation Rate of Mammalian Cells in Culture.

    PubMed

    Galán-Cobo, Ana; Ramírez-Lorca, Reposo; Serna, Ana; Echevarría, Miriam

    2015-01-01

    Abnormal AQP3 overexpression in tumor cells of different origins has been reported and a role for this enhanced AQP3 expression in cell proliferation and tumor processess has been indicated. To further understand the role AQP3 plays in cell proliferation we explore the effect that stable over expression of AQP3 produces over the proliferation rate and cell cycle of mammalian cells. The cell cycle was analyzed by flow cytometry with propidium iodide (PI) and the cell proliferation rate measured through cell counting and BrdU staining. Cells with overexpression of AQP3 (AQP3-o) showed higher proliferation rate and larger percentage of cells in phases S and G2/M, than wild type cells (wt). Evaluation of the cell response against arresting the cell cycle with Nocodazole showed that AQP3-o exhibited a less modified cell cycle pattern and lower Annexin V specific staining than wt, consistently with a higher resistance to apoptosis of AQP3-overexpressing cells. The cell volume and complexity were also larger in AQP3-o compared to wt cells. After transcriptomic analysis, RT-qPCR was performed to highlight key molecules implicated in cell proliferation which expression may be altered by overexpression of AQP3 and the comparative analysis between both type of cells showed significant changes in the expression of Zeb2, Jun, JunB, NF-kβ, Cxcl9, Cxcl10, TNF, and TNF receptors. We conclude that the role of AQP3 in cell proliferation seems to be connected to increments in the cell cycle turnover and changes in the expression levels of relevant genes for this process. Larger expression of AQP3 may confer to the cell a more tumor like phenotype and contributes to explain the presence of this protein in many different tumors. PMID:26367709

  14. Overexpression of AQP3 Modifies the Cell Cycle and the Proliferation Rate of Mammalian Cells in Culture

    PubMed Central

    Galán-Cobo, Ana; Ramírez-Lorca, Reposo; Serna, Ana; Echevarría, Miriam

    2015-01-01

    Abnormal AQP3 overexpression in tumor cells of different origins has been reported and a role for this enhanced AQP3 expression in cell proliferation and tumor processess has been indicated. To further understand the role AQP3 plays in cell proliferation we explore the effect that stable over expression of AQP3 produces over the proliferation rate and cell cycle of mammalian cells. The cell cycle was analyzed by flow cytometry with propidium iodide (PI) and the cell proliferation rate measured through cell counting and BrdU staining. Cells with overexpression of AQP3 (AQP3-o) showed higher proliferation rate and larger percentage of cells in phases S and G2/M, than wild type cells (wt). Evaluation of the cell response against arresting the cell cycle with Nocodazole showed that AQP3-o exhibited a less modified cell cycle pattern and lower Annexin V specific staining than wt, consistently with a higher resistance to apoptosis of AQP3-overexpressing cells. The cell volume and complexity were also larger in AQP3-o compared to wt cells. After transcriptomic analysis, RT-qPCR was performed to highlight key molecules implicated in cell proliferation which expression may be altered by overexpression of AQP3 and the comparative analysis between both type of cells showed significant changes in the expression of Zeb2, Jun, JunB, NF-kβ, Cxcl9, Cxcl10, TNF, and TNF receptors. We conclude that the role of AQP3 in cell proliferation seems to be connected to increments in the cell cycle turnover and changes in the expression levels of relevant genes for this process. Larger expression of AQP3 may confer to the cell a more tumor like phenotype and contributes to explain the presence of this protein in many different tumors. PMID:26367709

  15. Detection of irradiated quail meat by using DNA comet assay and evaluation of comets by image analysis

    NASA Astrophysics Data System (ADS)

    Erel, Yakup; Yazici, Nizamettin; Özvatan, Sumer; Ercin, Demet; Cetinkaya, Nurcan

    2009-09-01

    A simple technique of microgel electrophoresis of single cells (DNA comet assay) was used to detect DNA comets in irradiated quail meat samples. Obtained DNA comets were evaluated by both photomicrographic and image analysis. Quail meat samples were exposed to radiation doses of 0.52, 1.05, 1.45, 2.00, 2.92 and 4.00 kGy in gamma cell (gammacell 60Co, dose rate 1.31 kGy/h) covering the permissible limits for enzymatic decay and stored at 2 °C. The cells isolated from muscle (chest, thorax) in cold PBS were analyzed using the DNA comet assay on 1, 2, 3, 4, 7, 8 and 11 day post irradiation. The cells were lysed between 2, 5 and 9 min in 2.5% SDS and electrophorosis was carried out at a voltage of 2 V/cm for 2 min. After propidium iodide staining, the slides were evaluated through a fluorescent microscope. In all irradiated samples, fragmented DNA stretched towards the anode and damaged cells appeared as a comet. All measurement data were analyzed using BS 200 ProP with software image analysis (BS 200 ProP, BAB Imaging System, Ankara, Turkey). The density of DNA in the tails increased with increasing radiation dose. However, in non-irradiated samples, the large molecules of DNA remained relatively intact and there was only minor or no migration of DNA; the cells were round or had very short tails only. The values of tail DNA%, tail length and tail moment were significantly different and identical between 0.9 and 4.0 kGy dose exposure, and also among storage times on day 1, 4 and 8. In conclusion, the DNA Comet Assay EN 13784 standard method may be used not only for screening method for detection of irradiated quail meat depending on storage time and condition but also for the quantification of applied dose if it is combined with image analysis. Image analysis may provide a powerful tool for the evaluation of head and tail of comet intensity related with applied doses.

  16. Real-time detection of viable microorganisms by intracellular phototautomerism

    PubMed Central

    2010-01-01

    Background To date, the detection of live microorganisms present in the environment or involved in infections is carried out by enumeration of colony forming units on agar plates, which is time consuming, laborious and limited to readily cultivable microorganisms. Although cultivation-independent methods are available, they involve multiple incubation steps and do mostly not discriminate between dead or live microorganisms. We present a novel generic method that is able to specifically monitor living microorganisms in a real-time manner. Results The developed method includes exposure of cells to a weak acid probe at low pH. The neutral probe rapidly permeates the membrane and enters the cytosol. In dead cells no signal is obtained, as the cytosolic pH reflects that of the acidic extracellular environment. In live cells with a neutral internal pH, the probe dissociates into a fluorescent phototautomeric anion. After reaching peak fluorescence, the population of live cells decays. This decay can be followed real-time as cell death coincides with intracellular acidification and return of the probe to its uncharged non-fluorescent state. The rise and decay of the fluorescence signal depends on the probe structure and appears discriminative for bacteria, fungi, and spores. We identified 13 unique probes, which can be applied in the real-time viability method described here. Under the experimental conditions used in a microplate reader, the reported method shows a detection limit of 106 bacteria ml-1, while the frequently used LIVE/DEAD BacLight™ Syto9 and propidium iodide stains show detection down to 106 and 107 bacteria ml-1, respectively. Conclusions We present a novel fluorescence-based method for viability assessment, which is applicable to all bacteria and eukaryotic cell types tested so far. The RTV method will have a significant impact in many areas of applied microbiology including research on biocidal activity, improvement of preservation strategies and

  17. Effects of mistletoe (Viscum album L.) extracts Iscador on cell cycle and survival of tumor cells.

    PubMed

    Harmsma, Marjan; Ummelen, Monique; Dignef, Wendy; Tusenius, Karel Jan; Ramaekers, Frans C S

    2006-06-01

    The molecular and cellular mechanisms by which mistletoe (Viscum album L.) extracts exert cytotoxic and immunomodulatory anti-tumoral effects are largely unknown. In this study the hypothesis that Iscador preparations induce tumor regression by cell cycle inhibition and/or interference with apoptotic signaling pathways in cancer cells was investigated. Also a possible effect on angiogenesis, which is a prerequisite for tumor growth in vivo, is studied in endothelial cell cultures. Furthermore, it was examined which apoptotic signaling route(s) is (are) activated by Iscador by studying specific pro-apoptotic proteins in cultured cells. To characterize these properties, 9 human cancer cell lines of different origin, one epidermis derived cell line and 2 endothelial cell cultures were incubated with different concentrations of Iscador Quercus Spezial and Iscador Malus Spezial. Cell cycle kinetic parameters were measured by bromodeoxyuridine (BrdU) pulse labeling and tubulin staining. Apoptotic responses were detected by M30 Cyto-Death or Annexin V/propidium iodide assays. Characterization of the apoptotic pathway(s) was performed by staining cells for amongst others active caspase 3 and cytochrome C (mitochondrial pathway), as well as active caspase 8 (death receptor pathway). The sensitivity to Iscador treatment varies strongly between different cell lines and also ing those derived from small cell lung cancer, and adenocarcinoma of the lung and breast, as well as endothelial cell cultures, Iscador caused early cell cycle inhibition followed by apoptosis in a dose dependent manner. Amongst the low responders are cell lines derived from colorectal carcinoma. In general Iscador Malus exerted a stronger response than Iscador Quercus. Apoptosis was induced by activating the mitochondrial but not the death receptor dependent pathway, at least in case of Iscador Quercus. Iscador Malus also seemed to induce apoptosis via the death receptor route, which may explain the

  18. Induction of cell cycle arrest, DNA damage, and apoptosis by nimbolide in human renal cell carcinoma cells.

    PubMed

    Hsieh, Yi-Hsien; Lee, Chien-Hsing; Chen, Hsiao-Yun; Hsieh, Shu-Ching; Lin, Chia-Liang; Tsai, Jen-Pi

    2015-09-01

    Nimbolide is a tetranortriterpenoid isolated from the leaves and flowers of Azadirachta indica which has been shown to exhibit anticancer, antioxidant, anti-inflammatory, and anti-invasive properties in a variety of cancer cells. However, the anti-tumor effect on human renal cell carcinoma (RCC) cells is unknown. In this study, we found that nimbolide treatment had a cytotoxic effect on 786-O and A-498 RCC cells in a dose-dependent manner. According to flow cytometric analysis, nimbolide treatment resulted in G2/M arrest in 786-O and A-498 cells accompanied with an increase in the phosphorylation status of p53, cdc2, cdc25c, and decreased expressions of cyclin A, cyclin B, cdc2, and cdc25c. Nimbolide also caused DNA damage in a dose-dependent manner as determined by comet assay and measurement of γ-H2AX. In addition, apoptotic cells were observed in an Annexin V-FITC/propidium iodide double-stained assay. The activities of caspase-3, -9, and poly ADP-ribose polymerase (PARP) were increased, and the expression of pro-caspase-8 was decreased in nimbolide-treated 786-O and A-498 cells. Western blot analysis revealed that the levels of intrinsic-related apoptotic proteins Bax and extrinsic-related proteins (DR5, CHOP) were significantly increased in nimbolide-treated 786-O and A-498 cells. In addition, the expressions of Bcl-2 and Mcl-1 were decreased in 786-O and A-498 cells after nimbolide treatment. We conclude that nimbolide can inhibit the growth of human RCC cells by inducing G2/M phase arrest by modulating cell cycle-related proteins and cell apoptosis by regulating intrinsic and extrinsic caspase signaling pathways. Nimbolide may be a promising therapeutic strategy for the treatment of RCC. PMID:25916210

  19. Curcumin Inhibits Glyoxalase 1—A Possible Link to Its Anti-Inflammatory and Anti-Tumor Activity

    PubMed Central

    Santel, Thore; Pflug, Gabi; Hemdan, Nasr Y. A.; Schäfer, Angelika; Hollenbach, Marcus; Buchold, Martin; Hintersdorf, Anja; Lindner, Inge; Otto, Andreas; Bigl, Marina; Oerlecke, Ilka; Hutschenreuter, Antje; Sack, Ulrich; Huse, Klaus; Groth, Marco; Birkemeyer, Claudia; Schellenberger, Wolfgang; Gebhardt, Rolf; Platzer, Mathias; Weiss, Thomas; Vijayalakshmi, Mookambeswaran A.; Krüger, Monika; Birkenmeier, Gerd

    2008-01-01

    Background Glyoxalases (Glo1 and Glo2) are involved in the glycolytic pathway by detoxifying the reactive methylglyoxal (MGO) into D-lactate in a two-step reaction using glutathione (GSH) as cofactor. Inhibitors of glyoxalases are considered as anti-inflammatory and anti-carcinogenic agents. The recent finding that various polyphenols modulate Glo1 activity has prompted us to assess curcumin's potency as an Glo1 inhibitor. Methodology/Principal Findings Cultures of whole blood cells and tumor cell lines (PC-3, JIM-1, MDA-MD 231 and 1321N1) were set up to investigate the effect of selected polyphenols, including curcumin, on the LPS-induced cytokine production (cytometric bead-based array), cell proliferation (WST-1 assay), cytosolic Glo1 and Glo2 enzymatic activity, apoptosis/necrosis (annexin V-FITC/propidium iodide staining; flow cytometric analysis) as well as GSH and ATP content. Results of enzyme kinetics revealed that curcumin, compared to the polyphenols quercetin, myricetin, kaempferol, luteolin and rutin, elicited a stronger competitive inhibitory effect on Glo1 (Ki = 5.1±1.4 µM). Applying a whole blood assay, IC50 values of pro-inflammatory cytokine release (TNF-α, IL-6, IL-8, IL-1β) were found to be positively correlated with the Ki-values of the aforementioned polyphenols. Moreover, whereas curcumin was found to hamper the growth of breast cancer (JIMT-1, MDA-MB-231), prostate cancer PC-3 and brain astrocytoma 1321N1 cells, no effect on growth or vitality of human primary hepatocytes was elucidated. Curcumin decreased D-lactate release by tumor cells, another clue for inhibition of intracellular Glo1. Conclusions/Significance The results described herein provide new insights into curcumin's biological activities as they indicate that inhibition of Glo1 by curcumin may result in non-tolerable levels of MGO and GSH, which, in turn, modulate various metabolic cellular pathways including depletion of cellular ATP and GSH content. This may account

  20. Paracrine Neuroprotective Effects of Neural Stem Cells on Glutamate-Induced Cortical Neuronal Cell Excitotoxicity

    PubMed Central

    Geranmayeh, Mohammad Hossein; Baghbanzadeh, Ali; Barin, Abbas; Salar-Amoli, Jamileh; Dehghan, Mohammad Mehdi; Rahbarghazi, Reza; Azari, Hassan

    2015-01-01

    Purpose: Glutamate is a major excitatory neurotransmitter in mammalian central nervous system. Excessive glutamate releasing overactivates its receptors and changes calcium homeostasis that in turn leads to a cascade of intracellular events causing neuronal degeneration. In current study, we used neural stem cells conditioned medium (NSCs-CM) to investigate its neuroprotective effects on glutamate-treated primary cortical neurons. Methods: Embryonic rat primary cortical cultures were exposed to different concentrations of glutamate for 1 hour and then they incubated with NSCs-CM. Subsequently, the amount of cell survival in different glutamate excitotoxic groups were measured after 24 h of incubation by trypan blue exclusion assay and MTT assay. Hoechst and propidium iodide were used for determining apoptotic and necrotic cell death pathways proportion and then the effect of NSCs-CM was investigated on this proportion. Results: NSCs conditioned medium increased viability rate of the primary cortical neurons after glutamate-induced excitotoxicity. Also we found that NSCs-CM provides its neuroprotective effects mainly by decreasing apoptotic cell death rate rather than necrotic cell death rate. Conclusion: The current study shows that adult neural stem cells could exert paracrine neuroprotective effects on cortical neurons following a glutamate neurotoxic insult. PMID:26819924

  1. Melatonin Protects Human Adipose-Derived Stem Cells from Oxidative Stress and Cell Death

    PubMed Central

    Han, Xiaolian; Sivakumaran, Priyadharshini; Lim, Shiang Y.; Morrison, Wayne A.

    2016-01-01

    Background Adipose-derived stem cells (ASCs) have applications in regenerative medicine based on their therapeutic potential to repair and regenerate diseased and damaged tissue. They are commonly subject to oxidative stress during harvest and transplantation, which has detrimental effects on their subsequent viability. By functioning as an antioxidant against free radicals, melatonin may exert cytoprotective effects on ASCs. Methods We cultured human ASCs in the presence of varying dosages of hydrogen peroxide and/or melatonin for a period of 3 hours. Cell viability and apoptosis were determined with propidium iodide and Hoechst 33342 staining under fluorescence microscopy. Results Hydrogen peroxide (1–2.5 mM) treatment resulted in an incremental increase in cell death. 2 mM hydrogen peroxide was thereafter selected as the dose for co-treatment with melatonin. Melatonin alone had no adverse effects on ASCs. Co-treatment of ASCs with melatonin in the presence of hydrogen peroxide protected ASCs from cell death in a dose-dependent manner, and afforded maximal protection at 100 µM (n=4, one-way analysis of variance P<0.001). Melatonin co-treated ASCs displayed significantly fewer apoptotic cells, as demonstrated by condensed and fragmented nuclei under fluorescence microscopy. Conclusions Melatonin possesses cytoprotective properties against oxidative stress in human ASCs and might be a useful adjunct in fat grafting and cell-assisted lipotransfer. PMID:27218020

  2. Time-Lapse Imaging of Cell Death.

    PubMed

    Wallberg, Fredrik; Tenev, Tencho; Meier, Pascal

    2016-03-01

    The best approach to distinguish between necrosis and apoptosis is time-lapse video microscopy. This technique enables a biological process to be photographed at regular intervals over a period, which may last from a few hours to several days, and can be applied to cells in culture or in vivo. We have established two time-lapse microscopy methods based on different ways of calculating cell death: semiautomated and automated. In the semiautomated approach, cell death can be visualized by staining with combinations of Alexa Fluor 647-conjugated Annexin V and Sytox Green (SG), or Annexin V(FITC) and Propidium iodide (PI). The automated method is similar except that all cells are labeled with dyes. This allows faster quantification of data. To this end Cell Tracker Green is used to label all cells at time zero in combination with PI and Alexa Fluor 647-conjugated Annexin V. Necrotic cell death is accompanied by either simultaneous labeling with Annexin V and PI or SG (double-positive), or direct PI or SG staining. Additionally, necrotic cells display characteristic morphology, such as cytoplasmic swelling. In contrast to necrosis where membrane permeabilization is an early event, cells that die by apoptosis lose their membrane permeability relatively late. Therefore, the time between Annexin V staining and PI or SG uptake (double-positive) can be used to distinguish necrosis from apoptosis. This protocol describes the analysis of cell death by time-lapse imaging of HT1080 and L929 cells stained with these dyes, but it can be readily adapted to other cell types of interest. PMID:26933245

  3. Allyl-isatin suppresses cell viability, induces cell cycle arrest, and promotes cell apoptosis in hepatocellular carcinoma HepG2 cells.

    PubMed

    Bian, Weihua; An, Yukuan; Qu, Huiqing; Yang, Yue; Yang, Junhou; Xu, Yanyan

    2016-06-01

    The anticancer effect of the newly synthesized isatin derivative, N-allyl-isatin (Allyl-I), was evaluated in vitro with human hepatocellular carcinoma HepG2 cells. Cell viability was detected by cell counting kit-8 (CCK8) assay. Acridine orange (AO)/ethidium bromide (EB) double staining was used to observe the cell morphology. Flow cytometry was used to assess the effects of Allyl-I on the cell cycle, apoptosis rate, and mitochondrial membrane potential (MMP). Western blot analysis was performed to detect the influence of Ally1-I on the expression of cytochrome c (cyt c), Bax, Bcl-2, and cleaved caspase-3. Allyl-I significantly inhibited HepG2 cell viability in a time- and dose-dependent manner. Allyl-I can induce cell cycle arrest in HepG2 cells at the G2/M phase. Apoptotic nuclear morphological changes were observed after AO/EB double staining. Fluorescein isothiocyanate-conjugated Annexin V (Annexin V-FITC) and propidium iodide (PI) double staining showed that the apoptotic rates significantly increased in the presence of Allyl-I. Rhodamine 123 staining indicated that Allyl-I can decrease the MMP. Allyl-I also altered the expression of mitochondrial apoptosis-related proteins. Protein levels of cyt c and cleaved caspase-3 were upregulated following Allyl-I treatment. By contrast, the Bcl-2/Bax ratio decreased. Results suggest that Allyl-I suppresses cell viability, induces cell cycle arrest, and promotes cell apoptosis in HepG2 cells. Furthermore, the induction of apoptosis might be correlated with the mitochondrial pathway. PMID:26945926

  4. Cytotoxicity and genotoxicity of a trypanocidal drug quinapyramine sulfate loaded-sodium alginate nanoparticles in mammalian cells.

    PubMed

    Manuja, Anju; Kumar, Balvinder; Chopra, Meenu; Bajaj, Anshu; Kumar, Rajender; Dilbaghi, Neeraj; Kumar, Sandeep; Singh, Sandeep; Riyesh, T; Yadav, Suresh C

    2016-07-01

    We synthesized quinapyramine sulfate loaded-sodium alginate nanoparticles (QS-NPs) to reduce undesirable toxic effects of QS against the parasite Trypanosoma evansi, a causative agent of trypanosomosis. To determine the safety of the formulated nanoparticles, biocompatibility of QS-NPs was determined using Vero, Hela cell lines and horse erythrocytes in a dose-dependent manner. Our experiments unveiled a concentration-dependent safety/cytotoxicity (metabolic activity), genotoxicity (DNA damage, chromosomal aberrations), production of reactive oxygen species and hemolysis in QS-NPs treated cells. Annexin-V propidium iodide (PI) staining showed no massive apoptosis or necrosis. However, at very high doses (more than 300 times than the effective doses), we observed more toxicity in QS-NPs treated cells as compared to QS treated cells. QS-NPs were safe at effective trypanocidal doses and even at doses several times higher than the effective dose. PMID:27000439

  5. Islet Stellate Cells Isolated from Fibrotic Islet of Goto-Kakizaki Rats Affect Biological Behavior of Beta-Cell.

    PubMed

    Li, Feng-Fei; Chen, Bi-Jun; Li, Wei; Li, Ling; Zha, Min; Zhou, S; Bachem, M G; Sun, Zi-Lin

    2016-01-01

    We previously isolated islet stellate cells (ISCs) from healthy Wistar rat islets. In the present study, we isolated "already primed by diabetic environment" ISCs from islets of Goto-Kakizaki rats, determined the gene profile of these cells, and assessed the effects of these ISCs on beta-cell function and survival. We detected gene expression of ISCs by digital gene expression. INS-1 cell proliferation, apoptosis, and insulin production were measured after being treated with ISCs supernatant (SN). We observed the similar expression pattern of ISCs and PSCs, but 1067 differentially expressed genes. Insulin production in INS-1 cells cultured with ISC-SN was significantly reduced. The 5-ethynyl-2'-deoxyuridine-positive INS-1 cells treated with ISC-SN were decreased. Propidium iodide- (PI-) positive INS-1 cells were 2.6-fold higher than those in control groups. Caspase-3 activity was increased. In conclusion, ISCs presented in fibrotic islet of GK rats might be special PSCs, which impaired beta-cell function and proliferation and increased beta-cell apoptosis. PMID:26697502

  6. Effect of hyperthermic CO2-treated dendritic cell-derived exosomes on the human gastric cancer AGS cell line

    PubMed Central

    WANG, JINLIN; WANG, ZHIYONG; MO, YANXIA; ZENG, ZHAOHUI; WEI, PEI; LI, TAO

    2015-01-01

    The aim of the present study was to determine the antitumor effects of hyperthermic CO2 (HT-CO2)-treated dendritic cell (DC)-derived exosomes (Dex) on human gastric cancer AGS cells. Mouse-derived DCs were incubated in HT-CO2 at 43°C for 4 h. The exosomes in the cell culture supernatant were then isolated. Cell proliferation was analyzed using the cell counting kit-8 (CCK-8) assay. Cell apoptosis was observed using flow cytometry, Hoechst 33258 staining and the analysis of caspase-3 activity. In addition, the proliferation of tumor cells was evaluated in xenotransplant nude mice. HT-CO2 markedly inhibited cell proliferation, as assessed by the CCK-8 assay, and also induced apoptosis in a time-dependent manner, as demonstrated by Annexin V/propidium iodide flow cytometry, caspase-3 activity and morphological analysis using Hoechst fluorescent dye. It was also revealed that HT-CO2-treated Dex decreased the expression of heat shock protein 70 and inhibited tumor growth in nude mice. In conclusion, HT-CO2 exerted an efficacious immune-enhancing effect on DCs. These findings may provide a novel strategy for the elimination of free cancer cells during laparoscopic resection. However, the potential cellular mechanisms underlying this process require further investigation. PMID:26170979

  7. Hoechst fluorescence intensity can be used to separate viable bromodeoxyuridine-labeled cells from viable non-bromodeoxyuridine-labeled cells

    NASA Technical Reports Server (NTRS)

    Mozdziak, P. E.; Pulvermacher, P. M.; Schultz, E.; Schell, K.

    2000-01-01

    BACKGROUND: 5-Bromo-2'-deoxyuridine (BrdU) is a powerful compound to study the mitotic activity of a cell. Most techniques that identify BrdU-labeled cells require conditions that kill the cells. However, the fluorescence intensity of the membrane-permeable Hoechst dyes is reduced by the incorporation of BrdU into DNA, allowing the separation of viable BrdU positive (BrdU+) cells from viable BrdU negative (BrdU-) cells. METHODS: Cultures of proliferating cells were supplemented with BrdU for 48 h and other cultures of proliferating cells were maintained without BrdU. Mixtures of viable BrdU+ and viable BrdU- cells from the two proliferating cultures were stained with Hoechst 33342. The viable BrdU+ and BrdU- cells were sorted into different fractions from a mixture of BrdU+ and BrdU- cells based on Hoechst fluorescence intensity and the ability to exclude the vital dye, propidium iodide. Subsequently, samples from the original mixture, the sorted BrdU+ cell population, and the sorted BrdU- cell population were immunostained using an anti-BrdU monoclonal antibody and evaluated using flow cytometry. RESULTS: Two mixtures consisting of approximately 55% and 69% BrdU+ cells were sorted into fractions consisting of greater than 93% BrdU+ cells and 92% BrdU- cells. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. CONCLUSIONS: Hoechst fluorescence intensity in combination with cell sorting is an effective tool to separate viable BrdU+ from viable BrdU- cells for further study. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. Copyright 2000 Wiley-Liss, Inc.

  8. A rapid procedure for flow cytometric DNA analysis in cultures of normal and transformed epidermal cells.

    PubMed

    Tennenbaum, T; Giloh, H; Fusenig, N E; Kapitulnik, J

    1988-06-01

    A simple, rapid, and highly reproducible procedure for flow cytometric DNA analysis has been adapted for studying cell cycle kinetics in epidermal cell cultures. The preparation of cell nuclei and their staining with the fluorescent dye propidium iodide were performed directly on the culture dish, without prior suspension and fixation of the cells. Singly dispersed nuclei were produced by mild trypsinization of cells in the presence of the nonionic detergent Nonidet P-40 and spermine. The culture dishes could be kept frozen for prolonged periods of time before trypsinization and staining, without affecting either the recovery of nuclei or the cell cycle distribution profiles. This remarkable stability of cell nuclei greatly simplified the analysis of multiple samples in cell cycle kinetic studies. This method was used to analyze the cell cycle distribution in cultures of normal and transformed mouse epidermal cells, human colon carcinoma cells, primary bovine aortic endothelial cells, and fibroblastic and myogenic cell lines. This procedure should be very useful in studying growth kinetics, differentiation, and transformation of epidermal as well as other adherent cell types. PMID:2453587

  9. Cell Electrosensitization Exists Only in Certain Electroporation Buffers

    PubMed Central

    Dermol, Janja; Pakhomova, Olga N.; Pakhomov, Andrei G.; Miklavčič, Damijan

    2016-01-01

    Electroporation-induced cell sensitization was described as the occurrence of a delayed hypersensitivity to electric pulses caused by pretreating cells with electric pulses. It was achieved by increasing the duration of the electroporation treatment at the same cumulative energy input. It could be exploited in electroporation-based treatments such as electrochemotherapy and tissue ablation with irreversible electroporation. The mechanisms responsible for cell sensitization, however, have not yet been identified. We investigated cell sensitization dynamics in five different electroporation buffers. We split a pulse train into two trains varying the delay between them and measured the propidium uptake by fluorescence microscopy. By fitting the first-order model to the experimental results, we determined the uptake due to each train (i.e. the first and the second) and the corresponding resealing constant. Cell sensitization was observed in the growth medium but not in other tested buffers. The effect of pulse repetition frequency, cell size change, cytoskeleton disruption and calcium influx do not adequately explain cell sensitization. Based on our results, we can conclude that cell sensitization is a sum of several processes and is buffer dependent. Further research is needed to determine its generality and to identify underlying mechanisms. PMID:27454174

  10. Cell Electrosensitization Exists Only in Certain Electroporation Buffers.

    PubMed

    Dermol, Janja; Pakhomova, Olga N; Pakhomov, Andrei G; Miklavčič, Damijan

    2016-01-01

    Electroporation-induced cell sensitization was described as the occurrence of a delayed hypersensitivity to electric pulses caused by pretreating cells with electric pulses. It was achieved by increasing the duration of the electroporation treatment at the same cumulative energy input. It could be exploited in electroporation-based treatments such as electrochemotherapy and tissue ablation with irreversible electroporation. The mechanisms responsible for cell sensitization, however, have not yet been identified. We investigated cell sensitization dynamics in five different electroporation buffers. We split a pulse train into two trains varying the delay between them and measured the propidium uptake by fluorescence microscopy. By fitting the first-order model to the experimental results, we determined the uptake due to each train (i.e. the first and the second) and the corresponding resealing constant. Cell sensitization was observed in the growth medium but not in other tested buffers. The effect of pulse repetition frequency, cell size change, cytoskeleton disruption and calcium influx do not adequately explain cell sensitization. Based on our results, we can conclude that cell sensitization is a sum of several processes and is buffer dependent. Further research is needed to determine its generality and to identify underlying mechanisms. PMID:27454174

  11. Oridonin inhibits BxPC-3 cell growth through cell apoptosis.

    PubMed

    Xu, Bin; Shen, Wen; Liu, Xing; Zhang, Ting; Ren, Jun; Fan, Yongjun; Xu, Jian

    2015-03-01

    Oridonin, an ent-kaurene diterpenoid extracted from the traditional Chinese herb Rabdosia rubescens, has multiple biological and pharmaceutical functions and has been used clinically for many years. While the antitumor function of oridonin has been corroborated by numerous lines of evidence, its anticancer mechanism has not been well documented. In this study, the pancreatic cancer cell line BxPC-3 was used as a model to investigate a possible anticancer mechanism of oridonin through examining its effects on cell viability. The results showed that oridonin affected cell viability in a time- and dose-dependent manner. After exposure to different oridonin concentrations, growth rates and cell cycle arrest of BxPC-3 cells were significantly reduced compared with untreated cells, suggesting its effects on proliferation inhibition. Detailed signaling pathway analysis by western blot analysis revealed that low-dose oridonin treatment inhibited BxPC-3 cell proliferation by up-regulating p53 and down-regulating cyclin-dependent kinase 1 (CDK1), which led to cell cycle arrest in the G2/M phase. A high-dose oridonin not only arrested BxPC-3 cells in the G2/M phase but also induced cell accumulation in the S phase, presumably through γH2AX up-regulation and DNA damage. In addition, our results showed that a cell subpopulation was stained with propidium iodide after oridonin treatment. Protein quantification showed that cleaved poly(ADP-ribose) polymerase (PARP) expression was increased after a high-dose oridonin treatment, especially after long-term exposure. Accompanied by the increased level of deactivated PARP in BxPC-3 cells, the apoptosis initiators caspase-3 and caspase-7 expressions were also significantly increased, suggesting that caspase-mediated apoptosis contributed to cell death. PMID:25651847

  12. Necrosis of lung epithelial cells during infection with Mycobacterium tuberculosis is preceded by cell permeation.

    PubMed

    Dobos, K M; Spotts, E A; Quinn, F D; King, C H

    2000-11-01

    Mycobacterium tuberculosis establishes infection, progresses towards disease, and is transmitted from the alveolus of the lung. However, the role of the alveolar epithelium in any of these pathogenic processes of tuberculosis is unclear. In this study, lung epithelial cells (A549) were used as a model in which to examine cytotoxicity during infection with either virulent or avirulent mycobacteria in order to further establish the role of the lung epithelium during tuberculosis. Infection of A549 cells with M. tuberculosis strains Erdman and CDC1551 demonstrated significant cell monolayer clearing, whereas infection with either Mycobacterium bovis BCG or Mycobacterium smegmatis LR222 did not. Clearing of M. tuberculosis-infected A549 cells correlated to necrosis, not apoptosis. Treatment of M. tuberculosis-infected A549 cells with streptomycin, but not cycloheximide, demonstrated a significant reduction in the necrosis of A549 cell monolayers. This mycobacterium-induced A549 necrosis did not correlate to higher levels of intracellular or extracellular growth by the mycobacteria during infection. Staining of infected cells with propidium iodide demonstrated that M. tuberculosis induced increased permeation of A549 cell membranes within 24 h postinfection. Quantitation of lactate dehydrogenase (LDH) release from infected cells further demonstrated that cell permeation was specific to M. tuberculosis infection and correlated to A549 cellular necrosis. Inactivated M. tuberculosis or its subcellular fractions did not result in A549 necrosis or LDH release. These studies demonstrate that lung epithelial cell cytotoxicity is specific to infection by virulent mycobacteria and is caused by cellular necrosis. This necrosis is not a direct correlate of mycobacterial growth or of the expression of host cell factors, but is preceded by permeation of the A549 cell membrane and requires infection with live bacilli. PMID:11035739

  13. Active Targeting to Osteosarcoma Cells and Apoptotic Cell Death Induction by the Novel Lectin Eucheuma serra Agglutinin Isolated from a Marine Red Alga

    PubMed Central

    Hayashi, Keita; Walde, Peter; Miyazaki, Tatsuhiko; Sakayama, Kenshi; Nakamura, Atsushi; Kameda, Kenji; Masuda, Seizo; Umakoshi, Hiroshi; Kato, Keiichi

    2012-01-01

    Previously, we demonstrated that the novel lectin Eucheuma serra agglutinin from a marine red alga (ESA) induces apoptotic cell death in carcinoma. We now find that ESA induces apoptosis also in the case of sarcoma cells. First, propidium iodide assays with OST cells and LM8 cells showed a decrease in cell viability after addition of ESA. With 50 μg/ml ESA, the viabilities after 24 hours decreased to 54.7 ± 11.4% in the case of OST cells and to 41.7 ± 12.3% for LM8 cells. Second, using fluorescently labeled ESA and flow cytometric and fluorescence microscopic measurements, it could be shown that ESA does not bind to cells that were treated with glycosidases, indicating importance of the carbohydrate chains on the surface of the cells for efficient ESA-cell interactions. Third, Span 80 vesicles with surface-bound ESA as active targeting ligand were shown to display sarcoma cell binding activity, leading to apoptosis and complete OST cell death after 48 hours at 2 μg/ml ESA. The findings indicate that Span 80 vesicles with surface-bound ESA are a potentially useful drug delivery system not only for the treatment of carcinoma but also for the treatment of osteosarcoma. PMID:23346404

  14. Active Targeting to Osteosarcoma Cells and Apoptotic Cell Death Induction by the Novel Lectin Eucheuma serra Agglutinin Isolated from a Marine Red Alga.

    PubMed

    Hayashi, Keita; Walde, Peter; Miyazaki, Tatsuhiko; Sakayama, Kenshi; Nakamura, Atsushi; Kameda, Kenji; Masuda, Seizo; Umakoshi, Hiroshi; Kato, Keiichi

    2012-01-01

    Previously, we demonstrated that the novel lectin Eucheuma serra agglutinin from a marine red alga (ESA) induces apoptotic cell death in carcinoma. We now find that ESA induces apoptosis also in the case of sarcoma cells. First, propidium iodide assays with OST cells and LM8 cells showed a decrease in cell viability after addition of ESA. With 50 μg/ml ESA, the viabilities after 24 hours decreased to 54.7 ± 11.4% in the case of OST cells and to 41.7 ± 12.3% for LM8 cells. Second, using fluorescently labeled ESA and flow cytometric and fluorescence microscopic measurements, it could be shown that ESA does not bind to cells that were treated with glycosidases, indicating importance of the carbohydrate chains on the surface of the cells for efficient ESA-cell interactions. Third, Span 80 vesicles with surface-bound ESA as active targeting ligand were shown to display sarcoma cell binding activity, leading to apoptosis and complete OST cell death after 48 hours at 2 μg/ml ESA. The findings indicate that Span 80 vesicles with surface-bound ESA are a potentially useful drug delivery system not only for the treatment of carcinoma but also for the treatment of osteosarcoma. PMID:23346404

  15. Lithium increases proliferation of hippocampal neural stem/progenitor cells and rescues irradiation-induced cell cycle arrest in vitro.

    PubMed

    Zanni, Giulia; Di Martino, Elena; Omelyanenko, Anna; Andäng, Michael; Delle, Ulla; Elmroth, Kecke; Blomgren, Klas

    2015-11-10

    Radiotherapy in children causes debilitating cognitive decline, partly linked to impaired neurogenesis. Irradiation targets primarily cancer cells but also endogenous neural stem/progenitor cells (NSPCs) leading to cell death or cell cycle arrest. Here we evaluated the effects of lithium on proliferation, cell cycle and DNA damage after irradiation of young NSPCs in vitro.NSPCs were treated with 1 or 3 mM LiCl and we investigated proliferation capacity (neurosphere volume and bromodeoxyuridine (BrdU) incorporation). Using flow cytometry, we analysed apoptosis (annexin V), cell cycle (propidium iodide) and DNA damage (γH2AX) after irradiation (3.5 Gy) of lithium-treated NSPCs.Lithium increased BrdU incorporation and, dose-dependently, the number of cells in replicative phase as well as neurosphere growth. Irradiation induced cell cycle arrest in G1 and G2/M phases. Treatment with 3 mM LiCl was sufficient to increase NSPCs in S phase, boost neurosphere growth and reduce DNA damage. Lithium did not affect the levels of apoptosis, suggesting that it does not rescue NSPCs committed to apoptosis due to accumulated DNA damage.Lithium is a very promising candidate for protection of the juvenile brain from radiotherapy and for its potential to thereby improve the quality of life for those children who survive their cancer. PMID:26397227

  16. Lithium increases proliferation of hippocampal neural stem/progenitor cells and rescues irradiation-induced cell cycle arrest in vitro

    PubMed Central

    Omelyanenko, Anna; Andäng, Michael; Delle, Ulla; Elmroth, Kecke; Blomgren, Klas

    2015-01-01

    Radiotherapy in children causes debilitating cognitive decline, partly linked to impaired neurogenesis. Irradiation targets primarily cancer cells but also endogenous neural stem/progenitor cells (NSPCs) leading to cell death or cell cycle arrest. Here we evaluated the effects of lithium on proliferation, cell cycle and DNA damage after irradiation of young NSPCs in vitro. NSPCs were treated with 1 or 3 mM LiCl and we investigated proliferation capacity (neurosphere volume and bromodeoxyuridine (BrdU) incorporation). Using flow cytometry, we analysed apoptosis (annexin V), cell cycle (propidium iodide) and DNA damage (γH2AX) after irradiation (3.5 Gy) of lithium-treated NSPCs. Lithium increased BrdU incorporation and, dose-dependently, the number of cells in replicative phase as well as neurosphere growth. Irradiation induced cell cycle arrest in G1 and G2/M phases. Treatment with 3 mM LiCl was sufficient to increase NSPCs in S phase, boost neurosphere growth and reduce DNA damage. Lithium did not affect the levels of apoptosis, suggesting that it does not rescue NSPCs committed to apoptosis due to accumulated DNA damage. Lithium is a very promising candidate for protection of the juvenile brain from radiotherapy and for its potential to thereby improve the quality of life for those children who survive their cancer. PMID:26397227

  17. Adaptation to alkalosis induces cell cycle delay and apoptosis in cortical collecting duct cells: role of Aquaporin-2.

    PubMed

    Rivarola, Valeria; Flamenco, Pilar; Melamud, Luciana; Galizia, Luciano; Ford, Paula; Capurro, Claudia

    2010-08-01

    Collecting ducts (CD) not only constitute the final site for regulating urine concentration by increasing apical membrane Aquaporin-2 (AQP2) expression, but are also essential for the control of acid-base status. The aim of this work was to examine, in renal cells, the effects of chronic alkalosis on cell growth/death as well as to define whether AQP2 expression plays any role during this adaptation. Two CD cell lines were used: WT- (not expressing AQPs) and AQP2-RCCD(1) (expressing apical AQP2). Our results showed that AQP2 expression per se accelerates cell proliferation by an increase in cell cycle progression. Chronic alkalosis induced, in both cells lines, a time-dependent reduction in cell growth. Even more, cell cycle movement, assessed by 5-bromodeoxyuridine pulse-chase and propidium iodide analyses, revealed a G2/M phase cell accumulation associated with longer S- and G2/M-transit times. This G2/M arrest is paralleled with changes consistent with apoptosis. All these effects appeared 24 h before and were always more pronounced in cells expressing AQP2. Moreover, in AQP2-expressing cells, part of the observed alkalosis cell growth decrease is explained by AQP2 protein down-regulation. We conclude that in CD cells alkalosis causes a reduction in cell growth by cell cycle delay that triggers apoptosis as an adaptive reaction to this environment stress. Since cell volume changes are prerequisite for the initiation of cell proliferation or apoptosis, we propose that AQP2 expression facilitates cell swelling or shrinkage leading to the activation of channels necessary to the control of these processes. PMID:20432437

  18. Different Cell Viability Assays Reveal Inconsistent Results After Bleomycin Electrotransfer In Vitro.

    PubMed

    Jakštys, Baltramiejus; Ruzgys, Paulius; Tamošiūnas, Mindaugas; Šatkauskas, Saulius

    2015-10-01

    The aim of this study was to compare different and commonly used cell viability assays after CHO cells treatment with anticancer drug bleomycin (20 nM), high voltage (HV) electric pulses (4 pulses, 1200 V/cm, 100 µs, 1 Hz), and combination of bleomycin and HV electric pulses. Cell viability was measured using clonogenic assay, propidium iodide (PI) assay, MTT assay, and employing flow cytometry modality to precisely count cells in definite volume of the sample (flow cytometry assay). Results showed that although clonogenic cell viability drastically decreased correspondingly to 57 and 3 % after cell treatment either with HV pulses or combination of bleomycin and HV pulses (bleomycin electrotransfer), PI assay performed ~15 min after the treatments indicated nearly 100 % cell viability. MTT assay performed at 6-72 h time points after these treatments revealed that MTT cell viability is highly dependent on evaluation time point and decreased with later evaluation time points. Nevertheless, in comparison to clonogenic cell viability, MTT cell viability after bleomycin electrotransfer at all testing time points was significantly higher. Flow cytometry assay if used at later times, 2-3 days after the treatment, allowed reliable evaluation of cell viability. In overall, our results showed that in order to estimate cell viability after cell treatment with combination of the bleomycin and electroporation the most reliable method is clonogenic assay. Improper use of PI and MTT assays can lead to misinterpretation of the experimental results. PMID:26077843

  19. Gene transfer and protein dynamics in stem cells using single cell electroporation in a microfluidic device.

    PubMed

    Valero, A; Post, J N; van Nieuwkasteele, J W; Ter Braak, P M; Kruijer, W; van den Berg, A

    2008-01-01

    There is great interest in genetic modification of bone marrow-derived mesenchymal stem cells (MSC), not only for research purposes but also for use in (autologous) patient-derived-patient-used transplantations. A major drawback of bulk methods for genetic modifications of (stem) cells, like bulk-electroporation, is its limited yield of DNA transfection (typically then 10%). This is even more limited when cells are present at very low numbers, as is the case for stem cells. Here we present an alternative technology to transfect cells with high efficiency (>75%), based on single cell electroporation in a microfluidic device. In a first experiment we show that we can successfully transport propidium iodide (PI) into single mouse myoblastic C2C12 cells. Subsequently, we show the use of this microfluidic device to perform successful electroporation of single mouse myoblastic C2C12 cells and single human MSC with vector DNA encoding a green fluorescent-erk1 fusion protein (EGFP-ERK1 (MAPK3)). Finally, we performed electroporation in combination with live imaging of protein expression and dynamics in response to extracellular stimuli, by fibroblast growth factor (FGF-2). We observed nuclear translocation of EGFP-ERK1 in both cell types within 15 min after FGF-2 stimulation. Due to the successful and promising results, we predict that microfluidic devices can be used for highly efficient small-scale 'genetic modification' of cells, and biological experimentation, offering possibilities to study cellular processes at the single cell level. Future applications might be small-scale production of cells for therapeutic application under controlled conditions. PMID:18094762

  20. A Partially Purified Acinetobacter baumannii Phage Preparation Exhibits no Cytotoxicity in 3T3 Mouse Fibroblast Cells

    PubMed Central

    Henein, Alexandra E.; Hanlon, Geoffrey W.; Cooper, Callum J.; Denyer, Stephen P.; Maillard, Jean-Yves

    2016-01-01

    A surge in the level and scale of antibiotic resistance has prompted renewed interest in the application of bacteriophages to treat bacterial infections. However, concerns still exist over their efficacy and safety. Acinetobacter baumannii phage BS46, a member of the family Myoviridae, has previously been shown to be effective in murine models. The cytotoxic effect of this phage was evaluated in mouse fibroblast 3T3 cells using four different assays: trypan blue; staining with Hoechst and propidium iodide; lactate dehydrogenase release; and the MTS assay. The addition of phage concentrations up to 2 × 109 pfu/mL showed little to no impact on the viability of 3T3 cells after 24 h exposure using the different assays. This study demonstrates that phage BS46 is non-cytotoxic to 3T3 cells using four different assays and that appropriate quality assurance protocols for phage therapeutics are required. PMID:27536286

  1. A Partially Purified Acinetobacter baumannii Phage Preparation Exhibits no Cytotoxicity in 3T3 Mouse Fibroblast Cells.

    PubMed

    Henein, Alexandra E; Hanlon, Geoffrey W; Cooper, Callum J; Denyer, Stephen P; Maillard, Jean-Yves

    2016-01-01

    A surge in the level and scale of antibiotic resistance has prompted renewed interest in the application of bacteriophages to treat bacterial infections. However, concerns still exist over their efficacy and safety. Acinetobacter baumannii phage BS46, a member of the family Myoviridae, has previously been shown to be effective in murine models. The cytotoxic effect of this phage was evaluated in mouse fibroblast 3T3 cells using four different assays: trypan blue; staining with Hoechst and propidium iodide; lactate dehydrogenase release; and the MTS assay. The addition of phage concentrations up to 2 × 10(9) pfu/mL showed little to no impact on the viability of 3T3 cells after 24 h exposure using the different assays. This study demonstrates that phage BS46 is non-cytotoxic to 3T3 cells using four different assays and that appropriate quality assurance protocols for phage therapeutics are required. PMID:27536286

  2. Bis(phenylidenebenzeneamine)-1-disulfide Derivatives Induce Autophagy in Melanoma Cells Through a Mitochondria-mediated Pathway.

    PubMed

    Hu, Wan-Ping; Hsu, Chia-Chen; Wang, Ya-Ching; Senadi, Gopal Chandru; Kuo, Kung-Kai; Jen, Jen-Fon; Wang, Jeh-Jeng

    2015-11-01

    We have previously reported the efficient synthesis of bis(phenylidenebenzeneamine)-1-disulfide derivatives 1-8. In the present article, we delineated the signaling pathways involved in the anticancer effects of compound 2 on melanoma cells. The treatment of melanoma cells with compound 2 resulted in ROS generation, a decrease in ΔΨmt, ATP, and protein expression of mitochondrial respiratory chain subunits. In addition, no significant apoptotic or necrotic effect was seen following compound 2 treatment using an annexin V and propidium iodide (PI) double-staining. Nevertheless, autophagy-related proteins LC3 and Beclin 1 were enhanced by compound 2. Furthermore, compound 2 was also shown to reduce murine melanoma size in a mouse model. We revealed a novel bio-evaluation of bis(phenylidenebenzeneamine)-1-disulfide derivatives anti-tumor proliferation mechanism and suggest that these agents may have potential chemotherapeutic activity for melanoma cells. PMID:26504032

  3. Leptospermum flavescens Constituent-LF1 Causes Cell Death through the Induction of Cell Cycle Arrest and Apoptosis in Human Lung Carcinoma Cells

    PubMed Central

    Navanesan, Suerialoasan; Abdul Wahab, Norhanom; Manickam, Sugumaran; Sim, Kae Shin

    2015-01-01

    Leptospermum flavescens Sm. (Myrtaceae), locally known as ‘Senna makki’ is a smallish tree that is widespread and recorded to naturally occur in the montane regions above 900 m a.s.l from Burma to Australia. Although the species is recorded to be used traditionally to treat various ailments, there is limited data on biological and chemical investigations of L. flavescens. The aim of the present study was to investigate and understand the ability of L. flavescens in inducing cell death in lung cancer cells. The cytotoxic potentials of the extraction yields (methanol, hexane, ethyl acetate and water extracts as wells as a semi pure fraction, LF1) were evaluated against two human non-small cell lung carcinoma cell lines (A549 and NCI-H1299) using the MTT assay. LF1 showed the greatest cytotoxic effect against both cell lines with IC50 values of 7.12 ± 0.07 and 9.62 ± 0.50 μg/ml respectively. LF1 treated cells showed a sub-G1 region in the cell cycle analysis and also caused the presence of apoptotic morphologies in cells stained with acridine orange and ethidium bromide. Treatment with LF1 manifested an apoptotic population in cells that were evaluated using the Annexin V/ propidium iodide assay. Increasing dosage of LF1 caused a rise in the presence of activated caspase-3 enzymes in treated cells. Blockage of cell cycle progression was also observed in LF1-treated cells. These findings suggest that LF1 induces apoptosis and cell cycle arrest in treated lung cancer cells. Further studies are being conducted to isolate and identify the active compound as well to better understand the mechanism involved in inducing cell death. PMID:26287817

  4. A Natural Triterpene Derivative from Euphorbia kansui Inhibits Cell Proliferation and Induces Apoptosis against Rat Intestinal Epithelioid Cell Line in Vitro

    PubMed Central

    Cheng, Fangfang; Yang, Yanjing; Zhang, Li; Cao, Yudan; Yao, Weifeng; Tang, Yuping; Ding, Anwei

    2015-01-01

    Kansenone is a triterpene from the root of the traditional Chinese medicine, Euphorbia kansui. However, kansenone exerts serious toxicity, but the exact mechanism was not clear. In this work, the effects of kansenone on cell proliferation, cell cycle, cell damage, and cell apoptosis were investigated. The suppression of cell proliferation was assessed via the colorimetric MTT assay, and cell morphology was visualized via inverted microscopy after IEC-6 cells were incubated with different concentrations of kansenone. Reactive oxygen species (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA) content were detected for evaluating cell damage. RNase/propidium iodide (PI) labeling for evaluation of cell cycle distribution was performed by flow cytometry analysis. Annexin V-fluorescein isothiocyanate (FITC)/PI and Hoechst 33342/Annexin V-FITC/PI staining assay for cell apoptosis detection were performed using confocal laser scanning microscopy and high content screening. Moreover, apoptosis induction was further confirmed by transmission electron microscope (TEM) and JC-1 mitochondrial membrane potential, western blot and RT-PCR analysis. The results demonstrated that kansenone exerted high cytotoxicity, induced cell arrest at G0/G1 phase, and caused mitochondria damage. In addition, kansenone could up-regulate the apoptotic proteins Bax, AIF, Apaf-1, cytochrome c, caspase-3, caspase-9, caspase-8, FasR, FasL, NF-κB, and TNFR1 mRNA expression levels, and down-regulate the anti-apoptotic Bcl-2 family proteins, revealing that kansenone induces apoptosis through both the death receptor and mitochondrial pathways. PMID:26274958

  5. A Natural Triterpene Derivative from Euphorbia kansui Inhibits Cell Proliferation and Induces Apoptosis against Rat Intestinal Epithelioid Cell Line in Vitro.

    PubMed

    Cheng, Fangfang; Yang, Yanjing; Zhang, Li; Cao, Yudan; Yao, Weifeng; Tang, Yuping; Ding, Anwei

    2015-01-01

    Kansenone is a triterpene from the root of the traditional Chinese medicine, Euphorbia kansui. However, kansenone exerts serious toxicity, but the exact mechanism was not clear. In this work, the effects of kansenone on cell proliferation, cell cycle, cell damage, and cell apoptosis were investigated. The suppression of cell proliferation was assessed via the colorimetric MTT assay, and cell morphology was visualized via inverted microscopy after IEC-6 cells were incubated with different concentrations of kansenone. Reactive oxygen species (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA) content were detected for evaluating cell damage. RNase/propidium iodide (PI) labeling for evaluation of cell cycle distribution was performed by flow cytometry analysis. Annexin V-fluorescein isothiocyanate (FITC)/PI and Hoechst 33342/Annexin V-FITC/PI staining assay for cell apoptosis detection were performed using confocal laser scanning microscopy and high content screening. Moreover, apoptosis induction was further confirmed by transmission electron microscope (TEM) and JC-1 mitochondrial membrane potential, western blot and RT-PCR analysis. The results demonstrated that kansenone exerted high cytotoxicity, induced cell arrest at G0/G1 phase, and caused mitochondria damage. In addition, kansenone could up-regulate the apoptotic proteins Bax, AIF, Apaf-1, cytochrome c, caspase-3, caspase-9, caspase-8, FasR, FasL, NF-κB, and TNFR1 mRNA expression levels, and down-regulate the anti-apoptotic Bcl-2 family proteins, revealing that kansenone induces apoptosis through both the death receptor and mitochondrial pathways. PMID:26274958

  6. Umbelliferone exhibits anticancer activity via the induction of apoptosis and cell cycle arrest in HepG2 hepatocellular carcinoma cells.

    PubMed

    Yu, Shi-Min; Hu, Dong-Hui; Zhang, Jian-Jun

    2015-09-01

    Hepatocellular carcinoma (HCC) is a highly malignant tumor, associated with poor patient prognoses, and high rates of morbidity and mortality. To date, the therapeutic strategies available for the treatment of HCC remain limited. The present study aimed to elucidate the anticancer activity of umbelliferone, a naturally occurring coumarin derivative isolated from Ferula communis, against the HepG2 HCC cell line. A 3‑(4,5‑dimthylthaizol‑2‑yl)‑2,5, diphenyltetrazolium bromide assay was used to evaluate cell viability following umbelliferone treatment, and the effects of umbelliferone on cell cycle progression and apoptosis were evaluated using flow cytometry. The presence of morphological features characteristic of apoptosis, including cell shrinkage, membrane blebbing, nuclear condensation and apoptotic body formation, were evaluated in HepG2 cells following umbelliferone treatment. Cell cycle analysis conducted via propidium iodide (PI) staining indicated that umbelliferone treatment induced cell cycle arrest at S phase in HepG2 cells. Analysis with Annexin V and PI staining revealed that umbelliferone induced apoptotic events in HepG2 cells in a concentration‑dependant manner (0‑50 µM). Umbelliferone also induced dose‑dependant DNA fragmentation. In conclusion, umbelliferone was found to exhibit significant anticancer effects via the induction of apoptosis, cell cycle arrest and DNA fragmentation in HepG2 cancer cells. PMID:25997538

  7. Garcinol inhibits tumour cell proliferation, angiogenesis, cell cycle progression and induces apoptosis via NF-κB inhibition in oral cancer.

    PubMed

    Aggarwal, Sadhna; Das, Satya N

    2016-06-01

    Garcinol, a polyisoprenylated benzophenone is extracted from the rind of the fruit of Garcinia indica, a plant found extensively in tropical regions. Its ability to inhibit tumour growth has been demonstrated in certain cancers. In this study, we evaluated the potential anti-tumour effects of garcinol on oral squamous cell carcinoma (OSCC) cells. Three OSCC cell lines (SCC-4, SCC-9 and SCC-25) were treated with garcinol for 48 h and its effect on growth and proliferation, clonogenic survival, cell cycle and apoptosis was studied by MTT, clonogenic assay, propidium iodide (PI) staining and annexin-V binding assay, respectively. The alteration in expression of NF-κB and COX-2 was studied by western blot analysis and that of VEGF by ELISA. Garcinol treatment significantly (p < 0.001) inhibited the growth and proliferation and colony formation of OSCC cells with a concomitant induction of apoptosis and cell cycle arrest. It did not show toxic effect on normal cells. It significantly (p < 0.05) reduced the expression of NK-κB and COX-2 expression in treated cells as compared to untreated controls besides inhibiting VEGF expression. It appears that garcinol exerts anti-proliferative, pro-apoptotic, cell-cycle regulatory and anti-angiogenic effects on oral cancer cells through inhibition of NF-κB and COX-2. Thus, garcinol may be developed as a potential chemopreventive and/or chemotherapeutic agent for treatment of oral squamous cell carcinoma. PMID:26662963

  8. Phthalocyanine-mediated photodynamic therapy induces cell death and a G /G{sub 1} cell cycle arrest in cervical cancer cells

    SciTech Connect

    Haywood-Small, S.L. . E-mail: s.l.hankin@sheffield.ac.uk; Vernon, D.I.; Griffiths, J.; Schofield, J.; Brown, S.B.

    2006-01-13

    We have developed a series of novel photosensitizers which have potential for anticancer photodynamic therapy (PDT). Photosensitizers include zinc phthalocyanine tetra-sulphonic acid and a family of derivatives with amino acid substituents of varying alkyl chain length and degree of branching. Subcellular localization of these photosensitizers at the phototoxic IC{sub 5} concentration in human cervical carcinoma cells (SiHa Cells) was similar to that of the lysosomal dye Lucifer Yellow. Subsequent nuclear relocalization was observed following irradiation with 665 nm laser light. The PDT response was characterized using the Sulforhodamine B cytotoxicity assay. Flow cytometry was used for both DNA cell cycle and dual Annexin V-FITC/propidium iodide analysis. Phototoxicity of the derivatives was of the same order of magnitude as for tetrasulphonated phthalocyanine but with an overall trend of increased phototoxicity with increasing amino acid chain length. Our results demonstrate cell death, inhibition of cell growth, and G /G{sub 1} cell cycle arrest during the phthalocyanine PDT-mediated response.

  9. Apoptosis in esophagus and pancreas carcinoma cells induced by circulating microparticles is related to phosphatidyl serine and microparticle-associated caspases.

    PubMed

    Schneider, Jan; Chromik, Ansgar M; Uhl, Waldemar; Mügge, Andreas; Bulut, Daniel

    2012-06-01

    Circulating microparticles (MPs) are recently discussed as "biologically active", participating in the pathology of diseases rather than being a marker of damaging processes. It was the purpose of the present study to investigate the effects of MPs, as isolated from the blood of healthy volunteers, on the induction of apoptosis and necrosis in cultured KYSE-270 esophageal and ASPC1 pancreas carcinoma cells. MPs were obtained from the blood of 20 healthy volunteers (11 women; mean age 33.3 years). Viability, apoptosis, and necrosis were determined by flow cytometry using Annexin V/propidium iodide and tetramethylrhodamine ethyl ester perchlorate (TMRE)/propidium iodide for staining. Incubation of KYSE and ASPC1 carcinoma cells with MPs (1-20.000/μl) for 48 h reduced significantly viability of the cells, and induced apoptosis, but not necrosis. This apoptotic effect was significant at a concentration of ≥1.000 MPs/μl in both cell types. Pre-treatment of MPs with either the global caspase inhibitor ZVAD-FMK or Annexin V which blocks phosphatidyl serine in the outer membrane of MPs with high affinity, almost abolished MP-induced apoptosis. A specific enzyme assay as well Western blot analysis confirmed the presence (activity, protein) of the apoptotic enzyme caspase-3 in MPs. Incubation of carcinoma cells with MPs (20.000/μl) resulted in an increase in caspase-3 protein in carcinoma cells; this increase could be prevented by pre-treatment of MPs with Annexin V. It is suggested that MPs induce concentration-dependent apoptosis in KSYE esophageal and ASPC1 pancreas carcinoma cells in vitro by transferring caspases into target cells. This process probably requires a target cell-MP interaction, and membrane-bound anionic phosphatidyl serine may be involved. PMID:21452043

  10. Changes in cell death of peripheral blood lymphocytes isolated from children with acute lymphoblastic leukemia upon stimulation with 7 Hz, 30 mT pulsed electromagnetic field.

    PubMed

    Kaszuba-Zwoińska, Jolanta; Ćwiklińska, Magdalena; Balwierz, Walentyna; Chorobik, Paulina; Nowak, Bernadeta; Wójcik-Piotrowicz, Karolina; Ziomber, Agata; Malina-Novak, Kinga; Zaraska, Wiesław; Thor, Piotr J

    2015-03-01

    Pulsed electromagnetic field (PEMF) influenced the viability of proliferating in vitro peripheral blood mononuclear cells (PBMCs) isolated from Crohn's disease patients as well as acute myeloblastic leukemia (AML) patients by induction of cell death, but did not cause any vital changes in cells from healthy donors. Experiments with lymphoid U937 and monocytic MonoMac6 cell lines have shown a protective effect of PEMF on the death process in cells treated with death inducers. The aim of the current study was to investigate the influence of PEMF on native proliferating leukocytes originating from newly diagnosed acute lymphoblastic leukemia (ALL) patients. The effects of exposure to PEMF were studied in PBMCs from 20 children with ALL. PBMCs were stimulated with three doses of PEMF (7 Hz, 30 mT) for 4 h each with 24 h intervals. After the last stimulation, the cells were double stained with annexin V and propidium iodide dye to estimate viability by flow cytometric analysis. The results indicated an increase of annexin V positive as well as double stained annexin V and propidium iodide positive cells after exposure to threefold PEMF stimulation. A low-frequency pulsed electromagnetic field induces cell death in native proliferating cells isolated from ALL patients. The increased vulnerability of proliferating PBMCs to PEMF-induced interactions may be potentially applied in the therapy of ALL. The analysis of expression of apoptosis-related genes revealed changes in mRNA of some genes engaged in the intrinsic apoptotic pathway belonging to the Bcl-2 family and the pathway with apoptosis-inducing factor (AIF) abundance upon PEMF stimulation of PBMCs. PMID:26204398

  11. Differential survival of solitary and aggregated bacterial cells promotes aggregate formation on leaf surfaces

    PubMed Central

    Monier, J.-M.; Lindow, S. E.

    2003-01-01

    The survival of individual Pseudomonas syringae cells was determined on bean leaf surfaces maintained under humid conditions or periodically exposed to desiccation stress. Cells of P. syringae strain B728a harboring a GFP marker gene were visualized by epifluorescence microscopy, either directly in situ or after recovery from leaves, and dead cells were identified as those that were stained with propidium iodide in such populations. Under moist, conducive conditions on plants, the proportion of total live cells was always high, irrespective of their aggregated state. In contrast, the proportion of the total cells that remained alive on leaves that were periodically exposed to desiccation stress decreased through time and was only ≈15% after 5 days. However, the fraction of cells in large aggregates that were alive on such plants in both condition was much higher than more solitary cells. Immediately after inoculation, cells were randomly distributed over the leaf surface and no aggregates were observed. However, a very aggregated pattern of colonization was apparent within 7 days, and >90% of the living cells were located in aggregates of 100 cells or more. Our results strongly suggest that, although conducive conditions favor aggregate formation, such cells are much more capable of tolerating environmental stresses, and the preferential survival of cells in aggregates promotes a highly clustered spatial distribution of bacteria on leaf surfaces. PMID:14665692

  12. BYSTANDER RESPONSES IN THREE-DIMENSIONAL CULTURES CONTAINING RADIOLABELLED AND UNLABELLED HUMAN CELLS

    PubMed Central

    Pinto, M.; Azzam, E. I.; Howell, R. W.

    2010-01-01

    Research on the radiation-induced bystander effect has been carried out mainly in 2-D tissue culture systems. This study uses a 3-D model, wherein apparently normal human diploid fibroblasts (AG1522) are grown in a carbon scaffold, to investigate the induction of a G1 checkpoint in bystander cells present alongside radiolabelled cells. Cultures were simultaneously pulse-labelled with 3H-deoxycytidine (3HdC) to selectively irradiate a minor fraction of cells, and bromodeoxyuridine (BrdU) to identify the radiolabelled cells. After thorough washing of cultures, iododeoxyuridine (IdU) was administered to detect proliferating bystander cells. The cultures were harvested at various times thereafter, and cells were reacted with two monoclonal antibodies specific to IdU/BrdU or BrdU, respectively, stained with propidium iodide, and subjected to multi-parameter flow cytometry. Cell-cycle progression was followed in radiolabelled cells (BrdU+) that were chronically irradiated by low energy beta particles emitted by DNA-incorporated 3H, and in unlabelled bystander cells (BrdU−) by a flow cytometry based cumulative labelling index assay. As expected, radiolabelled cells were delayed, in a dose-dependent manner, in G2 and subsequently G1. No delay occurred in progression of bystander cells through G1, when the labelled cells were irradiated at dose rates up to 0.32 Gy h−1. PMID:17185313

  13. Mitochondria toxin-induced acute cochlear cell death indicates cellular activity-correlated energy consumption.

    PubMed

    Zou, Jing; Zhang, Ya; Zhang, Weikai; Poe, Dennis; Zhai, Suoqiang; Yang, Shiming; Pyykkö, Ilmari

    2013-09-01

    The different cell types within the cochlea may have a specific contribution to the pathological changes during metabolism failure, which may provide clues for developing novel strategies for inner ear therapy. In order to evaluate activity-correlated cell death during metabolism failure in the cochlea, 3-nitropropionic acid was used to irreversibly inhibit the respiratory chain. Dose-response of the cochlear cells to 3-nitropropionic acid was analyzed in vitro. 3-Nitropropionic acid was administered onto the round window of guinea pigs. Cell death was identified by terminal transferase labeling the free 3'OH breaks in the DNA strands in vivo and propidium iodide nuclear permeation in vitro. As a result, 23.6 and 96.3 % cell death were induced by 10 and 100 mM 3-nitropropionic acid, respectively, in vitro. In the guinea pigs, 500 mM 3-nitropropionic acid induced vestibular dysfunction and severe to profound hearing losses. The cells that are the most sensitive to 3-nitropropionic acid treatment include the stria marginal and intermediate cells, epithelial cells of the Reissner's membrane, and spiral ligament fibrocytes (types II and V). Moderate sensitive cells were satellite fibrocytes of the spiral limbic central zone, osteocytes of the cochlear shell, hair cells, and spiral ganglion cells. Reduction of neurofilament in the soma and periphery processes of spiral ganglion cells occurred after the exposure. These results may be relevant to the mechanisms of injury in sudden onset sensorineural hearing loss and hazardous substance exposure-induced hearing loss. PMID:23179932

  14. Induction of apoptosis and cell cycle arrest in human HCC MHCC97H cells with Chrysanthemum indicum extract

    PubMed Central

    Li, Zong-Fang; Wang, Zhi-Dong; Ji, Yuan-Yuan; Zhang, Shu; Huang, Chen; Li, Jun; Xia, Xian-Ming

    2009-01-01

    AIM: To investigate the effects of Chrysanthemum indicum extract (CIE) on inhibition of proliferation and on apoptosis, and the underlying mechanisms, in a human hepatocellular carcinoma (HCC) MHCC97H cell line. METHODS: Viable rat hepatocytes and human endothelial ECV304 cells were examined by trypan blue exclusion and MTT assay, respectively, as normal controls. The proliferation of MHCC97H cells was determined by MTT assay. The cellular morphology of MHCC97H cells was observed by phase contrast microscopy. Flow cytometry was performed to analyze cell apoptosis with annexin V/propidium iodide (PI), mitochondrial membrane potential with rhodamine 123 and cell cycle with PI in MHCC97H cells. Apoptotic proteins such as cytochrome C, caspase-9, caspase-3 and cell cycle proteins, including P21 and CDK4, were measured by Western blotting. RESULTS: CIE inhibited proliferation of MHCC97H cells in a time- and dose-dependent manner without cytotoxicity in rat hepatocytes and human endothelial cells. CIE induced apoptosis of MHCC97H cells in a concentration-dependent manner, as determined by flow cytometry. The apoptosis was accompanied by a decrease in mitochondrial membrane potential, release of cytochrome C and activation of caspase-9 and caspase-3. CIE arrested the cell cycle in the S phase by increasing P21 and decreasing CDK4 protein expression. CONCLUSION: CIE exerted a significant apoptotic effect through a mitochondrial pathway and arrested the cell cycle by regulation of cell cycle-related proteins in MHCC97H cells without an effect on normal cells. The cancer-specific selectivity shown in this study suggests that the plant extract could be a promising novel treatment for human cancer. PMID:19777612

  15. Overexpression of interleukin-18 protein reduces viability and induces apoptosis of tongue squamous cell carcinoma cells by activation of glycogen synthase kinase-3β signaling

    PubMed Central

    LIU, WEIWEI; HU, MIN; WANG, YUMEI; SUN, BAOZHEN; GUO, YU; XU, ZHIMIN; LI, JIA; HAN, BING

    2015-01-01

    The aim of this study was to investigate the effects of interleukin-18 (IL-18) expression on regulating the viability and apoptosis of tongue squamous cell carcinoma (TSCC) cells in vitro and examine the underlying molecular events. Human IL-18 cDNA was cloned into the vector pcDNA3.1 (+) and transfected into CRL-1623™ cells. Quantitative reverse transcription-PCR (RT-qPCR), western blot analysis, immunofluorescence, cell viability MTT assay, flow cytometric Annexin V/propidium iodide (PI), Giemsa staining, and caspase-3 activity assay were performed. The data showed that overexpression of IL-18 protein reduced TSCC cell viability by inducing apoptosis. Compared with cells transfected with the control vector, IL-18 expression activated caspase-3, -7, and -9 by inducing their cleavage and increased the expression of interferon (IFN)-γ and cytochrome c mRNA, but reduced cyclin D1 and A1 expression in TSCC cells. IL-18 expression upregulated the expression and phosphorylation of glycogen synthase kinase (GSK)-3β protein in CRL1623 cells, whereas the selective GSK-3β inhibitor kenpaullone antagonized the effects of IL-18 protein on TSCC cells in vitro. The results indicated that IL-18 played an important role in the inhibition of TSCC cell growth and may be further investigated as a novel therapeutic target against TSCC. PMID:25591548

  16. Cell death mechanisms vary with photodynamic therapy dose and photosensitizer

    NASA Astrophysics Data System (ADS)

    He, Jin; Oleinick, Nancy L.

    1995-03-01

    Mouse lymphoma L5178Y-R cells respond to photodynamic therapy (PDT) by undergoing rapid apoptosis, which is induced by PDT-activated signal transduction initiating in the damaged cellular membranes. To relate the level of PDT damage and photosensitizer to the mechanism of cell death, apoptosis has been detected by agarose gel electrophoresis of fragmented DNA and quantified by flow cytometry of cells after staining with Hoechst33342 and propidium iodide, a technique which can distinguish between live, apoptotic, and necrotic cells. When the silicon phthalocyanine Pc 4 or Pc 12 served as photosensitizer, lethal doses (as defined by clonogenic assay) of PDT induced apoptosis in essentially all cells, whereas supralethal doses prevented the characteristic degradation of DNA into oligonucleosomal fragments. In contrast with aluminum phthalocyanine (AlPc) cells died by apoptosis after all doses studied. It appears that high PDT doses with Pc 4 or Pc 12 damage enzymes needed to carry out the program of apoptosis; the absence of this effect with AlPc suggests either a different intracellular location or different photocytotoxic mechanism for the two photosensitizers.

  17. Antiproliferative and cell apoptosis-inducing activities of compounds from Buddleja davidii in Mgc-803 cells

    PubMed Central

    2012-01-01

    Background Buddleja davidii is widely distributed in the southwestern region of China. We have undertaken a systematic analysis of B. davidii as a Chinese traditional medicine with anticancer activity by isolating natural products for their activity against the human gastric cancer cell line Mgc-803 and the human breast cancer cell line Bcap-37. Results Ten compounds were extracted and isolated from B. davidii, among which colchicine was identified in B. davidii for the first time. The inhibitory activities of these compounds were investigated in Mgc-803, Bcap-37 cells in vitro by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, and the results showed that luteolin and colchicine had potent inhibitory activities against the growth of Mgc-803 cells. Subsequent fluorescence staining and flow cytometry analysis indicated that these two compounds could induce apoptosis in Mgc-803 cells. The results also showed that the percentages of early apoptotic cells (Annexin V+/PI-, where PI is propidium iodide) and late apoptotic cells (Annexin V+/PI+) increased in a dose- and time-dependent manner. After 36 h of incubation with luteolin at 20 μM, the percentages of cells were approximately 15.4% in early apoptosis and 43.7% in late apoptosis; after 36 h of incubation with colchicine at 20 μM, the corresponding values were 7.7% and 35.2%, respectively. Conclusions Colchicine and luteolin from B. davidii have potential applications as adjuvant therapies for treating human carcinoma cells. These compounds could also induce apoptosis in tumor cells. PMID:22938042

  18. Effect of Sterols Isolated from Myrtillocactus geometrizans on Growth Inhibition of Colon and Breast Cancer Cells

    PubMed Central

    Bolaños-Carrillo, Mario Augusto; Ventura-Gallegos, Jose Luis; Saldivar-Jiménez, Arturo David; Zentella-Dehesa, Alejandro; Martínez-Vázquez, Mariano

    2015-01-01

    Objective. To explore the effect of peniocerol and macdougallin on HCT-15 and MCF-7 cells proliferation, cell cycle, apoptosis, and PARP cleavage. Methods. HCT-15 and MCF-7 cells were treated with various concentrations of peniocerol and macdougallin (10–80 μM) during 24 or 48 h. Crystal Violet Assay was used to evaluate the inhibition effect. Cell cycle regulation was examined by a propidium iodide method. Cell apoptosis was detected through both Annexin–V FLUOS/PI double-labeled cytometry assays and Western blot was applied to assess PARP cleavage. Results. Peniocerol and macdougallin induced growth inhibition and apoptosis in vitro in a time- and dose-dependent manner. Moreover, peniocerol and macdougallin induced arrest of cell cycle-dependent manner and increased the proportion of cells in G0/G1 phase. PARP cleavage in HCT-15 and MCF-7 cells was induced by treatment with peniocerol and macdougallin after 36 hours. Conclusions. Our results showed that the mechanism of cytotoxicity displayed by peniocerol and macdougallin is related to cell cycle arrest and apoptosis in both cell lines. This is a significant observation because it helps to understand the way some oxysterols isolated from Myrtillocactus geometrizans develop their biological activities against cancer cells. PMID:26113867

  19. Paeoniflorin inhibits human pancreatic cancer cell apoptosis via suppression of MMP-9 and ERK signaling

    PubMed Central

    Yang, Nanmu; Cui, Hong; Han, Feng; Zhang, Ling; Huang, Tao; Zhou, Yi; Zhou, Jinxue

    2016-01-01

    Paeoniflorin exhibits anticancer, anti-inflammatory and antioxidation effects, as well as specific pharmacological effects on smooth muscle and the immune, cardiovascular and central nervous systems. The present study aimed to investigate the anticancer effects of paeoniflorin on pancreatic cancer cells and to elucidate the mechanisms by which these effects occur. In the present study, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays were performed to assess cell viability and cell cytotoxicity of BXPC-3 human pancreatic cancer cells, respectively. Cellular apoptosis and caspase-3/9 activities were analyzed using an Annexin V-fluorescein isothiocyanate/propidium iodide Apoptosis Detection kit, a DAPI staining assay and colorimetric kits, respectively. Matrix metalloproteinase-9 (MMP-9) and extracellular signal-regulated kinases (ERK) protein expression in BXPC-3 cells were also investigated using gelatin zymography assays and western blot analysis, respectively. In the present study, paeoniflorin was found to inhibit the cell viability and increase cell cytotoxicity of BXPC-3 cells in a dose- and time-dependent manner. In addition, cellular apoptosis, as well as caspase-3 and −9 activity of BXPC-3 cells was increased following paeoniflorin treatment. Notably, paeoniflorin reduced MMP-9 and ERK protein expression in BXPC-3 cells. These results indicate that paeoniflorin exhibits a potential anticancer effect by enhancing human pancreatic cancer cell apoptosis via the suppression of MMP-9 and ERK signaling. PMID:27446455

  20. Circulating IgM Requires Plasma Membrane Disruption to Bind Apoptotic and Non-Apoptotic Nucleated Cells and Erythrocytes

    PubMed Central

    Hesketh, Emily E.; Dransfield, Ian; Kluth, David C.; Hughes, Jeremy

    2015-01-01

    Autoimmunity is associated with defective phagocytic clearance of apoptotic cells. IgM deficient mice exhibit an autoimmune phenotype consistent with a role for circulating IgM antibodies in apoptotic cell clearance. We have extensively characterised IgM binding to non-apoptotic and apoptotic mouse thymocytes and human Jurkat cells using flow cytometry, confocal imaging and electron microscopy. We demonstrate strong specific IgM binding to a subset of Annexin-V (AnnV)+PI (Propidium Iodide)+ apoptotic cells with disrupted cell membranes. Electron microscopy studies indicated that IgM+AnnV+PI+ apoptotic cells exhibited morphologically advanced apoptosis with marked plasma membrane disruption compared to IgM-AnnV+PI+ apoptotic cells, suggesting that access to intracellular epitopes is required for IgM to bind. Strong and comparable binding of IgM to permeabilised non-apoptotic and apoptotic cells suggests that IgM bound epitopes are 'apoptosis independent' such that IgM may bind any cell with profound disruption of cell plasma membrane integrity. In addition, permeabilised erythrocytes exhibited significant IgM binding thus supporting the importance of cell membrane epitopes. These data suggest that IgM may recognize and tag damaged nucleated cells or erythrocytes that exhibit significant cell membrane disruption. The role of IgM in vivo in conditions characterized by severe cell damage such as ischemic injury, sepsis and thrombotic microangiopathies merits further exploration. PMID:26121639

  1. Dracorhodin perchlorate induces the apoptosis of glioma cells.

    PubMed

    Chen, Xin; Luo, Junjie; Meng, Linghu; Pan, Taifeng; Zhao, Binjie; Tang, Zhen-Gang; Dai, Yongjian

    2016-04-01

    Dracorhodin perchlorate (Dp), a synthetic analogue of the antimicrobial anthocyanin red pigment, has recently been shown to induce apoptotic cell death in various types of cancer cells. Yet, the inhibitory effect of Dp on human glioma cells remains uninvestigated. Therefore, in the present study, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry were used to detect cell viability and cell cycle progression in glioma U87MG and T98G cells, respectively. Annexin V-FITC/propidium iodide double staining and JC-1 staining were separately applied to determine cellular apoptosis and mitochondrial membrane potential damage in the cells. The expression levels of associated proteins involved in cell cycle progression and apoptosis were measured by western blotting. The activities of caspase‑9/-3 were determined by Caspase-Glo-9/3 assay. The results indicated that Dp treatment significantly inhibited cell proliferation in a dose- and time-dependent manner, and blocked cell cycle progression at the G1/S phase in the U87MG and T98G cells via the upregulation of p53 and p21 protein expression, and simultaneous downregulation of Cdc25A, Cdc2 and P-Cdc2 protein expression. Additionally, Dp treatment led to the loss of cellular mitochondrial membrane potential, and the release of cytochrome c, and strongly induced the occurence of apoptosis. Increased expression levels of Bim and Bax protein and the downregulated expression of Bcl-2 protein were observed. Caspase-9/-3 were activated and their activities were elevated after Dp treatment. These findings indicate that Dp inhibits cell proliferation, induces cell cycle arrest and apoptosis in glioma cells, and is a possible candidate for glioma treatment. PMID:26846469

  2. Induction of T-cell apoptosis by human herpesvirus 6.

    PubMed Central

    Inoue, Y; Yasukawa, M; Fujita, S

    1997-01-01

    The mechanisms of cell death in CD4+ T cells mediated by human herpesvirus 6 (HHV-6) were investigated. The frequency of cell death in the human CD4+ T-cell line JJHAN, which had been inoculated with HHV-6 variant A or B, appeared to be augmented by tumor necrosis factor alpha (TNF-alpha). Agarose gel electrophoresis of DNA from HHV-6-inoculated cells showed DNA fragmentation in multiples of the oligonucleosome length unit. The degree of DNA fragmentation increased when HHV-6-inoculated cells were cultured in the presence of TNF-alpha. Flow cytometry and Scatchard analysis of TNF receptors revealed an increase in the number of the p55 form of TNF receptors on JJHAN cells after HHV-6 inoculation. It also appeared that treatment with anti-Fas monoclonal antibody (MAb) induced marked apoptosis in HHV-6-inoculated cells. Transmission electron microscopy showed characteristics of apoptosis, such as chromatin condensation and fragmentation of nuclei, but virus particles were hardly detected in apoptotic cells. Two-color flow cytometric analysis using anti-HHV-6 MAb and propidium iodide revealed that DNA fragmentation was present predominantly in uninfected cells but not in productively HHV-6-infected cells. In addition, JJHAN cells incubated with UV light-irradiated and ultracentrifuged culture supernatant of HHV-6-infected cells appeared to undergo apoptosis. The present study demonstrated that both HHV-6 variants A and B induce apoptosis in CD4+ T cells by indirect mechanisms, as reported recently in human immunodeficiency virus type 1 infection. PMID:9094650

  3. Photosensitizer effect of 9-hydroxypheophorbide α on diode laser-irradiated laryngeal cancer cells: Oxidative stress-directed cell death and migration suppression

    PubMed Central

    He, Peijie; Bo, Shen; Chung, Phil-Sang; Ahn, Jin-Chul; Zhou, Liang

    2016-01-01

    The present study aimed to investigate the effect, and elucidate the potential mechanisms, of 9-hydroxypheophorbide α-based photodynamic therapy (9-HPbD-PDT) on apoptosis and necrosis induction, and migration suppression of laryngeal cancer AMC-HN-3 (HN-3) cells. Phototoxicity initiated by 9-HPbD-PDT on HN-3 cells was observed in a photosensitizer dose-dependent pattern. There was an initial increase of apoptotic cells coupled with gradual enhancement of reactive oxygen series (ROS) generation at lower doses of 9-HPbD. By contrast, at a higher dose of 9-HPbD, there was a clear increase of necrotic cells with a gradual decrease of ROS generation. Following PDT, an elevated percentage of apoptotic cells with shrinkage or condensing nuclei was observed using Hoechst 33342/propidium iodide double staining, and an upregulated expression of poly ADP-ribose polymerase was detected through western blotting. A disruption of the mitochondrial membrane potential was detected 2 h following PDT. Significant suppression of cell migration and downregulation of epidermal growth factor receptor (EGFR) expression were recorded following PDT. These results indicate that the distribution of photosensitizer leads to differences in the generation of ROS, which subsequently determines the type of cell death. Overall, mitochondrial activation under oxidative stress is important in the 9-HPbD-PDT-induced apoptosis of HN-3 cells. Migration suppression of HN-3 cells following PDT may be associated with the inhibited expression of EGFR, due to oxidative stress. PMID:27588136

  4. Heme Oxygenase-1 Induction Improves Cardiac Function following Myocardial Ischemia by Reducing Oxidative Stress

    PubMed Central

    Issan, Yossi; Kornowski, Ran; Aravot, Dan; Shainberg, Asher; Laniado-Schwartzman, Michal; Sodhi, Komal; Abraham, Nader G.; Hochhauser, Edith

    2014-01-01

    Background Oxidative stress plays a key role in exacerbating diabetes and cardiovascular disease. Heme oxygenase-1 (HO-1), a stress response protein, is cytoprotective, but its role in post myocardial infarction (MI) and diabetes is not fully characterized. We aimed to investigate the protection and the mechanisms of HO-1 induction in cardiomyocytes subjected to hypoxia and in diabetic mice subjected to LAD ligation. Methods In vitro: cultured cardiomyocytes were treated with cobalt-protoporphyrin (CoPP) and tin protoporphyrin (SnPP) prior to hypoxic stress. In vivo: CoPP treated streptozotocin-induced diabetic mice were subjected to LAD ligation for 2/24 h. Cardiac function, histology, biochemical damage markers and signaling pathways were measured. Results HO-1 induction lowered release of lactate dehydrogenase (LDH) and creatine phospho kinase (CK), decreased propidium iodide staining, improved cell morphology and preserved mitochondrial membrane potential in cardiomyocytes. In diabetic mice, Fractional Shortening (FS) was lower than non-diabetic mice (35±1%vs.41±2, respectively p<0.05). CoPP-treated diabetic animals improved cardiac function (43±2% p<0.01), reduced CK, Troponin T levels and infarct size compared to non-treated diabetic mice (P<0.01, P<0.001, P<0.01 respectively). CoPP-enhanced HO-1 protein levels and reduced oxidative stress in diabetic animals, as indicated by the decrease in superoxide levels in cardiac tissues and plasma TNFα levels (p<0.05). The increased levels of HO-1 by CoPP treatment after LAD ligation led to a shift of the Bcl-2/bax ratio towards the antiapoptotic process (p<0.05). CoPP significantly increased the expression levels of pAKT and pGSK3β (p<0.05) in cardiomyocytes and in diabetic mice with MI. SnPP abolished CoPP's cardioprotective effects. Conclusions HO-1 induction plays a role in cardioprotection against hypoxic damage in cardiomyocytes and in reducing post ischemic cardiac damage in the diabetic heart as proved by

  5. Are Early Somatic Embryos of the Norway Spruce (Picea abies (L.) Karst.) Organised?

    PubMed Central

    Petrek, Jiri; Zitka, Ondrej; Adam, Vojtech; Bartusek, Karel; Anjum, Naser A.; Pereira, Eduarda; Havel, Ladislav; Kizek, Rene

    2015-01-01

    Background Somatic embryogenesis in conifer species has great potential for the forestry industry. Hence, a number of methods have been developed for their efficient and rapid propagation through somatic embryogenesis. Although information is available regarding the previous process-mediated generation of embryogenic cells to form somatic embryos, there is a dearth of information in the literature on the detailed structure of these clusters. Methodology/Principal Findings The main aim of this study was to provide a more detailed structure of the embryogenic tissue clusters obtained through the in vitro propagation of the Norway spruce (Picea abies (L.) Karst.). We primarily focused on the growth of early somatic embryos (ESEs). The data on ESE growth suggested that there may be clear distinctions between their inner and outer regions. Therefore, we selected ESEs collected on the 56th day after sub-cultivation to dissect the homogeneity of the ESE clusters. Two colourimetric assays (acetocarmine and fluorescein diacetate/propidium iodide staining) and one metabolic assay based on the use of 2,3,5-triphenyltetrazolium chloride uncovered large differences in the metabolic activity inside the cluster. Next, we performed nuclear magnetic resonance measurements. The ESE cluster seemed to be compactly aggregated during the first four weeks of cultivation; thereafter, the difference between the 1H nuclei concentration in the inner and outer clusters was more evident. There were clear differences in the visual appearance of embryos from the outer and inner regions. Finally, a cluster was divided into six parts (three each from the inner and the outer regions of the embryo) to determine their growth and viability. The innermost embryos (centripetally towards the cluster centre) could grow after sub-cultivation but exhibited the slowest rate and required the longest time to reach the common growth rate. To confirm our hypothesis on the organisation of the ESE cluster, we

  6. CD52-Negative NK Cells Are Abundant in the Liver and Less Susceptible to Alemtuzumab Treatment

    PubMed Central

    Matsuura, Toshiharu; Muraoka, Izumi; Tryphonopoulos, Panagiotis; Fan, Ji; Tekin, Akin; Selvaggi, Gennaro; Levi, David; Ruiz, Phillip; Ricordi, Camillo; Vianna, Rodrigo; Ohdan, Hideki; Waldmann, Herman; Tzakis, Andreas G.; Nishida, Seigo

    2016-01-01

    Background T-cell depleting strategies have become an integral part of immunosuppressive regimens in organ transplantation. Alemtuzumab is a humanized monoclonal antibody against CD52, a cell-surface antigen on several immune cells. It has been suggested that lymphocyte depletion increases the risk of serious infections. However, this has not been observed with short-term alemtuzumab treatment in an organ transplant setting. For induction therapy using alemtuzumab following liver transplantation, we found that T- and B-cell numbers declined rapidly after alemtuzumab therapy; however, the natural killer (NK) cell number was sustained. NK cells are important effectors of innate immunity. Since the effects of alemtuzumab on NK cell functions, especially those of liver NK cells, are unknown, this study aimed to investigate this in detail. Methods To assess the effect of alemtuzumab on NK cells, samples were obtained from 7 organ donors and examined by flow cytometry using Annexin V and propidium iodide. Phenotypical and functional differences within subsets of NK cells with different levels of CD52 expression were determined by flow cytometry and in vitro cytotoxicity assays. Results CD52 expression on NK cells was lower than that on other lymphocyte subsets. The liver contained a large number of CD52− NK cells compared with the peripheral blood. In vitro treatment of liver-derived NK cells with alemtuzumab did not result in cell death. In contrast, co-incubation with alemtuzumab induced cell death in peripheral blood mononuclear cells and non-NK cells in the liver. Furthermore, CD52− liver NK cells were more cytotoxic and produced more IFN-γ than CD52+ NK cells after cytokine activation. Conclusion The liver contains a large number of CD52− NK cells. These cells are refractory to alemtuzumab and have robust activity. These findings indicate that CD52− NK cells persist and could protect against infection after alemtuzumab-based lymphocyte depletion. PMID

  7. Novel Cell Preservation Technique to Extend Bovine In Vitro White Blood Cell Viability

    PubMed Central

    Laurin, Emilie L.; McKenna, Shawn L. B.; Sanchez, Javier; Bach, Horacio; Rodriguez-Lecompte, Juan Carlos; Chaffer, Marcelo; Keefe, Greg P.

    2015-01-01

    Although cell-mediated immunity based diagnostics can be integral assays for early detection of various diseases of dairy cows, processing of blood samples for these tests is time-sensitive, often within 24 hours of collection, to maintain white blood cell viability. Therefore, to improve utility and practicality of such assays, the objective of this study was to assess the use of a novel white blood cell preservation technology in whole bovine blood. Blood samples from ten healthy cows were each divided into an unpreserved control sample and a test sample preserved with commercially-available cell transport medium. Samples were maintained at room temperature and stimulated with the mitogens pokeweed and concanavalinA, as well as with interleukin-12 p40. Stimulation was completed on days 1, 5, and 8 post-sampling. Viability of white blood cells was assessed through interferon gamma production determined with a commercial enzyme linked immunosorbent assay. In addition, mononuclear cell viability was assessed with propidium iodide flow cytometry. Greater interferon gamma production was observed on days 5 and 8 post-collection in preserved samples, with both pokeweed and concanavalinA stimulating positive interferon gamma production on day 5 post-collection. A greater proportion of the amount of interferon gamma produced on day 1 continued to be produced on days 5 and 8 post-collection with concanavalinA stimulation (with or without interleukin 12) as compared to pokeweed stimulation. Additionally, viable mononuclear cells were still present at eight days post-collection, with a higher mean proportion detected at days 5 and 8 in all stimulated preserved samples. This practical and simple method to extend in vitro white blood cell viability could benefit the efficient utilization of cell-based blood tests in ruminants. PMID:26447691

  8. Knockdown of GRP78 enhances cell death by cisplatin and radiotherapy in nasopharyngeal cells.

    PubMed

    Huang, Ying-Ying; Pu, Long-Jian; Song, Le-Le; Ma, Lin-Yan; Liu, Hao; Jiang, Chen-Chen

    2016-09-01

    Radiotherapy and adjuvant cisplatin chemotherapy are the mainstream approaches in the treatment of nasopharyngeal carcinoma (NPC). These have been shown to effectively improve the outcome and reduce tumor recurrence. However, radiotherapy a