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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. 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.

  3. 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

  4. Quantification of Electroporation-Mediated Propidium Iodide Delivery into 3T3 Cells

    NASA Astrophysics Data System (ADS)

    Sadik, Mohamed M.; Li, Jianbo; Shan, Jerry W.; Shreiber, David I.; Lin, Hao

    2010-11-01

    Electroporation is an effective means to deliver exogenous molecules into the cellular cytoplasm, while simultaneously maintaining cell viability and functionality. In this technique, an applied electric field transiently permeabilizes the cellular membrane to enable molecular exchange. The main objective of the current work is to identify the transport mechanisms involved during electroporation, and to quantify the amount of molecules delivered into the cellular cytoplasm. An optical diagnostic system is developed to examine the transport of Propidium Iodide (PI) into 3T3 mouse fibroblast cells. Upon entering the permeabilized cell, PI binds to DNA/RNA within the cytoplasm to emit fluorescence, which is measured to track the dynamic accumulation of the dye within the cell. The results show that the total fluorescence intensity increases with a decreasing buffer electrical conductivity. The data are compared with numerical simulations, which reveals good agreement. The experimental observations and numerical analysis demonstrate that: 1) Electrophoresis plays a dominant role in mediating the transport. 2) An electrokinetic mechanism, field-amplified sample stacking, controls the achievable delivery efficiency. The study in this work is an important step toward the quantification as well as the eventual improvement of this useful technique.

  5. 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...

  6. Comparative Analysis and Limitations of Ethidium Monoazide and Propidium Monoazide Treatments for the Differentiation of Viable and Nonviable Campylobacter Cells

    PubMed Central

    Seinige, Diana; Krischek, Carsten; Klein, Günter

    2014-01-01

    The lack of differentiation between viable and nonviable bacterial cells limits the implementation of PCR-based methods for routine diagnostic approaches. Recently, the combination of a quantitative real-time PCR (qPCR) and ethidium monoazide (EMA) or propidium monoazide (PMA) pretreatment has been described to circumvent this disadvantage. In regard to the suitability of this approach for Campylobacter spp., conflicting results have been reported. Thus, we compared the suitabilities of EMA and PMA in various concentrations for a Campylobacter viability qPCR method. The presence of either intercalating dye, EMA or PMA, leads to concentration-dependent shifts toward higher threshold cycle (CT) values, especially after EMA treatment. However, regression analysis resulted in high correlation coefficient (R2) values of 0.99 (EMA) and 0.98 (PMA) between Campylobacter counts determined by qPCR and culture-based enumeration. EMA (10 μg/ml) and PMA (51.10 μg/ml) removed DNA selectively from nonviable cells in mixed samples at viable/nonviable ratios of up to 1:1,000. The optimized EMA protocol was successfully applied to 16 Campylobacter jejuni and Campylobacter coli field isolates from poultry and indicated the applicability for field isolates as well. EMA-qPCR and culture-based enumeration of Campylobacter spiked chicken leg quarters resulted in comparable bacterial cell counts. The correlation coefficient between the two analytical methods was 0.95. Nevertheless, larger amounts of nonviable cells (>104) resulted in an incomplete qPCR signal reduction, representing a serious methodological limitation, but double staining with EMA considerably improved the signal inhibition. Hence, the proposed Campylobacter viability EMA-qPCR provides a promising rapid method for diagnostic applications, but further research is needed to circumvent the limitation. PMID:24487529

  7. Molecular pathogen detection in biosolids with a focus on quantitative PCR using propidium monoazide for viable cell enumeration.

    PubMed

    van Frankenhuyzen, Jessica K; Trevors, Jack T; Lee, Hung; Flemming, Cecily A; Habash, Marc B

    2011-12-01

    Sewage sludge is the solid, organic material remaining after wastewater is treated and discharged from a wastewater treatment plant. Sludge is treated to stabilize the organic matter and reduce the amount of human pathogens. Once government regulations are met, including material quality standards (e.g., E. coli levels and heavy metal content) sludge is termed "biosolids", which may be disposed of by land application according to regulations. Live-culture techniques have traditionally been used to enumerate select pathogens and/or indicator organisms to demonstrate compliance with regulatory requirements. However, these methods may result in underestimates of viable microorganisms due to several problems, including their inability to detect viable but non-culturable (VBNC) cells. Real-time quantitative polymerase chain reaction (qPCR) is currently under investigation as a fast, sensitive, and specific molecular tool for enumeration of pathogens in biosolids. Its main limitation is that it amplifies all target DNAs, including that from non-viable cells. This can be overcome by coupling qPCR with propidium monoazide (PMA), a microbial membrane-impermeant dye that binds to extracellular DNA and DNA in dead or membrane-compromised cells, inhibiting its amplification. PMA has successfully been used to monitor the presence of viable pathogens in several different matrices. In this review the use of PMA-qPCR is discussed as a suitable approach for viable microbial enumeration in biosolids. Recommendations for optimization of the method are made, with a focus on DNA extraction, dilution of sample turbidity, reagent concentration, and light exposure time. PMID:21963489

  8. A new method for the analysis of plasma cell DNA content in multiple myeloma samples using a CD38/propidium iodide double staining technique.

    PubMed

    Orfäo, A; García-Sanz, R; López-Berges, M C; Belén Vidriales, M; González, M; Caballero, M D; San Miguel, J F

    1994-12-01

    In the present paper a CD38/propidium iodide double staining technique is described which separately assesses the cell cycle distribution of myelomatous plasma cells from that of the residual normal hemopoietic cells. For this purpose, bone marrow (BM) cells from a group of 42 untreated multiple myeloma patients were analyzed. Of these, 23 cases were aneuploid (55%) and 19 diploid (45%). The use of the CD38/propidium iodide double staining method allowed a clear separation between CD38 strong positive cells from the remaining bone marrow populations, cell sorting experiments confirming that plasma cells were almost exclusively contained in the former fraction where they represented 97 +/- 2% of the total cells sorted. In all cases, the S-phase in plasma cells and in the remaining normal hemopoietic bone marrow cells was assessed, being higher in normal hemopoietic cells (8.0 +/- 6.3%) than in plasma cells (3.3 +/- 2.6%, P < 0.002). In addition, there was no correlation between the S-phase of the neoplastic and normal bone marrow cells (r = 0.22; P > 0.10); this work therefore shows that the assessment of the total proliferative rate of bone marrow samples does not reflect either the proliferation of normal cells or that of neoplastic plasma cells but will depend on the proliferative rate and the percentage of each population within the sample, which can be assessed by the technique described here. PMID:7533074

  9. 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.

  10. Venom present in sea anemone (Heteractis magnifica) induces apoptosis in non-small-cell lung cancer A549 cells through activation of mitochondria-mediated pathway.

    PubMed

    Ramezanpour, Mahnaz; da Silva, Karen Burke; Sanderson, Barbara J S

    2014-03-01

    Lung cancer is a major cause of cancer deaths throughout the world and the complexity of apoptosis resistance in lung cancer is apparent. Venom from Heteractis magnifica caused dose-dependent decreases in survival of the human non-small-cell lung cancer cell line, as determined by the MTT and Crystal Violet assays. The H. magnifica venom induced cell cycle arrest and induced apoptosis of A549 cells, as confirmed by annexin V/propidium iodide staining. The venom-induced apoptosis in A549 cells was characterized by cleavage of caspase-3 and a reduction in the mitochondrial membrane potential. Interestingly, crude extracts from H. magnifica had less effect on the survival of non-cancer cell lines. In the non-cancer cells, the mechanism via which cell death occurred was through necrosis not apoptosis. These findings are important for future work using H. magnifica venom for pharmaceutical development to treat human lung cancer. PMID:24190482

  11. Oxidative stress induced apoptosis of human lung carcinoma (A549) cells by a novel copper nanorod formulation.

    PubMed

    Thounaojam, Menaka C; Jadeja, Ravirajsinh N; Valodkar, Mayur; Nagar, Padamanabhi S; Devkar, Ranjitsinh V; Thakore, Sonal

    2011-11-01

    This study elucidates the process of synthesis of copper (Cu) nanorods using almond skin extract as stabilizing cum capping agent. These nanorods were (about 200 nm long and 40 nm wide) characterized by transmission electron microscopy (TEM). Further, cytotoxicity potential of these nanorods was evaluated in A549 cells (Human lung carcinoma cell line) via cell viability assay and extracellular lactate dehydrogenase (LDH) release. Also, reduced glutathione (GSH), lipid peroxidation (LPO), cellular oxidative stress (Rhodamine 123 florescence) and apoptosis (Annexin V FITC/Propidium iodide staining) were also investigated in control and treated cells. Results indicated that Cu nanorods induced apoptotic death of cancer cells by induction of oxidative stress, depletion of cellular antioxidants and mitochondrial dysfunction. This study reports a novel process of synthesis of almond skin extract capped Cu nanorods and its potential as an anticancer agent against A549 lung carcinoma cells. PMID:21820027

  12. FV peptide induces apoptosis in HEp 2 and HeLa cells: an insight into the mechanism of induction

    PubMed Central

    Sri Balasubashini, M; Karthigayan, S; Somasundaram, ST; Balasubramanian, T; Rukkumani, R; Menon, Venugopal P

    2006-01-01

    The present study is an attempt to evaluate the antiproliferative potential of peptide (7.6 kDa) from lionfish (Pterios volitans) venom on cultured HEp2 and HeLa cells. Different dose of purified peptide (1, 2 and 4 μg/ml) at different time points (12, 24 and 36 hrs) were tested for antiproliferative index of the peptide. Among them, 2 μg/ml at 24 hrs was found to effectively inhibit cancer cell growth in vitro and did not cause any adverse effect on normal human lymphocytes. Apoptosis was examined by propidium iodide staining, confirmed by the expression of caspase-8 and caspase-3, down regulation of Bcl-2 expression and DNA fragmentation in treated cells, when compared to untreated HEp2 and HeLa cells. Thus fish venom peptide was found to selectively induce apoptosis in cancer cell. PMID:17137521

  13. FV peptide induces apoptosis in HEp 2 and HeLa cells: an insight into the mechanism of induction.

    PubMed

    Sri Balasubashini, M; Karthigayan, S; Somasundaram, S T; Balasubramanian, T; Rukkumani, R; Menon, Venugopal P

    2006-01-01

    The present study is an attempt to evaluate the antiproliferative potential of peptide (7.6 kDa) from lionfish (Pterios volitans) venom on cultured HEp2 and HeLa cells. Different dose of purified peptide (1, 2 and 4 microg/ml) at different time points (12, 24 and 36 hrs) were tested for antiproliferative index of the peptide. Among them, 2 microg/ml at 24 hrs was found to effectively inhibit cancer cell growth in vitro and did not cause any adverse effect on normal human lymphocytes. Apoptosis was examined by propidium iodide staining, confirmed by the expression of caspase-8 and caspase-3, down regulation of Bcl-2 expression and DNA fragmentation in treated cells, when compared to untreated HEp2 and HeLa cells. Thus fish venom peptide was found to selectively induce apoptosis in cancer cell. PMID:17137521

  14. 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

  15. 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 optimize LAN parameters for future transfection experimentation.

  16. 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 impact of reagent handling and various staining and imaging parameters used to visually evaluate islets, consistent interpretation of islet cell membrane integrity and viability is dependent upon a number of factors. We discuss the utility and limitations of these reagents in evaluating islet cell membrane integrity and viability. PMID:20814586

  17. 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

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

    2008-03-01

    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(R)13, SYTO(R)24 and SYBR(R)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(R)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 impact of reagent handling and various staining and imaging parameters used to visually evaluate islets, consistent interpretation of islet cell membrane integrity and viability is dependent upon a number of factors. We discuss the utility and limitations of these reagents in evaluating islet cell membrane integrity and viability. PMID:20814586

  18. Methylantcinate A induces tumor specific growth inhibition in oral cancer cells via Bax-mediated mitochondrial apoptotic pathway.

    PubMed

    Tsai, Wan-Chi; Rao, Yerra Koteswara; Lin, Shih-Shen; Chou, Ming-Yung; Shen, Yi-Ting; Wu, Chih-Hao; Geethangili, Madamanchi; Yang, Chi-Chiang; Tzeng, Yew-Min

    2010-10-15

    An ergostane type triterpenoid methylantcinate A (MAA) isolated from the fruiting bodies of Antrodia camphorata inhibited the growth of oral cancer cell lines OEC-M1 and OC-2 in a dose-dependent manner, without cytotoxic to normal oral gingival fibroblast cells. The major mechanism of growth inhibition was apoptosis induction, as shown by flow cytometric analysis of annexin V-FITC and propidium iodide staining, caspase-3 activation and DNA fragmentation. The increased expression of pro-apoptotic Bax, poly-(ADP-ribose) polymerase cleavage, and activated caspase-3 and decreased expression of anti-apoptotic Bcl-2 and Bcl-xL were also observed. These results provide the first evidence that the anti-oral cancer effects of MAA may involve a mechanism through the mitochondrial dependent pathway. Thus, results reported here may offer further impulse to the development of MAA analogues as potential chemotherapeutic targets for oral cancer complications. PMID:20817519

  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. Assessing yeast viability from cell size measurements?

    PubMed

    Tibayrenc, Pierre; Preziosi-Belloy, Laurence; Roger, Jean-Michel; Ghommidh, Charles

    2010-08-20

    During microbial cell cultures, environmental conditions affect cell physiology and subsequently process efficiency. Physiological changes result in changing cell morphology, such as cell size variations. The aim of this work was to study cell size evolution of a Saccharomyces cerevisiae population exposed to various stresses during alcoholic batch fermentations, and to evaluate the potential use of cell size measurements to infer cell viability. During a reference culture, without perturbation, viability as assessed by propidium iodide staining (PI) remained 100% and mean cell diameter was found to be above 5microm. A rapid temperature shift from 33 to 43 degrees C at 50gl(-1) of ethanol resulted in an immediate arrest of growth and triggered a progressive loss of viability from 100% to 0% and a decrease of mean cell diameter from 5.2 to 3.7microm. Cell size distribution curves obtained with a cell counter showed an increasing subpopulation of significantly smaller cells. At single-cell level, combined microscopy size measurements and PI staining showed that this subpopulation was exclusively composed of dead cells. Similar results were obtained after acetic acid or furfural additions. Accordingly, a multivariate data analysis was achieved to estimate the ratio of dead cells from cell size distributions obtained using the cell counter. PMID:20599572

  1. 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

  2. Pleiotropic effects of cadmium in mesangial cells

    SciTech Connect

    Xiao Weiqun; Liu Ying; Templeton, Douglas M.

    2009-08-01

    The mesangial cell of the renal glomerulus is exposed to circulating toxic substances and is a target involved in the glomerular component of chronic occupational and environmental exposure to cadmium. We review evidence for the involvement of cadmium in mesangial cell pathology, including effects on cell signaling, oncogene expression, and cell death. Previously we have shown that cadmium can inhibit apoptosis initiated through both the extrinsic (death ligand receptor) and intrinsic (mitochondrial) pathways, whereas exposure of mesangial cells to 10 {mu}M CdCl{sub 2} for 6 h initiates caspase-independent cell death through both apoptotic and apoptotic-like (annexin V positive, propidium iodide staining) mechanisms. Apoptotic death is dependent upon activation of Ca{sup 2+}/calmodulin-dependent protein kinase II (CaMK-II). In the present study we show that low level exposure of mesangial cells to Cd{sup 2+} (0.5 {mu}M) initiates cell survival signals including PI3 kinase/Akt signaling, also dependent on CaMK-II, that are eventually overcome resulting in caspase-dependent cell death. These studies underscore the roles of cell signaling in various modes of cell death, and in particular the central role of CaMK-II in cadmium toxicology of the mesangial cell.

  3. Events associated with apoptotic effect of p-Coumaric acid in HCT-15 colon cancer cells

    PubMed Central

    Jaganathan, Saravana Kumar; Supriyanto, Eko; Mandal, Mahitosh

    2013-01-01

    AIM: To investigate the events associated with the apoptotic effect of p-Coumaric acid, one of the phenolic components of honey, in human colorectal carcinoma (HCT-15) cells. METHODS: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tertazolium-bromide assay was performed to determine the antiproliferative effect of p-Coumaric acid against colon cancer cells. Colony forming assay was conducted to quantify the colony inhibition in HCT 15 and HT 29 colon cancer cells after p-Coumaric acid treatment. Propidium Iodide staining of the HCT 15 cells using flow cytometry was done to study the changes in the cell cycle of treated cells. Identification of apoptosis was done using scanning electron microscope and photomicrograph evaluation of HCT 15 cells after exposing to p-Coumaric acid. Levels of reactive oxygen species (ROS) of HCT 15 cells exposed to p-Coumaric acid was evaluated using 2’, 7’-dichlorfluorescein-diacetate. Mitochondrial membrane potential of HCT-15 was assessed using rhodamine-123 with the help of flow cytometry. Lipid layer breaks associated with p-Coumaric acid treatment was quantified using the dye merocyanine 540. Apoptosis was confirmed and quantified using flow cytometric analysis of HCT 15 cells subjected to p-Coumaric acid treatment after staining with YO-PRO-1. RESULTS: Antiproliferative test showed p-Coumaric acid has an inhibitory effect on HCT 15 and HT 29 cells with an IC50 (concentration for 50% inhibition) value of 1400 and 1600 μmol/L respectively. Colony forming assay revealed the time-dependent inhibition of HCT 15 and HT 29 cells subjected to p-Coumaric acid treatment. Propidium iodide staining of treated HCT 15 cells showed increasing accumulation of apoptotic cells (37.45 ± 1.98 vs 1.07 ± 1.01) at sub-G1 phase of the cell cycle after p-Coumaric acid treatment. HCT-15 cells observed with photomicrograph and scanning electron microscope showed the signs of apoptosis like blebbing and shrinkage after p-Coumaric acid exposure. Evaluation of the lipid layer showed increasing lipid layer breaks was associated with the growth inhibition of p-Coumaric acid. A fall in mitochondrial membrane potential and increasing ROS generation was observed in the p-Coumaric acid treated cells. Further apoptosis evaluated by YO-PRO-1 staining also showed the time-dependent increase of apoptotic cells after treatment. CONCLUSION: These results depicted that p-Coumaric acid inhibited the growth of colon cancer cells by inducing apoptosis through ROS-mitochondrial pathway. PMID:24282361

  4. Bisphosphonates regulate cell proliferation, apoptosis and pro-osteoclastic expression in MG-63 human osteosarcoma cells

    PubMed Central

    CHANG, JUN; WANG, WEI; ZHANG, HUI; HU, YONG; YIN, ZONGSHENG

    2012-01-01

    Bisphosphonates are well established in the management of cancer-induced skeletal complications. Recent studies suggest that nitrogen-containing bisphosphonates (N-BPs) promote the apoptosis of cancer cells as well as osteoclasts in bone metastatic sites. To investigate whether N-BPs exhibit a direct antitumor effect on osteoclasts, the current study investigated the effects of zoledronic acid (ZOL) on MG-63 cells in vitro. MG-63 cells were treated with ZOL. The inhibitory effect of ZOL on the growth of MG-63 cells was measured by MTT assay. ZOL-induced apoptosis of the MG-63 cells was examined by Hoechst 33258 staining, electron microscopy, Annexin V-FITC and propidium iodide staining. Reverse-transcription polymerase chain reaction (RT-PCR) and western blotting analysis were employed to assess the expression of osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL). The MTT assay showed that ZOL induced a distinct dose- and time-dependent reduction of cell viability with an IC50 value of 52.37±1.0 μM for 72 h. Flow cytometric analysis further revealed that the cell apoptosis was induced by arrest of the cell cycle in the G1 phase. RT-PCR and western blot analysis demonstrated that ZOL upregulated OPG expression. These results suggest that ZOL has direct effects on osteosarcoma cell growth and apoptosis. Increased OPG expression is an indirect effect, possibly via changes in the local microenvironment. PMID:22844373

  5. Size-based cell sorting with a resistive pulse sensor and an electromagnetic pump in a microfluidic chip.

    PubMed

    Song, Yongxin; Li, Mengqi; Pan, Xinxiang; Wang, Qi; Li, Dongqing

    2015-02-01

    An electrokinetic microfluidic chip is developed to detect and sort target cells by size from human blood samples. Target-cell detection is achieved by a differential resistive pulse sensor (RPS) based on the size difference between the target cell and other cells. Once a target cell is detected, the detected RPS signal will automatically actuate an electromagnetic pump built in a microchannel to push the target cell into a collecting channel. This method was applied to automatically detect and sort A549 cells and T-lymphocytes from a peripheral fingertip blood sample. The viability of A549 cells sorted in the collecting well was verified by Hoechst33342 and propidium iodide staining. The results show that as many as 100 target cells per minute can be sorted out from the sample solution and thus is particularly suitable for sorting very rare target cells, such as circulating tumor cells. The actuation of the electromagnetic valve has no influence on RPS cell detection and the consequent cell-sorting process. The viability of the collected A549 cell is not impacted by the applied electric field when the cell passes the RPS detection area. The device described in this article is simple, automatic, and label-free and has wide applications in size-based rare target cell sorting for medical diagnostics. PMID:25146579

  6. Memory CD8+ T cells colocalize with IL-7+ stromal cells in bone marrow and rest in terms of proliferation and transcription

    PubMed Central

    Sercan Alp, Özen; Durlanik, Sibel; Schulz, Daniel; McGrath, Mairi; Grün, Joachim R; Bardua, Marcus; Ikuta, Koichi; Sgouroudis, Evridiki; Riedel, René; Zehentmeier, Sandra; Hauser, Anja E; Tsuneto, Motokazu; Melchers, Fritz; Tokoyoda, Koji; Chang, Hyun-Dong; Thiel, Andreas; Radbruch, Andreas

    2015-01-01

    It is believed that memory CD8+ T cells are maintained in secondary lymphoid tissues, peripheral tissues, and BM by homeostatic proliferation. Their survival has been shown to be dependent on IL-7, but it is unclear where they acquire it. Here we show that in murine BM, memory CD8+ T cells individually colocalize with IL-7+ reticular stromal cells. The T cells are resting in terms of global transcription and do not express markers of activation, for example, 4-1BB (CD137), IL-2, or IFN-γ, despite the expression of CD69 on about 30% of the cells. Ninety-five percent of the memory CD8+ T cells in BM are in G0 phase of cell cycle and do not express Ki-67. Less than 1% is in S/M/G2 of cell cycle, according to propidium iodide staining. While previous publications have estimated the extent of proliferation of CD8+ memory T cells on the basis of BrdU incorporation, we show here that BrdU itself induces proliferation of CD8+ memory T cells. Taken together, the present results suggest that CD8+ memory T cells are maintained as resting cells in the BM in dedicated niches with their survival conditional on IL-7 receptor signaling. PMID:25639669

  7. 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

  8. Survival of Host-Associated Bacteroidales Cells and Their Relationship with Enterococcus spp., Campylobacter jejuni, Salmonella enterica Serovar Typhimurium, and Adenovirus in Freshwater Microcosms as Measured by Propidium Monoazide-Quantitative PCR

    PubMed Central

    Bae, Sungwoo

    2012-01-01

    The ideal host-associated genetic fecal marker would be capable of predicting the presence of specific pathogens of concern. Flowthrough freshwater microcosms containing mixed feces and inocula of the pathogens Campylobacter jejuni, Salmonella enterica serovar Typhimurium, and adenovirus were placed at ambient temperature in the presence and absence of diurnal sunlight. The total Enterococcus DNA increased during the early periods (23 h) under sunlight exposure, even though cultivable Enterococcus and DNA in intact cells, as measured by propidium monoazide (PMA), decreased with first-order kinetics during the entire period. We found a significant difference in the decay of host-associated Bacteroidales cells between sunlight exposure and dark conditions (P value < 0.05), whereas the persistence of host-associated Bacteroidales DNA was comparable. The 2-log reduction times of adenovirus were 72 h for sunlight exposure and 99 h for dark conditions with similar decay rate constants (P value = 0.13). The persistences of fecal Bacteroidales cells and Campylobacter cells exposed to sunlight were similar, and host-associated Bacteroidales DNA and waterborne pathogen DNA were degraded at comparable rates (P values > 0.05). Overall, the ratio of quantitative PCR (qPCR) cycle threshold (CT) values with and without PMA treatment was indicative of the time elapsed since inoculation of the microcosm with (i) fecal material from different animal sources based on host-associated Bacteroidales and (ii) pure cultures of bacterial pathogens. The use of both PMA-qPCR and qPCR may yield more realistic information about recent sources of fecal contamination and result in improved prediction of waterborne pathogens and assessment of health risk. PMID:22139002

  9. 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

  10. The protective effect of dopamine against OGD/R injury-induced cell death in HT22 mouse hippocampal cells.

    PubMed

    Wang, Wenzhu; Zhao, Lixi; Bai, Fan; Zhang, Tong; Dong, Hao; Liu, Lixu

    2016-03-01

    Previous studies have shown that levo-dopamine (l-dopa) can improve the consciousness of certain patients with prolonged coma after cerebral ischemia-reperfusion injury, and promote cell growth in vivo. This study aimed to investigate whether l-dopa, which is used clinically to treat Parkinson's disease, might also ameliorate ischemia-reperfusion injury-induced cell death. The oxygen-glucose deprivation and re-oxygenation (OGD/R) model was used to mimic the ischemia-reperfusion pathological process in vitro. HT22 cells were treated with dopamine hydrochloride at different times (i.e., 2h prior to OGD, during the period of OGD, during the period of R, and throughout the period of OGD/R) and at different concentrations (i.e., 25?M, 50?M, 100?M). Lactate dehydrogenase (LDH) release, flow cytometry-annexin V, and propidium iodide staining with light microscopy showed that dopamine hydrochloride (added during re-oxygenation) promoted cell proliferation and facilitated maintenance of normal cell morphology. However, when present during oxygen-glucose deprivation for 18h and present throughout OGD/R, dopamine hydrochloride increased cell damage as manifested by shrinkage, rounding up, and reduced viability. In conclusion, dopamine protected HT22 cells from OGD/R injury-induced cell death only at a particular point in time, suggesting that it may be useful for treating severe ischemia-reperfusion brain injury. PMID:26867202

  11. A new fibrin sealant as a three-dimensional scaffold candidate for mesenchymal stem cells

    PubMed Central

    2014-01-01

    Introduction The optimization of an organic scaffold for specific types of applications and cells is vital to successful tissue engineering. In this study, we investigated the effects of a new fibrin sealant derived from snake venom as a scaffold for mesenchymal stem cells, to demonstrate the ability of cells to affect and detect the biological microenvironment. Methods The characterization of CD34, CD44 and CD90 expression on mesenchymal stem cells was performed by flow cytometry. In vitro growth and cell viability were evaluated by light and electron microscopy. Differentiation into osteogenic, adipogenic and chondrogenic lineages was induced. Results The fibrin sealant did not affect cell adhesion, proliferation or differentiation and allowed the adherence and growth of mesenchymal stem cells on its surface. Hoechst 33342 and propidium iodide staining demonstrated the viability of mesenchymal stem cells in contact with the fibrin sealant and the ability of the biomaterial to maintain cell survival. Conclusions The new fibrin sealant is a three-dimensional scaffolding candidate that is capable of maintaining cell survival without interfering with differentiation, and might also be useful in drug delivery. Fibrin sealant has a low production cost, does not transmit infectious diseases from human blood and has properties of a suitable scaffold for stem cells because it permits the preparation of differentiated scaffolds that are suitable for every need. PMID:24916098

  12. Study the oxidative injury of yeast cells by NADH autofluorescence

    NASA Astrophysics Data System (ADS)

    Liang, Ju; Wu, Wen-Lan; Liu, Zhi-Hong; Mei, Yun-Jun; Cai, Ru-Xiu; Shen, Ping

    2007-06-01

    Autofluorescence has an advantage over the extrinsic fluorescence of an unperturbed environment during investigation, especially in complex system such as biological cells and tissues. NADH is an important fluorescent substance in living cells. The time courses of intracellular NADH autofluorescence in the process of yeast cells exposed to H 2O 2 and ONOO - have been recorded in detail in this work. In the presence of different amounts of H 2O 2 and ONOO -, necrosis, apoptosis and reversible injury are initiated in yeast cells, which are confirmed by acridine orange/ethidum bromide and Annexin V/propidium iodide staining. It is found that intracellular NADH content increases momently in the beginning of the apoptotic process and then decreases continually till the cell dies. The most remarkable difference between the apoptotic and the necrotic process is that the NADH content in the latter case changes much more sharply. Further in the case of reversible injury, the time course of intracellular NADH content is completely different from the above two pathways of cell death. It just decreases to some degree firstly and then resumes to the original level. Based on the role of NADH in mitochondrial respiratory chain, the time course of intracellular NADH content is believed to have reflected the response of mitochondrial redox state to oxidative stress. Thus, it is found that the mitochondrial redox state changes differently in different pathways of oxidative injury in yeast cells.

  13. Calpains are involved in Entamoeba histolytica-induced death of HT-29 colonic epithelial cells.

    PubMed

    Jang, Yun Soo; Song, Kyoung-Ju; Kim, Ju Young; Lee, Young Ah; Kim, Kyeong Ah; Lee, Sang Kyou; Shin, Myeong Heon

    2011-06-01

    Entamoeba histolytica is an enteric tissue-invading protozoan parasite that can cause amebic colitis and liver abscess in humans. E. histolytica has the capability to kill colon epithelial cells in vitro; however, information regarding the role of calpain in colon cell death induced by ameba is limited. In this study, we investigated whether calpains are involved in the E. histolytica-induced cell death of HT-29 colonic epithelial cells. When HT-29 cells were co-incubated with E. histolytica, the propidium iodide stained dead cells markedly increased compared to that in HT-29 cells incubated with medium alone. This pro-death effect induced by ameba was effectively blocked by pretreatment of HT-29 cells with the calpain inhibitor, calpeptin. Moreover, knockdown of m- and -calpain by siRNA significantly reduced E. histolytica-induced HT-29 cell death. These results suggest that m- and -calpain may be involved in colon epithelial cell death induced by E. histolytica. PMID:21738275

  14. 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

  15. 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

  16. Ginsenoside compound K induces apoptosis in nasopharyngeal carcinoma cells via activation of apoptosis-inducing factor

    PubMed Central

    2014-01-01

    Background Nasopharyngeal carcinoma (NPC) has a high incidence rate in Southern China. Although there are conventional therapies, the side effects and toxicities are not always tolerable for patients. Recently, the tumoricidal effect of ginsenosides on different cancer cells has been studied. This study aims to investigate the anti-cancer effect of ginsenosides on NPC cells and their underlying mechanism. Methods The cytotoxicity of ginsenosides on NPC cell line HK-1 was measured by MTT assay. Apoptosis was detected by propidium iodide staining followed by flow cytometry. A xenograft tumor model was established by injecting nude mice with HK-1 cells. The activation of caspases and apoptosis-inducing factor (AIF) were evaluated by Western blot analysis. Nuclear translocation of AIF was also studied by immunofluorescence staining. Mitochondrial membrane potential was measured by JC-1 dye using flow cytometry. Results Four ginsenosides, 20 (S)-Rh2, compound K (CK), panaxadiol (PD) and protopanaxadiol (PPD), induced apoptotic cell death in HK-1 cells in a concentration-dependent manner. CK inhibited HK-1 xenograft tumor growth most extensively and depleted mitochondrial membrane potential depolarization and induced translocation of AIF from cytoplasm to nucleus in HK-1 cells. In addition, depletion of AIF by siRNA abolished CK-induced HK-1 cell death. Conclusion Ginsenoside CK-induced apoptosis of HK-1 cells was mediated by the mitochondrial pathway and could significantly inhibit tumor growth in vivo. PMID:24690317

  17. The histone deacetylase inhibitors vorinostat and romidepsin downmodulate IL-10 expression in cutaneous T-cell lymphoma cells

    PubMed Central

    Tiffon, CE; Adams, JE; van der Fits, L; Wen, S; Townsend, PA; Ganesan, A; Hodges, E; Vermeer, MH; Packham, G

    2011-01-01

    BACKGROUND AND PURPOSE Vorinostat and romidepsin are histone deacetylase inhibitors (HDI), approved for the treatment of cutaneous T-cell lymphoma (CTCL). However, the mechanism(s) by which these drugs exert their anti-cancer effects are not fully understood. Since CTCL is associated with immune dysregulation, we investigated whether these HDI modulated cytokine expression in CTCL cells. EXPERIMENTAL APPROACH CTCL cell lines and primary CTCL cells were treated in vitro with vorinostat or romidepsin, or with STAT3 pathway inhibitors. Cell cycle parameters and apoptosis were analysed by propidium iodide and annexin V/propidium iodide staining respectively. Cytokine expression was analysed using QRT-PCR and elisa assays. STAT3 expression/phosphorylation and transcriptional activity were analysed using immunoblotting and transfection/reporter assays respectively. KEY RESULTS Vorinostat and romidepsin strongly down-regulated expression of the immunosuppressive cytokine, interleukin (IL)-10, frequently overexpressed in CTCL, at both the RNA and protein level in CTCL cell lines and at the RNA level in primary CTCL cells. Vorinostat and romidepsin also increased expression of IFNG RNA and decreased expression of IL-2 and IL-4 RNA, although to a lesser extent compared to IL-10. Transient exposure to vorinostat was sufficient to suppress IL-10 secretion but was not sufficient to irreversibly commit cells to undergo cell death. STAT3 pathway inhibitors decreased production of IL-10 and vorinostat/romidepsin partially decreased STAT3-dependent transcription without effects on STAT3 expression or phosphorylation. CONCLUSIONS AND IMPLICATIONS These results demonstrate that HDI modulate cytokine expression in CTCL cells, potentially via effects on STAT3. Immunomodulation may contribute to the clinical activity of HDI in this disease. PMID:21198545

  18. Effects of ethanol on pancreatic beta-cell death: interaction with glucose and fatty acids.

    PubMed

    Dembele, Korami; Nguyen, K Hoa; Hernandez, Tiffany A; Nyomba, B L Grégoire

    2009-04-01

    Western lifestyle plays an important role in the prevalence of type 2 diabetes by causing insulin resistance and pancreatic beta-cell dysfunction, a prerequisite for the development of diabetes. High fat diet and alcohol are major components of the western diet. The aim of the present study was to investigate the effects of ethanol and fatty acids on beta-cell survival and metabolism. We treated the rat beta-cell line RINm5F with ethanol, a mixture of palmitic and oleic acids, or both. Reactive oxygen species (ROS) were determined by (5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate) (CM-H2DCFDA) fluorescence assay, and mitochondrial activity was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) reduction assay and by determining ATP production. Cell viability was assessed with a cell counter and trypan blue exclusion, and the mode of cell death by Hoechst33342 and propidium iodide staining. With both ethanol and fatty acid treatments, MTT reduction and ATP production decreased, whereas ROS production increased. Ethanol treatment had no effect on cell number, whereas fatty acid treatment reduced the cell number. Cell incubation with ethanol, fatty acids, or both increased the number of Hoechst 33342-positive nuclei. However, the majority of nuclei from fatty acid-treated cells were stained with propidium iodide, indicating a loss of plasma membrane integrity. We conclude that both ethanol and fatty acids generate cellular oxidative stress, and affect mitochondrial function in RINm5F beta-cells. However, ethanol causes beta-cell death by apoptosis, whereas fatty acids cause cell death predominantly by necrosis. It is not known whether these results are applicable to human beta-cells. PMID:18330713

  19. 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

  20. Growth arrest and non-apoptotic programmed cell death associated with the up-regulation of c-myc mRNA expression in T-47D breast tumor cells following exposure to Epipremnum pinnatum (L.) Engl. hexane extract.

    PubMed

    Tan, M L; Muhammad, T S Tengku; Najimudin, N; Sulaiman, S F

    2005-01-15

    Epipremnum pinnatum (L.) Engl. hexane extract produced a significant growth inhibition against T-47D breast carcinoma cells and analysis of cell death mechanisms indicated that the extract elicited a non-apoptotic programmed cell death. T-47D cells exposed to the extract at EC(50) concentration (72 h) for 24 h failed to demonstrate typical DNA fragmentation associated with apoptosis, as carried out using a modified TUNEL assay. In addition, acute exposure to the extract produced an insignificant regulation of caspase-3 and p53 mRNA expression but increased in the c-myc mRNA expression. Ultrastructural analysis using transmission electron microscope demonstrated distinct vacuolated cells, which strongly indicated a Type II non-apoptotic cell death although the changes in chromatin were also detected. The presence of non-apoptotic programmed cell death was then reconfirmed with annexin-V and propidium iodide staining. These findings suggested that up-regulation of c-myc mRNA expression may have contributed to the growth arrest and Type II non-apoptotic programmed cell death in the Epipremnum pinnatum (L.) Engl. hexane extract-treated T-47D cells. PMID:15619555

  1. 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

  2. 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

  3. 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

  4. Low Programmed Cell Death 5 Expression is a Prognostic Factor in Ovarian Cancer

    PubMed Central

    Gao, Li; Ye, Xue; Ma, Rui-Qiong; Cheng, Hong-Yan; Han, Hong-Jing; Cui, Heng; Wei, Li-Hui; Chang, Xiao-Hong

    2015-01-01

    Background: Ovarian cancer is a leading gynecological malignancy. We investigated the prognostic value of programmed cell death 5 (PDCD5) in patients with ovarian cancer. Methods: Expression levels of PDCD5 mRNA and protein were examined in six ovarian cancer cell lines (SKOV3, CAOV3, ES2, OV1, 3AO, and HOC1A) and one normal ovarian epithelial cell line (T29) using reverse transcription polymerase chain reaction, Western blotting, and flow cytometry. After inducing PDCD5 induction in SKOV3 cells or treating this cell line with taxol or doxorubicin (either alone or combined), apoptosis was measured by Annexin V-FITC/propidium iodide staining. Correlations between PDCD5 protein expression and pathological features, histological grade, FIGO stage, effective cytoreductive surgery, and serum cancer antigen-125 values were evaluated in patients with ovarian cancer. Results: PDCD5 mRNA and protein expression were downregulated in ovarian cancer cells. Recombinant human PDCD5 increased doxorubicin-induced apoptosis in SKOV3 cells (15.96 ± 2.07%, vs. 3.17 ± 1.45% in controls). In patients with ovarian cancer, PDCD5 expression was inversely correlated with FIGO stage, pathological grade, and patient survival (P < 0.05, R = 0.7139 for survival). Conclusions: PDCD5 expression is negatively correlated with disease progression and stage in ovarian cancer. Therefore, measuring PDCD5 expression may be a good method of determining the prognosis of ovarian cancer patients. PMID:25881604

  5. 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

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

    PubMed

    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

  7. 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

  8. shRNA-mediated RPS15A silencing inhibits U937 acute myeloid leukemia cell proliferation and enhances apoptosis.

    PubMed

    Li, Guangyao; Zhang, Li; Liu, Jizhu; Xiao, Taiwu; Liu, Guozhen; Wang, Jingxia; Hou, Ming

    2016-05-01

    Ribosomal protein S15a (RPS15A), which is a component of the 40S ribosomal subunit, is able to promote mRNA/ribosome interaction during the early stage of translation. Previous studies have demonstrated that RPS15A regulates cell growth and is involved in several types of human cancer. The aim of the present study was to investigate the role of RPS15A in acute myeloid leukemia (AML). Lentivirus‑delivered short hairpin RNA (shRNA) was used to silence RPS15A expression in the U937 AML cell line. Subsequently, the effects of RPS15A silencing on cell viability, cell cycle progression and apoptosis were investigated. The results indicated that RPS15A knockdown significantly inhibited cell growth. Furthermore, flow cytometric analysis demonstrated that the majority of U937 cells were arrested in G0/G1 phase and sub‑G1 phase after RPS15A knockdown, as determined using propidium iodide staining. In addition, U937 cells underwent apoptosis in response to RPS15A silencing, as determined using Annexin V/7‑aminoactinomycin D staining. In conclusion, the present study provides novel evidence indicating that RPS15A modulates AML cell growth in vitro, and may be considered a novel therapeutic target for the treatment of AML. PMID:27035327

  9. 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

  10. Increased sensitivity of damaged DNA to digestion with nuclease S1 as assessed in single cells by flow cytometry

    SciTech Connect

    Prosperi, E.; Supino, R.; Bottiroli, G. )

    1993-01-01

    DNA sensitivity to digestion with nuclease S1 was investigated in cells irradiated with gamma rays, or treated with the antitumor drug adriamycin (Adr). The nuclease-resistant DNA fraction was determined by propidium iodide staining. Treated cells were found to be more sensitive to nuclease digestion than the undamaged controls. Gamma ray-induced strand breaks were detectable at doses up to 10 Gy; an increase in the reaction temperature, from 37[degrees] to 63[degrees]C, was necessary in order to detect higher levels of damage. Nuclease S1 sensitivity in Adr-treated cells showed a single-peak, concentration-dependent relationship, in agreement with the known self-inhibitory effect exerted by high drug doses. Determination of DNA digestion could be performed in combination with other cellular parameters (e.g., protein content). Detection of drug-resistant cells in a heterogeneous population of small-cell lung carcinoma was achieved on the basis of the different sensitivity of the cells to enzymatic digestion. These results indicate that nuclease S1 may be a useful probe for studying in single cells DNA alterations induced by drugs or radiation. 31 refs., 8 figs.

  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. Cryopreservation of carrot (Daucus carota l.) cell suspensions and protoplasts by vitrification.

    PubMed

    Chen, Yong; Wang, Jun-Hui

    2003-01-01

    Carrot cell suspensions and protoplasts were successfully cryopreserved by vitrification. Cells were precultured in liquid Murashige and Skoog medium containing 0.175 M sucrose for 3 d and then in liquid MS medium containing 0.4 M sorbitol for 1 d. After loading of the precultured carrot cells in 25 % PVS2 at room temperature for 5 min and treatment with 100 % PVS2 at 0 degrees C for 7.5 min, they were quenched in liquid nitrogen. Optimal survival was 83.3 % (based on the triphenyl tetrazolium chloride reduction assay) following warming and unloading. Recovered cells retained the ability to regenerate plantlets in vitro. In the case of vitrification of protoplasts isolated from carrot cell suspensions, the optimal loading and dehydration durations were 5 min in 25% PVS2 and 3 min 100 % PVS2 respectively. Survival of 47 % of the untreated control (based on the FDA-PI (fluorescein diacetate-propidium iodide) staining) was achieved after cryopreservation. PMID:12644854

  13. Cabergoline protects SH-SY5Y neuronal cells in an in vitro model of ischemia.

    PubMed

    Miglio, Gianluca; Varsaldi, Federica; Francioli, Elisabetta; Battaglia, Angelo; Canonico, Pier Luigi; Lombardi, Grazia

    2004-04-12

    Dopamine receptor agonists are protective in different models of neurodegeneration by both receptor-dependent and -independent mechanisms. We used SH-SY5Y cells, differentiated into neuron-like type, to evaluate if cabergoline, a dopamine D2 receptor agonist endowed with anti-oxidant activity, protects the cells against ischemia (oxygen-glucose deprivation model). Cabergoline protected the cells from ischemia-induced cell death in a concentration-dependent manner (EC(50)=1.2 microM), as demonstrated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, lactate dehydrogenase (LDH) release, and fluorescein diacetate-propidium iodide staining. This effect, observed even when the drug was added after oxygen-glucose deprivation, was not mediated by either dopamine D2 receptor activation or anti-apoptotic Bcl-2 protein over-expression (Western blotting analysis), but was linked to a reduction in cellular free radical loading (2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining) and membrane lipid peroxidation (thiobarbituric acid-reacting test). In conclusion, cabergoline protects in vitro neurons against ischemia-induced cell death, suggesting its possible use in the therapy of other neurodegenerative diseases in addition to Parkinson's disease. PMID:15087238

  14. Effects of STAT3 Gene Silencing and Rapamycin on Apoptosis in Hepatocarcinoma Cells

    PubMed Central

    Zhang, Yi; Zhang, Jun-Wei; Lv, Guo-Yue; Xie, Shu-Li; Wang, Guang-Yi

    2012-01-01

    The PI3K/Akt/mTOR and JAK/STAT3 signaling pathways are important for regulating apoptosis, and are frequently activated in cancers. In this study, we targeted STAT3 and mTOR in human hepatocellular carcinoma Bel-7402 cells and examined the subsequent alterations in cellular apoptosis. The expression of STAT3 was silenced with small interfering RNA (siRNA)-expressing plasmid. The activity of mTOR was inhibited using rapamycin. Following treatment, Annexin V/propidium iodide staining followed by flow cytometry and Hoechst33258 immunofluorescence staining was used to examine cellular apoptosis. JC-1 staining was used to monitor depolarization of mitochondrial membrane (ΔΨm). Furthermore, the expression of activated caspase 3 protein was analyzed by Western blotting. Compared to non-treated or control siRNA-transfected cells, significantly higher levels of apoptosis were detected in siSTAT3-transfected or rapamycin-treated cells (P < 0.05), which was further enhanced in cells targeted for both molecules (P < 0.05). The pro-apoptotic effects were accompanied with concomitant depolarization of mitochondrial membrane and up-regulation of activated caspase 3. Combined treatments using rapamycin and STAT3 gene silencing significantly increases apoptosis in Bel-7402 cells, displaying more dramatic effect than any single treatment. This study provides evidence for targeting multiple molecules in cancer therapy. PMID:22408571

  15. 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

  16. 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

  17. 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

  18. The mechanism of NPC-14686-induced [Ca?]i rises and non-Ca?-triggered cell death in MG63 human osteosarcoma cells.

    PubMed

    Chien, Jau-Min; Chou, Chiang-Ting; Liang, Wei-Zhe; Kuo, Daih-Huang; Kuo, Chun-Chi; Ho, Chin-Man; Shieh, Pochuen; Jan, Chung-Ren

    2014-06-30

    NPC-14686 has been shown to have anti-inflammatory effect in previous studies, but the mechanisms are unclear. The effect of NPC-14686 on cytosolic Ca? concentrations ([Ca?]i) and viability in MG63 human osteosarcoma cells was explored. The Ca?-sensitive fluorescent dye fura-2 was applied to measure [Ca?]i. NPC-14686 at concentrations of 100-500 ?M induced a [Ca?]i rise in a concentration-dependent manner. The response was reduced by 80% by removing Ca?. NPC-14686 induced Mn? influx leading to quenching of fura-2 fluorescence. NPC-14686-evoked Ca? entry was suppressed by nifedipine, econazole, SK&F96365, and protein kinase C inhibitor. Inhibition of phospholipase C with U73122 abolished NPC-14686-induced [Ca?]i rise. At 20-50 ?M, NPC-14686 decreased cell viability, which was not reversed by chelating cytosolic Ca? 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 NPC-14686 (30-50 ?M) induced apoptosis in a concentration-dependent manner. NPC-14686 also increased levels of reactive oxygen species. Together, in human osteosarcoma cells, NPC-14686 induced a [Ca?]i rise by inducing phospholipase C-dependent Ca? release from the endoplasmic reticulum and Ca? entry via protein kinase C-sensitive store-operated Ca? channels. NPC-14686 induced cell death that might involve apoptosis via mitochondrial pathways. PMID:24826784

  19. Intracellular pH distribution as a cell health indicator in Saccharomyces cerevisiae

    PubMed Central

    Aabo, Thomas; Glckstad, Jesper; Siegumfeldt, Henrik; Arneborg, Nils

    2011-01-01

    Internal pH regulation is vital for many cell functions, including transport mechanisms and metabolic enzyme activity. More specifically, transport mechanisms are to a wide degree governed by internal pH distributions. We introduce the term standard deviation of the intracellular pH (s.d.(pHint)) to describe the internal pH distributions. The cellular pH distributional response to external stress such as heat has not previously been determined. In this study, the intracellular pH (pHi) and the s.d.(pHint) of Saccharomyces cerevisiae cells exposed to supralethal temperatures were measured using fluorescence ratio imaging microscopy (FRIM). An exponential decline in pHi was observed after an initial small decline. For the first time, we report the use of FRIM for determining in vivo plasma membrane proton permeability coefficients in yeast. Furthermore, the exponential decay of pHi and the rupture of the cell plasma membrane, as measured by propidium iodide staining, at 70C were not simultaneous but were separated by a significant temporal difference. Finally, a nonlinear relationship between the pHi and s.d.(pHint) was found; i.e. the s.d.(pHint) was significantly more sensitive to supralethal temperatures than pHi. s.d.(pHint) is therefore proposed as an early health/vitality indicator in S. cerevisiae cells exposed to heat stress. PMID:21527496

  20. Cytoprotection by the NO-donor SNAP against ischemia/reoxygenation injury in mouse embryonic stem cell-derived cardiomyocytes.

    PubMed

    Grbe, A; Varga, Z V; Plczi, J; Rungarunlert, S; Klincumhom, N; Pirity, M K; Madonna, R; Eschenhagen, T; Dinnys, A; Csont, T; Ferdinandy, P

    2014-03-01

    Embryonic stem cell (ESC)-derived cardiomyocytes are a promising cell source for the screening for potential cytoprotective molecules against ischemia/reperfusion injury, however, little is known on their behavior in hypoxia/reoxygenation conditions. Here we tested the cytoprotective effect of the NO-donor SNAP and its downstream cellular pathway. Mouse ESC-derived cardiomyocytes were subjected to 150-min simulated ischemia (SI) followed by 120-min reoxygenation or corresponding non-ischemic conditions. The following treatments were applied during SI or normoxia: the NO-donor S-Nitroso-N-acetyl-D,L-penicillamine (SNAP), the protein kinase G (PKG) inhibitor, the KATP channel blocker glibenclamide, the particulate guanylate cyclase activator brain type natriuretic peptide (BNP), and a non-specific NO synthase inhibitor (N-Nitro-L-arginine, L-NNA) alone or in different combinations. Viability of cells was assayed by propidium iodide staining. SNAP attenuated SI-induced cell death in a concentration-dependent manner, and this protection was attenuated by inhibition of either PKG or KATP channels. However, SI-induced cell death was not affected by BNP or by L-NNA. We conclude that SNAP protects mESC-derived cardiomyocytes against SI/R injury and that soluble guanylate-cyclase, PKG, and KATP channels play a role in the downstream pathway of SNAP-induced cytoprotection. The present mESC-derived cardiomyocyte based screening platform is a useful tool for discovery of cytoprotective molecules. PMID:24078218

  1. 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.

  2. 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

  3. 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

  4. Degradation of miR-21 induces apoptosis and inhibits cell proliferation in human hepatocellular carcinoma.

    PubMed

    Najafi, Z; Sharifi, M; Javadi, G

    2015-11-01

    MicroRNAs (miRNAs) are small non-coding RNAs, 18-25 nucleotides long and have an important role in post-transcriptional regulation of gene. Several aspects of cellular activities such as cell growth, proliferation and differentiation are regulated by miRNAs. In many cancers and malignancies, up- or downregulation of different miRNAs has been reported. In human hepatocellular carcinoma (HCC), upregulation of miR-21 has been reported in human in vitro studies. Here, we made an assessment of the effect of miR-21 degradation on viability and apoptosis of HCC cell line (HepG2) using locked nucleic acid (LNA). At different time points (24, 48, 72 h) after LNA-anti-miR-21 transfection, 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide assay and Annexin/propidium iodide staining were performed. The results show that miR-21 degradation can decrease the viability of cells, mainly by induction of apoptosis and necrosis. These findings suggest that degradation of miR-21 could be used as a novel approach in treatment of HCC. PMID:26427512

  5. Involvement of Mst1 in tumor necrosis factor-{alpha}-induced apoptosis of endothelial cells

    SciTech Connect

    Ohtsubo, Hideki; Ichiki, Toshihiro Imayama, Ikuyo; Ono, Hiroki; Fukuyama, Kae; Hashiguchi, Yasuko; Sadoshima, Junichi; Sunagawa, Kenji

    2008-03-07

    Mammalian sterile 20-kinase 1 (Mst1), a member of the sterile-20 family protein kinase, plays an important role in the induction of apoptosis. However, little is know about the physiological activator of Mst1 and the role of Mst1 in endothelial cells (ECs). We examined whether Mst1 is involved in the tumor necrosis factor (TNF)-{alpha}-induced apoptosis of ECs. Western blot analysis revealed that TNF-{alpha} induced activation of caspase 3 and Mst1 in a time- and dose-dependent manner. TNF-{alpha}-induced Mst1 activation is almost completely prevented by pretreatment with Z-DEVD-FMK, a caspase 3 inhibitor. Nuclear staining with Hoechst 33258 and fluorescence-activated cell sorting of propidium iodide-stained cells showed that TNF-{alpha} induced apoptosis of EC. Diphenyleneiodonium, an inhibitor of NADPH oxidase, and N-acetylcysteine, a potent antioxidant, also inhibited TNF-{alpha}-induced activation of Mst1 and caspase 3, as well as apoptosis. Knockdown of Mst1 expression by short interfering RNA attenuated TNF-{alpha}-induced apoptosis but not cleavage of caspase 3. These results suggest that Mst1 plays an important role in the induction of TNF-{alpha}-induced apoptosis of EC. However, positive feedback mechanism between Mst1 and caspase 3, which was shown in the previous studies, was not observed. Inhibition of Mst1 function may be beneficial for maintaining the endothelial integrity and inhibition of atherogenesis.

  6. Toxicity and antibacterial assessment of chitosancoated silver nanoparticles on human pathogens and macrophage cells

    PubMed Central

    Jena, Prajna; Mohanty, Soumitra; Mallick, Rojee; Jacob, Biju; Sonawane, Avinash

    2012-01-01

    Background Pathogenic bacteria are able to develop various strategies to counteract the bactericidal action of antibiotics. Silver nanoparticles (AgNPs) have emerged as a potential alternative to conventional antibiotics because of their potent antimicrobial properties. The purpose of this study was to synthesize chitosan-stabilized AgNPs (CS-AgNPs) and test for their cytotoxic, genotoxic, macrophage cell uptake, antibacterial, and antibiofilm activities. Methods AgNPs were synthesized using chitosan as both a stabilizing and a reducing agent. Antibacterial activity was determined by colony-forming unit assay and scanning electron microscopy. Genotoxic and cytotoxic activity were determined by DNA fragmentation, comet, and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays. Cellular uptake and intracellular antibacterial activity were tested on macrophages. Results CS-AgNPs exhibited potent antibacterial activity against different human pathogens and also impeded bacterial biofilm formation. Scanning electron microscopy analysis indicated that CS-AgNPs kill bacteria by disrupting the cell membrane. CS-AgNPs showed no significant cytotoxic or DNA damage effect on macrophages at the bactericidal dose. Propidium iodide staining indicated active endocytosis of CS-AgNPs resulting in reduced intracellular bacterial survival in macrophages. Conclusion The present study concludes that at a specific dose, chitosan-based AgNPs kill bacteria without harming the host cells, thus representing a potential template for the design of antibacterial agents to decrease bacterial colonization and to overcome the problem of drug resistance. PMID:22619529

  7. Amyloid-like aggregates of neuronal tau induced by formaldehyde promote apoptosis of neuronal cells

    PubMed Central

    Nie, Chun Lai; Wang, Xing Sheng; Liu, Ying; Perrett, Sarah; He, Rong Qiao

    2007-01-01

    Background The microtubule associated protein tau is the principle component of neurofibrillar tangles, which are a characteristic marker in the pathology of Alzheimer's disease; similar lesions are also observed after chronic alcohol abuse. Formaldehyde is a common environmental contaminant and also a metabolite of methanol. Although many studies have been done on methanol and formaldehyde intoxication, none of these address the contribution of protein misfolding to the pathological mechanism, in particular the effect of formaldehyde on protein conformation and polymerization. Results We found that unlike the typical globular protein BSA, the natively-unfolded structure of human neuronal tau was induced to misfold and aggregate in the presence of ~0.01% formaldehyde, leading to formation of amyloid-like deposits that appeared as densely staining granules by electron microscopy and atomic force microscopy, and bound the amyloid-specific dyes thioflavin T and Congo Red. The amyloid-like aggregates of tau were found to induce apoptosis in the neurotypic cell line SH-SY5Y and in rat hippocampal cells, as observed by Hoechst 33258 staining, assay of caspase-3 activity, and flow cytometry using Annexin V and Propidium Iodide staining. Further experiments showed that Congo Red specifically attenuated the caspase-3 activity induced by amyloid-like deposits of tau. Conclusion The results suggest that low concentrations of formaldehyde can induce human tau protein to form neurotoxic aggregates, which could play a role in the induction of tauopathies. PMID:17241479

  8. 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

  9. Engagement of the leukocyte-associated Ig-like receptor-1 induces programmed cell death and prevents NF-kappaB nuclear translocation in human myeloid leukemias.

    PubMed

    Poggi, A; Pellegatta, F; Leone, B E; Moretta, L; Zocchi, M R

    2000-10-01

    Leukocyte-associated Ig-like receptor-1 (LAIR-1) is a surface molecule that functions as an inhibitory receptor on natural killer cells, T lymphocytes and monocytes. Here, we provide evidence that occupancy of LAIR-1 on human myelomonocytic leukemic cell lines inhibits proliferation and leads to programmed cell death (PCD), evaluated by propidium iodide staining and transmission electron microscopy. Interestingly, PCD elicited via LAIR-1 was not blocked by different caspase inhibitors, at variance with apoptosis induced via CD95/Fas, which was prevented by the caspase-1 and caspase-8 specific inhibitors. In addition, we show that the p65 subunit of the nuclear factor kappaB (NF-kappaB), constitutively expressed in the nucleus of these cell lines, was retained in the cytoplasm upon engagement of LAIR-1. This was evident already 8 h after LAIR-1 occupancy, when apoptosis was not yet detectable by fluorometric or ultrastructural analysis. Moreover, a reduction in inhibitor kappaBalpha phosphorylation was observed after LAIR-1 engagement. As blocking of NF-kappaB activation has been shown to rescue sensitivity to anti-cancer drugs in solid tumors, we suggest that LAIR-1 may represent a possible target for pharmacological approaches aimed to potentiate anti-leukemic therapy. PMID:11069054

  10. Involvement of Nrf2-Mediated Upregulation of Heme Oxygenase-1 in Mollugin-Induced Growth Inhibition and Apoptosis in Human Oral Cancer Cells

    PubMed Central

    Lee, Young-Man; Auh, Q-Schick; Lee, Deok-Won; Kim, Jun-Yeol; Jung, Ha-Jin; Lee, Seung-Ho; Kim, Eun-Cheol

    2013-01-01

    Although previous studies have shown that mollugin, a bioactive phytochemical isolated from Rubia cordifolia L. (Rubiaceae), exhibits antitumor effects, its biological activity in oral cancer has not been reported. We thus investigated the effects and putative mechanism of apoptosis induced by mollugin in human oral squamous cell carcinoma cells (OSCCs). Results show that mollugin induces cell death in a dose-dependent manner in primary and metastatic OSCCs. Mollugin-induced cell death involved apoptosis, characterized by the appearance of nuclear shrinkage, flow cytometric analysis of sub-G1 phase arrest, and annexin V-FITC and propidium iodide staining. Western blot analysis and RT-PCR revealed that mollugin suppressed activation of NF-κB and NF-κB-dependent gene products involved in antiapoptosis (Bcl-2 and Bcl-xl), invasion (MMP-9 and ICAM-1), and angiogenesis (FGF-2 and VEGF). Furthermore, mollugin induced the activation of p38, ERK, and JNK and the expression of heme oxygenase-1 (HO-1) and nuclear factor E2–related factor 2 (Nrf2). Mollugin-induced growth inhibition and apoptosis of HO-1 were reversed by an HO-1 inhibitor and Nrf2 siRNA. Collectively, this is the first report to demonstrate the effectiveness of mollugin as a candidate for a chemotherapeutic agent in OSCCs via the upregulation of the HO-1 and Nrf2 pathways and the downregulation of NF-κB. PMID:23738323

  11. Cilengitide targets pediatric glioma and neuroblastoma cells through cell detachment and anoikis induction.

    PubMed

    Leblond, Pierre; Dewitte, Amélie; Le Tinier, Florence; Bal-Mahieu, Christine; Baroncini, Marc; Sarrazin, Thierry; Lartigau, Eric; Lansiaux, Amélie; Meignan, Samuel

    2013-09-01

    The prognosis of children with high-grade glioma or high-risk neuroblastoma remains poor. Cilengitide is a selective antagonist of αvβ3 and αvβ5 integrins, which are involved in tumor growth and development of metastasis. We have evaluated the effects of cilengitide on pediatric glioma and neuroblastoma cell lines for the first time. Expression levels of αvβ3 and αvβ5 were determined by flow cytometry in three neuroblastoma and five pediatric glioma cell lines compared with adult U87-MG before and after irradiation. Cell detachment, cytotoxicity, and cell growth under nonadhesive conditions were measured using the MTS assay. Cell death and apoptosis were assessed by annexin-V/propidium iodide staining. The varying αvβ3 and αvβ5 expression levels were unrelated to tumor grade. Irrespective of the αvβ5 expression level, the pediatric cells expressing αvβ3 were dose dependently sensitive to cilengitide. UW479 cells expressed only αvβ5 integrin and were not sensitive to cilengitide, suggesting that cilengitide's action largely depends on αvβ3 inhibition. Cell detachment resulted in a higher cytotoxicity in pediatric glioma compared with U87-MG cells, which seem able to grow despite the significant cilengitide-induced cell detachment. Growth kinetics on polyHEMA showed that only pediatric glioma cells were sensitive to anoikis and so died after cilengitide-induced detachment. Furthermore, irradiation of glioma cells increased αvβ3 expression slightly but not cilengitide sensitivity. Cilengitide's action on glioma and neuroblastoma cells appears to be dependent on αvβ3 expression and sensitivity to anoikis. Cilengitide is able to target pediatric glioma and neuroblastoma cells in vitro directly and efficiently. Tumor context could validate these promising observations. PMID:23728220

  12. Overexpression of heme oxygenase-1 in human pulmonary epithelial cells results in cell growth arrest and increased resistance to hyperoxia.

    PubMed Central

    Lee, P J; Alam, J; Wiegand, G W; Choi, A M

    1996-01-01

    Heme oxygenase (HO) catalyzes the rate-limiting step in the degradation of heme to biliverdin, which is reduced by biliverdin reductase to bilirubin. Heme oxygenase-1 (HO-1) is inducible not only by its heme substrate, but also by a variety of agents causing oxidative stress. Although much is known about the regulation of HO-1 expression, the functional significance of HO-1 induction after oxidant insult is still poorly understood. We hypothesize and provide evidence that HO-1 induction serves to protect cells against oxidant stress. Human pulmonary epithelial cells (A549 cells) stably transfected with the rat HO-1 cDNA exhibit marked increases of HO-1 mRNA levels which were correlated with increased HO enzyme activity. Cells that overexpress HO-1 (A549-A4) exhibited a marked decrease in cell growth compared with wild-type A549 (A549-WT) cells or A549 cells transfected with control DNA (A549-neo). This slowing of cell growth was associated with an increased number of cells in G0/G1 phase during the exponential growth phase and decreased entry into the S phase, as determined by flow cytometric analysis of propidium iodide-stained cells and pulse experiments with bromodeoxyuridine. Furthermore, the A549-A4 cells accumulated at the G2/M phase and failed to progress through the cell cycle when stimulated with serum, whereas the A549-neo control cells exhibited normal cell cycle progression. Interestingly, the A549-A4 cells also exhibited marked resistance to hyperoxic oxidant insult. Tin protoporphyrin, a selective inhibitor of HO, reversed the growth arrest and ablated the increased survival against hyperoxia observed in the A549-A4 cells overexpressing HO-1. Taken together, our data suggest that overexpression of HO-1 results in cell growth arrest, which may facilitate cellular protection against non-heme-mediated oxidant insult such as hyperoxia. Images Fig. 1 Fig. 4 PMID:8816811

  13. 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

  14. 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

  15. Endothelial cell apoptosis in brown adipose tissue of rats induced by hyperinsulinaemia: the possible role of TNF-α

    PubMed Central

    Markelic, M.; Velickovic, K.; Golic, I.; Otasevic, V.; Stancic, A.; Jankovic, A.; Vucetic, M.; Buzadzic, B.; Korac, B.; Korac, A.

    2011-01-01

    The aim of the present study was to investigate whether hyperinsulinaemia, which frequently precedes insulin resistance syndrome (obesity, diabetes), induces apoptosis of endothelial cells (ECs) in brown adipose tissue (BAT) and causes BAT atrophy and also, to investigate the possible mechanisms underlying ECs death. In order to induce hyperinsuli-naemia, adult male rats of Wistar strain were treated with high dose of insulin (4 U/kg, intraperitonely) for one or three days. Examinations at ultrastructural level showed apoptotic changes of ECs, allowing us to point out that changes mainly but not exclusively, occur in nuclei. Besides different stages of condensation and alterations of the chromatin, nuclear fragmentation was also observed. Higher number of ECs apoptotic nuclei in the BAT of hyperinsulinaemic rats was also confirmed by propidium iodide staining. Immunohistochemical localization of tumor necrosis factor-alpha (TNF-α) revealed increased expression in ECs of BAT of hyperinsulinaemic animals, indicating its possible role in insulin-induced apoptotic changes. These results suggest that BAT atrophy in hyperinsulinaemia is a result of endothelial and adipocyte apoptosis combined, rather than any of functional components alone. PMID:22297440

  16. Validation of SYTO 9/propidium iodide uptake for rapid detection of viable but noncultivable Legionella pneumophila.

    PubMed

    Gião, M S; Wilks, S A; Azevedo, N F; Vieira, M J; Keevil, C W

    2009-07-01

    Legionella pneumophila is an ubiquitous environmental microorganism that can cause Legionnaires' disease or Pontiac fever. As a waterborne pathogen, it has been found to be resistant to chlorine disinfection and survive in drinking water systems, leading to potential outbreaks of waterborne disease. In this work, the effect of different concentrations of free chlorine was studied (0.2, 0.7, and 1.2 mg l(-1)), the cultivability of cells assessed by standard culture techniques (buffered charcoal yeast extract agar plates) and viability using the SYTO 9/propidium iodide fluorochrome uptake assay (LIVE/DEAD BacLight). Results demonstrate that L. pneumophila loses cultivability after exposure for 30 min to 0.7 mg l(-1) of free chlorine and in 10 min when the concentration is increased to 1.2 mg l(-1). However, the viability of the cells was only slightly affected even after 30 min exposure to the highest concentration of chlorine; good correlation was obtained between the rapid SYTO 9/propidium iodide fluorochrome uptake assay and a longer cocultivation with Acanthamoeba polyphaga assay, confirming that these cells could still recover their cultivability. These results raise new concerns about the assessment of drinking water disinfection efficiency and indicate the necessity of further developing new validated rapid methods, such as the SYTO 9/propidium iodide uptake assay, to assess viable but noncultivable L. pneumophila cells in the environment. PMID:19043657

  17. Sonoporation-Induced Apoptosis and Cell Cycle Arrest: Initial Findings

    NASA Astrophysics Data System (ADS)

    Zhong, Wenjing; Sit, Wai Hung; Wan, Jennifer M. F.; Yu, Alfred C. H.

    2011-09-01

    Sonoporation is known to be able to temporarily permeabilize cells, but during this process it may have traumatic impact on cell viability. In this work, we found that sonoporation may induce apoptosis and G2/M-phase cell cycle arrest in some cells hours after ultrasonic exposure in vitro. Methods: Suspensions of HL-60 leukemia cells were prepared (106 cells/ml), and a 1% v/v microbubble solution was added to induce sonoporation during ultrasound exposure. They were then placed 7 cm away from a 2.54 cm-diameter, 1 MHz unfocused ultrasound probe, and these samples were insonated for 1 min with ultrasound pulses (10% duty cycle, 1 kHz pulse repetition frequency). In this study, two levels of peak negative ultrasound pressure were used: 0.3 MPa and 0.5 MPa. After exposure, the cell suspensions were further incubated. They were harvested after 4 h, 8 h, 12 h and 24 h to analyze the cell-cycle distribution (sub-G1, G0/G1, S, G2/M) at these time points using propidium iodide staining and flow cytometry. Results: Some sonoporation-treated cells had undergone apoptosis by 4h, and the largest number of apoptotic cells (sub-G1 phase) was observed after 12h (0.3 MPa group: 25.0%; 0.5 MPa group: 27.2%). Also, after experiencing sonoporation, some viable cells were stopped in the G2/M phase without undergoing cytokinesis, and the maximum G2/M population rise was seen after 12h (0.3 MPa group: +12.2%; 0.5 MPa group: +14.7%). This was accompanied by decreases in the populations of G0/G1-phase and S-phase.

  18. 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

  19. 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

  20. Thymoquinone Induces Cell Death in Human Squamous Carcinoma Cells via Caspase Activation-Dependent Apoptosis and LC3-II Activation-Dependent Autophagy

    PubMed Central

    Yu, Cheng-Chia; Lai, Yi-Yeh; Chen, Pei-Ni

    2014-01-01

    Background Thymoquinone (TQ), an active component of Nigella sativa or black cumin, elicits cytotoxic effects on various cancer cell lines. However, the anti-cancer effects of TQ on head and neck squamous cell carcinoma (HNSCC) remain unclear. Methodology/Principal Findings In this study, TQ elicited a strong cytotoxic effect on SASVO3, a highly malignant HNSCC cell line. The mechanisms of this cytotoxic effect were concentration dependent. TQ also induced apoptotic cell death in SASVO3 cells as indicated by an increase in Bax expression and caspase-9 activation. Apoptosis was possibly caspase-9 dependent because the exposure of cells to a caspase-9 inhibitor partially prevented cell death. The exposed cells also showed increased levels of autophagic vacuoles and LC3-II proteins, which are specific autophagy markers. Cell viability assay results further revealed that bafilomycin-A1, an autophagy inhibitor, enhanced TQ cytotoxicity; by comparison, Annexin V and propidium-iodide staining assay results showed that this inhibitor did not promote apoptosis. TQ treatment also increased the accumulation of autophagosomes. Using a lentivirus-shRNA system for LC3 silencing, we found that cell viability was eradicated in autophagy-defective cells. An in vivo BALB/c nude mouse xenograft model further showed that TQ administered by oral gavage reduced tumor growth via induced autophagy and apoptosis. Conclusions These findings indicated that TQ induced cell death in oral cancer cells via two distinct anti-neoplastic activities that can induce apoptosis and autophagy. Therefore, TQ is a promising candidate in phytochemical-based, mechanistic, and pathway-targeted cancer prevention strategies. PMID:25000169

  1. 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

  2. Genistein induces G2/M cell cycle arrest and apoptosis via ATM/p53-dependent pathway in human colon cancer cells

    PubMed Central

    ZHANG, ZHIYU; WANG, CHONG-ZHI; DU, GUANG-JIAN; QI, LIAN-WEN; CALWAY, TYLER; HE, TONG-CHUAN; DU, WEI; YUAN, CHUN-SU

    2013-01-01

    Soybean isoflavones have been used as a potential preventive agent in anticancer research for many years. Genistein is one of the most active flavonoids in soybeans. Accumulating evidence suggests that genistein alters a variety of biological processes in estrogen-related malignancies, such as breast and prostate cancers. However, the molecular mechanism of genistein in the prevention of human colon cancer remains unclear. Here we attempted to elucidate the anticarcinogenic mechanism of genistein in human colon cancer cells. First we evaluated the growth inhibitory effect of genistein and two other isoflavones, daidzein and biochanin A, on HCT-116 and SW-480 human colon cancer cells. In addition, flow cytometry was performed to observe the morphological changes in HCT-116/SW-480 cells undergoing apoptosis or cell cycle arrest, which had been visualized using Annexin V-FITC and/or propidium iodide staining. Real-time PCR and western blot analyses were also employed to study the changes in expression of several important genes associated with cell cycle regulation. Our data showed that genistein, daidzein and biochanin A exhibited growth inhibitory effects on HCT-116/SW-480 colon cancer cells and promoted apoptosis. Genistein showed a significantly greater effect than the other two compounds, in a time- and dose-dependent manner. In addition, genistein caused cell cycle arrest in the G2/M phase, which was accompanied by activation of ATM/p53, p21waf1/cip1 and GADD45α as well as downregulation of cdc2 and cdc25A demonstrated by q-PCR and immunoblotting assay. Interestingly, genistein induced G2/M cell cycle arrest in a p53-dependent manner. These findings exemplify that isoflavones, especially genistein, could promote colon cancer cell growth inhibition and facilitate apoptosis and cell cycle arrest in the G2/M phase. The ATM/p53-p21 cross-regulatory network may play a crucial role in mediating the anticarcinogenic activities of genistein in colon cancer. PMID:23686257

  3. Expression of pituitary tumor-transforming 2 in human glioblastoma cell lines and its role in glioblastoma tumorigenesis

    PubMed Central

    GUO, YUNBAO; SHAO, YIMENG; CHEN, JING; XU, SONGBAI; ZHANG, XINGDONG; LIU, HAIYAN

    2016-01-01

    The present study aimed to investigate the association between the expression of pituitary tumor-transforming 2 (PTTG2), and cell proliferation, invasion and apoptosis in glioblastoma. The U251 human glioblastoma cell line was transfected with the pcDNA-PTTG2 and small interfering (si)RNA-PTTG2 plasmids using Lipofectamine 2000. The expression of PTTG2 in U251 glioblastoma cells was determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. The association between PTTG2 expression, and cell proliferation, invasion and apoptosis in vitro were investigated using an MTT assay, Matrigel Transwell assay and flow cytometry combined with Annexin V/propidium iodide staining, respectively. RT-qPCR and western blot analysis demonstrated that PTTG2 mRNA and protein expression were significantly overexpressed and significantly suppressed following transfection with pcDNA-PTTG2 and short interfering RNA (siRNA)-PTTG2 plasmids, respectively (P<0.05). In addition, the cell proliferation rate and invasive cell number in cells with overexpressed PTTG2 were significantly higher compared with cells in the untreated group, and the invasive cell number in the siRNA-PTTG2 group was significantly lower than the untreated group (P<0.05). Flow cytometry analysis demonstrated that, compared with the untreated group, the quantity of apoptotic cells in PTTG2 overexpression group was significantly reduced, and the quantity of apoptotic cells in the siRNA-PTTG2 group was increased. Similar results were obtained with regards to the expression level of caspase-3. The results of the present study indicate that PTTG2 overexpression promotes cell proliferation and invasion during glioblastoma progression. In addition, the results suggest that PTTG2 overexpression inhibits cell apoptosis in glioblastoma by affecting caspase-3-dependent signaling pathways. It can therefore be suggested that PTTG2 may serve as a novel therapeutic target for treating glioblastoma.

  4. 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

  5. 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

  6. 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

  7. Alteration of Drug Sensitivity in Human Colon Cancer Cells after Exposure to Heat: Implications for Liver Metastasis Therapy using RFA and Chemotherapy

    PubMed Central

    Makizumi, Ryouji; Yang, Weng-Lang; Owen, Randall P.; Sharma, Rohit R.; Ravikumar, T. S.

    2008-01-01

    Radiofrequency ablation (RFA) is gaining popularity for treating colorectal liver metastases by inducing image guided tumor hyperthermia. In order to reduce tumor recurrence, adjuvant therapies have been administered post-RFA. We hypothesized that tumor cells escaping RFA cytotoxicity by being in the sublethal zones of tumor might develop differential behavior toward cytotoxic drugs. Here, we used cultured human colorectal cancer cells to evaluate the interaction between heat treatment and chemotherapeutic agents. Human colon cancer cell lines HT29 and HCT116 were subjected to temperatures of 42° to 50°C for 15 min, in combination with 5-fluorouracil, oxaliplatin, or irinotecan at different sequences. Cytotoxicity was determined by MTT assay. The cell cycle progression was analyzed by flow cytometry with propidium iodide staining. The expression of several genes associated with drug sensitivity was quantitated by real-time RT-PCR before and after heat treatment. Either heat treatment at 45°C by simultaneous or pre-treatment with three different chemotherapeutic agents didn't affect the cytotoxicity of the combined treatment to HT29 and HCT116 cells, except for irinotecan treatment in HCT116 cells. However, when pre-exposure to 45°C, HCT116 cells, but not HT29 cells, developed resistance to these three drugs. In an analysis of cell cycle profile after the drug followed heat treatment, a longer delay in cell cycle progression in HCT116 cells was observed in comparison to HT29 cells. Furthermore, HCT116 and HT29 cells exhibited different expression profiles of several drug-related genes in response to heat treatment at 45°C. An observation of a differential response to the drug and heat treatment sequences between two human colon cancer cell lines suggests that tumor heterogeneity and selection of chemotherapeutic agents need to be under consideration in the clinical setting. PMID:19079666

  8. Red but not dead? Membranes of stressed Saccharomyces cerevisiae are permeable to propidium iodide.

    PubMed

    Davey, H M; Hexley, P

    2011-01-01

    Flow cytometric monitoring of propidium iodide (PI) uptake is a well-established and rapid method for monitoring cell death and is used on the basis that the intact membrane of viable cells excludes the propidium ion and that loss of this permeability barrier represents irreparable damage and thus cell death. These assumptions are typically based on analysis of live and killed cells. Here we have identified stress levels that lead to a loss of viability of a proportion of Saccharomyces cerevisiae cells and under these conditions we show that there is a subpopulation of cells that can take up PI during and immediately following exposure to stress but that a short incubation allows repair of the membrane damage such that subsequent exposure to PI does not result in staining. Irrespective of the stress applied, approximately 7% of cells exhibited the ability to repair. These results indicate that the level of damage that the yeast cell membrane can sustain and yet retain the ability to repair is greater than previously recognized and care must therefore be taken in using the terms 'PI-positive' and 'dead' synonymously. We discuss these findings in the context of the potential for such environmental stress-induced, transient membrane permeability to have evolutionary implications via the facilitation of horizontal gene transfer. PMID:21199254

  9. NF-kappa B modulation is involved in celastrol induced human multiple myeloma cell apoptosis.

    PubMed

    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

  10. Artesunate inhibits the growth and induces apoptosis of human gastric cancer cells by downregulating COX-2.

    PubMed

    Zhang, Ping; Luo, He-Sheng; Li, Ming; Tan, Shi-Yun

    2015-01-01

    Artesunate, a derivative of artemisinin isolated from Artemisia annua L., has been traditionally used to treat malaria, and artesunate has demonstrated cytotoxic effects against a variety of cancer cells. However, there is little available information about the antitumor effects of artesunate on human gastric cancer cells. In the present study, we investigated the antitumor effect of artesunate on human gastric cancer cells and whether its antitumor effect is associated with reduction in COX-2 expression. The effects of artesunate on the growth and apoptosis of gastric cancer cells were investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometric analysis of annexin V-fluorescein isothiocyanate/propidium iodide staining, rhodamine 123 staining, and Western blot analysis. Results indicate that artesunate exhibits antiproliferative effects and apoptosis-inducing activities. Artesunate markedly inhibited gastric cancer cell proliferation in a time- and dose-dependent manner and induced apoptosis in gastric cancer cells a dose-dependent manner, which was associated with a reduction in COX-2 expression. Treatment with the selective COX-2 inhibitor celecoxib, or transient transfection of gastric cancer cells with COX-2 siRNA, also inhibited cell proliferation and induced apoptosis. Furthermore, the treatment with artesunate promoted the expression of proapoptotic factor Bax and suppressed the expression of antiapoptotic factor Bcl-2. In addition, caspase-3 and caspase-9 were activated, and artesunate induced loss of mitochondrial membrane potential, suggesting that the apoptosis is mediated by mitochondrial pathways. These results demonstrate that artesunate has an effect on anti-gastric cancer cells. One of the antitumor mechanisms of artesunate may be that its inhibition of COX-2 led to reduced proliferation and induction of apoptosis, connected with mitochondrial dysfunction. Artesunate might be a potential therapeutic agent for gastric cancer. PMID:25945055

  11. Efficient intracellular delivery of molecules with high cell viability using nanosecond-pulsed laser-activated carbon nanoparticles.

    PubMed

    Sengupta, Aritra; Kelly, Sean C; Dwivedi, Nishant; Thadhani, Naresh; Prausnitz, Mark R

    2014-03-25

    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

  12. 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

  13. Cytotoxic and apoptogenic effects of Strobilanthes crispa Blume extracts on nasopharyngeal cancer cells.

    PubMed

    Koh, Rhun Yian; Sim, Yi Chi; Toh, Hwee Jin; Liam, Liang Kuan; Ong, Rachael Sze Lynn; Yew, Mei Yeng; Tiong, Yee Lian; Ling, Anna Pick Kiong; Chye, Soi Moi; Ng, Khuen Yen

    2015-10-01

    The chemotherapeutic agents used to treat nasopharyngeal cancer (NPC) exhibit low efficacy. Strobilanthes crispa Blume is widely used for its anticancer, diuretic and anti‑diabetic properties. The present study aimed to determine the cytotoxic and apoptogenic effects of S. crispa on CNE‑1 NPC cells. A 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5 diphenyl tetrazolium bromide assay was used to evaluate the cytotoxic effects of S. crispa against CNE‑1 cells. The rate of apoptosis was determined using propidium iodide staining and caspase assays. Ethyl acetate, hexane and chloroform extracts of S. crispa leaves all exhibited cytotoxic effects on CNE‑1 cells, at a half maximal inhibitory concentration (IC50) of 119, 123.5 and 161.7 µg/ml, respectively. In addition, hexane, chloroform and ethyl acetate extracts of S. crispa stems inhibited CNE‑1 cell proliferation, at a IC50 of 49.4, 148.3 and 163.5 µg/ml, respectively. Flow cytometric analysis revealed an increased proportion of cells in the sub G1 phase and a decreased proportion of cells in the G2/M phase, following treatment with the extracts. However, the extracts did not alter the activities of caspase ‑3/7, ‑8 and ‑9. No cytotoxic effect was observed when the cells were treated with the methanol and water extracts of S. crispa stems and leaves. In conclusion, the S. crispa extracts were cytotoxic against CNE‑1 cells and these extracts were able to induce apoptosis, independent of caspase activation. PMID:26239257

  14. Pathological Cyclic Strain-Induced Apoptosis in Human Periodontal Ligament Cells through the RhoGDIα/Caspase-3/PARP Pathway

    PubMed Central

    Wang, Tingle; Song, Meng; Chen, Wantao

    2013-01-01

    Aim Human periodontal ligament (PDL) cells incur changes in morphology and express proteins in response to cyclic strain. However, it is not clear whether cyclic strain, especially excessive cyclic strain, induces PDL cell apoptosis and if so, what mechanism(s) are responsible. The aim of the present study was to elucidate the molecular mechanisms by which pathological levels of cyclic strain induce human PDL cell apoptosis. Materials and Methods Human PDL cells were obtained from healthy premolar tissue. After three to five passages in culture, the cells were subjected to 20% cyclic strain at a frequency of 0.1 Hz for 6 or 24 h using an FX-5000T system. Morphological changes of the cells were assessed by inverted phase-contrast microscopy, and apoptosis was detected by fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide staining followed by flow cytometry. Protein expression was evaluated by Western blot analysis. Results The number of apoptotic human PDL cells increased in a time-dependent manner in response to pathological cyclic strain. The stretched cells were oriented parallel to each another with their long axes perpendicular to the strain force vector. Cleaved caspase-3 and poly-ADP-ribose polymerase (PARP) protein levels increased in response to pathological cyclic strain over time, while Rho GDP dissociation inhibitor alpha (RhoGDIα) decreased. Furthermore, knock-down of RhoGDIα by targeted siRNA transfection increased stretch-induced apoptosis and upregulated cleaved caspase-3 and PARP protein levels. Inhibition of caspase-3 prevented stretch-induced apoptosis, but did not change RhoGDIα protein levels. Conclusion The overall results suggest that pathological-level cyclic strain not only influenced morphology but also induced apoptosis in human PDL cells through the RhoGDIα/caspase-3/PARP pathway. Our findings provide novel insight into the mechanism of apoptosis induced by pathological cyclic strain in human PDL cells. PMID:24130754

  15. 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 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

  16. 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

  17. 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

  18. 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

  19. Antroquinonol, a natural ubiquinone derivative, induces a cross talk between apoptosis, autophagy and senescence in human pancreatic carcinoma cells.

    PubMed

    Yu, Chia-Chun; Chiang, Po-Cheng; Lu, Pin-Hsuan; Kuo, Mao-Tien; Wen, Wu-Che; Chen, Peini; Guh, Jih-Hwa

    2012-08-01

    Pancreatic cancer is a malignant neoplasm of the pancreas. A mutation and constitutive activation of K-ras occurs in more than 90% of pancreatic adenocarcinomas. A successful approach for the treatment of pancreatic cancers is urgent. Antroquinonol, a ubiquinone derivative isolated from a camphor tree mushroom, Antrodia camphorata, induced a concentration-dependent inhibition of cell proliferation in pancreatic cancer PANC-1 and AsPC-1 cells. Flow cytometric analysis of DNA content by propidium iodide staining showed that antroquinonol induced G1 arrest of the cell cycle and a subsequent apoptosis. Antroquinonol inhibited Akt phosphorylation at Ser(473), the phosphorylation site critical for Akt kinase activity, and blocked the mammalian target of rapamycin (mTOR) phosphorylation at Ser(2448), a site dependent on mTOR activity. Several signals responsible for mTOR/p70S6K/4E-BP1 signaling cascades have also been examined to validate the pathway. Moreover, antroquinonol induced the down-regulation of several cell cycle regulators and mitochondrial antiapoptotic proteins. In contrast, the expressions of K-ras and its phosphorylation were significantly increased. The coimmunoprecipitation assay showed that the association of K-ras and Bcl-xL was dramatically augmented, which was indicative of apoptotic cell death. Antroquinonol also induced the cross talk between apoptosis, autophagic cell death and accelerated senescence, which was, at least partly, explained by the up-regulation of p21(Waf1/Cip1) and K-ras. In summary, the data suggest that antroquinonol induces anticancer activity in human pancreatic cancers through an inhibitory effect on PI3-kinase/Akt/mTOR pathways that in turn down-regulates cell cycle regulators. The translational inhibition causes G1 arrest of the cell cycle and an ultimate mitochondria-dependent apoptosis. Moreover, autophagic cell death and accelerated senescence also explain antroquinonol-mediated anticancer effect. PMID:21840189

  20. Zerumbone protects INS-1 rat pancreatic beta cells from high glucose-induced apoptosis through generation of reactive oxygen species.

    PubMed

    Wang, Changyin; Zou, Shibo; Cui, Zhengjun; Guo, Pengfei; Meng, Qingnan; Shi, Xun; Gao, Ya; Yang, Gaoyuan; Han, Zhaofeng

    2015-05-01

    The aim of this study is to explore the effect of zerumbone, a natural sesquiterpene isolated from Zingiber zerumbet Smith, on high glucose-induced cytotoxicity in pancreatic β cells. INS-1 rat pancreatic β cells were treated with 33 mM glucose with or without different concentrations of zerumbone and cell viability and apoptosis were assessed. The involvement of reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK) signaling in the action of zerumbone was examined. Notably, zerumbone significantly (P < 0.05) prevented the reduction of cell viability induced by high glucose. Such protection was in a concentration-dependent fashion up to 60 μM of zerumbone. Annexin-V/propidium iodide staining analysis showed that zerumbone impaired the apoptotic response of high glucose-treated INS-1 cells, which was coupled with a significant decline in cleaved caspase-3 and caspase-9. Pretreatment with the ROS inhibitor N-acetylcysteine abrogated the phosphorylation of p38 and JNK induced by high glucose. Zerumbone significantly (P < 0.05) decreased the generation of ROS and the phosphorylation of p38 and JNK MAPKs in high glucose-treated INS-1 cells. Pharmacological activation of p38 and JNK with anisomycin reversed the anti-apoptotic effect of zerumbone. Additionally, simultaneous inhibition of p38 and JNK significantly (P < 0.05) reduced the apoptotic response in high glucose-treated INS-1 cells. In conclusion, zerumbone confers protection against high glucose-induced apoptosis of INS-1 pancreatic β cells, largely through interfering with ROS production and p38 and JNK activation. Zerumbone may have potential therapeutic effects against hyperglycemia-induced β cell damage in diabetes. PMID:25769956

  1. 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

  2. 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.

  3. 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

  4. 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

  5. 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

  6. Naproxen-induced Ca2+ movement and death in MDCK canine renal tubular cells.

    PubMed

    Cheng, H-H; Chou, C-T; Sun, T-K; Liang, W-Z; Cheng, J-S; Chang, H-T; Tseng, H-W; Kuo, C-C; Chen, F-A; Kuo, D-H; Shieh, P; Jan, C-R

    2015-11-01

    Naproxen is an anti-inflammatory drug that affects cellular calcium ion (Ca(2+)) homeostasis and viability in different cells. This study explored the effect of naproxen on [Ca(2+)](i) and viability in Madin-Darby canine kidney cells (MDCK) canine renal tubular cells. At concentrations between 50 μM and 300 μM, naproxen induced [Ca(2+)](i) rises in a concentration-dependent manner. This Ca(2+) signal was reduced partly when extracellular Ca(2+) was removed. The Ca(2+) signal was inhibited by a Ca(2+) channel blocker nifedipine but not by store-operated Ca(2+) channel inhibitors (econazole and SKF96365), a protein kinase C (PKC) activator phorbol 12-myristate 13-acetate, and a PKC inhibitor GF109203X. In Ca(2+)-free medium, pretreatment with 2,5-di-tert-butylhydroquinone or thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+) pumps, partly inhibited naproxen-induced Ca(2+) signal. Inhibition of phospholipase C with U73122 did not alter naproxen-evoked [Ca(2+)](i) rises. At concentrations between 15 μM and 30 μM, naproxen killed cells in a concentration-dependent manner, which was not reversed by prechelating cytosolic Ca(2+) with the acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl. Annexin V/propidium iodide staining data suggest that naproxen induced apoptosis. Together, in MDCK renal tubular cells, naproxen induced [Ca(2+)](i) rises by inducing Ca(2+) release from multiple stores that included the endoplasmic reticulum and Ca(2+) entry via nifedipine-sensitive Ca(2+) channels. Naproxen induced cell death that involved apoptosis. PMID:25636639

  7. 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

  8. Pseudolaric acid B exerts antitumor activity via suppression of the Akt signaling pathway in HeLa cervical cancer cells.

    PubMed

    Li, Mingqun; Hong, Li

    2015-08-01

    Pseudolaric acid B (PAB) is a diterpene acid isolated from the bark of the root and trunk of Pseudolarix kaempferi Gordon (Pinaceae), which has demonstrated cytotoxic effects against various types of cancer. However, the mechanisms underlying the anticancer effects of PAB have remained to be elucidated. In the present study, the effects of PAB on the viability and apoptosis of HeLa cells were investigated by MTT assay, flow cytometric analysis of Annexin V-fluorescein isothiocyanate/propidium iodide staining, Rhodamine 123 staining and western blot analysis. The results demonstrated that PAB had antiproliferative and apoptosis-inducing effects on HeLa cells. PAB markedly inhibited HeLa cell viability in a time- and concentration-dependent manner. Flow cytometric analysis indicated that PAB induced apoptosis in HeLa cells in a dose-dependent manner. Treatment with PAB suppressed the expression of anti-apoptotic factor B cell lymphoma-2, and promoted the expression of pro-apoptotic factor Bcl-2-associated X protein. In addition, PAB induced an increase in Caspase-3 activity and loss of mitochondrial membrane potential, suggesting that this apoptosis may be mediated by mitochondrial pathways. Furthermore, the results of western blot analysis indicated that PAB was able to reduce Akt phosphorylation, thereby inhibiting the Akt pathway. These results suggested that PAB inhibited cell proliferation and induced apoptosis in HeLa cells, and that the anti-tumor effects of PAB were associated with inhibition of the Akt pathway. In conclusion, the results of the present study suggested that PAB may represent a novel therapeutic strategy for the treatment of human cervical cancer. However, additional studies are required to investigate the underlying apoptotic mechanisms. PMID:25891953

  9. 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

  10. 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

  11. Selective detection of live bacteria combining propidium monoazide sample treatment with microarray technology.

    PubMed

    Nocker, Andreas; Mazza, Alberto; Masson, Luke; Camper, Anne K; Brousseau, Roland

    2009-03-01

    The use of DNA-based molecular detection tools for bacterial diagnostics is hampered by the inability to distinguish signals originating from live and dead cells. The detection of live cells is typically most relevant in molecular diagnostics. DNA-intercalating dyes like ethidium monoazide and propidium monoazide (PMA) offer a possibility to selectively remove cells with compromised cell membranes from the analysis. Once these dyes enter a cell, they bind to DNA and can be covalently crosslinked to it by light exposure. PCR amplification of such modified DNA is strongly inhibited. In this study we evaluated the suitability of propidium monoazide treatment to exclude isopropanol-killed cells from detection in defined mixtures using diagnostic microarray technology. The organisms comprised Pseudomonas aeruginosa, Listeria monocytogenes, Salmonella typhimurium, Serratia marcescens, and Escherichia coli O157:H7. PCR products obtained from amplification of chaperonin 60 genes (cpn60; coding for GroEL) were hybridized to a custom-designed microarray containing strain-specific cpn60-based 35-mer oligonucleotide probes. Results were compared with data from quantitative PCR, which confirmed that PMA could successfully inhibit amplification of DNA from killed cells in the mixtures. Although microarray data based on analysis of end-point PCR amplicons is not quantitative, results showed a significant signal reduction when targeting killed cells and consistently agreed with qPCR results. Treatment of samples with PMA in combination with diagnostic microarray detection can therefore be considered beneficial when analyzing mixtures of intact and membrane-compromised cells. Minimization of detection signals deriving from dead cells will render data more relevant in studies including pathogen risk assessment. PMID:19103234

  12. 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

  13. 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

  14. Isocudraxanthone K Induces Growth Inhibition and Apoptosis in Oral Cancer Cells via Hypoxia Inducible Factor-1α

    PubMed Central

    Shin, Mee-Ran; Lee, Hwa-Jeong; Auh, Q-Schick; Lee, Young-Man; Kim, Youn-Chul; Kim, Eun-Cheol

    2014-01-01

    Isocudraxanthone K (IK) is a novel, natural compound from a methanol extract of the root bark of Cudrania tricuspidata. It has not been shown previously that IK possessed antitumor activity. We investigated the antitumor effects and molecular mechanism of IK and related signal transduction pathway(s) in oral squamous cell carcinoma cells (OSCCCs). The MTT assay revealed that IK had an antiproliferative effect on OSCCCs, in a dose- and time-dependent manner. IK induced apoptosis in OSCCCs, as identified by a cell-cycle analysis, annexin V-FITC and propidium iodide staining, and the nuclear morphology in cell death. IK caused time-dependent phosphorylation of Akt, p38, and ERK (extracellular signal-regulated kinase). In addition, IK increased the cytosolic to nuclear translocation of nuclear factor-κB (NF-κB) p65 and the degradation and phosphorylation of IκB-α in HN4 and HN12 cells. Furthermore, IK treatment downregulated hypoxia-inducible factor 1α (HIF-1α) and its target gene, vascular endothelial growth factor (VEGF). Cobalt chloride (CoCl2), a HIF-1α activator, attenuated the IK-induced growth-inhibiting and apoptosis-inducing effects, and blocked IK-induced expression of apoptosis regulatory proteins, such as Bax, Bcl-2, caspase-3, caspase-8, and caspase-9, and cytochrome c. Collectively, these data provide the first evidence of antiproliferative and apoptosis-inducing effects of IK as a HIF-1α inhibitor and suggest it may be a drug candidate for chemotherapy against oral cancer. PMID:25105148

  15. 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

  16. Postexposure application of Fas receptor small-interfering RNA to suppress sulfur mustard-induced apoptosis in human airway epithelial cells: implication for a therapeutic approach.

    PubMed

    Keyser, Brian M; Andres, Devon K; Nealley, Eric; Holmes, Wesley W; Benton, Betty; Paradiso, Danielle; Appell, Ashley; Carpin, Chris; Anderson, Dana R; Smith, William J; Ray, Radharaman

    2013-01-01

    Sulfur mustard (SM) is a vesicant chemical warfare and terrorism agent. Besides skin and eye injury, respiratory damage has been mainly responsible for morbidity and mortality after SM exposure. Previously, it was shown that suppressing the death receptor (DR) response by the dominant-negative Fas-associated death domain protein prior to SM exposure blocked apoptosis and microvesication in skin. Here, we studied whether antagonizing the Fas receptor (FasR) pathway by small-interfering RNA (siRNA) applied after SM exposure would prevent apoptosis and, thus, airway injury. Normal human bronchial/tracheal epithelial (NHBE) cells were used as an in vitro model with FasR siRNA, FasR agonistic antibody CH11, and FasR antagonistic antibody ZB4 as investigative tools. In NHBE cells, both SM (300 M) and CH11 (100 ng/ml) induced caspase-3 activation, which was inhibited by FasR siRNA and ZB4, indicating that SM-induced apoptosis was via the Fas response. FasR siRNA inhibited SM-induced caspase-3 activation when added to NHBE cultures up to 8 hours after SM. Results using annexin V/propidium iodide-stained cells showed that both apoptosis and necrosis were involved in cell death due to SM; FasR siRNA decreased both apoptotic and necrotic cell populations. Bronchoalveolar lavage fluid (BALF) of rats exposed to SM (1 mg/kg, 50 minutes) revealed a significant (P < 0.05) increase in soluble Fas ligand and active caspase-3 in BALF cells. These findings suggest an intervention of Fas-mediated apoptosis as a postexposure therapeutic strategy with a therapeutic window for SM inhalation injury and possibly other respiratory diseases involving the Fas response. PMID:23129783

  17. Blockage of miR-92a-3p with locked nucleic acid induces apoptosis and prevents cell proliferation in human acute megakaryoblastic leukemia.

    PubMed

    Sharifi, M; Salehi, R

    2016-01-01

    MicroRNAs (miRNAs) are non-coding RNAs involved in post-transcriptional regulation of gene expression. In many cancers, up- or downregulation of different miRNAs is reported. In acute myeloid leukemia, upregulation of miR-92a-3p was reported in human in vitro studies. We performed blockage of miR-29a-3p in human acute megakaryoblastic leukemia cell line (M-07e) by using locked nucleic acid (LNA) and cell proliferation; apoptosis and necrosis were assessed. At different time points after LNA-anti-miR92a-3p transfection, miR-92a-3p quantitation was assessed by qRT-real-time PCR, MTT assay and annexin/propidium iodide staining were performed. The data were processed using the ANOVA test. At all three time points, the expression of miR-92a-3p was lower in the LNA-anti-miR group compared with the control groups. Cell viability between LNA-Anti-miR and the control group was statistically significant. Blockage of miR-92a-3p was associated with increment of the ratio of apoptotic cells in the LNA-anti-miR group was higher than the other group. The ratio of necrotic cells in the LNA-antimiR group was higher than the other groups. These assessments indicate that miR-92a-3p blockage can decrease the viability of M-07e cells, which is mainly due to induction of apoptosis and necrosis. Our findings could open up a path to a miRNA based therapeutic approach for treatment of acute megakaryoblastic leukemia. PMID:26658357

  18. Fructose induces mitochondrial dysfunction and triggers apoptosis in skeletal muscle cells by provoking oxidative stress.

    PubMed

    Jaiswal, Natasha; Maurya, Chandan K; Arha, Deepti; Avisetti, Deepa R; Prathapan, Ayyappan; Raj, Palayyan S; Raghu, Kozhiparambil G; Kalivendi, Shasi V; Tamrakar, Akhilesh Kumar

    2015-07-01

    Mitochondrial dysfunction in skeletal muscle has been implicated in the development of insulin resistance, a major characteristic of type 2 diabetes. There is evidence that oxidative stress results from the increased production of reactive oxygen species and reactive nitrogen species leads to mitochondrial dysfunction, tissue damage, insulin resistance, and other complications observed in type 2 diabetes. It has been suggested that intake of high fructose contributes to insulin resistance and other metabolic disturbances. However, there is limited information about the direct effect of fructose on the mitochondrial function of skeletal muscle, the major metabolic determinant of whole body insulin activity. Here, we assessed the effect of fructose exposure on mitochondria-mediated mechanisms in skeletal muscle cells. Exposure of L6 myotubes to high fructose stimulated the production of mitochondrial reactive oxygen species and nitric oxide (NO), and the expression of inducible NO synthase. Fructose-induced oxidative stress was associated with increased translocation of nuclear factor erythroid 2-related factor-2 to the nucleus, decreases in mitochondrial DNA content and mitochondrial dysfunctions, as evidenced by decreased activities of citrate synthase and mitochondrial dehydrogenases, loss of mitochondrial membrane potential, decreased activity of the mitochondrial respiratory complexes, and impaired mitochondrial energy metabolism. Furthermore, positive Annexin-propidium iodide staining and altered expression of Bcl-2 family members and caspases in L6 myotubes indicated that the cells progressively became apoptotic upon fructose exposure. Taken together, these findings suggest that exposure of skeletal muscle cells to fructose induced oxidative stress that decreased mitochondrial DNA content and triggered mitochondrial dysfunction, which caused apoptosis. PMID:25913123

  19. (R)-(+)-α-Lipoic acid protected NG108-15 cells against H2O2-induced cell death through PI3K-Akt/GSK-3β pathway and suppression of NF-κβ-cytokines

    PubMed Central

    Kamarudin, Muhamad Noor Alfarizal; Mohd Raflee, Nur Afiqah; Syed Hussein, Sharifah Salwa; Lo, Jia Ye; Supriady, Hadi; Abdul Kadir, Habsah

    2014-01-01

    Alpha-lipoic acid, a potent antioxidant with multifarious pharmacological benefits has been reported to be neuroprotective in several neuronal models and used to treat neurological disorders such as Alzheimer’s disease. Nonetheless, conclusive mechanisms of alpha-lipoic acid for its protective effects particularly in NG108-15 cells have never been investigated. In this study, the intricate neuroprotective molecular mechanisms by (R)-(+)-alpha-lipoic acid (R-LA) against H2O2-induced cell death in an in vitro model of neurodegeneration were elucidated. Pretreatment with R-LA (2 hours) significantly increased NG108-15 cell viability as compared to H2O2-treated cells and mitigated the induction of apoptosis as evidenced by Hoechst 33342/propidium iodide staining. R-LA (12.5–50 μM) aggrandized the reduced glutathione over glutathione disulfide ratio followed by a reduction in the intracellular reactive oxygen species level and an increase in mitochondrial membrane potential following H2O2 exposure. Moreover, pretreatment with R-LA stimulated the activation of PI3K-Akt through mTORC1 and mTORC2 components (mTOR, rictor and raptor) and production of antiinflammatory cytokine, IL-10 which led to the inactivation of glycogen synthase kinase-3β (GSK-3β) and reduction of both Bax/Bcl2 and Bax/Bcl-xL ratios, accompanied by inhibition of the cleaved caspase-3. Additionally, this observation was preceded by the suppression of NF-κβ p65 translocation and production of proinflammatory cytokines (IL-6 and TNF-α). The current findings accentuate new mechanistic insight of R-LA against apoptogenic and brain inflammatory factors in a neuronal model. These results further advocate the therapeutic potential of R-LA for the treatment of neurodegenerative diseases. PMID:25336920

  20. 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

  1. (R)-(+)-α-lipoic acid protected NG108-15 cells against H₂O₂-induced cell death through PI3K-Akt/GSK-3β pathway and suppression of NF-κβ-cytokines.

    PubMed

    Kamarudin, Muhamad Noor Alfarizal; Mohd Raflee, Nur Afiqah; Hussein, Sharifah Salwa Syed; Lo, Jia Ye; Supriady, Hadi; Abdul Kadir, Habsah

    2014-01-01

    Alpha-lipoic acid, a potent antioxidant with multifarious pharmacological benefits has been reported to be neuroprotective in several neuronal models and used to treat neurological disorders such as Alzheimer's disease. Nonetheless, conclusive mechanisms of alpha-lipoic acid for its protective effects particularly in NG108-15 cells have never been investigated. In this study, the intricate neuroprotective molecular mechanisms by (R)-(+)-alpha-lipoic acid (R-LA) against H2O2-induced cell death in an in vitro model of neurodegeneration were elucidated. Pretreatment with R-LA (2 hours) significantly increased NG108-15 cell viability as compared to H2O2-treated cells and mitigated the induction of apoptosis as evidenced by Hoechst 33342/propidium iodide staining. R-LA (12.5-50 μM) aggrandized the reduced glutathione over glutathione disulfide ratio followed by a reduction in the intracellular reactive oxygen species level and an increase in mitochondrial membrane potential following H2O2 exposure. Moreover, pretreatment with R-LA stimulated the activation of PI3K-Akt through mTORC1 and mTORC2 components (mTOR, rictor and raptor) and production of antiinflammatory cytokine, IL-10 which led to the inactivation of glycogen synthase kinase-3β (GSK-3β) and reduction of both Bax/Bcl2 and Bax/Bcl-xL ratios, accompanied by inhibition of the cleaved caspase-3. Additionally, this observation was preceded by the suppression of NF-κβ p65 translocation and production of proinflammatory cytokines (IL-6 and TNF-α). The current findings accentuate new mechanistic insight of R-LA against apoptogenic and brain inflammatory factors in a neuronal model. These results further advocate the therapeutic potential of R-LA for the treatment of neurodegenerative diseases. PMID:25336920

  2. 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

  3. Use of image cytometry for quantification of pathogenic fungi in association with host cells.

    PubMed

    Berkes, Charlotte; Chan, Leo Li-Ying; Wilkinson, Alisha; Paradis, Benjamin

    2013-01-01

    Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans commonly employ infection of mammalian hosts or host cells (i.e. macrophages) followed by yeast quantification using colony forming unit analysis or flow cytometry. While colony forming unit enumeration has been the most commonly used method in the field, this technique has disadvantages and limitations, including slow growth of some fungal species on solid media and low and/or variable plating efficiencies, which is of particular concern when comparing growth of wild-type and mutant strains. Flow cytometry can provide rapid quantitative information regarding yeast viability, however, adoption of flow cytometric detection for pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Here, we demonstrate an image-based cytometric methodology using the Cellometer Vision (Nexcelom Bioscience, LLC) for the quantification of viable pathogenic yeasts in co-culture with macrophages. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome, and here, we quantitatively assess the growth of H. capsulatum yeasts in RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with image cytometry. Our method faithfully recapitulates growth trends as measured by traditional colony forming unit enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of live macrophages with a GFP-expressing strain of C. albicans. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens in association with host cells. PMID:23851941

  4. 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

  5. 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 survive nsPEFs. The ability of the CHO cytoskeleton to recover and complete mitosis after nsPEF-induced damage in G2/M phase may be integral to the cell line's higher tolerance of nsPEF exposure.

  6. 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. J. Cell. Biochem. 117: 106-117, 2016. © 2015 Wiley Periodicals, Inc. PMID:26058460

  7. 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.

  8. 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 of cutaneous leishmaniasis. PMID:26674781

  9. 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 and TM4 cells to AgNPs inhibited proliferation and self-renewal of SSCs. Conclusion Our results suggest that AgNPs exhibit size-dependent nanoreprotoxicity in male somatic cells and SSCs, strongly suggesting that applications of AgNPs in commercial products must be carefully evaluated. Further studies of AgNPs-induced nanoreprotoxicity in animal models are required. PMID:25733828

  10. 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.

  11. 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 during LBH589 treatment. Our results provide new clues to the proapoptotic mechanism of LBH589. PMID:26176219

  12. The molecular chaperone alphaA-crystallin enhances lens epithelial cell growth and resistance to UVA stress.

    PubMed

    Andley, U P; Song, Z; Wawrousek, E F; Bassnett, S

    1998-11-20

    alphaA-Crystallin (alphaA) is a member of the small heat shock protein (sHSP) family and has the ability to prevent denatured proteins from aggregating in vitro. Lens epithelial cells express relatively low levels of alphaA, but in differentiated fiber cells, alphaA is the most abundant soluble protein. The lenses of alphaA-knock-out mice develop opacities at an early age, implying a critical role for alphaA in the maintenance of fiber cell transparency. However, the function of alpha-crystallin in the lens epithelium is unknown. To investigate the physiological function of alphaA in lens epithelial cells, we used the following two systems: alphaA knock-out (alphaA(-/-)) mouse lens epithelial cells and human lens epithelial cells that overexpress alphaA. The growth rate of alphaA(-/-) mouse lens epithelial cells was reduced by 50% compared with wild type cells. Cell cycle kinetics, measured by fluorescence-activated cell sorter analysis of propidium iodide-stained cells, indicated a relative deficiency of alphaA(-/-) cells in the G2/M phases. Exposure of mouse lens epithelial cells to physiological levels of UVA resulted in an increase in the number of apoptotic cells in the cultures. Four hours after irradiation the fraction of apoptotic cells in the alphaA(-/-) cultures was increased 40-fold over wild type. In cells lacking alphaA, UVA exposure modified F-actin, but actin was protected in cells expressing alphaA. Stably transfected cell lines overexpressing human alphaA were generated by transfecting extended life span human lens epithelial cells with the mammalian expression vector construct pCI-neoalphaA. Cells overexpressing alphaA were resistant to UVA stress, as determined by clonogenic survival. alphaA remained cytoplasmic after exposure to either UVA or thermal stress indicating that, unlike other sHSPs, the protective effect of alphaA was not associated with its relocalization to the nucleus. These results indicate that alphaA has important cellular functions in the lens over and above its well characterized role in refraction. PMID:9813033

  13. 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

  14. 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. Furthermore, cell cytotoxicity and autophagy were induced upon treatment with sodium formate. Z-VAD-fmk and SP600125 were able to effectively circumvent the effects of sodium formate on cell viability. These results suggested that the cytotoxicity induced by sodium formate induces the activation of the JNK signaling pathway, leading to caspase-dependent apoptosis. Increased levels of autophagy were also observed during the process of 661W cell damage induced by sodium formate. PMID:26676762

  15. 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

  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:26998114

  17. 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 130μM 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)≤200mgmL(-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

  18. Effect of non-esterified fatty acids on bovine theca cell steroidogenesis and proliferation in vitro.

    PubMed

    Vanholder, T; Lmr Leroy, J; Van Soom, A; Maes, D; Coryn, M; Fiers, T; de Kruif, A; Opsomer, G

    2006-03-01

    Elevated serum non-esterified fatty acid (NEFA) levels associated with a negative energy balance (NEB) may affect ovarian function and hence reproductive performance in high-yielding dairy cows. We have investigated the individual and combined effects of the three major NEFAs on bovine theca cell proliferation and steroidogenesis in vitro. Theca cells from healthy large follicles (>8 mm) obtained from slaughterhouse ovaries were cultured in serum free medium in the presence of 0, 50, 150 and 200 microM of palmitic acid (PA; C16:0); 0, 50, 150 and 250 microM of stearic acid (SA; C18:0); and/or 0, 50, 150 and 250 microM of oleic acid (OA; C18:1). Progesterone and androstenedione concentrations were measured in spent medium after 48 h of culture and cell numbers were determined spectrophotometrically per culture well. Cell viability was assessed by annexin-V FITC/propidium iodide staining. Only the treatment with 200 microM of PA inhibited cell proliferation (P<0.001) when tested individually, both of the mixtures tested (M1=100 microM of PA, 130 microM of SA and 140 microM of OA; M2=200 microM PA, 260 microM of SA and 280 microM of OA) reduced cell numbers (P<0.001). Progesterone and androstenedione production, both per well and per 10(4) cells, were not affected by any of the treatments, with the exception of M2. This mixture reduced progesterone production per well and per 10(4) cells (P<0.05). The effects observed were most likely caused by the cytotoxic action of the NEFAs, as demonstrated by the increased percentage of early apoptotic (M1) and late apoptotic/necrotic cells (M1 and M2) in the combination treatments (P<0.05). When combined, elevated physiological concentrations of PA, SA and OA can modulate theca cell proliferation and steroidogenesis in vitro by reducing theca cell viability. These NEFAs may be one of the mediators through which NEB compromises ovarian functioning and thus fertility in high-yielding dairy cows. PMID:15978752

  19. 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. PMID:20469912

  20. 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 of HIF-1α siRNA may be strengthened by DDP. The present data indicated that HIF-1α siRNA is important in the inhibition of NSCLC cells. Additionally, the effects of HIF-1α siRNA may be strengthened by DDP, which suggests that HIF-1α siRNA may be combined with DDP for the treatment of tumors. PMID:26998070

  1. Propidium Monoazide Coupled with PCR Predicts Infectivity of Enteric Viruses in Swine Manure and Biofertilized Soil.

    PubMed

    Fongaro, Gislaine; Hernández, Marta; García-González, María Cruz; Barardi, Célia Regina Monte; Rodríguez-Lázaro, David

    2016-03-01

    The use of propidium monoazide (PMA) coupled with real-time PCR (RT-qPCR or qPCR for RNA or DNA viruses, respectively) was assessed to discriminate infectious enteric viruses in swine raw manure, swine effluent from anaerobic biodigester (AB) and biofertilized soils. Those samples were spiked either with infectious and heat-inactivated human adenovirus-2 (HAdV-2) or mengovirus (vMC0), and PMA-qPCR/RT-qPCR allowed discriminating inactivated viruses from the infective particles, with significant reductions (>99.9 %). Then, the procedure was further assayed to evaluate the presence and stability of two non-cultivable viruses (porcine adenovirus and rotavirus A) in natural samples (swine raw manure, swine effluent from AB and biofertilized soils); it demonstrated viral inactivation during the storage period at 23 °C. As a result, the combination of PMA coupled to real-time PCR can be a promising alternative for prediction of viral infectivity in comparison to more labour-intensive and costly techniques such as animal or tissue-culture infectivity methods, and for those viruses that do not have currently available cell culture techniques. PMID:26742766

  2. Involvement of mitogen-activated protein kinase pathway in T-2 toxin-induced cell cycle alteration and apoptosis in human neuroblastoma cells.

    PubMed

    Agrawal, Mona; Bhaskar, A S B; Lakshmana Rao, P V

    2015-01-01

    T-2 toxin is the most toxic trichothecene and a frequent contaminant in many agriculture products. Dietary ingestion represents the most common route of T-2 toxin exposure in humans. T-2 toxin exposure leads to many pathological conditions like nervous disorders, cardiovascular alterations, immune depression and dermal inflammation. However, the neuronal toxicity of T-2 toxin in vitro remains unclear. In the present study, we investigated the mechanism of T-2 toxin-induced apoptosis in human neuroblastoma cells (IMR-32). T-2 toxin was cytotoxic at a low concentration of 10 ng/ml. The 50% inhibitory concentration (IC50) of T-2 toxin was found to be 40 ng/ml as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, crystal violet dye exclusion test and lactate dehydrogenase (LDH) leakage. T-2 toxin increased intracellular reactive oxygen species generation as early as 15 min and peaked at 60 min as analyzed by flow cytometry. Annexin V + propidium iodide staining showed time-dependent increase in percent apoptotic cells. DNA gel electrophoresis showed oligonucleosomal DNA fragmentation typical of apoptotic cells. Additionally, casapse-3 activation and PARP cleavage indicated involvement of mitochondrial mediated caspase-dependent pathway of apoptosis. Cell cycle analysis revealed time-dependent increase in sub-G1 population of cells and significant up-regulation of CDK2, CDK6, cyclin A and p21 messenger RNA (mRNA) levels. Exposure to T-2 toxin induced the phosphorylation of extracellular signal-regulated kinase (ERK), p38-mitogen-activated protein kinase and c-jun N-terminal kinases (JNK). Analysis of human phospho-mitogen-activated protein kinase (MAPK) antibody array revealed time-dependent increase in phosphorylation. Upstream of ERK pathway Grb2, Ras and Raf and downstream transcription factors c-fos and c-jun were significantly up-regulated. Z-VAD-FMK and MAPK inhibitors (PD 98059, SB 203580 and ZM 336372) exposure prior to T-2 toxin treatment significantly decreased percent of apoptotic cells compared to only T-2 toxin-exposed cells. Results of the present study show that T-2 toxin at nanogram concentrations can induce apoptosis in human neuronal cells through multiple signal transduction pathways. The study provides possible leads for developing therapeutic approaches to prevent T-2 toxin-induced neurotoxicity. PMID:25084755

  3. 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 pre-stimulation. PMID:27124409

  4. B7-H3 silencing by RNAi inhibits tumor progression and enhances chemosensitivity in U937 cells

    PubMed Central

    Zhang, Wei; Wang, Jing; Wang, Yanfang; Dong, Fei; Zhu, Mingxia; Wan, Wenli; Li, Haishen; Wu, Feifei; Yan, Xinxing; Ke, Xiaoyan

    2015-01-01

    Background The role of B7-H3 in acute monocytic leukemia U937 cells has not been thoroughly investigated. Materials and methods B7-H3 knockdown in the U937 cell line was performed using small hairpin (sh)RNA lentivirus transduction. The effects on cell proliferation, cycle, migration, and invasion were investigated by Cell Counting Kit-8 assay, methyl cellulose colony-forming assay, propidium iodide staining, and Transwell assays in vitro. Changes in cell growth inhibition and apoptosis, when combined with chemotherapy drugs, were determined using the Cell Counting Kit-8 and Annexin V-FITC/PI assays. U937 xenograft models were used to assess the effects of B7-H3 on tumorigenicity and the therapeutic effect of B7-H3 knockdown in combination with chemotherapy drugs in vivo. Results Downregulation of B7-H3 significantly decreased U937 cell growth and colony-forming ability. The mean inhibition rate of tumor growth with B7-H3 knockdown was 59.4%, and the expression of both Ki-67 and PCNA in xenografts was significantly reduced. After B7-H3 silencing, the U937 cell cycle was arrested at the G0/G1 phase. The cell migration rate of B7-H3 knockdown cells was reduced more than fivefold, and invasion capacity decreased by 86.7%. B7-H3 RNAi profoundly increased the antitumor effect of chemotherapy in vitro and in vivo. On day 19, inhibition rates of tumor growth in B7-H3 shRNA combined with idarubicin, cytarabine, and idarubicin plus cytarabine were 70.5%, 80.0%, and 90.0%, respectively (P=0.006, P=0.004, and P=0.016, respectively). Conclusion B7-H3 may promote U937 cell progression, and shRNA targeting B7-H3 significantly enhances sensitivity to chemotherapeutic drugs. These results may provide new insight into the function of B7-H3 and a promising therapeutic approach targeting B7-H3 in acute monocytic leukemia. PMID:26203263

  5. Cytotoxic effects of commercial wheatgrass and fiber towards human acute promyelocytic leukemia cells (HL60).

    PubMed

    Alitheen, Noorjahan Banu; Oon, Chuah Li; Keong, Yeap Swee; Chuan, Tan Kee; Li, Ho Ket; Yong, Ho Wan

    2011-07-01

    Cytotoxicity, the possible selective activity upon HL60 as well as the anti-proliferation effect of local health supplement wheatgrass and mixture of fibers were investigated in vitro using various cancerous cell line and normal blood cell culture. The IC(50) of wheatgrass-treated HL60 (17.5 ± 1.1, 12.5 ± 0.3, and 16 ± 0.5 microgram/ml for 24, 48 and 72 h, respectively) and fibers-treated HL60 (86.0 ± 5.5, 35.0 ± 2.5, and 52.5 ± 4.5 microgram/ml for 24, 48 and 72 h, respectively) showed that both extracts possessed optimum effect after 48 hours of treatment. No significant cytotoxic effect was observed on other type of cells. For trypan blue dye exclusion method, wheatgrass reduced the number of viable cells by 13.5% (±1.5), 47.1% (±3.6), and 64.9% (±2.7) after 24, 48 and 72 h exposure, respectively. Mixture of fibers reduced the number of viable cells by 36.4% (±2.3), 57.1% (±3.1), and 89.0% (±3.4) after 24, 48 and 72 h exposure, respectively, indicated that necrosis is also an alternative to the apoptotic mechanism of cell death. Annexin-V/propidium iodide staining revealed that both extracts induced apoptosis where early apoptosis had been detected concurrently with the reduction of percentage of cell viability. Cell cycle analysis revealed that in HL60, the percentage of apoptosis increased with time (wheatgrass: 16.0% ± 2.4, 45.3% ± 3.4 and 39.6% ± 4.1; mixture of fibers: 14.6% ± 1.8, 45.4% ± 2.3 and 45.9% ± 1.2) after exposure for 24, 48 and 72 h, respectively at the concentration of 100 microgram/ml and showed optimum effect at 48 hours. Thus, these health products can be a potential alternative supplement for leukaemia patients. PMID:21715255

  6. Cannabinoid receptor 1 blockade protects human retinal pigment epithelial cells from oxidative injury

    PubMed Central

    Wei, Yan; Wang, Xu; Zhao, Feng; Zhao, Pei-quan

    2013-01-01

    Background Because oxidative stress is assumed to be a key mechanism in the pathological process of age-related macular degeneration (AMD), increasing numbers of studies have focused on discovering new pathways and treatments for reducing oxidative damage. Our work investigates the potential role of the cannabinoid receptor 1 (CB1) in oxidative stress of primary human retinal pigment epithelial (RPE) cells, a cellular model of AMD. Methods Primary human RPE cells were cultured and exposed to hydrogen peroxide for 24 h to induce oxidative damage. The expression of and changes in the CB1 receptor were determined with western blot assay and confocal imaging. The CB1 receptor in the RPE cells was inhibited with small interfering RNA (siRNA) or rimonabant (SR141716). Cell viability, apoptosis, and reactive oxygen species production were measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and sulforhodamine B assay, annexin V and propidium iodide staining, and the dichlorofluorescein fluorescence assay, respectively. Intracellular superoxide dismutase activity was assayed with a commercially available assay kit. Phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) protein expression and activation of signaling molecules were assessed with western blot analysis. Results We showed that human RPE cells express the CB1 receptor. In addition, oxidative stress upregulates the expression of the CB1 receptor. Deleting the CB1 receptor or treating with the CB1 receptor antagonist rimonabant (SR141716) rescued RPE cells from hydrogen peroxideinduced oxidative damage. Rimonabant pretreatment effectively reduced the apoptosis of RPE cells, inhibited the generation of intracellular reactive oxygen species and elevated the activity of superoxide dismutase. In addition, rimonabant significantly strengthened the oxidative stress-induced activation of the PI3K/Akt signaling pathway. Conclusions The results demonstrate the expression and regulation of CB1 receptors in human RPE cells. Inhibiting the CB1 receptor may be an effective therapeutic strategy for AMD by downregulating oxidative stress signaling and facilitating PI3K/Akt activation. PMID:23441106

  7. 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 ?g/ml: 50.201.55% vs. 0 ?g/ml: 2.620.19%; P<0.01). Cordycepin inhibited EA.hy926 cell migration (percentage of wound healing area, 2,000 ?g/ml: 3.450.29% vs. 0 ?g/ml: 85.480.84%; P<0.05), as well as tube formation (total length of tubular structure, 1,000 ?g/ml: 10739 ?m vs. 0 ?g/ml: 93656 ?m; 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

  8. 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) and genetic (siRNA targeting BIP and CHOP) methods, the induction of BIP, PDI, IRE1a, and LC3-II was blocked, but PARP cleavage was markedly enhanced. Discussion Taken together, these results suggested that sodium butyrate-induced autophagy was mediated by endoplasmic reticulum stress, and that preventing autophagy by blocking the endoplasmic reticulum stress response enhanced sodium butyrate-induced apoptosis. These results provide novel insights into the anti-tumor mechanisms of butyric acid. PMID:26784903

  9. Arsenic sulfide, the main component of realgar, a traditional Chinese medicine, induces apoptosis of gastric cancer cells in vitro and in vivo

    PubMed Central

    Zhang, Lian; Tian, Wei; Kim, Sungkyoung; Ding, Wenping; Tong, Yingying; Chen, Siyu

    2015-01-01

    Background Arsenic sulfide (As4S4), the main component of realgar, a traditional Chinese medicine, has shown antitumor efficacy in several tumor types, especially for acute promyelocytic leukemia. In this study, we aimed to explore the efficacy and mechanism of As4S4 in gastric cancer. Methods The effect of As4S4 on cell proliferation and apoptosis of gastric cancer cells was investigated by MTT assay, 4′,6-diamidino-2-phenylindole (DAPI) staining, and annexin V–fluorescein isothiocyanate/propidium iodide staining using gastric cancer cell lines AGS (harboring wild-type p53) and MGC803 (harboring mutant p53) in vitro. The expression of apoptosis-related proteins was measured by Western blotting, real-time polymerase chain reaction, and immunohistochemistry analysis. Mouse xenograft models were established by inoculation with MGC803 cells, and the morphology and the proportion of apoptotic cells in tumor tissues were detected by hematoxylin and eosin staining and TdT-mediated dUTP nick end labeling (TUNEL) assay, respectively. Results As4S4 inhibited the proliferation and induced apoptosis of AGS and MGC803 cells in a time- and dose-dependent manner. As4S4 upregulated the expression of Bax and MDM2 while downregulated the expression of Bcl-2. The expression of p53 increased significantly in the AGS cells but did not readily increase in the MGC803 cells, which harbored mutant p53. Pifithrin-α, a p53 inhibitor, blocked the modulation of As4S4 on AGS cells, but not on MGC803 cells. Using xenograft as a model, we showed that As4S4 suppressed tumor growth and induced apoptosis in vivo and that the expression of p53 increased accordingly. Conclusion As4S4 is a potent cytotoxic agent for gastric cancer cells, as it induced apoptosis both in vitro and in vivo through a p53-dependent pathway. Our data indicate that As4S4 may have therapeutic potential in gastric cancer. PMID:25565771

  10. CXC195 induces apoptosis and endoplastic reticulum stress in human hepatocellular carcinoma cells by inhibiting the PI3K/Akt/mTOR signaling pathway.

    PubMed

    Chen, Xiao-Liang; Fu, Jian-Ping; Shi, Jun; Wan, Ping; Cao, Hong; Tang, Zhi-Mou

    2015-12-01

    CXC195 exhibits strong protective effects against neuronal apoptosis by exerting antioxidant activity. However, the pharmacological function of CXC195 in cancer remains to be elucidated. The present study demonstrated that CXC195 exhibited significant cytotoxic effects, and induced cell cycle arrest and apoptosis in HepG2 human hepatocellular carcinoma (HCC) cell lines. Following treatment of HepG2 cells with 150 µΜ CXC195 for 24 , cell viability and the apoptotic rate were assessed using an MTT assay and Annexin V/propidium iodide staining followed by flow cytometric analysis. Molecular markers of the cell cycle, apoptosis, mitochondrial function and endoplasmic reticulum (ER) stress were analyzed by western blot or polymerase chain reaction analysis. Caspase activation, cytochrome c and apoptosis‑inducing factor release, and analysis of the B cell lymphoma 2 (Bcl‑2)‑associated X protein/Bcl‑2 ratio demonstrated that the anticancer effects of CXC195 in HepG2 cells were mediated by caspase and mitochondria‑dependent apoptosis. CXC195 also induced the expression of ER stress‑associated proteins, including CCAAT‑enhancer‑binding protein homologous protein, and glucose‑regulated proteins 94 and 78, and led to the activation of multiple branches of ER stress transducers, including inositol‑requiring enzyme 1α‑apoptosis signal‑regulating kinase‑p38/c‑Jun N‑terminal kinase, and protein kinase R‑like endoplasmic reticulum kinase‑eukaryotic translation initiation factor 2α‑activating transcription factor (ATF)4 and ATF6, in the HepG2 cells. In addition, CXC195 inhibited the phosphorylation of phosphoinositide 3‑kinase (PI3K), Akt and mammalian target of rapamycin (mTOR) in the HepG2 cells. These effects were enhanced following treatment with selected inhibitors of PI3K (LY294002), Akt (SH‑6) and mTOR (rapamycin). Furthermore, these inhibitors enhanced the pro‑apoptotic effects of CXC195 in the HepG2 cells. In conclusion, the results of the present study indicated that CXC195 induced apoptosis and ER stress in HepG2 cells through the inhibition of the PI3K/Akt/mTOR signaling pathway. PMID:26496900

  11. 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 have identified several novel targets for RASSF1A tumor suppressor gene both at the RNA and the protein levels in two different cellular backgrounds. The identified targets are involved in diverse cellular processes; this should help toward understanding mechanisms that contribute to RASSF1A biological activity. PMID:14500366

  12. The cathelicidin-BF Lys16 mutant Cbf-K16 selectively inhibits non-small cell lung cancer proliferation in vitro.

    PubMed

    Tian, Yuwei; Wang, Hui; Li, Bing; Ke, Mengyun; Wang, Jing; Dou, Jie; Zhou, Changlin

    2013-11-01

    The 30-amino acid antimicrobial peptide Cbf-K16 is a cathelicidin-BF (BF-30) Lys16 mutant derived from the snake venom of Bungarus fasciatus. Our previous study found that BF-30 selectively inhibited the proliferation of the metastatic melanoma cell line B16F10 in vitro and in vivo, but had a negligible effect on human lung cells. In the present study, it was demonstrated for the first time that Cbf-K16 selectively inhibits the proliferation of lung carcinoma cells in vitro, with low toxicity to normal cells. The half-maximal inhibitory concentrations (IC50) of Cbf-K16 against H460 human non-small cell lung carcinoma cells and mouse Lewis lung cancer cells were only 16.5 and 10.5 µM, respectively, which were much less compared to that of BF-30 (45 and 40.3 µM). Data using a transmission electron microscope (TEM) assay showed that, at 20 and 40 µM, Cbf-K16 induced the rupture of the cytoplasmic membrane, which was consistent with data obtained from lactate dehydrogenase (LDH) release assays. The LDH release increased from 17.8 to 52.9% as the duration and dosage of Cbf-K16 increased. Annexin V-fluorescein and propidium iodide staining assays indicated that there were no obvious apoptotic effects at the different dosages and times tested. In H460 cells, the rate of genomic DNA binding increased from 51.9 to 86.8% as the concentration of Cbf-K16 increased from 5 to 10 µM. These data indicate that Cbf-K16 selectively inhibits the proliferation of lung carcinoma cells via cytoplasmic membrane permeabilization and DNA binding, rather than apoptosis. Although Cbf-K16 displayed significant cytotoxic activity (40 µM) against tumor cells, in splenocytes no significant inhibitory effect was observed and hemolysis was only 5.6%. These results suggest that Cbf-K16 is a low-toxicity anti-lung cancer drug candidate. PMID:23982315

  13. Propidium monoazide combined with real-time quantitative PCR underestimates heat-killed Listeria innocua.

    PubMed

    Løvdal, Trond; Hovda, Maria Befring; Björkblom, Benny; Møller, Simon G

    2011-05-01

    The combination of propidium monoazide (PMA) and quantitative real-time PCR (qPCR) significantly overestimated the fraction of viable Listeria innocua as compared to plate counts and confocal fluorescence microscopy. Our data imply that PMA-qPCR must be used with caution as an analytical tool for the differentiation between viable and dead bacteria. PMID:21324348

  14. 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...

  15. 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-qPCR. PMID:23060868

  16. The Overexpression of Hypomethylated miR-663 Induces Chemotherapy Resistance in Human Breast Cancer Cells by Targeting Heparin Sulfate Proteoglycan 2 (HSPG2)*

    PubMed Central

    Hu, Haiyan; Li, Shuqin; Cui, Xiuying; Lv, Xiaobin; Jiao, Yu; Yu, Fengyan; Yao, Herui; Song, Erwei; Chen, Yongsong; Wang, Minghui; Lin, Ling

    2013-01-01

    MicroRNAs are involved in regulating the biology of cancer cells, but their involvement in chemoresistance is not fully understood. We found that miR-663 was up-regulated in our induced multidrug-resistant MDA-MB-231/ADM cell line and that this up-regulation was closely related to chemosensitivity. In the present study, we aimed to clarify the role of miR-663 in regulating the chemoresistance of breast cancer. MicroRNA microarray and quantitative RT-PCR assays were used to identify differentially expressed microRNAs. Cell apoptosis was evaluated by annexin V/propidium iodide staining, TUNEL, and reactive oxygen species generation analysis. The expression of miR-663 and HSPG2 in breast cancer tissues was detected by in situ hybridization and immunohistochemistry. The potential targets of miR-663 were defined by a luciferase reporter assay. Bisulfite sequencing PCR was used to analyze the methylation status. We found that miR-663 was significantly elevated in MDA-MB-231/ADM cells, and the down-regulation of miR-663 sensitized MDA-MB-231/ADM cells to both cyclophosphamide and docetaxel. The overexpression of miR-663 in breast tumor tissues was associated with chemoresistance; in MDA-MB-231 cells, this chemoresistance was accompanied by the down-regulation of HSPG2, which was identified as a target of miR-663. MDA-MB-231/ADM contained fewer methylated CpG sites than its parental cell line, and miR-663 expression in MDA-MB-231 cells was reactivated by 5-aza-29-deoxycytidine treatment, indicating that DNA methylation may play a functional role in the expression of miR-663. Our findings suggest that the overexpression of hypomethylated miR-663 induced chemoresistance in breast cancer cells by down-regulating HSPG2, thus providing a potential target for the development of an microRNA-based approach for breast cancer therapy. PMID:23436656

  17. 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 occur through both the extrinsic and intrinsic apoptosis pathways with involvement of the NF-κB and HSP70 signaling pathways. PMID:25395836

  18. 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, and laminin or activating β1-integrin/FAK/AKT pathway might be potential treatments to prevent RGC loss in glaucomatous optic neuropathy. PMID:27064044

  19. 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.

  20. 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 the S phase was detected with each compound separately, while combined treatment only increased the production of fragmented DNA. Both compounds induced apoptosis through the extrinsic pathway after 24 hours; however, after 48 hours, the intrinsic pathway was activated by topotecan treatment only. In conclusion, thymoquinone increased the effectiveness of the chemotherapeutic reagent topotecan by inhibiting proliferation and lowering toxicity through p53- and Bax/Bcl2-independent mechanisms. PMID:26848703

  1. 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 demonstrated that two of the constituents of Cyperus longus, viridiflorol and longiverbenone, should be investigated further as possible promising chemotherapeutic agents in cancer treatment. PMID:26893742

  2. Selective Quantification of Viable Escherichia coli Bacteria in Biosolids by Quantitative PCR with Propidium Monoazide Modification ▿

    PubMed Central

    Taskin, Bilgin; Gozen, Ayse Gul; Duran, Metin

    2011-01-01

    Quantitative differentiation of live cells in biosolids samples, without the use of culturing-based approaches, is highly critical from a public health risk perspective, as recent studies have shown significant regrowth and reactivation of indicator organisms. Persistence of DNA in the environment after cell death in the range of days to weeks limits the application of DNA-based approaches as a measure of live cell density. Using selective nucleic acid intercalating dyes like ethidium monoazide (EMA) and propidium monoazide (PMA) is one of the alternative approaches to detecting and quantifying viable cells by quantitative PCR. These compounds have the ability to penetrate only into dead cells with compromised membrane integrity and intercalate with DNA via their photoinducible azide groups and in turn inhibit DNA amplification during PCRs. PMA has been successfully used in different studies and microorganisms, but it has not been evaluated sufficiently for complex environmental samples such as biosolids. In this study, experiments were performed with Escherichia coli ATCC 25922 as the model organism and the uidA gene as the target sequence using real-time PCR via the absolute quantification method. Experiments with the known quantities of live and dead cell mixtures showed that PMA treatment inhibits PCR amplification from dead cells with over 99% efficiency. The results also indicated that PMA-modified quantitative PCR could be successfully applied to biosolids when the total suspended solids (TSS) concentration is at or below 2,000 mg·liter−1. PMID:21602375

  3. 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 major breakthrough bringing new insight of the potential use of the polysaccharides as health-care food or medicine to provide significant natural defense against human cancer. PMID:23691187

  4. 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

  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 HepG2 cells. These results suggested that DMQ and the H-Ethyl acetate fraction of Hedyotis Diffusa Willd showed potential anticancer effects. Furthermore, the mechanisms of action may involve mitochondrial apoptotic and death receptor pathways. PMID:27064569

  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 on HepG2 cells. These results suggested that DMQ and the H-Ethyl acetate fraction of Hedyotis Diffusa Willd showed potential anticancer effects. Furthermore, the mechanisms of action may involve mitochondrial apoptotic and death receptor pathways. PMID:27064569

  7. 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

  8. 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 compared to sham treated cells. CHO cells undergoing mitosis after exposure also exhibit improper separation of chromatids which could indicate loss of function of the mitotic spindle checkpoint. Activation and loss of function of checkpoints in CHO but not Jurkat cells after nsPEF exposure suggests that activation of cell cycle checkpoints could be important in defining the character of cell line specific recovery after nsPEF exposure. Moreover, the increased sensitivity in G2/M phase exhibited by both cell lines indicates that cell cycle phase is an important consideration during nsPEF exposure, particularly when aiming to induce apoptosis.

  9. Effect of exposure to stress conditions on propidium monoazide (PMA)-qPCR based Campylobacter enumeration in broiler carcass rinses.

    PubMed

    Duarte, A; Botteldoorn, N; Coucke, W; Denayer, S; Dierick, K; Uyttendaele, M

    2015-06-01

    Campylobacter quantification by qPCR is unable to distinguish viable vs. dead cells in contrast to the culture-based ISO 10272-2 reference method. Propidium monoazide (PMA) has been used to overcome this disadvantage. A Campylobacter PMA-qPCR enumeration method was evaluated for its consistency and compared to the culture-based enumeration for both artificially and natural contaminated broiler carcass rinses. The PMA effect was further evaluated on stressed cells. Five conditions, commonly encountered during the slaughter process and storage (acid, heat, cold, oxidation and freezing), were inflicted to the broiler carcass rinses artificially contaminated with Campylobacter jejuni or Campylobacter coli. A better correlation between the reference method and the qPCR enumeration was obtained when PMA was used. The two cultured-based methods used showed a significant CFU reduction for heat, cold and acid stresses although the PMA-qPCR enumeration showed that viable bacteria were underestimated. Freezing showed the highest reduction effect, while the reduction extend was also overestimated by the microbiological enumeration procedure. Exposure to a mild oxidative stress was the only stress condition applied at temperatures permitting adaptation of Campylobacter and did not lead to either reduction in CFU nor in the PMA-qPCR signal. PMID:25791007

  10. Propidium monoazide combined with real-time quantitative PCR to quantify viable Alternaria spp. contamination in tomato products.

    PubMed

    Crespo-Sempere, Ana; Estiarte, Nria; Marn, Sonia; Sanchis, Vicente; Ramos, Antonio J

    2013-08-01

    Alternaria is a common contaminating genus of fungi in fruits, grains, and vegetables that causes severe economic losses to farmers and the food industry. Furthermore, it is claimed that Alternaria spp. are able to produce phytotoxic metabolites, and mycotoxins that are unsafe for human and animal health. DNA amplification techniques are being increasingly applied to detect, identify, and quantify mycotoxigenic fungi in foodstuffs, but the inability of these methods to distinguish between viable and nonviable cells might lead to an overestimation of mycotoxin-producing living cells. A promising technique to overcome this problem is the pre-treatment of samples with nucleic acid intercalating dyes, such as propidium monoazide (PMA), prior to quantitative PCR (qPCR). PMA selectively penetrates cells with a damaged membrane inhibiting DNA amplification during qPCRs. In our study, a primer pair (Alt4-Alt5) to specifically amplify and quantify Alternaria spp. by qPCR was designed. Quantification data of qPCR achieved a detection limit of 10(2)conidia/g of tomato. Here, we have optimized for the first time a DNA amplification-based PMA sample pre-treatment protocol for detecting viable Alternaria spp. cells. Artificially inoculated tomato samples treated with 65?M of PMA, showed a reduction in the signal by almost 7cycles in qPCR between live and heat-killed Alternaria spp. conidia. The tomato matrix had a protective effect on the cells against PMA toxicity, reducing the efficiency to distinguish between viable and nonviable cells. The results reported here indicate that the PMA-qPCR method is a suitable tool for quantifying viable Alternaria cells, which could be useful for estimating potential risks of mycotoxin contamination. PMID:23796654

  11. Rapid Detection of Viable Salmonellae in Produce by Coupling Propidium Monoazide with Loop-Mediated Isothermal Amplification ▿

    PubMed Central

    Chen, Siyi; Wang, Fei; Beaulieu, John C.; Stein, Rebecca E.; Ge, Beilei

    2011-01-01

    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 salmonellae in produce by coupling a simple propidium monoazide sample treatment with loop-mediated isothermal amplification (PMA-LAMP). We first designed and optimized a LAMP assay targeting Salmonella. Second, the performance of PMA-LAMP for detecting and quantifying viable salmonellae was determined. Finally, the assay was evaluated in experimentally contaminated produce items (cantaloupe, spinach, and tomato). Under the optimized condition, PMA-LAMP consistently gave negative results for heat-killed Salmonella cells with concentrations up to 108 CFU/ml (or CFU/g in produce). The detection limits of PMA-LAMP were 3.4 to 34 viable Salmonella cells in pure culture and 6.1 × 103 to 6.1 × 104 CFU/g in spiked produce samples. In comparison, PMA-PCR was up to 100-fold less sensitive. The correlation between LAMP time threshold (TT) values and viable Salmonella cell numbers was high (R2 = 0.949 to 0.993), with a quantification range (102 to 105 CFU/reaction in pure culture and 104 to 107 CFU/g in produce) comparable to that of PMA in combination with quantitative real-time PCR (PMA-qPCR). The complete PMA-LAMP assay took about 3 h to complete when testing produce samples. In conclusion, this rapid, accurate, and simple method to detect and quantify viable Salmonella cells in produce may present a useful tool for the produce industry to better control potential microbial hazards in produce. PMID:21498750

  12. Detection and quantification of viable Bacillus cereus group species in milk by propidium monoazide quantitative real-time PCR.

    PubMed

    Cattani, Fernanda; Barth, Valdir C; Nasário, Jéssica S R; Ferreira, Carlos A S; Oliveira, Sílvia D

    2016-04-01

    The Bacillus cereus group includes important spore-forming bacteria that present spoilage capability and may cause foodborne diseases. These microorganisms are traditionally evaluated in food using culturing methods, which can be laborious and time-consuming, and may also fail to detect bacteria in a viable but nonculturable state. The purpose of this study was to develop a quantitative real-time PCR (qPCR) combined with a propidium monoazide (PMA) treatment to analyze the contamination of UHT milk by B. cereus group species viable cells. Thirty micrograms per milliliter of PMA was shown to be the most effective concentration for reducing the PCR amplification of extracellular DNA and DNA from dead cells. The quantification limit of the PMA-qPCR assay was 7.5×10(2)cfu/mL of milk. One hundred thirty-five UHT milk samples were analyzed to evaluate the association of PMA to qPCR to selectively detect viable cells. The PMA-qPCR was able to detect B. cereus group species in 44 samples (32.6%), whereas qPCR without PMA detected 78 positive samples (57.8%). Therefore, the PMA probably inhibited the amplification of DNA from cells that were killed during UHT processing, which avoided an overestimation of bacterial cells when using qPCR and, thus, did not overvalue potential health risks. A culture-based method was also used to detect and quantify B. cereus sensu stricto in the same samples and showed positive results in 15 (11.1%) samples. The culture method and PMA-qPCR allowed the detection of B. cereus sensu stricto in quantities compatible with the infective dose required to cause foodborne disease in 3 samples, indicating that, depending on the storage conditions, even after UHT treatment, infective doses may be reached in ready-to-consume products. PMID:26830746

  13. 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 an appropriate method for discriminating between live and dead cells to enumerate Legionella for regulatory purposes. PMID:25288885

  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 -20C or if the collected cells were stored at 4C 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 ?g/ml colistin for 5 min. PMID:25283547

  15. Evaluation of Propidium Monoazide Real-Time PCR for Early Detection of Viable Mycobacterium tuberculosis in Clinical Respiratory Specimens

    PubMed Central

    Kim, Young Jin; Lee, Sun Min; Park, Byung Kyu; Kim, Sung Soo; Yi, Jongyoun; Kim, Hyung Hoi; Lee, Eun Yup

    2014-01-01

    Background Conventional acid-fast bacilli (AFB) staining cannot differentiate viable from dead cells. Propidium monoazide (PMA) is a photoreactive DNA-binding dye that inhibits PCR amplification by DNA modification. We evaluated whether PMA real-time PCR is suitable for the early detection of viable Mycobacterium tuberculosis (MTB) in clinical respiratory specimens. Methods A total of 15 diluted suspensions from 5 clinical MTB isolates were quadruplicated and subjected to PMA treatment and/or heat inactivation. Eighty-three AFB-positive sputum samples were also tested to compare the ΔCT values (CT value in PMA-treated sputum samples-CT value in non-PMA-treated sputum samples) between culture-positive and culture-negative specimens. Real-time PCR was performed using Anyplex MTB/NTM Real-Time Detection (Seegene, Korea), and the CT value changes after PMA treatment were compared between culture-positive and culture-negative groups. Results In MTB suspensions, the increase in the CT value after PMA treatment was significant in dead cells (P=0.0001) but not in live cells (P=0.1070). In 14 culture-negative sputum samples, the median ΔCT value was 5.3 (95% confidence interval [CI], 4.1-8.2; P<0.0001), whereas that in 69 culture-positive sputum samples was 1.1 (95% CI, 0.7-2.0). In the ROC curve analysis, the cutoff ΔCT value for maximum sensitivity (89.9%) and specificity (85.7%) for differentiating dead from live cells was 3.4. Conclusions PMA real-time PCR is a useful approach for differentiating dead from live bacilli in AFB smear-positive sputum samples. PMID:24790907

  16. Quantification of viable bacteria in wastewater treatment plants by using propidium monoazide combined with quantitative PCR (PMA-qPCR).

    PubMed

    Li, Dan; Tong, Tiezheng; Zeng, Siyu; Lin, Yiwen; Wu, Shuxu; He, Miao

    2014-02-01

    The detection of viable bacteria in wastewater treatment plants (WWTPs) is very important for public health, as WWTPs are a medium with a high potential for waterborne disease transmission. The aim of this study was to use propidium monoazide (PMA) combined with the quantitative polymerase chain reaction (PMA-qPCR) to selectively detect and quantify viable bacteria cells in full-scale WWTPs in China. PMA was added to the concentrated WWTP samples at a final concentration of 100 micromol/L and the samples were incubated in the dark for 5 min, and then lighted for 4 min prior to DNA extraction and qPCR with specific primers for Escherichia coli and Enterococci, respectively. The results showed that PMA treatment removed more than 99% of DNA from non-viable cells in all the WWTP samples, while matrices in sludge samples markedly reduced the effectiveness of PMA treatment. Compared to qPCR, PMA-qPCR results were similar and highly linearly correlated to those obtained by culture assay, indicating that DNA from non-viable cells present in WWTP samples can be eliminated by PMA treatment, and that PMA-qPCR is a reliable method for detection of viable bacteria in environmental samples. This study demonstrated that PMA-qPCR is a rapid and selective detection method for viable bacteria in WWTP samples, and that WWTPs have an obvious function in removing both viable and non-viable bacteria. The results proved that PMA-qPCR is a promising detection method that has a high potential for application as a complementary method to the standard culture-based method in the future. PMID:25076521

  17. 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...

  18. 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...

  19. 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 of LC3), although the level of Bcl‑2 was decreased. Furthermore, cell cytotoxicity and autophagy were induced upon treatment with sodium formate. Z‑VAD‑fmk and SP600125 were able to effectively circumvent the effects of sodium formate on cell viability. These results suggested that the cytotoxicity induced by sodium formate induces the activation of the JNK signaling pathway, leading to caspase‑dependent apoptosis. Increased levels of autophagy were also observed during the process of 661W cell damage induced by sodium formate. PMID:26676762

  20. Comparative analysis of bacterial community composition in bulk tank raw milk by culture-dependent and culture-independent methods using the viability dye propidium monoazide.

    PubMed

    Weber, Mareike; Geißert, Janina; Kruse, Myriam; Lipski, André

    2014-11-01

    Microbial diversity of 3 raw milk samples after 72 h of storage at 4 °C in a bulk tank was analyzed by culture-dependent and -independent methods. The culture-dependent approach was based on the isolation of bacteria on complex and selective media, chemotaxonomic differentiation of isolates, and subsequent identification by 16S rRNA gene sequencing. The culture-independent approach included the treatment of raw milk with the dye propidium monoazide before direct DNA extraction by mechanic and enzymatic cell lysis approaches, and cloning and sequencing of the 16S rRNA genes. The selective detection of viable bacteria improved the comparability between bacterial compositions of raw milk based on culture-dependent and -independent methods, which was the major objective of this study. Several bacterial species of the phyla Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria were detected by the culture-dependent method, whereas mainly bacteria of the phylum Proteobacteria as well as low proportions of the phyla Bacteroidetes and Actinobacteria were detected by the culture-independent method. This led to the conclusion that the phylum Firmicutes was strongly discriminated by the culture-independent approach. Generally, species richness detected by the culture-dependent method was higher than that detected by the culture-independent method for all samples. However, few taxa could be detected solely by the direct DNA-based method. In conclusion, the combination of culture-dependent and -independent methods led to the detection of the highest bacterial diversity for the raw milk samples analyzed. It was shown that DNA extraction from raw milk as the essential step in culture-independent methods causes the discrimination of taxa by incomplete cell lysis. Treatment of raw milk with the viability dye propidium monoazide was optimized for the application in raw milk without former removal of milk ingredients and proved to be a suitable tool to ensure comparability of bacterial diversity depicted by both methods. PMID:25242425

  1. Rapid and Accurate Detection of Bacteriophage Activity against Escherichia coli O157:H7 by Propidium Monoazide Real-Time PCR

    PubMed Central

    Niu, Yan D.; Li, Jinquan; McAllister, Tim A.

    2014-01-01

    Conventional methods to determine the efficacy of bacteriophage (phage) for biocontrol of E. coli require several days, due to the need to culture bacteria. Furthermore, cell surface-attached phage particles may lyse bacterial cells during experiments, leading to an overestimation of phage activity. DNA-based real-time quantitative polymerase chain reaction (qPCR) is a fast, sensitive, and highly specific means of enumerating pathogens. However, qPCR may underestimate phage activity due to its inability to distinguish viable from nonviable cells. In this study, we evaluated the suitability of propidium monoazide (PMA), a microbial membrane-impermeable dye that inhibits amplification of extracellular DNA and DNA within dead or membrane-compromised cells as a means of using qPCR to identify only intact E. coli cells that survive phage exposure. Escherichia coli O157:H7 strain R508N and 4 phages (T5-like, T1-like, T4-like, and O1-like) were studied. Results compared PMA-qPCR and direct plating and confirmed that PMA could successfully inhibit amplification of DNA from compromised/damaged cells E. coli O157:H7. Compared to PMA-qPCR, direct plating overestimated (P < 0.01) phage efficacy as cell surface-attached phage particles lysed E. coli O157:H7 during the plating process. Treatment of samples with PMA in combination with qPCR can therefore be considered beneficial when assessing the efficacy of bacteriophage for biocontrol of E. coli O157:H7. PMID:25530959

  2. Rapid and accurate detection of bacteriophage activity against Escherichia coli O157:H7 by propidium monoazide real-time PCR.

    PubMed

    Liu, Hui; Niu, Yan D; Li, Jinquan; Stanford, Kim; McAllister, Tim A

    2014-01-01

    Conventional methods to determine the efficacy of bacteriophage (phage) for biocontrol of E. coli require several days, due to the need to culture bacteria. Furthermore, cell surface-attached phage particles may lyse bacterial cells during experiments, leading to an overestimation of phage activity. DNA-based real-time quantitative polymerase chain reaction (qPCR) is a fast, sensitive, and highly specific means of enumerating pathogens. However, qPCR may underestimate phage activity due to its inability to distinguish viable from nonviable cells. In this study, we evaluated the suitability of propidium monoazide (PMA), a microbial membrane-impermeable dye that inhibits amplification of extracellular DNA and DNA within dead or membrane-compromised cells as a means of using qPCR to identify only intact E. coli cells that survive phage exposure. Escherichia coli O157:H7 strain R508N and 4 phages (T5-like, T1-like, T4-like, and O1-like) were studied. Results compared PMA-qPCR and direct plating and confirmed that PMA could successfully inhibit amplification of DNA from compromised/damaged cells E. coli O157:H7. Compared to PMA-qPCR, direct plating overestimated (P < 0.01) phage efficacy as cell surface-attached phage particles lysed E. coli O157:H7 during the plating process. Treatment of samples with PMA in combination with qPCR can therefore be considered beneficial when assessing the efficacy of bacteriophage for biocontrol of E. coli O157:H7. PMID:25530959

  3. Propidium monoazide combined with real-time PCR for selective detection of viable Staphylococcus aureus in milk powder and meat products.

    PubMed

    Zhang, Zhihong; Liu, Wenting; Xu, Hengyi; Aguilar, Zoraida P; Shah, Nagendra P; Wei, Hua

    2015-03-01

    Staphylococcus aureus is a spherical, gram-positive, pathogenic bacterium commonly associated with bovine mastitis and clinical infections. It is also recognized as a pathogen that causes outbreaks of food poisoning. The objective of this study was to develop and evaluate a rapid and reliable technique that combines propidium monoazide (PMA) staining with real-time quantitative (q)PCR to detect and quantify viable cells of Staph. aureus in milk powder and meat products. The inclusivity and exclusivity of the assay were evaluated using 58 strains belonging to 14 species. Serial dilutions of Staph. aureus cells were used to establish a standard curve and to confirm the effect of PMA treatment. Milk powder and meat products were used as the spiked foods, and the ability of PMA-qPCR to eliminate nonviable cells was determined in milk powder. Furthermore, meat products were inoculated with different concentrations of Staph. aureus and 10(5) cfu/g of Bacillus cereus and Salmonella enterica to test the interference by nontarget microorganisms. When PMA treatment was applied before DNA extraction, we were able to eliminate false-positive results with little effect on viable cells. The PMA-qPCR assay was specific and more sensitive than conventional PCR, and the level of detection was 3.0×10(2) cfu/g in spiked milk powder. Additionally, we observed no significant interference for the detection of viable Staph. aureus from other nontarget bacteria. The PMA-qPCR protocol is an effective and rapid method to quantify viable cells of Staph. aureus in food samples. The PMA-qPCR assay is specific and reliable, offering a valuable diagnostic tool for routine analysis in food and clinical diagnostic research at a reasonable cost. PMID:25582587

  4. 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 by PCR. This PMA-qPCR assay provides a sensitive, specific, and efficient method for detecting live Salmonella cells in foods and environmental samples and may have an impact on the accurate microbiological monitoring of Salmonella in foods and environment samples. PMID:24289661

  5. Rapid Quantification of Viable Campylobacter Bacteria on Chicken Carcasses, Using Real-Time PCR and Propidium Monoazide Treatment, as a Tool for Quantitative Risk Assessment?

    PubMed Central

    Josefsen, M. H.; Lfstrm, C.; Hansen, T. B.; Christensen, L. S.; Olsen, J. E.; Hoorfar, J.

    2010-01-01

    A number of intervention strategies against Campylobacter-contaminated poultry focus on postslaughter reduction of the number of cells, emphasizing the need for rapid and reliable quantitative detection of only viable Campylobacter bacteria. We present a new and rapid quantitative approach to the enumeration of food-borne Campylobacter bacteria that combines real-time quantitative PCR (Q-PCR) with simple propidium monoazide (PMA) sample treatment. In less than 3 h, this method generates a signal from only viable and viable but nonculturable (VBNC) Campylobacter bacteria with an intact membrane. The method's performance was evaluated by assessing the contributions to variability by individual chicken carcass rinse matrices, species of Campylobacter, and differences in efficiency of DNA extraction with differing cell inputs. The method was compared with culture-based enumeration on 50 naturally infected chickens. The cell contents correlated with cycle threshold (CT) values (R2 = 0.993), with a quantification range of 1 102 to 1 107 CFU/ml. The correlation between the Campylobacter counts obtained by PMA-PCR and culture on naturally contaminated chickens was high (R2 = 0.844). The amplification efficiency of the Q-PCR method was not affected by the chicken rinse matrix or by the species of Campylobacter. No Q-PCR signals were obtained from artificially inoculated chicken rinse when PMA sample treatment was applied. In conclusion, this study presents a rapid tool for producing reliable quantitative data on viable Campylobacter bacteria in chicken carcass rinse. The proposed method does not detect DNA from dead Campylobacter bacteria but recognizes the infectious potential of the VBNC state and is thereby able to assess the effect of control strategies and provide trustworthy data for risk assessment. PMID:20562292

  6. 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 conditions. PMID:24358142

  7. 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...

  8. Propidium monoazide (PMA) and ethidium bromide monoazide (EMA) improve DNA array and high-throughput sequencing of porcine reproductive and respiratory syndrome virus identification.

    PubMed

    Bellehumeur, Christian; Boyle, Brian; Charette, Steve J; Harel, Josée; L'Homme, Yvan; Masson, Luke; Gagnon, Carl A

    2015-09-15

    Pan-viral DNA array (PVDA) and high-throughput sequencing (HTS) are useful tools to identify novel viruses of emerging diseases. However, both techniques have difficulties to identify viruses in clinical samples because of the host genomic nucleic acid content (hg/cont). Both propidium monoazide (PMA) and ethidium bromide monoazide (EMA) have the capacity to bind free DNA/RNA, but are cell membrane-impermeable. Thus, both are unable to bind protected nucleic acid such as viral genomes within intact virions. However, EMA/PMA modified genetic material cannot be amplified by enzymes. In order to assess the potential of EMA/PMA to lower the presence of amplifiable hg/cont in samples and improve virus detection, serum and lung tissue homogenates were spiked with porcine reproductive and respiratory virus (PRRSV) and were processed with EMA/PMA. In addition, PRRSV RT-qPCR positive clinical samples were also tested. EMA/PMA treatments significantly decreased amplifiable hg/cont and significantly increased the number of PVDA positive probes and their signal intensity compared to untreated spiked lung samples. EMA/PMA treatments also increased the sensitivity of HTS by increasing the number of specific PRRSV reads and the PRRSV percentage of coverage. Interestingly, EMA/PMA treatments significantly increased the sensitivity of PVDA and HTS in two out of three clinical tissue samples. Thus, EMA/PMA treatments offer a new approach to lower the amplifiable hg/cont in clinical samples and increase the success of PVDA and HTS to identify viruses. PMID:26129867

  9. 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

  10. 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

  11. Cryptosporidium Propidium Monoazide-PCR, a Molecular Biology-Based Technique for Genotyping of Viable Cryptosporidium Oocysts▿

    PubMed Central

    Brescia, Cristin C.; Griffin, Shannon M.; Ware, Michael W.; Varughese, Eunice A.; Egorov, Andrey I.; Villegas, Eric N.

    2009-01-01

    Cryptosporidium is an important waterborne protozoan parasite that can cause severe diarrhea and death in the immunocompromised. The current methods used to monitor for Cryptosporidium oocysts in water are the microscopy-based USEPA methods 1622 and 1623. These methods assess total levels of oocysts in source waters, but do not determine oocyst viability or genotype. Recently, propidium monoazide (PMA) has been used in conjunction with molecular diagnostic tools to identify species and assess the viability of bacteria. The goal of this study was the development of a Cryptosporidium PMA-PCR (CryptoPMA-PCR) assay that includes PMA treatment prior to PCR analysis in order to prevent the amplification of DNA from dead oocysts. The results demonstrated that PMA penetrates only dead oocysts and blocks amplification of their DNA. The CryptoPMA-PCR assay can also specifically detect live oocysts within a mixed population of live and dead oocysts. More importantly, live oocysts, not dead oocysts, were detected in raw waste or surface water samples spiked with Cryptosporidium oocysts. This proof-of-concept study is the first to demonstrate the use of PMA for pre-PCR treatment of Cryptosporidium oocysts. The CryptoPMA-PCR assay is an attractive approach to specifically detect and genotype viable Cryptosporidium oocysts in the water, which is critical for human health risk assessment. PMID:19749067

  12. 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-immunostaining. Osteoblastogenesis was estimated by measurement of alkaline phosphatase (ALP) activity and production of mineralized matrix (von-Kossa staining, Alizarin Red staining). During the process of osteoblastic cell differentiation, the expression of the bone specific marker genes osteocalcin (OCN) and osteopontin (OPN) were recorded by quantitative real time reverse transcription PCR (qRT-PCR). Compared with standard culture conditions, the osteogenic marker genes OCN and OPN were highly expressed during the differentiation process induced either by osteo-inductive media additives (50 µg/ml ascorbic acid, 10 mmol/l β-glycero phosphate) or by sparsely ionizing radiation (X-rays). After 21 days of postirradiation incubation sparsely ionizing radiation could be shown to induce the formation of bone-like nodules (von-Kossa staining) for OCT-1 and MC3T3-E1 S4 cells but nor for MC3T3- E1 S24 cells. Ionizing radiation leads to a cell cycle arrest which is resolved in a dose and time dependent way. This was accompanied by a dose dependent regulation of the cyclin kinase inhibitor CDKN1A (p21/WAF) and transforming growth factor beta 1 (TGF-β1). TGF-β1 is known to affect osteoblast differentiation, matrix formation and mineralization. Modulation of its expression could influence the expression of main osteogenic transcription factors. For exposure with high LET radiation a pronounced cell cycle block was evident. The expression of the osteogenic marker genes OCN and Osterix (OSX) was increased in the OCT-1 cells with differentiation potential for exposure to α particles and accelerated carbon and argon ions. The results on the expression of differentiation markers during radiation-induced premature differentiation of bone cells of the osteoblast lineage show that densely ionizing radiation results in expression of proteins essential for bone formation and consequently in an increase in bone volume. Such an effect has been observed in in-vivo carbon ion irradiated rats. As radiation dependent permanent cell cycle blocks lead to a depletion of proliferation-competent cells from the osteoblastic precursor pool in the body, a gradual decrease of bone mass in weightlessness may be attributed to synergistic effects of radiation and weightlessness.

  13. 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 correlated with environmental factors known to induce variation in pigmentation. PMID:18158306

  14. Apoptotic and necrotic influence of dental resin polymerization initiators in human gingival fibroblast cultures.

    PubMed

    Masuki, Kouhei; Nomura, Yuji; Bhawal, Ujjal Kumar; Sawajiri, Masahiko; Hirata, Isao; Nahara, Yukinori; Okazaki, Masayuki

    2007-11-01

    The aim of this study was to examine the apoptotic and necrotic influence of four dental resin polymerization initiators--namely benzoyl peroxide (BPO), camphorquinone (CQ), dimethylaminoethyl methacrylate (DMAEMA), and dimethyl-para-toluidine (DMPT)--on human gingival fibroblast (HGF) cells. To this end, the growth inhibition of HGF cells with 1 mM BPO, CQ, and DMAEMA, and 500 microM DMPT was evaluated using Cell Counting Kit-8. Then, cell cycle analysis by flow cytometry was used to assess propidium iodide-stained cells (distribution of cells in G0/G1, S, G2/M phases). All four dental resin polymerization initiators induced G0/G1 cell cycle arrest. As for the patterns of cell death (necrosis and/or apoptosis), they were analyzed using Annexin V-FITC/PI staining with flow cytometry. All four dental resin polymerization initiators most likely induced necrosis. PMID:18203492

  15. 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

  16. 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.

  17. Magnetic nano-beads based separation combined with propidium monoazide treatment and multiplex PCR assay for simultaneous detection of viable Salmonella Typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes in food products.

    PubMed

    Yang, Youjun; Xu, Feng; Xu, Hengyi; Aguilar, Zoraida P; Niu, Ruijiang; Yuan, Yong; Sun, Jichang; You, Xingyong; Lai, Weihua; Xiong, Yonghua; Wan, Cuixiang; Wei, Hua

    2013-06-01

    We developed a rapid and reliable technique for simultaneous detection of Salmonella Typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes that can be used in food products. Magnetic nano-beads (MNBs) based immunomagnetic separation (IMS) was used to separate the target bacterial cells while multiplex PCR (mPCR) was used to amplify the target genes. To detect only the viable bacteria, propidium monoazide (PMA) was applied to selectively suppress the DNA detection from dead cells. The results showed the detection limit of IMS-PMA-mPCR assay was about 10(2) CFU/ml (1.2 × 10(2) CFU/ml for S. Typhimurium, 4.0 × 10(2) CFU/ml for E. coli O157:H7 and 5.4 × 10(2) CFU/ml for L. monocytogenes) in pure culture and 10(3) CFU/g (5.1 × 10(3) CFU/g for S. Typhimurium, 7.5 × 10(3) CFU/g for E. coli O157:H7 and 8.4 × 10(3) CFU/g for L. monocytogenes) in spiking food products samples (lettuce, tomato and ground beef). This report has demonstrated for the first time, the effective use of rapid and reliable IMS combined with PMA treatment and mPCR assay for simultaneous detection of viable S. Typhimurium, E. coli O157:H7 and L. monocytogenes in spiked food samples. It is anticipated that the present approach will be applicable to simultaneous detection of the three target microorganisms for practical use. PMID:23541211

  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. 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...

  1. 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)-...

  2. Discrimination of viable from non-viable gram-negative bacterial pathogens in airborne particles using propidium monoazide-assisted qPCR.

    PubMed

    Kaushik, Rajni; Balasubramanian, Rajasekhar

    2013-04-01

    The presence of bacterial pathogens in airborne particulate matter (PM) has been of considerable concern from the public health standpoint. Conventional culture-based methods are tedious, time consuming and are unable to quantify stressed viable but non-culturable (VBNC) populations of these pathogens. This study reports the optimization, validation and application of a new and rapid quantitative method for enumeration of four live potential Gram-negative bacterial pathogens (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Aeromonas hydrophila) in PM of biomass burning origin. This method makes use of an intercalating dye (propidium monoazide, PMA) in conjunction with real-time PCR (qPCR) analysis following DNA extraction from PM samples for distinguishing viable from non-viable potential bacterial pathogens. This method was not affected by the complex matrix of the environmental samples, nor by any PCR inhibition effects. The number of viable pathogens ranged from 0 to 8×10(4) gene copies/m(3) in PM. With the exception of A. hydrophilia, all the three pathogens were found to be present in PM. The correlation between the counts obtained using the PMA-qPCR (modified qPCR) and those from the culture-based method was very high with R(2)~1.0 and p value<0.0001. PMID:23428754

  3. 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

  4. 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

  5. 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)

  6. Pyruvate kinase, muscle isoform 2 promotes proliferation and insulin secretion of pancreatic ?-cells via activating Wnt/CTNNB1 signaling

    PubMed Central

    Wang, Suijun; Yang, Zhen; Gao, Ying; Li, Quanzhong; Su, Yong; Wang, Yanfang; Zhang, Yun; Man, Hua; Liu, Hongxia

    2015-01-01

    Failure of pancreatic ?-cells is closely associated with type 2 diabetes mellitus (T2DM), an intractable disease affecting numerous patients. Pyruvate kinase, muscle isoform 2 (PKM2) is a potential modulator of insulin secretion in ?-cells. This study aims at revealing roles and possible mechanisms of PKM2 in pancreatic ?-cells. Mouse pancreatic ?-cell line NIT-1 was used for high glucose treatment and PKM2 overexpression by its specific expression vector. Cell proliferation by Thiazolyl blue assay, cell apoptosis by annexin V-fluorescein isothiocyanate/prodium iodide staining and insulin secretion assay by ELISA were performed in each group. The mRNA and protein levels of related factors were analyzed by real-time quantitative PCR and western blot. Results showed that Pkm2 was inhibited under high glucose conditions compared to the untreated cells (P < 0.01). Its overexpression significantly suppressed NIT-1 cell apoptosis (P < 0.01), and induced cell proliferation (P < 0.05) and insulin secretion (P < 0.05). Related factors showed consistent mRNA expression changes. Protein levels of ?-catenin (CTNNB1), insulin receptor substrate 1 (IRS1) and IRS2 were all promoted by PKM2 overexpression (P < 0.01), indicating the activated Wnt/CTNNB1 signaling. These results indicated the inductive roles of PKM2 in pancreatic ?-cell NIT-1, including promoting cell proliferation and insulin secretion, and inhibiting cell apoptosis, which might be achieved via activating the Wnt/CTNNB1 signaling and downstream factors. This study offers basic information on the role and mechanism of PKM2 in pancreatic ?-cells, and lays the foundation for using PKM2 as a potential therapeutic target in T2DM. PMID:26823761

  7. Bioactive chemicals from carrot (Daucus carota) juice extracts for the treatment of leukemia.

    PubMed

    Zaini, Rana; Clench, Malcolm R; Le Maitre, Christine L

    2011-11-01

    Overwhelming evidence indicates that consumption of fruits and vegetables with antioxidant properties correlates with reduced risk for cancers, including leukemia. Carrots contain beneficial agents, such as β-carotene and polyacetylenes, which could be effective in the treatment of leukemia. This study investigated the effect of carrot juice extracts on myeloid and lymphoid leukemia cell lines together with normal hematopoietic stem cells. Leukemia cell lines and nontumor control cells were treated with carrot juice extracts for up to 72 hours in vitro. Induction of apoptosis was investigated by using annexin V/propidium iodide staining followed by flow cytometric analysis, and results were confirmed by using 4'-6-diamidino-2-phenylindole morphology. Effects on cellular proliferation were investigated via cell cycle analysis and cell counts. Treatment of leukemia cell lines with carrot juice extract induced apoptosis and inhibited progression through the cell cycle. Lymphoid cell lines were affected to a greater extent than were myeloid cell lines, and normal hematopoietic stem cells were less sensitive than most cell lines. This study has shown that extracts from carrots can induce apoptosis and cause cell cycle arrest in leukemia cell lines. The findings suggest that carrots may be an excellent source of bioactive chemicals for the treatment of leukemia. PMID:21864090

  8. Knockdown of protein phosphatase 5 inhibits ovarian cancer growth in vitro

    PubMed Central

    ZHENG, XIAOJIAO; ZHANG, LIANXIAO; JIN, BOHONG; ZHANG, FUBIN; ZHANG, DUOYI; CUI, LINING

    2016-01-01

    Ovarian cancer is the most common cause of gynecological cancer-related mortality. Serine/threonine protein phosphatase 5 (PP5, PPP5C) has been recognized to be involved in the regulation of multiple cellular signaling cascades that control diverse cellular processes, including cell growth, differentiation, proliferation, motility and apoptosis. In this study, to evaluate the functional role of PP5 in ovarian cancer cells, lentivirus-mediated RNA interference (RNAi) was applied to silence PPP5C in the human ovarian cancer cell line CAOV-3. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell colony forming ability was measured by colony formation. Cell cycle progression was determined by propidium iodide staining and flow cytometry. The results demonstrated that lentivirus-mediated RNAi specifically suppressed the expression of PPP5C at the mRNA and protein levels in CAOV-3 cells. Further investigations revealed that PP5 knockdown significantly inhibited the proliferation and colony formation of CAOV-3 cells. Moreover, the cell cycle of CAOV-3 cells was arrested at the G0/G1 phase following PP5 knockdown. This study highlights the crucial role of PP5 in promoting ovarian cancer cell proliferation, and provides a foundation for further study into the clinical potential of lentiviral-mediated delivery of PP5 RNAi therapy for the treatment of ovarian cancer. PMID:26870184

  9. Inhibition of ovarian cancer proliferation and invasion by pachymic acid

    PubMed Central

    Gao, Ai-Hua; Zhang, Liang; Chen, Xin; Chen, Ying; Xu, Zhen-Zhen; Liu, Ya-Nan; Zhang, Hong

    2015-01-01

    To determine the effect of pachymic acid (PA) on proliferation, cell cycle, and invasion in human ovarian carcinoma cell lines HO-8910 and explore some possible mechanisms, HO-8910 cells was treated with different concentrations of PA (0.5, 1, 2 μM). CCK-8 assay, propidium iodide staining, was applied to measuring the growth inhibiting rates of HO-8910 cells. Cell cycle was measured by flow cytometry. In addition, the activity of PA against HO-8910 cells invasion was evaluated in transwell assay. Western blot detected the proteins expression of E-cadherin, β-catenin and COX-2 of different groups treated with PA in different concentrations (0.5, 1, 2 μM) for 48 h. Our results showed that PA could effectively inhibit the in vitro growth of HO-8910 cells in dose-dependent manners in 72 h, suppressed migration and invasion of HO-8910 cells in concentration-dependent manners at 24 h, caused the increased accumulation of G1 phase cells, and caused down-regulation of β-catenin and COX-2 and up-regulation of E-cadherin expression level. Taken together, it could conclude that PA might inhibit proliferation and invasion of ovarian carcinoma cell through decreasing β-catenin and COX-2 expression and increasing E-cadherin exprssion. PMID:25973134

  10. Tumor-targeting novel manganese complex induces ROS-mediated apoptotic and autophagic cancer cell death

    PubMed Central

    LIU, JIA; GUO, WENJIE; LI, JING; LI, XIANG; GENG, JI; CHEN, QIUYUN; GAO, JING

    2015-01-01

    In this study, the antitumor activity of the novel manganese (II) compound, Adpa-Mn {[(Adpa)Mn(Cl)(H2O)] (Adpa=bis(2-pyridylmethyl)amino-2-propionic acid)}, and its possible mechanisms of action were investigated. In vitro, the growth inhibitory effects of Adpa-Mn (with IC50 values lower than 15 μM) on tumor cell lines were examined by MTT assay. We found that this compound was more selective against cancer cells than the popular chemotherapeutic reagent, cisplatin. We then found that Adpa-Mn achieved its selectivity against cancer cells through the transferrin (Tf)-transferrin receptor (TfR) system, which is highly expressed in tumor cells. Furthermore, Adpa-Mn induced both apoptosis and autophagy, as indicated by chromatin condensation, the activation of poly(ADP-ribose) polymerase (PARP), Annexin V/prop-idium iodide staining, an enhanced fluorescence intensity of monodansylcadaverine (MDC), as well as the elevated expression of the autophagy-related protein, microtubule-associated protein 1 light chain 3 (LC3). In addition, Adpa-Mn induced the generation of intracellular reactive oxygen species (ROS) and its anticancer effects were significantly reduced following pre-treatment with the antioxidant, N-acetyl cysteine, indicating that ROS triggered cell death. In vivo, the induction of apoptosis and autophagy in tumor tissue was confirmed following treatment with Adpa-Mn, which contributed to its significant antitumor activity against hepatocellular carcinoma (Hep-A cell) xenografts at 10 mg/kg. Taken together, these data suggest the possible use of Adpa-Mn as a novel anticancer drug. PMID:25604962

  11. A microneedle array able to inject tens of thousands of cells simultaneously

    NASA Astrophysics Data System (ADS)

    Teichert, Gregory H.; Burnett, Sandra; Jensen, Brian D.

    2013-09-01

    This paper presents a biological microelectromechanical system for injecting foreign particles into thousands of cells simultaneously. The system inserts an array of microneedles into a monolayer of cells, and the foreign particles enter the cells by diffusion. The needle array is fabricated using a series of deep reactive ion etches and produces about 4 million needles that average 1 μm in diameter and 8 μm in length with 10 μm spacing. The insertion of the needles is controlled through a compliant suspension. The compliant suspension was designed to provide for needle motion into the cells while restraining rotations or transverse motions that could result in tearing of the cell membranes. Testing was performed using propidium iodide, a membrane impermeable dye, injected into HeLa cells. Average cell survivability was found to be 97.7%, and up to 97.9% of the surviving cells received the propidium iodide.

  12. 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

  13. Evaluation of in vitro anti-proliferative and immunomodulatory activities of compounds isolated from Curcuma longa

    PubMed Central

    Yue, Grace G. L.; Chan, Ben C. L.; Hon, Po-Ming; Lee, Mavis Y. H.; Fung, Kwok-Pui; Leung, Ping-Chung; Lau, Clara B. S.

    2010-01-01

    The rhizome of Curcuma longa (CL) has been commonly used in Asia as a potential candidate for the treatment of different diseases, including inflammatory disorders and cancers. The present study evaluated the anti-proliferative activities of the isolated compounds (3 curcuminoids and 2 turmerones) from CL, using human cancer cell lines HepG2, MCF-7 and MDA-MB-231. The immunomodulatory activities of turmerones (α and aromatic) isolated from CL were also examined using human peripheral blood mononuclear cells (PBMC). Our results showed that the curcuminoids (curcumin, demethoxycurcumin and bisdemethoxycurcumin) and α-turmerone significantly inhibited proliferation of cancer cells in dose-dependent manner. The IC50 values of these compounds in cancer cells ranged from 11.0–41.8 μg/ml. Alpha-turmerone induced MDA-MB-231 cells to undergo apoptosis, which was confirmed by annexin-V & propidium iodide staining, and DNA fragmentation assay. The caspase cascade was activated as shown by a significant decrease of procaspases-3, -8 and -9 in α-turmerone treated cells. Both α-turmerone and aromatic-turmerone showed stimulatory effects on PBMC proliferation and cytokine production. The anti-proliferative effect of α-turmerone and immunomodulatory activities of ar-turmerone were shown for the first time. The findings revealed the potential use of CL crude extract (containing curcuminoids and volatile oil including turmerones) as chemopreventive agent. PMID:20438793

  14. DBS-relevant electric fields increase hydraulic conductivity of in vitro endothelial monolayers

    NASA Astrophysics Data System (ADS)

    Lopez-Quintero, S. V.; Datta, A.; Amaya, R.; Elwassif, M.; Bikson, M.; Tarbell, J. M.

    2010-02-01

    Deep brain stimulation (DBS) achieves therapeutic outcome through generation of electric fields (EF) in the vicinity of energized electrodes. Targeted brain regions are highly vascularized, and it remains unknown if DBS electric fields modulate blood-brain barrier (BBB) function, either through electroporation of individual endothelial cells or electro-permeation of barrier tight junctions. In our study, we calculated the intensities of EF generated around energized Medtronic 3387 and 3389 DBS leads by using a finite element model. Then we designed a novel stimulation system to study the effects of such fields with DBS-relevant waveforms and intensities on bovine aortic endothelial cell (BAEC) monolayers, which were used as a basic analog for the blood-brain barrier endothelium. Following 5 min of stimulation, we observed a transient increase in endothelial hydraulic conductivity (Lp) that could be related to the disruption of the tight junctions (TJ) between cells, as suggested by zonula occludens-1 (ZO-1) protein staining. This 'electro-permeation' occurred in the absence of cell death or single cell electroporation, as indicated by propidium iodide staining and cytosolic calcein uptake. Our in vitro results, using uniform fields and BAEC monolayers, thus suggest that electro-permeation of the BBB may occur at electric field intensities below those inducing electroporation and within intensities generated near DBS electrodes. Further studies are necessary to address potential BBB disruption during clinical studies, with safety and efficacy implications.

  15. 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.

  16. UVA Irradiation of Dysplastic Keratinocytes: Oxidative Damage versus Antioxidant Defense

    PubMed Central

    Nechifor, Marina T.; Niculiţe, Cristina M.; Urs, Andreea O.; Regalia, Teodor; Mocanu, Mihaela; Popescu, Alexandra; Manda, Gina; Dinu, Diana; Leabu, Mircea

    2012-01-01

    UVA affects epidermal cell physiology in a complex manner, but the harmful effects have been studied mainly in terms of DNA damage, mutagenesis and carcinogenesis. We investigated UVA effects on membrane integrity and antioxidant defense of dysplastic keratinocytes after one and two hours of irradiation, both immediately after exposure, and 24 h post-irradiation. To determine the UVA oxidative stress on cell membrane, lipid peroxidation was correlated with changes in fatty acid levels. Membrane permeability and integrity were assessed by propidium iodide staining and lactate dehydrogenase release. The effects on keratinocyte antioxidant protection were investigated in terms of catalase activity and expression. Lipid peroxidation increased in an exposure time-dependent manner. UVA exposure decreased the level of polyunsaturated fatty acids, which gradually returned to its initial value. Lactate dehydrogenase release showed a dramatic loss in membrane integrity after 2 h minimum of exposure. The cell ability to restore membrane permeability was noted at 24 h post-irradiation (for one hour exposure). Catalase activity decreased in an exposure time-dependent manner. UVA-irradiated dysplastic keratinocytes developed mechanisms leading to cell protection and survival, following a non-lethal exposure. The surviving cells gained an increased resistance to apoptosis, suggesting that their pre-malignant status harbors an abnormal ability to control their fate. PMID:23222638

  17. Evans Blue Staining of Cardiomyocytes Induced by Myocardial Contrast Echocardiography in Rats: Evidence for Necrosis Instead of Apoptosis

    PubMed Central

    Miller, Douglas L.; Li, Peng; Dou, Chunyan; Armstrong, William F.; Gordon, David

    2008-01-01

    High Mechanical Index (MI) echocardiography with contrast agent has been shown to induce Evans blue staining of cardiomyocytes, seen one day after exposure, in addition to contraction band necrosis, seen immediately after exposure. This research examined the roles of necrosis vs. apoptosis in these bioeffects. Myocardial contrast echocardiography at high MI with 1:4 ECG triggering was performed in anesthetized rats at 1.5 MHz. Histologically observable cell injury was accumulated by infusing a high dose of 50 μl/kg Definity® via tail vein for 5 min at the start of 10 min of scanning. Evans blue dye or propidium iodide was injected as an indicator of cardiomyocyte plasma membrane integrity. Histological sections were stained using the TUNEL method for labeling nuclei with DNA degradation (e.g. apoptosis). Evans blue fluorescent cells were counted on frozen sections or on hematoxylin-stained and TUNEL labeled paraffin sections. In addition, transmission electron microscopy was used to assess potential apoptotic nuclei. Hypercontraction and propidium iodide staining were observed immediately after imaging-exposure. Although TUNEL positive cells were evident after 4 h, these also had indications of contraction band necrosis and features of apoptosis were not confirmed by electron microscopy. Inflammatory cell infiltration was evident after 24 h. A second, more subtle injury was recognized by Evans blue staining, with minimal inflammatory cell infiltration at the morphologically intact stained cells after 24 h. Apoptosis was not detected by the TUNEL method in the cardiomyocytes stained with Evans blue at 24 h. However, Evans blue stained cell numbers declined after 48 h, with continued inflammatory cell infiltration. The initial insult for Evans blue stained cardiomyocytes apparently induced partial permeability of the plasma membrane, which led to gradual degeneration (but not apoptosis) and necrosis after 24–48 h. PMID:17689176

  18. 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

  19. 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.

  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. 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

  2. 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

  3. 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

  4. 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.

  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. 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.

  7. 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

  8. Assessment of metal extract toxicity on human lymphocytes cultured in vitro.

    PubMed

    Granchi, D; Ciapetti, G; Savarino, L; Cavedagna, D; Donati, M E; Pizzoferrato, A

    1996-06-01

    In this study the toxic effects of chromium, nickel, and cobalt extracts on in vitro cultured lymphocytes were evaluated. Graphite furnace atomic absorption spectrometry was used to measure the ion concentration. After serial dilution of the extracts, the viability of lymphocytes at 24, 48, and 72 h was estimated by flow cytometry, including propidium iodide staining and light scatter property assessment, and by MTT reduction test. The results of the investigation allowed us to conclude that 1) standardization of the procedure for preparing extracts is fundamental to obtaining repeatability of results; 2) the toxicity of an extract cannot be evaluated with a single viability assay; a combination of functional and structural tests is required; 3) when methods based on enzymatic reactions are performed, e.g. MTT test, it is advisable to replace the extract containing metal ions with fresh medium in order to avoid any interference with viability testing; 4) the amount of Co and Ni in the extract is similar, but the Cr release is very poor; 5) the lower toxicity of Cr extract probably is due to the lower ion concentration; 6) the assessment of 50% cytotoxic concentration (TC50) allows quantification of materials toxicity and comparison of various metals; and 7) the determination of a noncytotoxic concentration, i.e., a concentration lower than TC10, is required for subsequent investigation of cell functions because such studies can be carried out only on viable cell population. PMID:8731206

  9. Multifunctional immunological monitoring of HIV positive patients: a novel staging system.

    PubMed

    del Llano, A M; Zorrilla, C; Lavergne, J A

    1993-12-01

    A tri-functional in vitro evaluation has been utilized to analyze peripheral blood mononuclear cells (BMNC) from HIV-infected patients, which allows for the classification of these individuals into convenient stages, according to the number of in vitro parameters affected. The classifying functional parameters are: the mitochondrial metabolic activity of freshly isolated BMNC, measured by an MTT reduction assay, the detection of apoptosis in 72 hour cultures of these cells assessed by propidium iodide staining and dual parametric flow cytometric analysis, and their proliferative response to pokeweed mitogen. Our results indicate that HIV-infected patients at different stages of their clinical disease, can present dysfunctions in one, two or three of the above-mentioned parameters. Based on these results, patients can be classified into four newly-described stages which are Stage 0, including uninfected controls and all patients with unaffected parameters, and Stages 1, 2 and 3, including patients having one, two or all three parameters affected, respectively. This type of immunological evaluation and classification of HIV-infected patients has the potential of becoming a predictive tool in the longitudinal follow-up of their HIV infection. PMID:8140207

  10. Comparison of vitrification and conventional freezing for cryopreservation of caprine embryos.

    PubMed

    Araújo-Lemos, Paula F B; Freitas Neto, Leopoldo M; Moura, Marcelo T; Melo, Janaína V; Lima, Paulo F; Oliveira, Marcos A L

    2015-08-01

    The experiment aimed to compare conventional freezing and different vitrification protocols for cryopreservation of caprine embryos at morphological, ultrastructural, and functional levels. Caprine embryos produced in vivo were allocated randomly to three groups: (1) conventional freezing with ethylene glycol (EG); (2) dimethyl sulfoxide + EG (DMSO/EG) vitrification; and (3) dimethylformamide + EG (DMF/EG) vitrification. All groups were scored for cell viability (propidium iodide staining and ultrastructural levels) and re-expansion rate after thawing or warming. Embryos subjected to DMSO/EG vitrification showed higher cell viability (73.33%), compared with DMF/EG vitrification and conventional freezing group embryos (40.00 and 66.66%, respectively). The ultrastructural study revealed that vitrified embryos had greater preservation of cellular structure than embryos from conventional freezing with EG. DMSO/EG vitrification resulted in higher rates of re-expansion in vitro (47.36%) than DMF/EG vitrification (31.58%), and conventional freezing (25.00%). In conclusion, caprine embryos produced in vivo are better cryopreserved after vitrification than conventional freezing, therefore we conclude that DMSO/EG vitrification is the most effective protocol for cryopreservation. PMID:24964134

  11. 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

  12. Xenon preconditioning reduces brain damage from neonatal asphyxia in rats.

    PubMed

    Ma, Daqing; Hossain, Mahmuda; Pettet, Garry K J; Luo, Yan; Lim, Ta; Akimov, Stanislav; Sanders, Robert D; Franks, Nicholas P; Maze, Mervyn

    2006-02-01

    Xenon attenuates on-going neuronal injury in both in vitro and in vivo models of hypoxic-ischaemic injury when administered during and after the insult. In the present study, we sought to investigate whether the neuroprotective efficacy of xenon can be observed when administered before an insult, referred to as 'preconditioning'. In a neuronal-glial cell coculture, preexposure to xenon for 2 h caused a concentration-dependent reduction of lactate dehydrogenase release from cells deprived of oxygen and glucose 24 h later; xenon's preconditioning effect was abolished by cycloheximide, a protein synthesis inhibitor. Preconditioning with xenon decreased propidium iodide staining in a hippocampal slice culture model subjected to oxygen and glucose deprivation. In an in vivo model of neonatal asphyxia involving hypoxic-ischaemic injury to 7-day-old rats, preconditioning with xenon reduced infarction size when assessed 7 days after injury. Furthermore, a sustained improvement in neurologic function was also evident 30 days after injury. Phosphorylated cAMP (cyclic adenosine 3',5'-monophosphate)-response element binding protein (pCREB) was increased by xenon exposure. Also, the prosurvival proteins Bcl-2 and brain-derived neurotrophic factor were upregulated by xenon treatment. These studies provide evidence for xenon's preconditioning effect, which might be caused by a pCREB-regulated synthesis of proteins that promote survival against neuronal injury. PMID:16034370

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

    TOXLINE Toxicology Bibliographic Information

    Nutku E; Zhuang Q; Soussi-Gounni A; Aris F; Mazer BD; 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.

  14. 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.

  15. 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

  16. Maintenance of Membrane Integrity and Permeability Depends on a Patched-Related Protein in Caenorhabditis elegans.

    PubMed

    Choi, Myung-Kyu; Son, Sangwon; Hong, Mingi; Choi, Min Sung; Kwon, Jae Young; Lee, Junho

    2016-04-01

    Membrane integrity is critical for cell survival, defects of which cause pathological symptoms such as metabolic diseases. In this study, we used ethanol sensitivity of the nematodeCaenorhabditis elegansto identify genetic factors involved in membrane integrity. InC. elegans, acute exposure to a high concentration (7% v/v) of ethanol changes membrane permeability, as measured by propidium iodide staining, and causes paralysis. We used the timing of complete paralysis as an indicator for alteration of membrane integrity in our genetic screen, and identifiedptr-6as a gene that confers ethanol resistance when mutated.PTR-6is a patched-related protein and contains a sterol sensing domain. Inhibition of two PTR-encoding genes,ptr-15andptr-23, andmboa-1, encoding an Acyl Co-A: cholesterol acyltransferase homolog, restored ethanol sensitivity of theptr-6mutant, suggesting that theseptrgenes andmboa-1are involved in the maintenance of membrane integrity and permeability. Our results suggest thatC. eleganscan be used as a model system to identify factors involved in metabolic diseases and to screen for therapeutic drugs. PMID:26857627

  17. 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

  18. Rapid quantification of pathogenic fungi by Cellometer image-based cytometry.

    PubMed

    Berkes, Charlotte A; Chan, Leo Li-Ying; Wilkinson, Alisha; Paradis, Benjamin

    2012-12-01

    The objective of this study was to develop an image-based cytometric methodology for the quantification of viable pathogenic yeasts, which can offer increased sensitivity and efficiency when compared to the traditional colony forming unit (CFU) assay. Live/dead yeast quantification by flow cytometry has been previously demonstrated, however, adoption of flow cytometric detection of pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome. Here, we quantitatively assess the growth of H. capsulatum yeasts within RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with Cellometer image-based cytometry; this method faithfully recapitulates growth trends as measured by traditional CFU enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of bone marrow-derived macrophages with a GFP-expressing strain of C. albicans. To demonstrate that image-based cytometry can be used as a tool to assess the susceptibility of fungi to antifungal drugs, we perform dose response experiments with the antifungal drugs amphotericin B and itraconazole and show that image-based cytometry allows rapid assessment of the kinetics of cytotoxicity induced by these antifungals. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens, either alone or in association with host cells. PMID:22985717

  19. 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

  20. Orphenadrine prevents 3-nitropropionic acid-induced neurotoxicity in vitro and in vivo

    PubMed Central

    Pubill, David; Verdaguer, Ester; Canudas, Anna Ma; Sureda, Francesc Xavier; Escubedo, Elena; Camarasa, Jordi; Palls, Merce; Camins, Antoni

    2001-01-01

    Previous studies indicate that 3-nitropropionic acid (3-NPA) neurotoxicity involves the excitotoxic activation of N-methyl-D-aspartate (NMDA) receptors. Thus, we examined the effect of orphenadrine (an anticholinergic drug with NMDA receptor antagonist properties) on 3-NPA neurotoxicity in both cultured rat cerebellar granule cells (CGCs) and in rats. Orphenadrine protected CGCs from 3-NPA-induced mortality, as assessed by both the neutral red viability assay and laser scanning cytometry, using propidium iodide staining. For rats, two indirect markers of neuronal damage were used: the binding of [3H]-PK 11195 to the peripheral-type benzodiazepine receptor (PBR), a microglial marker, and expression of the 27?kD heat-shock protein (HSP27), a marker of activated astroglia. Systemic administration of 3-NPA (30?mg?kg?1 per day for 3 days) induced a 170% increase in [3H]-PK 11195 binding, and expression of HSP27. Both the increase in [3H]-PK 11195 and HSP 27 expression were prevented by previous administration of 30?mg?kg?1 per day of orphenadrine for 3 days. Lower doses (10 and 20?mg?kg?1) had no protective effect. Orphenadrine also reduced 3-NPA-induced mortality in a dose-dependent manner. We propose that orphenadrine or orphenadrine-like drugs could be used to treat neurodegenerative disorders mediated by overactivation of NMDA receptors. PMID:11159722

  1. Neuroprotection from tissue inhibitor of metalloproteinase-1 and its nanoparticles.

    PubMed

    Chaturvedi, Mayank; Figiel, Izabela; Sreedhar, Bojja; Kaczmarek, Leszek

    2012-12-01

    Matrix metalloproteinases (MMPs) are family of zinc dependent endopeptidases, which cleave extracellular matrix proteins, and play an important role in tissue remodelling in physiological and pathological processes. There is enhanced expression of MMPs, in particular MMP-9, during numerous pathological conditions, including epilepsy and ischemic stroke. Therefore, inhibition of MMP-9 is considered as a potential therapeutic target. Tissue Inhibitor of Matrix Metalloproteinase-1 (TIMP-1) is a 28kDa endogenous inhibitor of MMP-9. In this study we examined recombinant mouse TIMP-1 for its in-vitro neuroprotective effects, against Kainic Acid (KA) induced excitotoxicity in organotypic hippocampal slice culture (OHC) model. We also studied, sustained release effects of TIMP-1 in OHC by using poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs). TIMP-1 and TIMP-1 PLGA NPs were added to the slice cultures at different time points, i.e., 30min before treatment with KA and 6h after KA treatment. Propidium iodide staining was used to reveal cell toxicity in the cultures. In addition, neurotoxicity was assessed using standard lactate dehydrogenase (LDH) release assay. Gelatinolytic activity in conditioned cultured medium of OHC was accessed by a fluorescent substrate assay. Briefly, our result show that TIMP-1 provided significant level of neuroprotection, especially when given before 30min of KA and released from the NPs. Since gelatinolytic activity assay showed a decrease in MMP-9 activity, it can be suggested that this neuroprotection might be mediated by the gelatinase inhibition. PMID:22892277

  2. 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

  3. Silver ions disrupt K+ homeostasis and cellular integrity in intact barley (Hordeum vulgare L.) roots

    PubMed Central

    Coskun, Devrim; Britto, Dev T.; Jean, Yuel-Kai; Schulze, Lasse M.; Becker, Alexander; Kronzucker, Herbert J.

    2012-01-01

    The heavy metals silver, gold, and mercury can strongly inhibit aquaporin-mediated water flow across plant cell membranes, but critical examinations of their side effects are rare. Here, the short-lived radiotracer 42K is used to demonstrate that these metals, especially silver, profoundly change potassium homeostasis in roots of intact barley (Hordeum vulgare L.) plants, by altering unidirectional K+ fluxes. Doses as low as 5??M AgNO3 rapidly reduced K+ influx to 5% that of controls, and brought about pronounced and immediate increases in K+ efflux, while higher doses of Au3+ and Hg2+ were required to produce similar responses. Reduced influx and enhanced efflux of K+ resulted in a net loss of >40% of root tissue K+ during a 15?min application of 500??M AgNO3, comprising the entire cytosolic potassium pool and about a third of the vacuolar pool. Silver also brought about major losses of UV-absorbing compounds, total electrolytes, and NH4+. Co-application, with silver, of the channel blockers Cs+, TEA+, or Ca2+, did not affect the enhanced efflux, ruling out the involvement of outwardly rectifying ion channels. Taken together with an examination of propidium iodide staining under confocal microscopy, the results indicate that silver ions affect K+ homeostasis by directly inhibiting K+ influx at lower concentrations, and indirectly inhibiting K+ influx and enhancing K+ efflux, via membrane destruction, at higher concentrations. Ni2+, Cd2+, and Pb2+, three heavy metals not generally known to affect aquaporins, did not enhance K+ efflux or cause propidium iodide incorporation. The study reveals strong and previously unknown effects of major aquaporin inhibitors and recommends caution in their application. PMID:21948852

  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. Cytotoxic Effects of Loperamide Hydrochloride on Canine Cancer Cells

    PubMed Central

    REGAN, Rebecca Cohen; GOGAL, Robert Michael; BARBER, James Perry; TUCKFIELD, Richard Cary; HOWERTH, Elizabeth Wynne; LAWRENCE, Jessica Ann

    2014-01-01

    Loperamide is a peripheral opiate agonist that can cause apoptosis and G2/M arrest in human cancer cell lines and may sensitize cells to chemotherapy. The objectives of this study were to investigate the effects of loperamide on viability, apoptosis and cell cycle kinetics in canine cancer cells and to establish whether the drug sensitizes cells to doxorubicin. Cell viability was assessed using Alamar Blue. Cell death and cell cycle were studied using flow cytometry with 7-Aminoactinomycin-D (7-AAD) and propidium iodide (PI), respectively. Loperamide decreased cell viability in a dose-dependent fashion and was most effective against canine osteosarcoma cells. In all cell lines, it induced a dose and time dependent apoptosis and resulted in accumulation in G0/G1. When co-incubated with doxorubicin, loperamide induced a synergistic cell kill in canine carcinoma cells. Investigation is warranted into the role of loperamide in the treatment of canine cancer. PMID:25649936

  7. 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

  8. Cigarette smoke-induced necroptosis and DAMP release trigger neutrophilic airway inflammation in mice.

    PubMed

    Pouwels, Simon D; Zijlstra, G Jan; van der Toorn, Marco; Hesse, Laura; Gras, Renee; Ten Hacken, Nick H T; Krysko, Dmitri V; Vandenabeele, Peter; de Vries, Maaike; van Oosterhout, Antoon J M; Heijink, Irene H; Nawijn, Martijn C

    2016-02-15

    Recent data indicate a role for airway epithelial necroptosis, a regulated form of necrosis, and the associated release of damage-associated molecular patterns (DAMPs) in the development of chronic obstructive pulmonary disease (COPD). DAMPs can activate pattern recognition receptors (PRRs), triggering innate immune responses. We hypothesized that cigarette smoke (CS)-induced epithelial necroptosis and DAMP release initiate airway inflammation in COPD. Human bronchial epithelial BEAS-2B cells were exposed to cigarette smoke extract (CSE), and necrotic cell death (membrane integrity by propidium iodide staining) and DAMP release (i.e., double-stranded DNA, high-mobility group box 1, heat shock protein 70, mitochondrial DNA, ATP) were analyzed. Subsequently, BEAS-2B cells were exposed to DAMP-containing supernatant of CS-induced necrotic cells, and the release of proinflammatory mediators [C-X-C motif ligand 8 (CXCL-8), IL-6] was evaluated. Furthermore, mice were exposed to CS in the presence and absence of the necroptosis inhibitor necrostatin-1, and levels of DAMPs and inflammatory cell numbers were determined in bronchoalveolar lavage fluid. CSE induced a significant increase in the percentage of necrotic cells and DAMP release in BEAS-2B cells. Stimulation of BEAS-2B cells with supernatant of CS-induced necrotic cells induced a significant increase in the release of CXCL8 and IL-6, in a myeloid differentiation primary response gene 88-dependent fashion. In mice, exposure of CS increased the levels of DAMPs and numbers of neutrophils in bronchoalveolar lavage fluid, which was statistically reduced upon treatment with necrostatin-1. Together, we showed that CS exposure induces necrosis of bronchial epithelial cells and subsequent DAMP release in vitro, inducing the production of proinflammatory cytokines. In vivo, CS exposure induces neutrophilic airway inflammation that is sensitive to necroptosis inhibition. PMID:26719146

  9. Inhibitory effects of cocoa tea (Camellia ptilophylla) in human hepatocellular carcinoma HepG2 in vitro and in vivo through apoptosis.

    PubMed

    Yang, Xiao-rong; Wang, Yuan-yuan; La, Kai-Kai; Peng, Li; Song, Xiao-hong; Shi, Xiang-gang; Zhu, Xiao-feng; Leung, Ping-chung; Ko, Chun-hay; Ye, Chuang-xing

    2012-09-01

    Cocoa tea (Camellia ptilophylla), a naturally decaffeinated tea commonly consumed as a healthy beverage in southern China, has been recently found to be a potential candidate for the treatment of different diseases, including obesity and cancers. The present study aimed to evaluate the anti-liver cancer activities of green cocoa tea infusion (GCTI) in vitro and in vivo using human hepatocarcinoma cell line HepG2 cells and nude mice xenograft model. The apoptotic activities of GCTI were assessed using flow cytometry, Western blotting and immunohistochemical analysis. Our results showed that GCTI significantly inhibited the proliferation of HepG2 cells in a dose-dependent manner (IC₅₀ values=292 μg/ml at 72 h). GCTI induced HepG2 cells to undergo apoptosis, which was demonstrated by cell cycle analysis and annexin-V and propidium iodide staining. The caspase cascade was activated as shown by significant proteolytic cleavage of caspase-3 and PARP in GCTI-treated cells in a dose- and time-dependent manner. In addition, GCTI increased the expression of cell cycle inhibitory proteins (p21, p27 and p53) and the Bax-to-Bcl-2 ratio to induce apoptosis. The antiproliferative effect of GCTI was confirmed in HepG2 xenograft nude mice. The tumor growth was effectively inhibited by GCTI in a dose-dependent manner as indicated by the decrease in tumor volume and tumor weight after 4 weeks of treatment. Administration of GCTI increased terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and caspase-3-positive cells in the tumor section. In conclusion, these results revealed that GCTI may be a potential and promising agent of natural resource to treat liver cancer. PMID:22018604

  10. 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.

  11. Pravastatin induces cell cycle arrest and decreased production of VEGF and bFGF in multiple myeloma cell line.

    PubMed

    Trojan, P J J; Bohatch-Junior, M S; Otuki, M F; Souza-Fonseca-Guimarães, F; Svidnicki, P V; Nogaroto, V; Fernandes, D; Krum, E A; Favero, G M

    2016-02-01

    Multiple myeloma (MM) is a B cell bone marrow neoplasia characterized by inflammation with an intense secretion of growth factors that promote tumor growth, cell survival, migration and invasion. The aim of this study was to evaluate the effects of pravastatin, a drug used to reduce cholesterol, in a MM cell line.Cell cycle and viability were determinate by Trypan Blue and Propidium Iodide. IL6, VEGF, bFGF and TGFβ were quantified by ELISA and qRT-PCR including here de HMG CoA reductase. It was observed reduction of cell viability, increase of cells in G0/G1 phase of the cell cycle and reducing the factors VEGF and bFGF without influence on 3-Methyl-Glutaryl Coenzyme A reductase expression.The results demonstrated that pravastatin induces cell cycle arrest in G0/G1 and decreased production of growth factors in Multiple Myeloma cell line. PMID:26909624

  12. 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 compounds exert a considerable anticancer effect. Therefore, these compounds may serve as protectors of normal cells during chemotherapy without significantly diminishing the effect of the applied chemotherapeutic. PMID:25137576

  13. 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

  14. 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

  15. 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

  16. Pharmacological Improvement and Preclinical Evaluation of Methotrexate-Loaded Lipid-Core Nanocapsules in a Glioblastoma Model.

    PubMed

    Figueiró, Fabrício; de Oliveira, Catiúscia P; Rockenbach, Liliana; Mendes, Franciane B; Bergamin, Letícia S; Jandrey, Elisa Helena F; Edelweiss, Maria Isabel; Guterres, Silvia S; Pohlmann, Adriana R; Battastini, Ana Maria O

    2015-10-01

    Glioblastoma multiforme is a devastating cerebral tumor with an exceedingly poor prognosis. Methotrexate (MTX) is a folic acid analogue that inhibits DNA synthesis by binding to dihydrofolate reductase. Biodegradable nanoparticles are emerging as a promising system for drug delivery to specific tissues. The aims of the current study were pharmacological improvement and preclinical evaluation of MTX-loaded lipid-core nanocapsules (MTX-LNCs) in a glioblastoma model. Cell viability was assessed using the MTT assay, and the cell cycle was characterized by flow cytometry analysis of propidium iodide staining. Apoptosis was measured using an AnnexinV kit and by examining active caspase-3 immunocontent. In vivo glioma implantation was performed in rats, followed by measurement of the tumor size and tumoral apoptosis, BCL-2 immunohistochemistry and analyses of toxicological parameters. MTX-LNCs with increased encapsulation efficiency were successfully prepared. Our in vitro results showed a decrease in glioma cell viability after MTX-LNC treatment that was preceded by cell cycle arrest, leading the cells to undergo apoptotic death, as indicated by AnnexinV staining and increased active caspase-3 protein levels. In the in vivo glioma model, we observed a decrease in the tumor size and an increase in apoptosis in the tumor microenvironment (based on the AnnexinV assay and BCL-2 measurement). MTX-LNC treatment decreased the leukocyte number but altered neither toxicological tissue marker expression nor metabolic parameters. The present results reveal that MTX-LNCs represented an efficient formulation in a preclinical model of glioma and are a potential candidate for clinical trials. PMID:26502643

  17. 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.

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

    PubMed

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

    2015-10-21

    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. PMID:26406535

  19. 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

  20. 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.

  1. Flow Cytometric Characteristics of Sperm Cells Isolated from Pollen of Zea mays L. 1

    PubMed Central

    Zhang, Guichang; Campenot, Mary K.; McGann, Locksley E.; Cass, David D.

    1992-01-01

    Sperm cells have been isolated from pollen of maize (Zea mays L.) and purified with Percoll density centrifugation. Their flow cytometric characteristics were determined on a FACScan flow cytometer with the fluorescent dyes, fluorescein diacetate and propidium iodide. Freshly isolated sperm cells appeared as a dot cluster on the forward scatter and side scatter dot plot. This dot cluster contained 85 to 95% of the 10 thousand counts collected. More than 98% of cells from the cluster were fluorescein diacetate positive, with no propidium iodide positivity, indicating high cell viability. After 5 hours in 15% (w/v) sucrose at room temperature (23°C), scattering properties, cell number, and percentage of fluorescein diacetate-positive cells remained the same. In contrast, Brewbaker and Kwack salts in 15% sucrose resulted in the emergence of a new cell population, as well as a decrease in cell number at 5 hours. Further investigations with individual components of the Brewbaker and Kwack salts showed that calcium was mainly responsible for the deleterious effects. These results demonstrate the utility of flow cytometry as a tool to determine viability and to monitor morphological changes of plant sperm cells and to challenge current views on the ability of Brewbaker and Kwack salts to maintain viability of isolated sperm cells. ImagesFigure 1 PMID:16668883

  2. 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

  3. 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

  4. 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

  5. Degradation of Giardia lamblia cysts in mixed human and swine wastes.

    PubMed Central

    Deng, M Y; Cliver, D O

    1992-01-01

    This study was conducted to determine the persistence of Giardia lamblia cysts in mixed septic tank effluent and swine manure slurry and to correlate fluorescein diacetate-propidium iodide staining of G. lamblia cysts with their morphology under low-voltage scanning electron microscopy. Under field conditions, G. lamblia cysts were degraded more rapidly in the mixed waste than in the control Dulbecco's phosphate-buffered saline (PBS). For total and viable cysts, the mixed waste had D values (time for a 90% reduction in number of cysts) of 18.3 and 15.5 days, and the Dulbecco's PBS control had D values of 41.6 and 26.8 days. The rates of cyst degradation in septic tank effluent and in Dulbecco's PBS were similar. Increasing the proportion of swine manure slurry in the mixed waste favored degradation of the parasite. These results indicate that the mixed waste treatment was the predominant factor affecting the cyst persistence and that it was swine manure slurry that played the role of degrading the parasite. Visualization of viable and nonviable Giardia cysts with low-voltage scanning electron microscopy revealed an excellent correlation between the viability of the cysts determined by fluorescein diacetate-propidium iodide staining and their electron microscopic morphology. Images PMID:1381171

  6. Inter-laboratory comparison of the in vivo comet assay including three image analysis systems.

    PubMed

    Plappert-Helbig, Ulla; Guérard, Melanie

    2015-12-01

    To compare the extent of potential inter-laboratory variability and the influence of different comet image analysis systems, in vivo comet experiments were conducted using the genotoxicants ethyl methanesulfonate and methyl methanesulfonate. Tissue samples from the same animals were processed and analyzed-including independent slide evaluation by image analysis-in two laboratories with extensive experience in performing the comet assay. The analysis revealed low inter-laboratory experimental variability. Neither the use of different image analysis systems, nor the staining procedure of DNA (propidium iodide vs. SYBR® Gold), considerably impacted the results or sensitivity of the assay. In addition, relatively high stability of the staining intensity of propidium iodide-stained slides was found in slides that were refrigerated for over 3 months. In conclusion, following a thoroughly defined protocol and standardized routine procedures ensures that the comet assay is robust and generates comparable results between different laboratories. PMID:26248301

  7. 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.

  8. Thymoquinone Inhibits Tumor Growth and Induces Apoptosis in a Breast Cancer Xenograft Mouse Model: The Role of p38 MAPK and ROS

    PubMed Central

    Woo, Chern Chiuh; Hsu, Annie; Kumar, Alan Prem; Sethi, Gautam; Tan, Kwong Huat Benny

    2013-01-01

    Due to narrow therapeutic window of cancer therapeutic agents and the development of resistance against these agents, there is a need to discover novel agents to treat breast cancer. The antitumor activities of thymoquinone (TQ), a compound isolated from Nigella sativa oil, were investigated in breast carcinoma in vitro and in vivo. Cell responses after TQ treatment were assessed by using different assays including MTT assay, annexin V-propidium iodide staining, Mitosox staining and Western blot. The antitumor effect was studied by breast tumor xenograft mouse model, and the tumor tissues were examined by histology and immunohistochemistry. The level of anti-oxidant enzymes/molecules in mouse liver tissues was measured by commercial kits. Here, we show that TQ induced p38 phosphorylation and ROS production in breast cancer cells. These inductions were found to be responsible for TQs anti-proliferative and pro-apoptotic effects. Moreover, TQ-induced ROS production regulated p38 phosphorylation but not vice versa. TQ treatment was found to suppress the tumor growth and this effect was further enhanced by combination with doxorubicin. TQ also inhibited the protein expression of anti-apoptotic genes, such as XIAP, survivin, Bcl-xL and Bcl-2, in breast cancer cells and breast tumor xenograft. Reduced Ki67 and increased TUNEL staining were observed in TQ-treated tumors. TQ was also found to increase the level of catalase, superoxide dismutase and glutathione in mouse liver tissues. Overall, our results demonstrated that the anti-proliferative and pro-apoptotic effects of TQ in breast cancer are mediated through p38 phosphorylation via ROS generation. PMID:24098377

  9. 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

  10. 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 4.17μM against A-549, HeLa, Hep G2 and MCF-7 respectively, while for compound 13q, IC50 were 15.47, 5.30, 6.15 and 13.39μM 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±0.0μM respectively, while compound 10 was the most active with IC50 of 9.0±2.8μM, 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

  11. Immunogenic cell death by oncolytic herpes simplex virus type 1 in squamous cell carcinoma cells.

    PubMed

    Takasu, A; Masui, A; Hamada, M; Imai, T; Iwai, S; Yura, Y

    2016-04-01

    Molecules essential for the induction of immunogenic cell death (ICD) are called damage-associated molecular patterns (DAMPs). The effects of oncolytic herpes simplex virus type 1 (HSV-1) on the production of DAMPs were examined in squamous cell carcinoma (SCC) cells. The cytopathic effects of HSV-1 RH2 were observed in mouse SCCVII cells infected at a high multiplicity of infection (MOI), and the amounts of viable cells were decreased. After being infected with RH2, ATP and high mobility group box 1 (HMGB1) were released extracellulary, while calreticulin (CRT) translocated to the cell membrane. A flow-cytometric analysis revealed an increase in the number of annexin-V and propidium iodide (PI)-stained cells; and the amount of cleaved poly (ADP-ribose) polymerase (PARP) was increased. The killing effect of RH2 was reduced by pan-caspase inhibitor z-VAD-fmk and the caspase-1 inhibitor z-YVAD-fmk, suggesting the involvement of apoptosis and pyroptosis. In C3H mice bearing synergic SCCVII tumors, the growth of tumors injected with the supernatant of RH2-infected cells was less than that of tumors injected with phosphate-buffered saline (PBS). These results indicate that oncolytic HSV-1 RH2 produces DAMPs from SCC cells to induce cell death. This may contribute to the enhancement of tumor immunity by oncolytic HSV-1. PMID:26987291

  12. 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

  13. 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

  14. 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.

  15. 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

  16. A novel anticancer effect of Astragalus saponins: Transcriptional activation of NSAID-activated gene.

    PubMed

    Auyeung, Kathy K W; Cho, Chi-Hin; Ko, Joshua K S

    2009-09-01

    Astragalus membranaceus has been used to ameliorate the side effects of antineoplastic drugs because of its immunomodulating nature. We had recently demonstrated that total Astragalus saponins (AST) possess anticarcinogenic and proapoptotic properties in human colon cancer cells and tumor xenograft. In this study, we identified NSAID-activated gene (NAG-1) as a potential molecular target of AST. The growth-inhibitory and proapoptotic effects of AST were assessed in a panel of human cancer cell lines. Hoechst 33342 nuclear staining, Annexin V-FITC/propidium iodide staining, Western immunoblotting, real-time PCR, luciferase reporter assay and electrophoretic mobility shift assay were conducted to determine the association of NAG-1 and related transcription factors with AST during its regulation of apoptotic activities. Moreover, the combined proapoptotic and NAG-1 promoting activities of AST and/or inhibitors of the PI3K-Akt pathway were also examined. AST caused overexpression of NAG-1, leading to PARP cleavage and apoptosis. The induction of NAG-1 promoter activity by the drug was associated with increased gene expression, in addition to prior increase in Egr-1 expression and DNA binding activity. AST-induced NAG-1 activation was intensified when PI3K inhibitor LY294002 or Akt inhibitor was co-treated and reversed by NAG-1 siRNA transfection. Nevertheless, the extent of NAG-1 induction could not be altered by the ERK inhibitor PD98059. Our results indicate that NAG-1 is a potential molecular target of AST in its antitumorigenic and proapoptotic actions, which would have additive effects when used along with PI3K-Akt inhibitors. The information obtained could facilitate future development of a novel target-specific chemotherapeutic agent with known molecular pathway. PMID:19384947

  17. Cytotoxicity of inorganic mercury in murine T and B lymphoma cell lines: involvement of reactive oxygen species, Ca(2+) homeostasis, and cytokine gene expression.

    PubMed

    Kim, Sang Hyun; Sharma, Raghubir P

    2003-08-01

    Mercury is a highly toxic heavy metal; exposure to mercury in humans and animals causes damage in several organs or systems including the immune system. To characterize the toxicity of mercury in the immune cells, the cytotoxic effects of inorganic mercury were studied in two distinct lymphoma lines, the murine T lymphoma (EL4) and B lymphoma (A20) cells. Mercury concentration-dependently decreased cell viability, membrane integrity, and proliferation in both EL4 and A20 cells. Mercury increased the reactive oxygen species (ROS) production in both EL4 and A20 cells, and pretreatment with antioxidants reversed mercury-induced ROS generation. Pretreatment of cells with antioxidants N-acetylcysteine (NAC) and silymarin decreased mercury-induced lactate dehydrogenase (LDH) release in both types of cells; however, Ca(2+) channel blocker lanthanum (La(2+)) decreased it only in A20 cells. The mode of cytotoxicity was a mixture of both apoptosis and necrosis. Mercury-induced apoptosis and necrosis in the two cell lines were indicated by staining with Hoechst 33258, propidium iodide, and co-staining with annexin V and propidium iodide. Both mercury-induced apoptosis and necrosis were attenuated by antioxidants. Mercury increased gene expression of IL-4 and TNFalpha in EL4 cells; these cytokines were not expressed in A20 cells. Data suggested different pathways of mercury-induced cytotoxicity in T and B lymphoma cells and involvement of ROS, Ca(2+) homeostasis, and inflammatory cytokine gene expression. PMID:12849721

  18. 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

  19. 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

  20. 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.

  1. Antifungal Activity of the Noncytotoxic Human Peptide Hepcidin 20 against Fluconazole-Resistant Candida glabrata in Human Vaginal Fluid

    PubMed Central

    Del Gaudio, Gaetano; Lombardi, Lisa; Maisetta, Giuseppantonio; Esin, Semih; Batoni, Giovanna; Sanguinetti, Maurizio; Senesi, Sonia

    2013-01-01

    Vaginal infections caused by Candida glabrata are difficult to eradicate due to this species' scarce susceptibility to azoles. Previous studies have shown that the human cationic peptide hepcidin 20 (Hep-20) exerts fungicidal activity in sodium phosphate buffer against a panel of C. glabrata clinical isolates with different levels of susceptibility to fluconazole. In addition, the activity of the peptide was potentiated under acidic conditions, suggesting an application in the topical treatment of vaginal infections. To investigate whether the peptide activity could be maintained in biological fluids, in this study the antifungal activity of Hep-20 was evaluated by a killing assay in (i) a vaginal fluid simulant (VFS) and in (ii) human vaginal fluid (HVF) collected from three healthy donors. The results obtained indicated that the activity of the peptide was maintained in VFS and HVF supplemented with EDTA. Interestingly, the fungicidal activity of Hep-20 was enhanced in HVF compared to that observed in VFS, with a minimal fungicidal concentration of 25 μM for all donors. No cytotoxic effect on human cells was exerted by Hep-20 at concentrations ranging from 6.25 to 100 μM, as shown by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide tetrazolium salt (XTT) reduction assay and propidium iodide staining. A piece of indirect evidence of Hep-20 stability was also obtained from coincubation experiments of the peptide with HVF at 37°C for 90 min and for 24 h. Collectively, these results indicate that this peptide should be further studied as a novel therapeutic agent for the topical treatment of vaginal C. glabrata infections. PMID:23796919

  2. Fourier Transform Infrared spectroscopy discloses different types of cell death in flow cytometrically sorted cells.

    PubMed

    Le Roux, K; Prinsloo, L C; Meyer, D

    2015-10-01

    Fourier Transform Infrared (FTIR) spectroscopy is a label free methodology showing promise in characterizing different types of cell death. Cervical adenocarcinoma (HeLa) and African monkey kidney (Vero) cells were treated with a necrosis inducer (methanol), novel apoptotic inducers (diphenylphosphino gold (I) complexes) and positive control, auranofin. Following treatment, cells stained with annexin-V and propidium iodide were sorted using a Fluorescence Activated Cell Sorter (FACS Aria) to obtain populations consisting of either viable, necrotic or apoptotic cells. Transmission Electron Microscopy confirmed successful sorting of all three populations. Four bands were identified which could discriminate between viable and necrotic cells namely 989 cm(-1), 2852 cm(-1), 2875 cm(-1) and 2923 cm(-1). In HeLa cells viable and induced apoptosis could be distinguished by 1294 cm(-1), while four bands were different in Vero cells namely; 1626 cm(-1), 1741 cm(-1), 2852 cm(-1) 2923 cm(-1). Principal Component Analysis showed separation between the different types of cell death and the loadings plots indicated an increase in an additional band at 1623 cm(-1) in dead cells. FTIR spectroscopy can be developed into an invaluable tool for the assessment of specific types of chemically induced cell death with notably different molecular signatures depending on whether the cells are cancerous and mechanism of cell death. PMID:26254093

  3. A cell-based screening assay for Natural Killer cell activity.

    PubMed

    Blom, W Marty; van Nielen, Wim G L; de Groene, Els M; Albers, Ruud

    2009-06-01

    Natural Killer (NK) cells are important in the first response against viruses and tumours. Compounds that modulate human NK cell activity offer interesting prophylactic and therapeutic options, however, a systematic screening tool is lacking. Development of suitable NK cell lines or receptor-based assays is hindered by the highly complicated regulation of the different NK cell subsets by multiple receptors. Here, we describe a cell-based flowcytometric activity assay adapted to identify NK cell modulating compounds. Fresh human peripheral blood mononuclear cells (PBMC) were incubated with NK-sensitive K562 target cells labelled with 5-(6)-carboxyfluorescein succinimidyl ester, followed by DNA-labelling with propidium iodide to identify dead cells. The assay demonstrated a good performance with an average Z'-factor of 0.6 and over 95% of the assays fulfilled the quality criteria, suggesting that it is possible to use a complex system with two different cell types to screen compounds. A large number of (natural) compounds and extracts were tested and normalized to the positive control, Interleukin-2. Promising and less promising compounds were distinguished. Effectiveness of compounds was based on the augmentation of NK cell activity as well as the number of responding subjects. To conclude the assay is robust, reliable and can be used for functional screening of natural compounds modulating NK cell activity. PMID:19293002

  4. 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.

  5. 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.

  6. 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

  7. Two Molecularly Distinct G2/M Checkpoints Are Induced by Ionizing Irradiation

    PubMed Central

    Xu, Bo; Kim, Seong-Tae; Lim, Dae-Sik; Kastan, Michael B.

    2002-01-01

    Cell cycle checkpoints are among the multiple mechanisms that eukaryotic cells possess to maintain genomic integrity and minimize tumorigenesis. Ionizing irradiation (IR) induces measurable arrests in the G1, S, and G2 phases of the mammalian cell cycle, and the ATM (ataxia telangiectasia mutated) protein plays a role in initiating checkpoint pathways in all three of these cell cycle phases. However, cells lacking ATM function exhibit both a defective G2 checkpoint and a prolonged G2 arrest after IR, suggesting the existence of different types of G2 arrest. Two molecularly distinct G2/M checkpoints were identified, and the critical importance of the choice of G2/M checkpoint assay was demonstrated. The first of these G2/M checkpoints occurs early after IR, is very transient, is ATM dependent and dose independent (between 1 and 10 Gy), and represents the failure of cells which had been in G2 at the time of irradiation to progress into mitosis. Cell cycle assays that can distinguish mitotic cells from G2 cells must be used to assess this arrest. In contrast, G2/M accumulation, typically assessed by propidium iodide staining, begins to be measurable only several hours after IR, is ATM independent, is dose dependent, and represents the accumulation of cells that had been in earlier phases of the cell cycle at the time of exposure to radiation. G2/M accumulation after IR is not affected by the early G2/M checkpoint and is enhanced in cells lacking the IR-induced S-phase checkpoint, such as those lacking Nbs1 or Brca1 function, because of a prolonged G2 arrest of cells that had been in S phase at the time of irradiation. Finally, neither the S-phase checkpoint nor the G2 checkpoints appear to affect survival following irradiation. Thus, two different G2 arrest mechanisms are present in mammalian cells, and the type of cell cycle checkpoint assay to be used in experimental investigation must be thoughtfully selected. PMID:11809797

  8. 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

  9. Impact of buffering hypercapnic acidosis on cell wounding in ventilator-injured rat lungs

    PubMed Central

    Caples, Sean M.; Rasmussen, Deborah L.; Lee, Won Y.; Wolfert, Marla Z.; Hubmayr, Rolf D.

    2009-01-01

    We measured the effects of raising perfusate pH on ventilator-induced cell wounding and repair in ex vivo mechanically ventilated hypercapnic rat lungs. Lungs were randomized to one of three perfusate groups: 1) unbuffered hypercapnic acidosis, 2) bicarbonate-buffered hypercapnia, or 3) tris-hydroxymethyl aminomethane (THAM)-buffered hypercapnia. The membrane-impermeant label propidium iodide was added to the perfusate either during or after injurious ventilation providing a means to subsequently identify transiently wounded and permanently wounded cells in optical sections of subpleural alveoli. Normalizing perfusate pH in hypercapnic preparations attenuated ventilator-induced cell injury, particularly in THAM-buffered preparations. This was observed despite greater amounts of edema and impaired lung mechanics compared with other treatment groups. Protective effects of buffering of hypercapnic acidosis on injury and repair were subsequently confirmed in a cell scratch model. We conclude that buffering of hypercapnic acidosis attenuates plasma cell injury induced by mechanical hyperinflation. PMID:18996901

  10. 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

  11. Coordination of Intercellular Ca2+ Signaling in Endothelial Cell Tubes of Mouse Resistance Arteries

    PubMed Central

    Socha, Matthew J.; Domeier, Timothy L.; Behringer, Erik J.; Segal, Steven S.

    2012-01-01

    Objective To test the hypothesis that Ca2+ responses to G-protein coupled receptor (GPCR) activation are coordinated between neighboring endothelial cells of resistance arteries. Methods Endothelial cell tubes were freshly isolated from superior epigastric arteries of C57BL/6 mice. Intercellular coupling was tested using microinjection of propidium iodide. Following loading with fluo-4 dye, intracellular Ca2+ responses to ACh were imaged with confocal microscopy. Results Cell-to-cell transfer of propidium iodide confirmed functional gap junction channels. 1 μM ACh evoked Ca2+ responses [9.8±0.8/min, (F/F0)=3.11±0.2] which pseudo-linescan analysis revealed to be composed of Ca2+ waves and spatially-restricted Ca2+ release events. 100 nM ACh induced Ca2+ responses of lower frequency (4.5±0.7/min) and amplitude (F/F0=1.95±0.11) composed primarily of spatially-restricted events. The interval between Ca2+ waves in Adjacent cells (0.79±0.12 s) was shorter (P<0.05) than between Nonadjacent cells (1.56±0.25 s). Spatially-restricted Ca2+ release events had similar frequencies and latencies between Adjacent and Nonadjacent cells. Inhibiting intracellular Ca2+ release with 2-APB, Xestospongin C or thapsigargin eliminated Ca2+ responses. Conclusions With moderate GPCR stimulation, localized Ca2+ release events predominate among cells. Greater GPCR stimulation evokes coordinated intercellular Ca2+ waves via the endoplasmic reticulum. Calcium signaling during GPCR activation is complex among cells, varying with stimulus intensity and proximity to actively signaling cells. PMID:22860994

  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. Monitoring the progression of cell death and the disassembly of dying cells by flow cytometry.

    PubMed

    Jiang, Lanzhou; Tixeira, Rochelle; Caruso, Sarah; Atkin-Smith, Georgia K; Baxter, Amy A; Paone, Stephanie; Hulett, Mark D; Poon, Ivan K H

    2016-04-01

    The use of annexin A5 (A5) and either propidium iodide or 7-aminoactinomycin D (PI/7-AAD) stains to measure cell death by flow cytometry has been considered the gold standard by most investigators. However, this widely used method often makes the assumption that there are only three types of particles in a sample: viable, apoptotic and necrotic cells. To study the progression of cell death in greater detail, in particular how apoptotic cells undergo fragmentation to generate membrane-bound vesicles known as apoptotic bodies, we established a flow cytometry-based protocol to accurately and rapidly measure the cell death process. This protocol uses a combination of A5 and TO-PRO-3 (a commercially available nucleic acid-binding dye that stains early apoptotic and necrotic cells differentially), and a logical seven-stage analytical approach to distinguish six types of particles in a sample, including apoptotic bodies and cells at three different stages of cell death. The protocol requires 1-5 h for sample preparation (including induction of cell death), 20 min for staining and 5 min for data analysis. PMID:26938116

  14. Freezing behavior of adherent neuron-like cells and morphological change and viability of post-thaw cells.

    PubMed

    Uemura, Makoto; Ishiguro, Hiroshi

    2015-04-01

    Freezing of nerve cells forming a neuronal network has largely been neglected, despite the fact that the cryopreservation of nerve cells benefits the study of cells in the areas of medicine and poison screening. Freezing of nerve cells is also attractive for studying cell morphology because of the characteristic long, thread-like neurites extending from the cell body. In the present study, freezing of neuron-like cells adhering to the substrate (differentiated PC12 cells), in physiological saline, was investigated in order to understand the fundamental freezing and thawing characteristics of nerve cells with neurites. The microscopic freezing behavior of cells under different cooling rates was observed. Next, the post-thaw morphological changes in the cells, including the cytoskeleton, were investigated and post-thaw cell viability was evaluated by dye exclusion using propidium iodide. Two categories of morphological changes, beading and shortening of the neurites, were found and quantified. Also, the morphological changes of neurites due to osmotic stress from sodium chloride were studied to gain a better understanding of causation. The results showed that morphological changes and cell death were promoted with a decrease in end temperature during freezing. PMID:25645578

  15. 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 on leaf surfaces and the implications for biological control of pathogenic and other deleterious microorganisms is discussed. PMID:16151141

  16. Synergistic Interactions between HDAC and Sirtuin Inhibitors in Human Leukemia Cells

    PubMed Central

    Cea, Michele; Soncini, Debora; Fruscione, Floriana; Raffaghello, Lizzia; Garuti, Anna; Emionite, Laura; Moran, Eva; Magnone, Mirko; Zoppoli, Gabriele; Reverberi, Daniele; Caffa, Irene; Salis, Annalisa; Cagnetta, Antonia; Bergamaschi, Micaela; Casciaro, Salvatore; Pierri, Ivana; Damonte, Gianluca; Ansaldi, Filippo; Gobbi, Marco; Pistoia, Vito; Ballestrero, Alberto; Patrone, Franco

    2011-01-01

    Aberrant histone deacetylase (HDAC) activity is frequent in human leukemias. However, while classical, NAD+-independent HDACs are an established therapeutic target, the relevance of NAD+-dependent HDACs (sirtuins) in leukemia treatment remains unclear. Here, we assessed the antileukemic activity of sirtuin inhibitors and of the NAD+-lowering drug FK866, alone and in combination with traditional HDAC inhibitors. Primary leukemia cells, leukemia cell lines, healthy leukocytes and hematopoietic progenitors were treated with sirtuin inhibitors (sirtinol, cambinol, EX527) and with FK866, with or without addition of the HDAC inhibitors valproic acid, sodium butyrate, and vorinostat. Cell death was quantified by propidium iodide cell staining and subsequent flow-cytometry. Apoptosis induction was monitored by cell staining with FITC-Annexin-V/propidium iodide or with TMRE followed by flow-cytometric analysis, and by measuring caspase3/7 activity. Intracellular Bax was detected by flow-cytometry and western blotting. Cellular NAD+ levels were measured by enzymatic cycling assays. Bax was overexpressed by retroviral transduction. Bax and SIRT1 were silenced by RNA-interference. Sirtuin inhibitors and FK866 synergistically enhanced HDAC inhibitor activity in leukemia cells, but not in healthy leukocytes and hematopoietic progenitors. In leukemia cells, HDAC inhibitors were found to induce upregulation of Bax, a pro-apoptotic Bcl2 family-member whose translocation to mitochondria is normally prevented by SIRT1. As a result, leukemia cells become sensitized to sirtuin inhibitor-induced apoptosis. In conclusion, NAD+-independent HDACs and sirtuins cooperate in leukemia cells to avoid apoptosis. Combining sirtuin with HDAC inhibitors results in synergistic antileukemic activity that could be therapeutically exploited. PMID:21818379

  17. 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

  18. Experimental Adaptation of Rotaviruses to Tumor Cell Lines.

    PubMed

    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

  19. 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

  20. 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

  1. 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

  2. Effects of shear stress cultivation on cell membrane disruption and intracellular calcium concentration in sonoporation of endothelial cells

    PubMed Central

    Park, Juyoung; Fan, Zhenzhen; Deng, Cheri X.

    2010-01-01

    Microbubble facilitated ultrasound (US) application can enhance intracellular delivery of drugs and genes in endothelial cells cultured in static condition by transiently disrupting the cell membrane, or sonoporation. However, endothelial cells in vivo that are constantly exposed to blood flow may exhibit different sonoporation characteristics. This study investigates the effects of shear stress cultivation on sonoporation of endothelial cells in terms of membrane disruption and changes in the intracellular calcium concentration ([Ca2+]i). Sonoporation experiments were conducted using murine brain microvascular endothelial (bEnd.3) cells and human umbilical vein endothelial cells (HUVECs) cultured under static or shear stress (5 dyne/cm2 for 5 days) condition in a microchannel environment. The cells were exposed to a short US tone burst (1.25 MHz, 8 μs duration, 0.24 MPa) in the presence of Definity™ microbubbles to facilitate sonoporation. Membrane disruption was assessed by propidium iodide (PI) and changes in [Ca2+]i measured by fura-2AM. Results from this study show that shear stress cultivation significantly reduced the impact of ultrasound-driven microbubbles activities on endothelial cells. Cells cultured under shear stress condition exhibited much lower percentage with membrane disruption and changes in [Ca2+]i compared to statically cultured cells. The maximum increases of PI uptake and [Ca2+]i were also significantly lower in the shear stress cultured cells. In addition, the extent of [Ca2+]i waves in shear cultured HUVECs was reduced compared to the statically cultured cells. PMID:20863503

  3. K562 cells display different vulnerability to H₂O₂ induced oxidative stress in differing cell cycle phases.

    PubMed

    Akcakaya, Handan; Dal, Fulya; Tok, Sabiha; Cinar, Suzan-Adin; Nurten, Rustem

    2015-02-01

    Oxidative stress can be defined as the increase of oxidizing agents like reactive oxygen and nitrogen species, or the imbalance between the antioxidative defense mechanism and oxidants. Cell cycle checkpoint response can be defined as the arrest of the cell cycle functioning after damaging chemical exposure. This temporary arrest may be a period of time given to the cells to repair the DNA damage before entering the cycle again and completing mitosis. In order to determine the effects of oxidative stress on several cell cycle phases, human erytroleukemia cell line (K562) was synchronized with mimosine and genistein, and cell cycle analysis carried out. Synchronized cells were exposed to oxidative stress with hydrogen peroxide (H2O2) at several concentrations and different times. Changes on mitochondria membrane potential (ΔΨm) of K562 cells were analyzed in G1, S, and G2 /M using Rhodamine 123 (Rho 123). To determine apoptosis and necrosis, stressed cells were stained with Annexin V (AnnV) and propidium iodide (PI) for flow cytometry. Changes were observed in the ΔΨm of synchronized and asynchronized cells that were exposed to oxidative stress. Synchronized cells in S phase proved resistant to the effects of oxidative stress and synchronized cells at G2 /M phase were sensitive to the effects of H2O2 -induced oxidative stress at 500 μM and above. PMID:25181960

  4. Amygdalin induces apoptosis in human cervical cancer cell line HeLa cells.

    PubMed

    Chen, Yu; Ma, Jinshu; Wang, Fang; Hu, Jie; Cui, Ai; Wei, Chengguo; Yang, Qing; Li, Fan

    2013-02-01

    Amygdalin, a naturally occurring substance, has been suggested to be efficacious as an anticancer substance. The effect of amygdalin on cervical cancer cells has never been studied. In this study, we found that the viability of human cervical cancer HeLa cell line was significantly inhibited by amygdalin. 4,6-Diamino-2-phenyl indole (DAPI) staining showed that amygdalin-treated HeLa cells developed typical apoptotic changes. The development of apoptosis in the amygdalin-treated HeLa cells were confirmed by double staining of amygdalin-treated HeLa cells with annexin V-FITC and propidium iodide (PI) along with increase in caspase-3 activity in these cells. Further studies indicated that antiapoptotic protein Bcl-2 was downregulated whereas proapoptotic Bax protein was upregulated in the amygdalin-treated HeLa cells implying involvement of the intrinsic pathway of apoptosis. In vivo, amygdalin administration inhibited the growth of HeLa cell xenografts through a mechanism of apoptosis. The results in the present study suggest that amygdalin may offer a new therapeutic option for patients with cervical cancer. PMID:23137229

  5. Single-cell variability in growing Saccharomyces cerevisiae cell populations measured with automated flow cytometry.

    PubMed

    Kacmar, James; Zamamiri, Abdelqader; Carlson, Ross; Abu-Absi, Nicholas R; Srienc, Friedrich

    2004-04-29

    Cell cultures normally are heterogeneous due to factors such as the cell cycle, inhomogeneous cell microenvironments, and genetic differences. However, distributions of cell properties usually are not taken into account in the characterization of a culture when only population averaged values are measured. In this study, the cell size, green fluorescence protein (Gfp) content, and viability after automated staining with propidium iodide (PI) are monitored at the single-cell level in Saccharomyces cerevisiae cultures growing in a batch bioreactor using an automated flow injection flow cytometer system. To demonstrate the wealth of information that can be obtained with this system, three cultures containing three different plasmids are compared. The first plasmid is a centromeric plasmid expressing under the control of a TEF2 promoter the S65T mutant form of Gfp. The other two plasmids are 2 microm plasmids and express the FM2 mutant of Gfp under the control of either the TEF1 or the TEF2 promoter. The automated sampling, cell preparation, and analysis permitted frequent quantification of the culture characteristics. The time course of the data representing not only population average values but also their variability, provides a detailed and reproducible "fingerprint" of the culture dynamics. The data demonstrate that small changes in the genetic make up of the recombinant system can result in large changes in the culture Gfp production and viability. Thus, the developed instrumentation is valuable for rapidly testing promoter strength, plasmid stability, cell viability, and culture variability. PMID:15066762

  6. Morphological changes of post-isolation of caprine pancreatic islet.

    PubMed

    Hani, Homayoun; Allaudin, Zeenathul Nazariah; Tengku Ibrahim, Tengku Azmi; Mohd-Lila, Mohd-Azmi; Sarsaifi, Kazhal; Camalxaman, Siti Nazrina; Othman, Abas Mazni

    2015-02-01

    Pancreatic islet transplantation is commonly used to treat diabetes. Cell isolation and purification methods can affect the structure and function of the isolated islet cells. Thus, the development of cell isolation techniques that preserve the structure and function of pancreatic islet cells is essential for enabling successful transplantation procedures. The impact of purification procedures on cell function can be assessed by performing ultrastructure and in vivo studies. Thus, the aim of this study was to evaluate the effect of caprine islets purification procedure on islet cell ultrastructure and functional integrity prior to and post-isolation/purification. The islets were isolated from caprine pancreas by using an optimized collagenase XI-S concentration, and the cells were subsequently purified using Euro-Ficoll density gradient. In vitro viability of islets was determined by fluorescein diacetate and propidium iodide staining. Static incubation was used to assess functionality and insulin production by islet cells in culture media when exposed to various levels of glucose. Pancreatic tissues were examined by using light microscopy, fluorescence microscopy, scanning, and transmission electron microscopy. In vivo viability and functionality of caprine islets were assessed by evaluating the transplanted islets in diabetic mice. Insulin assay of glucose-stimulated insulin secretion test showed that the insulin levels increased with increasing concentration of glucose. Thus, purified islets stimulated with high glucose concentration (25 mM) secreted higher levels of insulin (0.542 ± 0.346 μg/L) than the insulin levels (0.361 ± 0.219, 0.303 ± 0.234 μg/L) secreted by exposure to low glucose concentrations (1.67 mM). Furthermore, insulin levels of recipient mice were significantly higher (p < 0.001) than those prior to xenotransplantation. In addition, following islets transplantation, there was significant enhancement in blood glucose levels of diabetic recipient mice. Overall, although the purified caprine islets had minor deformations in the plasma membrane and changes in cell integrity of peripheral region, the alterations did not significantly alter the functionality and viability of the purified islets. PMID:25303943

  7. 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 Hsp70 level in IL-1β-stimulated SW1353 cells. Our results suggested that SGTB positively regulate the activation of caspase-3 by negatively regulating the activity of Hsp70 and might promote chondrocyte apoptosis in OA. This study may provide a novel insight into the pathophysiology of OA and a potential therapeutic target for its treatment. PMID:26586481

  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. Caspofungin kills Candida albicans by causing both cellular apoptosis and necrosis.

    PubMed

    Hao, Binghua; Cheng, Shaoji; Clancy, Cornelius J; 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 target this process may augment the benefits of antifungal agents. PMID:23114781

  10. 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

  11. Dihydromyricetin induces cell apoptosis via a p53-related pathway in AGS human gastric cancer cells.

    PubMed

    Ji, F J; Tian, X F; Liu, X W; Fu, L B; Wu, Y Y; Fang, X D; Jin, H Y

    2015-01-01

    The aim of the present study was to determine the anti-proliferative and pro-apoptotic effects of dihydromyricetin (DHM) on the AGS human gastric cancer cells and their underlying mechanisms. The effects of DHM on AGS cells were evaluated by using 3-(4, 5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase, and Annexin V/propidium iodide (PI) double-staining assays. The underlying mechanisms were determined by using quantitative real-time polymerase chain reaction. The results demonstrated that DHM significantly (P < 0.05) inhibited AGS cell proliferation and induced cell cytotoxicity in a dose- and time-dependent manner. Additionally, Annexin V/PI double-staining assay showed that DHM promoted cell apoptosis in both, early and late stages. Furthermore, DHM also regulated the expression of apoptotic genes such as p53 and B-cell lymphoma-2 (bcl-2) in a dose- and time-dependent manner. In conclusion, this is the first report demonstrating the anticancer and pro-apoptosis effects of DHM on AGS human gastric cancer cells. The results strongly suggest that DHM may be a potential therapeutic candidate for the treatment of gastric cancer. PMID:26634523

  12. Evaluation of the photobiomodulation in L929 cell culture.

    PubMed

    Marques, Juliana Mangolin; Pacheco-Soares, Cristina; Da Silva, Newton Soares

    2014-12-01

    Low-level laser therapy has become an important tool for bio-modulation process. It can induce stimulatory or inhibitory effects according to cell behavior at specific irradiation. Our objective was to determine L929 cell line response to irradiation at λ 685 and 830 nm, concentrations of 5 and 10% fetal bovine serum and different energy densities of 0.1, 0.5, 1, 2, 3, 5, 7, 10, 20, and 30 J/cm(2). Thus, cells were plated at 1 × 10(5) cells/mL and irradiated with semiconductor laser As-Ga-Al. Twenty-four hours after irradiation, cells were subjected to MTT, neutral red, crystal violet tests, and cell staining was performed using the kit Alexa Fluor 488 Annexin V-FITC and propidium iodide. Our results showed that low-level laser therapy stimulates effect when the energy density is 5 to 3030 J/cm(2) and inhibits effects on energy density 0.1 to 3 J/cm(2). This inhibitory effect was evidenced by the absence of dead cells labeled, decreased cell density, and by the absorption of neutral red in intact cells. The study also demonstrated that fetal bovine serum, at different concentrations, did not affect response of the cells after irradiation. PMID:25092826

  13. Inhibition of VDAC1 prevents Ca?-mediated oxidative stress and apoptosis induced by 5-aminolevulinic acid mediated sonodynamic therapy in THP-1 macrophages.

    PubMed

    Chen, Haibo; Gao, Weiwei; Yang, Yang; Guo, Shuyuan; Wang, Huan; Wang, Wei; Zhang, Shuisheng; Zhou, Qi; Xu, Haobo; Yao, Jianting; Tian, Zhen; Li, Bicheng; Cao, Wenwu; Zhang, Zhiguo; Tian, Ye

    2014-12-01

    Ultrasound combined with endogenous protoporphyrin IX derived from 5-aminolevulinic acid (ALA-SDT) is known to induce apoptosis in multiple cancer cells and macrophages. Persistent retention of macrophages in the plaque has been implicated in the pathophysiology and progression of atherosclerosis. Here we investigated the effects of inhibition of voltage-dependent anion channel 1 (VDAC1) on ALA-SDT-induced THP-1 macrophages apoptosis. Cells were pre-treated with VDAC1 inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) disodium salt for 1 h or downregulated VDAC1 expression by small interfering RNA and exposed to ultrasound. Cell viability was assessed by MTT assay, and cell apoptosis along with necrosis was evaluated by Hoechst 33342/propidium iodide staining and flow cytometry. Levels of cytochrome c release was assessed by confocal microscope and Western blot. The levels of full length caspases, caspase activation, and VDAC isoforms were analyzed by Western blot. Intracellular reactive oxygen species generation, mitochondrial membrane permeability, and intracellular Ca(2+) [Ca(2+)]i levels were measured with fluorescent probes. We confirmed that the pharmacological inhibition of VDAC1 by DIDS notably prevented ALA-SDT-induced cell apoptosis in THP-1 macrophages. Additionally, DIDS significantly inhibited intracellular ROS generation and apoptotic biochemical changes such as inner mitochondrial membrane permeabilization, loss of mitochondrial membrane potential, cytochrome c release and activation of caspase-3 and caspase-9. Moreover, ALA-SDT elevated the [Ca(2+)]i levels and it was also notably reduced by DIDS. Furthermore, both of intracellular ROS generation and cell apoptosis were predominately inhibited by Ca(2+) chelating reagent BAPTA-AM. Intriguingly, ALA-treatment markedly augmented VDAC1 protein levels exclusively, and the downregulation of VDAC1 expression by specific siRNA also significantly abolished cell apoptosis. Altogether, these results suggest that VDAC1 plays a crucial role in ALA-SDT-induced THP-1 macrophages apoptosis, and targeting VDAC1 is a potential way regulating macrophages apoptosis, a finding that may be relevant to therapeutic strategies against atherosclerosis. PMID:25342393

  14. Bacteroides fragilis induce necrosis on mice peritoneal macrophages: In vitro and in vivo assays

    SciTech Connect

    Vieira, J.M.B.D.; Laboratorio de Biologia de Anaerobios, IMPPG, UFRJ, Rio de Janeiro ; 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.

  15. Overexpression of AQP3 Modifies the Cell Cycle and the Proliferation Rate of Mammalian Cells in Culture

    PubMed Central

    Galn-Cobo, Ana; Ramrez-Lorca, Reposo; Serna, Ana; Echevarra, 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-k?, 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

  16. Dose-related effects of psoralen and ultraviolet light on the cell cycle of murine melanoma cells

    SciTech Connect

    Varga, J.M.; Wiesehahn, G.; Bartholomew, J.C.; Hearst, J.E.

    1982-06-01

    Cloudman (S91) murine melanoma cells were treated with 4'-hydroxymethyltrioxsalen (HMT), a bifunctional psoralen and exposed to long-wavelength (365 nm) ultraviolet light. DNA content of the cells stained with propidium iodide was measured by flow cytometry, and cell cycle phases were delineated from the DNA histograms by using a curve-fitting routine. Researchers found that HMT in combination with long-wavelength (365 nm) ultraviolet irradiation blocked melanoma cells in different phases of the cell cycle, depending on the dose of long-wavelength (365 nm) ultraviolet light and the concentration of HMT. The binding of (/sup 3/H)HMT to DNA was measured parallel with cell cycle analyses. Treatments with HMT at concentrations corresponding to about 1 HMT bound per 10(6) base pairs of DNA led to the accumulation of cells with predominantly G2 DNA content. At higher concentrations (2 to 3 HMT/10(6) base pairs), the cells were blocked in the S and G1 phases. In conclusion, it was shown that extremely sparse substitution of HMT to DNA blocks melanoma cells in the G2 phase or other phases of the cell cycle in a dose-dependent manner.

  17. Bothropoides pauloensis venom effects on isolated perfused kidney and cultured renal tubular epithelial cells.

    PubMed

    Marinho, Aline D; Morais, Isabel C O; Lima, Dânya B; Jorge, Antônio R C; Jorge, Roberta J B; Menezes, Ramon R P P B; Mello, Clarissa P; Pereira, Gustavo J S; Silveira, João A M; Toyama, Marcos H; Orzáez, Mar; Martins, Alice M C; Monteiro, Helena S A

    2015-12-15

    Snake envenomation (Bothrops genus) is common in tropical countries and acute kidney injury is one of the complications observed in Bothrops snakebite with relevant morbidity and mortality. Here, we showed that Bothropoides pauloensis venom (BpV) decreased cell viability (IC50 of 7.5 μg/mL). Flow cytometry with annexin V and propidium iodide showed that cell death occurred predominantly by apoptosis and late apoptosis, through caspases 3 and 7 activation, mitochondrial membrane potential collapse and ROS overproduction. BpV reduced perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate, percentage of sodium, chloride or potassium tubular transportation. These findings demonstrated that BpV cytotoxicity on renal epithelial cells might be responsible for the nephrotoxicity observed in isolated kidney. PMID:26410111

  18. 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 membrane permeation and stability. The assay allows for high-throughput applications and has great potential for rapid monitoring of microbial content in air, liquids or on surfaces. PMID:20565844

  19. 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.

  20. 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

  1. 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

  2. Clostridium perfringens beta-toxin induces necrostatin-inhibitable, calpain-dependent necrosis in primary porcine endothelial cells.

    PubMed

    Autheman, Delphine; Wyder, Marianne; Popoff, Michel; D'Herde, Katharina; Christen, Stephan; Posthaus, Horst

    2013-01-01

    Clostridium perfringens ?-toxin (CPB) is a ?-barrel pore-forming toxin and an essential virulence factor of C. perfringens type C strains, which cause fatal hemorrhagic enteritis in animals and humans. We have previously shown that CPB is bound to endothelial cells within the intestine of affected pigs and humans, and that CPB is highly toxic to primary porcine endothelial cells (pEC) in vitro. The objective of the present study was to investigate the type of cell death induced by CPB in these cells, and to study potential host cell mechanisms involved in this process. CPB rapidly induced lactate dehydrogenase (LDH) release, propidium iodide uptake, ATP depletion, potassium efflux, a marked rise in intracellular calcium [Ca(2+)]i, release of high-mobility group protein B1 (HMGB1), and caused ultrastructural changes characteristic of necrotic cell death. Despite a certain level of caspase-3 activation, no appreciable DNA fragmentation was detected. CPB-induced LDH release and propidium iodide uptake were inhibited by necrostatin-1 and the two dissimilar calpain inhibitors PD150606 and calpeptin. Likewise, inhibition of potassium efflux, chelation of intracellular calcium and treatment of pEC with cyclosporin A also significantly inhibited CPB-induced LDH release. Our results demonstrate that rCPB primarily induces necrotic cell death in pEC, and that necrotic cell death is not merely a passive event caused by toxin-induced membrane disruption, but is propagated by host cell-dependent biochemical pathways activated by the rise in intracellular calcium and inhibitable by necrostatin-1, consistent with the emerging concept of programmed necrosis ("necroptosis"). PMID:23734212

  3. Flow-cytometric analyses of viability biomarkers in pesticide-exposed sperm of three aquatic invertebrates.

    PubMed

    Favret, Karen P; Lynn, John W

    2010-05-01

    Toxicity studies on sperm often use fertilization success as the end point. This type of assay can be affected by sperm density, egg quality, and sperm-egg compatibility. Testing sperm viability biomarkers with flow cytometry is a fast, high-throughput technique for seminal analysis. In this study, we detected sperm viability biomarkers with several fluorescent reporter dyes using flow cytometry in three aquatic invertebrates (Crassostrea virginica, Dreissena polymorpha, and Lytechinus variegatus) after exposure to a pesticide and herbicide. The pesticide, Bayluscide, appeared to affect mitochondrial membrane potential in the sperm of all three species, as measured with MitoTracker Red CMXRos. A decrease in the percentage of sperm stained with SYBR-14 (indicating uncompromised plasma membrane) was observed in C. virginica and D. polymorpha sperm exposed to Bayluscide, but propidium iodide staining (indicating compromised plasma membranes) appeared to be inhibited by Bayluscide. Acrosome-reacted sperm, as measured by FITC-PNA, decreased after Bayluscide exposure in C. virginica and D. polymorpha sperm. The herbicide, Roundup Ready To-Use-Plus, did not affect the overall percentages of sperm stained with MitoTracker but did cause an increase in MitoTracker fluorescence intensity at 16 mg/L in D. polymorpha. Roundup also caused significant decreases in SYBR-14 staining, significant increases in propidium iodide staining, and significant increases in FITC-PNA staining in D. polymorpha sperm. By not having to rely on egg availability and optimal sperm density, sperm toxicity can be more accurately assessed with flow cytometry as being directly correlated to sperm viability rather than the possibility of altered toxicity results due to sperm-to-egg compatibility. PMID:19876686

  4. 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

  5. Time-Lapse Imaging of Cell Death.

    PubMed

    Wallberg, Fredrik; Tenev, Tencho; Meier, Pascal

    2016-01-01

    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

  6. 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

  7. Selective cloning of hybridoma cells for enhanced immunoglobulin production using flow cytometric cell sorting and automated laser nephelometry.

    PubMed

    Marder, P; Maciak, R S; Fouts, R L; Baker, R S; Starling, J J

    1990-01-01

    Techniques for selective cloning of murine hybridoma cells by flow cytometric cell sorting and use of automated laser nephelometry to determine the resultant clones' immunoglobulin secretion levels are described. Using a commercially available attachment to a fluorescence-activated cell sorter, individual hybridoma cells were successfully distributed into microtiter wells in an automated manner based on their forward angle light scatter properties and their reaction to fluorescein-conjugated anti-mouse-IgG. The techniques were used to estimate successfully the frequency of immunoglobulin-secreting cells in established cultures. In addition, heterogeneity of cell surface immunoglobulin expression was observed and utilized as a criterion for flow sorting of new hybridoma variants. In these studies, clones derived from high (anti-IgG) intensity sorting regions yielded cultures with enhanced immunoglobulin secretion levels, as determined by automated laser nephelometry. Furthermore, the surface immunoglobulin phenotype of the derived clones was conserved in subsequent progeny. Finally, it was established that inclusion of propidium iodide in the hybridoma cell sorting mixtures improved cloning efficiency by facilitating enhanced discrimination and elimination of nonviable cells. Our results indicate that flow cytometric-assisted single cell deposition provides positive attributes of several traditional hybridoma cloning techniques and, in addition, furnishes a tool for steering the cloning process toward selection of enhanced immunoglobulin producing cultures. PMID:2344798

  8. Islet Stellate Cells Isolated from Fibrotic Islet of Goto-Kakizaki Rats Affect Biological Behavior of Beta-Cell

    PubMed Central

    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

  9. Cyclic GMP protects human macrophages against peroxynitrite-induced apoptosis

    PubMed Central

    Shaw, Catherine A; Webb, David J; Rossi, Adriano G; Megson, Ian L

    2009-01-01

    Background Nitric oxide (NO) can be both pro- and anti-apoptotic in various cell types, including macrophages. This apparent paradox may result from the actions of NO-related species generated in the microenvironment of the cell, for example the formation of peroxynitrite (ONOO-). In this study we have examined the ability of NO and ONOO- to evoke apoptosis in human monocyte-derived macrophages (MDM?), and investigated whether preconditioning by cyclic guanosine monophosphate (cGMP) is able to limit apoptosis in this cell type. Methods Characterisation of the NO-related species generated by (Z)-1- [2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO) and 1,2,3,4-oxatriazolium, 5-amino-3-(3,4-dichlorophenyl)-, chloride (GEA-3162) was performed by electrochemistry using an isolated NO electrode and electron paramagnetic resonance (EPR) spectrometry. Mononuclear cells were isolated from peripheral blood of healthy volunteers and cultured to allow differentiation into MDM?. Resultant MDM? were treated for 24 h with DETA/NO (100 1000 ?M) or GEA-3162 (10 300 ?M) in the presence or absence of BAY 412272 (1 ?M), isobutylmethylxanthine (IBMX; 1 ?M), 1H- [1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 20 ?M) or 8-bromo-cGMP (1 mM). Apoptosis in MDM? was assessed by flow cytometric analysis of annexin V binding in combination with propidium iodide staining. Results Electrochemistry and EPR revealed that DETA/NO liberated free NO radical, whilst GEA-3162 concomitantly released NO and O2-, and is therefore a ONOO- generator. NO (DETA/NO) had no effect on cell viability, but ONOO- (GEA-3162) caused a concentration-dependent induction of apoptosis in MDM?. Preconditioning of MDM? with NO in combination with the phosphodiesterase inhibitor, 3-Isobutyl-1-methylxanthine (IBMX), or the NO-independent stimulator of soluble guanylate cyclase, BAY 412272, significantly attenuated ONOO--induced apoptosis in a cGMP-dependent manner. Conclusion These results demonstrate disparities between the ability of NO and ONOO- to induce apoptosis in human MDM?. Furthermore, this study provides evidence for a novel cGMP-dependent pre-conditioning mechanism to limit ONOO--induced apoptosis in human MDM?. PMID:19422695

  10. 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 for curcumin's potency as an anti-inflammatory and anti-tumor agent. The findings support the use of curcumin as a potential therapeutic agent. PMID:18946510

  11. 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.

  12. Epidermal Langerhans cells are resistant to the permeabilizing effects of extracellular ATP: in vitro evidence supporting a protective role of membrane ATPase.

    PubMed

    Girolomoni, G; Santantonio, M L; Pastore, S; Bergstresser, P R; Giannetti, A; Cruz, P D

    1993-03-01

    Extracellular adenosine 5'-triphosphate (ATPo) can induce pore formation in cell membranes, leading to cell permeabilization and eventual cell death. In this study, we examined the sensitivity of human epidermal Langerhans cells to ATP-induced permeabilization and tested the possibility that the Mg(++)- or Ca(++)-dependent plasma membrane ectonucleotidase (mATPase) on Langerhans cells provides protection against the cytotoxic effects of ATPo. Membrane permeability was assessed by using the fluorescent tracer propidium iodide, which confers red nuclear fluorescence to permeabilized cells. Langerhans cells were identified within human epidermal cell suspensions with fluorescein isothiocyanate-conjugated MoAb against CD1a or human leukocyte antigen-DR (HLA-DR) antigens. Cultured human keratinocytes and J774 macrophages were both highly sensitive to permeabilization induced by incubation with ATP (0.5 to 20 mM at 37 degrees C), whereas Langerhans cells were relatively resistant. The non-hydrolyzable ATP analog, adenosine 5'-(beta,gamma-imido) triphosphate, but not other nucleotides such as ADP, AMP, GTP, or UTP, was also able to induce permeabilization comparable to that of ATP, thereby suggesting that ATP hydrolysis is not required for this effect. ATP4- is the moiety most likely responsible for permeabilization, because propidium iodide uptake occurred only when the pH of the medium was > or = 7.4. Permeabilization induced by ATP was augmented by chelation of divalent cations with ethylene-diamine-tetraacetic acid and by the addition of lanthanum or cerium (0.01 to 1 mM). Finally, incubation with the adenosine analog, 5'-p-fluorosulfonylbenzoyl-adenosine (1 mM), inhibited mATPase staining of Langerhans cells in human epidermal sheets, but markedly augmented ATP-induced permeabilization of Langerhans cells. The results indicate that epidermal LC are resistant to the lytic effects of ATPo and that mATPase is involved in such resistance. PMID:8440905

  13. 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

  14. Detachment and reorientation of cells using near-infrared laser microbeam

    NASA Astrophysics Data System (ADS)

    Gu, Ling; Ingle, Ninad; Mohanty, Samarendra K.

    2011-11-01

    Reorientation of adhering cell(s) with respect to other cell(s) has not been yet possible, thus limiting study of controlled interaction between cells. Here, we report cell detachment upon irradiation with a focused near-infrared laser beam, and reorientation of adherent cells. The detached cell was transported along the axial direction by scattering force and trapped at a higher plane inside the media using the same laser beam by a gravito-optical trap. The trapped cell could then be repositioned by movement of the sample stage and reoriented by rotation of the astigmatic trapping beam. The height at which the cell was stably held was found to depend on the laser beam power. Viability of the detached and manipulated cell was found not to be compromised as confirmed by propidium iodide fluorescence exclusion assay. The reoriented cell was allowed to reattach to the substrate at a controlled distance and orientation with respect to other cells. Further, the cell was found to retain its shape even after multiple detachments and manipulation using the laser beam. This technique opens up new avenues for noncontact modification of cellular orientations that will enable study of intercellular interactions and design of engineered tissue.

  15. Acetoacetate protects neuronal cells from oxidative glutamate toxicity.

    PubMed

    Noh, Hae Sook; Hah, Young-Sool; Nilufar, Rashidova; Han, Jaehee; Bong, Jae-Hwan; Kang, Sang Soo; Cho, Gyeong Jae; Choi, Wan Sung

    2006-03-01

    Glutamate cytotoxicity contributes to neuronal degeneration in many central nervous system (CNS) diseases, such as epilepsy and ischemia. We previously reported that a high-fat and low-carbohydrate diet, the ketogenic diet (KD), protects against kainic acid-induced hippocampal cell death in mice. We hypothesized based on these findings that ketosis resulting from KD might inhibit glutamate cytotoxicity, resulting in inhibition of hippocampal neuronal cell death. Therefore, we investigated the role of ketone bodies [acetoacetate (AA) and beta-hydroxybutyrate (beta-OHB)] both in a mouse hippocampal cell line (HT22) and in rat primary hippocampal neurons. As a result, we found that pretreatment with 5 mM lithium AA and 4 mM Na beta-OHB protected the HT22 hippocampal cell line and primary hippocampal neuronal culture against 5 mM glutamate toxicity and that up to 2 hr of pretreatment with 5 mM AA had a protective effect against 5 mM glutamate toxicity in the HT22 cell line. Pretreatment with 5 mM AA decreased ROS production of HT22 cell line at 2 and 8 hr exposure of glutamate, and it decreased the appearance of annexin V-positive HT22 cells, which are indicative of an early stage of apoptosis, and propidium iodide-positive HT22 cells, which are indicative of necrosis. PMID:16435389

  16. Sex hormone modulation of cell growth and apoptosis of the human monocytic/macrophage cell line

    PubMed Central

    Cutolo, Maurizio; Capellino, Silvia; Montagna, Paola; Ghiorzo, Paola; Sulli, Alberto; Villaggio, Barbara

    2005-01-01

    Sex hormones seem to modulate the immune/inflammatory responses by different mechanisms in female and male rheumatoid arthritis patients. The effects of 17β-oestradiol and of testosterone were tested on the cultured human monocytic/macrophage cell line (THP-1) activated with IFN-γ in order to investigate their role in cell proliferation and apoptosis. Activated human THP-1 cells were cultured in the presence of 17β-oestradiol and testosterone (final concentration, 10 nM). The evaluation of markers of cell proliferation included the NF-κB DNA-binding assay, the NF-κB inhibition complex, the proliferating cell nuclear antigen expression and the methyl-tetrazolium salt test. Apoptosis was detected by the annexin V-propidium assay and by the cleaved poly-ADP ribose polymerase expression. Specific methods included flow analysis cytometry scatter analysis, immunocytochemistry and western blot analysis. Cell growth inhibition and increased apoptosis were observed in testosterone-treated THP-1 cells. Increased poly-ADP ribose polymerase-cleaved expression and decreased proliferating cell nuclear antigen expression, as well as an increase of IκB-α and a decrease of the IκB-α phosphorylated form (ser 32), were found in testosterone-treated THP-1 cells. However, the NF-κB DNA binding was found increased in 17β-oestradiol-treated THP-1 cells. The treatment with staurosporine (enhancer of apoptosis) induced decreased NF-κB DNA binding in all conditions, but particularly in testosterone-treated THP-1 cells. Treatment of THP-1 by sex hormones was found to influence cell proliferation and apoptosis. Androgens were found to increase the apoptosis, and oestrogens showed a protective trend on cell death – both acting as modulators of the NF-κB complex. PMID:16207329

  17. Preservation of tomcat (Felis catus) semen in variable temperatures.

    PubMed

    Siemieniuch, Marta; Dubiel, Andrzej

    2007-05-01

    The aim of our study was to estimate the viability of cat epididymal sperm in short time storage at +4 degrees C and in long term storage at -196 degrees C and to assess the percentage of live sperm in fresh semen using eosin/nigrosin staining compared to the flow cytometry method. The testes with epididymides were obtained after routine castration procedure. The sperm for further research were collected after flushing the epididymides using extender consist of: Tris 2.4 g, citric acid 1.4 g, glucose 0.8 g, 0.06% (w/v) Na-benzylpenicillin, 0.1% (w/v) streptomycin sulphate and distilled water. Half of each sample was equilibrated with the dilution and loaded in 0.25 ml plastic straws. The straws were placed on a rack in liquid nitrogen vapour at -120 degrees C for 10 min, plunged in liquid nitrogen for 10 min, replaced to marked goblets and loaded into canes for long term storage in liquid nitrogen at -196 degrees C. Sixty percent of motile spermatozoa was accomplished after thawing. However, the percentage of the sperm with intact acrosomes was decreased and the share of cells with midpiece and tail defects was increased. The storage of sperm flushed from epididymides at +4 degrees C for a short time and the usage of sperm during 2-3 days after collection seems to be better than cryopreservation. In our study, normospermia was present in 72.7 +/- 8.8% of fresh semen. The most common defect was the presence of distal droplets, imperfect heads or abnormal acrosomal outline. The motility of fresh sperm flushed from epididymides achieved 77.9 +/- 6.8%. The viability of sperm amounting to 52.5 +/- 13.8% was achieved on third day of conservation in the liquid extender. The percentage of viable sperm in fresh epididymal spermatozoa was 84.9 +/- 7.8%. Compared to these results, the percentage of live cells using SYBR-14/propidium iodide staining was insignificantly lower (82.2 +/- 8%). The live, non-apoptotic cells were 79.0 +/- 7.8%. The share of live, early-apoptotic spermatozoa and late-apoptotic spermatozoa was, respectively, 2 +/- 1.4% and 1.5 +/- 0.9%. The viability of sperm estimated by eosin/nigrosin staining was confirmed by the flow cytometry method. There was no statistical differences between the staining. The usage of apoptosis detection kit revealed, that the percentage of early-apoptotic and late-apoptotic cells was insignificant. PMID:16725286

  18. 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; Andng, 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 (?H2AX) 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

  19. 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

  20. Cell cycle distribution of hypoxia and progression of hypoxic tumour cells in vivo.

    PubMed Central

    Webster, L.; Hodgkiss, R. J.; Wilson, G. D.

    1998-01-01

    Hypoxia was assessed in three murine tumour models in vivo by measuring the incorporation of 7-(4'-(2-nitroimidazole-1-yl)-butyl)-theophylline (NITP), an immunologically identifiable hypoxia marker that binds bioreductively to cells under low-oxygen conditions. Proliferating cells were labelled in the same tumours by administering the thymidine analogue bromodeoxyuridine (BrdUrd). The relative hypoxia in each cell cycle phase of cells isolated from tumours was assessed by addition of propidium iodide with analysis by flow cytometry. There was no relationship between tumour volume and hypoxia in either the anaplastic sarcoma SaF or the poorly differentiated carcinoma CaNT and only a slight negative correlation in moderately well-differentiated carcinoma Rh. The G1/G0 phase contained the greatest number of aneuploid hypoxic cells (aneuploid hypoxia ranging from less than 1% up to 40%, 38% and 71% in SaF, CaNT and Rh respectively), although there were significant amounts of hypoxia present in S- and G2/M phases for all three tumours examined. However, the highest proportion of hypoxia occurred in the G2/M phase, in which up to 60% of the cells were hypoxic. Simultaneous measurement of hypoxia, proliferation and DNA content using a novel triple-staining flow cytometry method showed that hypoxic cells could actively participate in the cell cycle. In addition, the cell cycle distribution of NITP and BrdUrd labelling showed that hypoxic cells could progress through the cell cycle, although their rate of progression was slower than that of better oxygenated cells. PMID:9460993

  1. Electrodelivery of drugs into cancer cells in the presence of poloxamer 188.

    PubMed

    Tsoneva, Iana; Iordanov, Iordan; Berger, Annette J; Tomov, Toma; Nikolova, Biliana; Mudrov, Nikola; Berger, Martin R

    2010-01-01

    In the present study it is shown that poloxamer 188, added before or immediately after an electrical pulse used for electroporation, decreases the number of dead cells and at the same time does not reduce the number of reversible electropores through which small molecules (cisplatin, bleomycin, or propidium iodide) can pass/diffuse. It was suggested that hydrophobic sections of poloxamer 188 molecules are incorporated into the edges of pores and that their hydrophilic parts act as brushy pore structures. The formation of brushy pores may reduce the expansion of pores and delay the irreversible electropermeability. Tumors were implanted subcutaneously in both flanks of nude mice using HeLa cells, transfected with genes for red fluorescent protein and luciferase. The volume of tumors stopped to grow after electrochemotherapy and the use of poloxamer 188 reduced the edema near the electrode and around the subcutaneously growing tumors. PMID:20706647

  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. 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

  6. 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

  7. 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.

  8. Plasma cells of the chicken Harderian gland.

    PubMed

    Scott, T R; Savage, M L; Olah, I

    1993-07-01

    The chicken Harderian gland (HG) is densely populated in its subepithelial spaces with plasma cells (PC). These immune cells produce and secrete Ig of the IgA, IgG, and IgM classes. Such Ig secretion into the tears affords the upper respiratory tract with protective antibodies. The immunological role of the HG is quite interesting; yet this gland is a site of unusual PC proliferation. Studies of the gland utilizing bromodeoxyuridine (BrdUrd) incorporation into DNA and propidium iodide (PI) staining of PC DNA have verified previous suggestions in the literature that PC of the chicken HG proliferate. Both isolated PC suspensions and frozen sections of the HG from chicks aged 6 to 9 wk reveal that BrdUrd is incorporated into PC DNA. Furthermore, flow cytometric analysis of PI-stained PC indicates a relatively high percentage of PC in S phase of the cell cycle. Continued studies are examining possible mechanisms controlling proliferation and differentiation of PC in the HG. It is believed that the stromal elements of the HG produce and secrete a factor(s) that influences PC proliferation and differentiation. Isolation and characterization of this influencing factor(s) will allow for the possible systemic application of the factor(s) for enhancement of immune responses. PMID:8346152

  9. 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

  10. 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

  11. 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

  12. 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

  13. Immunosuppressive activity of an aqueous Viola tricolor herbal extract

    PubMed Central

    Sauer, Barbara; Huber, Roman; Gruber, Christian W.; Gründemann, Carsten

    2014-01-01

    Ethnopharmacological relevance Heartsease (Viola tricolor L.), a member of the Violaceae family, has a long history as a medicinal plant and has been documented in the Pharmacopoeia of Europe. Due to its anti-inflammatory properties it is regarded as a traditional remedy against skin diseases, for example for the treatment of scabs, itching, ulcers, eczema or psoriasis, and it is also used in the treatment of inflammation of the lungs and chest such as bronchitis or asthma. Because T-cells play an important role in the pathological process of inflammatory diseases we investigated the effect of an aqueous Viola extract on lymphocyte functions and explored the ‘active’ principle of the extract using bioactivity-guided fractionation. Material and Methods An aqueous Viola extract was prepared by C18 solid-phase extraction. Effects on proliferation of activated lymphocytes (using the cell membrane permeable fluorescein dye CFSE), apoptosis and necrosis (using annexin V and propidium iodide staining), interleukin-2 (IL-2) receptor expression (using fluorochrome-conjugated antibodies) and IL-2 cytokine secretion (using an ELISA-based bead array system) were measured by flow cytometry. Influence on lymphocyte polyfunctionality was characterized by Viola extract-induced production of IFN-γ and TNF-α, as well as its influence on lymphocyte degranulation activity. Fractionation and phytochemical analysis of the extract were performed by RP-HPLC and mass spectrometry. Results The aqueous Viola extract inhibited proliferation of activated lymphocytes by reducing IL-2 cytokine secretion without affecting IL-2 receptor expression. Similarly, effector functions were affected as indicated by the reduction of IFN-γ and TNF-α production; degranulation capacity of activated lymphocytes remained unaffected. Bioassay-guided fractionation and phytochemical analysis of the extract led to identification of circular plant peptides, so called cyclotides, as bioactive components. Conclusion An aqueous Viola extract contains bioactive cyclotides, which inhibit proliferation of activated lymphocytes in an IL-2 dependent manner. The findings provide a rationale for use of herbal Viola preparations in the therapy of disorders related to an overactive immune system. However, further studies to evaluate its clinical potency and potential risks have to be performed. PMID:24216163

  14. 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.

  15. 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

  16. 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

  17. 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

  18. 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

  19. Vitamin C suppresses cell death in MCF-7 human breast cancer cells induced by tamoxifen.

    PubMed

    Subramani, Tamilselvan; Yeap, Swee Keong; Ho, Wan Yang; Ho, Chai Ling; Omar, Abdul Rahman; Aziz, Suraini Abdul; Rahman, Nik Mohd Afizan Nik Abd; Alitheen, Noorjahan Banu

    2014-02-01

    Vitamin C is generally thought to enhance immunity and is widely taken as a supplement especially during cancer treatment. Tamoxifen (TAM) has both cytostatic and cytotoxic properties for breast cancer. TAM engaged mitochondrial oestrogen receptor beta in MCF-7 cells and induces apoptosis by activation of pro-caspase-8 followed by downstream events, including an increase in reactive oxygen species and the release of pro-apoptotic factors from the mitochondria. In addition to that, TAM binds with high affinity to the microsomal anti-oestrogen-binding site and inhibits cholesterol esterification at therapeutic doses. This study aimed to investigate the role of vitamin C in TAM-mediated apoptosis. Cells were loaded with vitamin C by exposure to dehydroascorbic acid, thereby circumventing in vitro artefacts associated with the poor transport and pro-oxidant effects of ascorbic acid. Pre-treatment with vitamin C caused a dose-dependent attenuation of cytotoxicity, as measured by acridine-orange/propidium iodide (AO/PI) and Annexin V assay after treatment with TAM. Vitamin C dose-dependently protected cancer cells against lipid peroxidation caused by TAM treatment. By real-time PCR analysis, an impressive increase in FasL and tumour necrosis factor-α (TNF-α) mRNA was detected after TAM treatment. In addition, a decrease in mitochondrial transmembrane potential was observed. These results support the hypothesis that vitamin C supplementation during cancer treatment may detrimentally affect therapeutic response. PMID:24266867

  20. 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

  1. Apoptosis and Desquamation of Urothelial Cells in Tissue Remodeling During Rat Postnatal Development

    PubMed Central

    Erman, Andreja; Zupančič, Daša; Jezernik, Kristijan

    2009-01-01

    Postnatal rat urothelium was studied from day 0 to day 14, when intense cell loss as part of tissue remodeling was expected. The morphological and biochemical characteristics of urothelial cells in the tissue and released cells were investigated by light and electron microscopy, by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) assay, by annexin V/propidium iodide assay, and by immunofluorescent detection of active caspases and tight-junction protein occludin. Intense apoptosis and massive desquamation were detected between postnatal days 7 and 10. During this period, active caspases and TUNEL-positive cells were found in the urothelium. Disassembled cell–cell junctions were detected between cells. The majority of desquamated cells expressed no apoptotic cell morphology, but were active caspase positive and TUNEL positive. Ann+/PI− apoptotic bodies and desquamated Ann+/PI+ cells were detected in the lumen. These results indicate that apoptosis and desquamation participate in urothelial cell loss in the rat early postnatal period, indispensable for fast urothelial remodeling during development. (J Histochem Cytochem 57:721–730, 2009) PMID:19365092

  2. Microfluidic Device for Stem Cell Differentiation and Localized Electroporation of Postmitotic Neurons

    PubMed Central

    Kang, Wonmo; Giraldo-Vela, Juan P.; Nathamgari, S. Shiva P.; McGuire, Tammy; McNaughton, Rebecca L.; Kessler, John A.; Espinosa, Horacio D.

    2014-01-01

    New techniques to deliver of nucleic acids and other molecules for gene editing and gene expression profiling, which can be performed with minimal perturbation to cell growth or differentiation, are essential for advancing biological research. Studying cells in their natural state, with temporal control, is particularly important for primary cells that are derived by differentiation from stem cells and are adherent, e.g., neurons. Existing high-throughput transfection methods either require cells to be in suspension or are highly toxic and limited to a single transfection per experiment. Here we present a microfluidic device that couples on-chip culture of adherent cells and transfection by localized electroporation. Integrated microchannels allow long-term cell culture on the device and repeated temporal transfection. The microfluidic device was validated by first performing electroporation of HeLa and HT1080 cells, with transfection efficiencies of ~95% for propidium iodide and up to 50% for plasmids. Application to primary cells was demonstrated by on-chip differentiation of neural stem cells and transfection of postmitotic neurons with a green fluorescent protein plasmid. PMID:25205561

  3. Autophagy proteins play cytoprotective and cytocidal roles in leucine starvation-induced cell death in Saccharomyces cerevisiae

    PubMed Central

    Dziedzic, Slawomir A.; Caplan, Allan B.

    2012-01-01

    Autophagy is essential for prolonging yeast survival during nutrient deprivation; however, this report shows that some autophagy proteins may also be accelerating population death in those conditions. While leucine starvation caused YCA1-mediated apoptosis characterized by increased annexin V staining, nitrogen deprivation triggered necrotic death characterized by increased propidium iodide uptake. Although a Δatg8 strain died faster than its parental strain during nitrogen starvation, this mutant died slower than its parent during leucine starvation. Conversely, a Δatg11 strain died slower than its parent during nitrogen starvation, but faster during leucine starvation. Curiously, although GFP-Atg8 complemented the Δatg8 mutation, this protein made ATG8 cells more sensitive to nitrogen starvation, and less sensitive to leucine starvation. These results were difficult to explain if autophagy only extended life but could be an indication that a second form of autophagy could concurrently facilitate either apoptotic or necrotic cell death. PMID:22361650

  4. Polymorphonuclear cell apoptosis in exudates generated by polymers.

    PubMed

    Fabre, T; Belloc, F; Dupuy, B; Schappacher, M; Soum, A; Bertrand-Barat, J; Baquey, C; Durandeau, A

    1999-03-15

    Flow cytometry was used to quantify apoptotic and necrotic polymorphonuclear (PMN) cells in an exudate generated by biomaterials, and the results were compared with determinations of spontaneous apoptosis and necrosis in PMN cells from the bloodstream. The exudate formed inside cylindrical tubes subcutaneously implanted in the dorsal region of rats was collected over a 1-week period. A rapid and simple staining procedure based on the spectral properties of the bisbenzemide Hoechst 33342 was used to identify apoptotic PMN cells. Quantification of permeabilized PMN cells stained by propidium iodide was possible in the same unfixed specimens. The percentages of apoptotic and permeabilized PMN cells in peripheral rat blood were low (1.8 +/-0 0.5% and 1.7 +/- 0.7%, respectively), similar to results found in humans. In exudates generated by polyvinyl chloride (PVC), the percentages of apoptotic and permeabilized PMN cells were higher than in the blood. The percentage of PMN cells undergoing apoptosis progressively increased with time and reached a maximum at day 2 (27% +/- 6%). The percentage of permeabilized cells progressively increased with time and was much higher than the percentage of apoptotic cells on days 4 and 8. Apoptosis and necrosis of PMN cells at day 2 were inhibited when tubes were filled with 10% serum. Selective inhibition of apoptosis with a caspase inhibitor in vivo indicated that apoptosis and necrosis are two separate pathways leading to the death of PMN cells in the exudate. At day 2, polyurethane (PU) was associated with a lower rate of apoptosis than PVC or a random copolymer of trimethylene carbonate (TMC) and epsiloncaprolactone (ECL). Apoptosis was interpreted as an organized cell removal process that limits inflammation. Apoptosis was the natural route of PMN cell death at the early stage of inflammation. PMID:10397947

  5. Microscopic elucidation of abundant endophytic bacteria colonizing the cell wall–plasma membrane peri-space in the shoot-tip tissue of banana

    PubMed Central

    Thomas, Pious; Reddy, Krishna M.

    2013-01-01

    This study was aimed at generating microscopic evidence of intra-tissue colonization in banana in support of the previous findings on widespread association of endophytic bacteria with the shoot tips of field-grown plants and micropropagated cultures, and to understand the extent of tissue colonization. Leaf-sheath tissue sections (∼50–100 µm) from aseptically gathered shoot tips of cv. Grand Naine were treated with Live/Dead bacterial viability kit components SYTO 9 (S9) and propidium iodide (PI) followed by epifluorescence or confocal laser scanning microscopy (CLSM). The S9, which targets live bacteria, showed abundant green-fluorescing particles along the host cell periphery in CLSM, apparently in between the plasma membrane and the cell wall. These included non-motile and occasional actively motile single bacterial cells seen in different x–y planes and z-stacks over several cell layers, with the fluorescence signal similar to that of pure cultures of banana endophytes. Propidium iodide, which stains dead bacteria, did not detect any, but post-ethanol treatment, both PI and 4′,6-diamidino-2-phenylindole detected abundant bacteria. Propidium iodide showed clear nuclear staining, as did S9 to some extent, and the fluorophores appeared to detect bacteria at the exclusion of DNA-containing plant organelles as gathered from bright-field and phase-contrast microscopy. The S9–PI staining did not work satisfactorily with formalin- or paraformaldehyde-fixed tissue. The extensive bacterial colonization in fresh tissue was further confirmed with the suckers of different cultivars, and was supported by transmission electron microscopy. This study thus provides clear microscopic evidence of the extensive endophytic bacterial inhabitation in the confined cell wall–plasma membrane peri-space in shoot tissue of banana with the organisms sharing an integral association with the host. The abundant tissue colonization suggests a possible involvement of endophytes in the biology of the host besides recognizing cell wall–plasma membrane peri-space as a major niche for plant-associated bacteria.

  6. Soluble epoxide hydrolase inhibitor trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid is neuroprotective in rat model of ischemic stroke.

    PubMed

    Shaik, Jafar Sadik B; Ahmad, Muzamil; Li, Wenjin; Rose, Marie E; Foley, Lesley M; Hitchens, T Kevin; Graham, Steven H; Hwang, Sung Hee; Hammock, Bruce D; Poloyac, Samuel M

    2013-12-01

    Soluble epoxide hydrolase (sEH) diminishes vasodilatory and neuroprotective effects of epoxyeicosatrienoic acids by hydrolyzing them to inactive dihydroxy metabolites. The primary goals of this study were to investigate the effects of acute sEH inhibition by trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB) on infarct volume, functional outcome, and changes in cerebral blood flow (CBF) in a rat model of ischemic stroke. Focal cerebral ischemia was induced in rats for 90 min followed by reperfusion. At the end of 24 h after reperfusion rats were euthanized for infarct volume assessment by triphenyltetrazolium chloride staining. Brain cortical sEH activity was assessed by ultra performance liquid chromatography-tandem mass spectrometry. Functional outcome at 24 and 48 h after reperfusion was evaluated by arm flexion and sticky-tape tests. Changes in CBF were assessed by arterial spin-labeled-MRI at baseline, during ischemia, and at 180 min after reperfusion. Neuroprotective effects of t-AUCB were evaluated in primary rat neuronal cultures by Cytotox-Flour kit and propidium iodide staining. t-AUCB significantly reduced cortical infarct volume by 35% (14.5 2.7% vs. 41.5 4.5%), elevated cumulative epoxyeicosatrienoic acids-to-dihydroxyeicosatrienoic acids ratio in brain cortex by twofold (4.40 1.89 vs. 1.97 0.85), and improved functional outcome in arm-flexion test (day 1: 3.28 0.5 s vs. 7.50 0.9 s; day 2: 1.71 0.4 s vs. 5.28 0.5 s) when compared with that of the vehicle-treated group. t-AUCB significantly reduced neuronal cell death in a dose-dependent manner (vehicle: 70.9 7.1% vs. t-AUCB0.1?M: 58 5.11% vs. t-AUCB0.5?M: 39.9 5.8%). These findings suggest that t-AUCB may exert its neuroprotective effects by affecting multiple components of neurovascular unit including neurons, astrocytes, and microvascular flow. PMID:24043255

  7. Soluble epoxide hydrolase inhibitor trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid is neuroprotective in rat model of ischemic stroke

    PubMed Central

    Shaik, Jafar Sadik B.; Ahmad, Muzamil; Li, Wenjin; Rose, Marie E.; Foley, Lesley M.; Hitchens, T. Kevin; Graham, Steven H.; Hwang, Sung Hee; Hammock, Bruce D.

    2013-01-01

    Soluble epoxide hydrolase (sEH) diminishes vasodilatory and neuroprotective effects of epoxyeicosatrienoic acids by hydrolyzing them to inactive dihydroxy metabolites. The primary goals of this study were to investigate the effects of acute sEH inhibition by trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB) on infarct volume, functional outcome, and changes in cerebral blood flow (CBF) in a rat model of ischemic stroke. Focal cerebral ischemia was induced in rats for 90 min followed by reperfusion. At the end of 24 h after reperfusion rats were euthanized for infarct volume assessment by triphenyltetrazolium chloride staining. Brain cortical sEH activity was assessed by ultra performance liquid chromatography-tandem mass spectrometry. Functional outcome at 24 and 48 h after reperfusion was evaluated by arm flexion and sticky-tape tests. Changes in CBF were assessed by arterial spin-labeled-MRI at baseline, during ischemia, and at 180 min after reperfusion. Neuroprotective effects of t-AUCB were evaluated in primary rat neuronal cultures by Cytotox-Flour kit and propidium iodide staining. t-AUCB significantly reduced cortical infarct volume by 35% (14.5 ± 2.7% vs. 41.5 ± 4.5%), elevated cumulative epoxyeicosatrienoic acids-to-dihydroxyeicosatrienoic acids ratio in brain cortex by twofold (4.40 ± 1.89 vs. 1.97 ± 0.85), and improved functional outcome in arm-flexion test (day 1: 3.28 ± 0.5 s vs. 7.50 ± 0.9 s; day 2: 1.71 ± 0.4 s vs. 5.28 ± 0.5 s) when compared with that of the vehicle-treated group. t-AUCB significantly reduced neuronal cell death in a dose-dependent manner (vehicle: 70.9 ± 7.1% vs. t-AUCB0.1μM: 58 ± 5.11% vs. t-AUCB0.5μM: 39.9 ± 5.8%). These findings suggest that t-AUCB may exert its neuroprotective effects by affecting multiple components of neurovascular unit including neurons, astrocytes, and microvascular flow. PMID:24043255

  8. In Vitro and In Vivo Investigation of the Angiogenic Effects of Liraglutide during Islet Transplantation

    PubMed Central

    Langlois, Allan; Mura, Carole; Bietiger, William; Seyfritz, Elodie; Dollinger, Camille; Peronet, Claude; Maillard, Elisa; Pinget, Michel; Jeandidier, Nathalie; Sigrist, Séverine

    2016-01-01

    Introduction This study investigated the angiogenic properties of liraglutide in vitro and in vivo and the mechanisms involved, with a focus on Hypoxia Inducible Factor-1α (HIF-1α) and mammalian target of rapamycin (mTOR). Materials and Methods Rat pancreatic islets were incubated in vitro with 10 μmol/L of liraglutide (Lira) for 12, 24 and 48 h. Islet viability was studied by fluorescein diacetate/propidium iodide staining and their function was assessed by glucose stimulation. The angiogenic effect of liraglutide was determined in vitro by the measure of vascular endothelial growth factor (VEGF) secretion using enzyme-linked immunosorbent assay and by the evaluation of VEGF and platelet-derived growth factor-α (PDGFα) expression with quantitative polymerase chain reaction technic. Then, in vitro and in vivo, angiogenic property of Lira was evaluated using immunofluorescence staining targeting the cluster of differentiation 31 (CD31). To understand angiogenic mechanisms involved by Lira, HIF-1α and mTOR activation were studied using western blotting. In vivo, islets (1000/kg body-weight) were transplanted into diabetic (streptozotocin) Lewis rats. Metabolic control was assessed for 1 month by measuring body-weight gain and fasting blood glucose. Results Islet viability and function were respectively preserved and enhanced (p<0.05) with Lira, versus control. Lira increased CD31-positive cells, expression of VEGF and PDGFα (p<0.05) after 24 h in culture. Increased VEGF secretion versus control was also observed at 48 h (p<0.05). Moreover, Lira activated mTOR (p<0.05) signalling pathway. In vivo, Lira improved vascular density (p<0.01), body-weight gain (p<0.01) and reduced fasting blood glucose in transplanted rats (p<0.001). Conclusion The beneficial effects of liraglutide on islets appeared to be linked to its angiogenic properties. These findings indicated that glucagon-like peptide-1 analogues could be used to improve transplanted islet revascularisation. PMID:26974949

  9. 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 investigated the effect of cluster orientation on the cultivation medium and the influence of the change of the cluster’s three-dimensional orientation on its development. Maintaining the same position when transferring ESEs into new cultivation medium seemed to be necessary because changes in the orientation significantly affected ESE growth. Conclusions and Significance This work illustrated the possible inner organisation of ESEs. The outer layer of ESEs is formed by individual somatic embryos with high metabolic activity (and with high demands for nutrients, oxygen and water), while an embryonal group is directed outside of the ESE cluster. Somatic embryos with depressed metabolic activity were localised in the inner regions, where these embryonic tissues probably have a very important transport function. PMID:26624287

  10. 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 the increased levels of pAKT with a concomitant inhibition of pGSK3β leading to preserved mitochondrial membrane potential. PMID:24658657

  11. The effect of Lfcin-B on non-small cell lung cancer H460 cells is mediated by inhibiting VEGF expression and inducing apoptosis.

    PubMed

    Wang, Shusheng; Tu, Jiancheng; Zhou, Cuijie; Li, Jianwei; Huang, Long; Tao, Lei; Zhao, Lei

    2015-02-01

    Lfcin-B, an antimicrobial peptide found in various exocrine secretions of mammals, showed antitumor effects. However, the effect and relative mechanism of Lfcin-B on non-small cell lung cancer is unclear. In this study, assay of cell viability, quantitative real-time PCR, Western blot, annexin V/propidium iodide assay, flow cytometry and tumor-xenograft model were applied to elucidate the mechanism of Lfcin-B on non-small cell lung cancer NCI-H460 (H460) cells. Lfcin-B significantly suppressed the proliferation of H460 cells in vitro. Additionally, the transcription and translation of the VEGF gene in H460 cells were restrained after exposure to Lfcin-B. Moreover, the apoptosis of H460 cells was induced by Lfcin-B through stimulating caspase-3, caspase-9 and preventing survivin expression on both the transcription and translation level. Meanwhile, Lfcin-B increased the production of reactive oxygen species and suppressed the RNA of antioxidant enzymes (GPX1, GPX2, SOD3 and catalase) in H460 cells. Finally, Lfcin-B significantly prevented the tumor growth in the H460-bearing mice model. These results indicated that Lfcin-B could be a potential candidate for the treatment of lung cancer. PMID:24691828

  12. Butanol-Partitioned Extraction from Aqueous Extract of Gracilaria tenuistipitata Inhibits Cell Proliferation of Oral Cancer Cells Involving Apoptosis and Oxidative Stress.

    PubMed

    Yeh, Chi-Chen; Li, Kun-Tzu; Tang, Jen-Yang; Wang, Hui-Ru; Liu, Jing-Ru; Huang, Hurng-Wern; Chang, Fang-Rong; Tsai, Cheng-En; Lo, I-Wen; Huang, Ming-Yii; Chang, Hsueh-Wei

    2016-05-01

    We have previously found that the aqueous extract of Gracilaria tenuistipitata (AEGT) and its partitioned fractions had antioxidant properties in biochemical assays. Although the butanol-partitioned fraction of AEGT (AEGT-pBuOH) had a stronger antioxidant performance than AEGT, its biological effects are still unknown. In this study, the cellular responses of oral cancer cells to AEGT-pBuOH were monitored in terms of cell viability, cell cycle progression, apoptosis, and oxidative stress responses. In an ATP content assay, the cell viability of oral cancer cells treated with AEGT-pBuOH was dose responsively inhibited (p < 0.005). For flow cytometry, AEGT-pBuOH was also found to dose responsively induce cell cycle disturbance by propidium iodide (PI) staining and to induce apoptosis by annexin V/PI and pan-caspase staining (p < 0.005). In AEGT-pBuOH-treated oral cancer cells, the reactive oxygen species (ROS) was increased and mitochondrial membrane potential was decreased in a dose-response manner (p < 0.005). These results suggest that AEGT-pBuOH inhibited the proliferation and induced apoptosis of oral cancer cells involving the ROS generation and mitochondrial depolarization. PMID:27138906

  13. An improved flow cytometry-based natural killer cytotoxicity assay involving calcein AM staining of effector cells.

    PubMed

    Jang, Youn-Young; Cho, Duck; Kim, Sang-Ki; Shin, Dong-Jun; Park, Min-Ho; Lee, Je-Jung; Shin, Myung-Geun; Shin, Jong-Hee; Suh, Soon-Pal; Ryang, Dong-Wook

    2012-01-01

    Several flow cytometric methods for measuring natural killer cell activity have been developed. Commonly used protocols involve the staining of target cells with various fluorescent dyes. However, these protocols are not applicable to certain experimental settings. Therefore, we used Calcein AM (CAM), which has been reported to be the most suitable dye for use in target cell staining protocols, as a means of developing an improved flow cytometry-based NK cytotoxicity assay involving effector cell staining. Peripheral blood mononuclear cells (PBMCs) isolated by gradient density centrifugation and expanded NK cells were used as effector cells. Cytotoxicity against K562 cells and several hematologic cancer cell lines was measured by a flow cytometry-based method using CAM and propidium iodide. The new assay was compared with a standard (51)Cr release assay (CRA) in terms of its ability to measure the cytotoxicity of NK cells in PBMCs and expanded NK cells against K562 cells. The optimal concentration of CAM for staining effector cells was 0.05 μM, and CAM fluorescence intensity in effector cells was maintained for 4 hours. CAM staining had no significant effect on NK cell activity in human PBMCs or expanded NK cells. Comparison of the CRA and this new assay using K562 cells revealed a good correlation (PBMCs, r = 0.894; expanded NK cells, r = 0.887). Distinct separation between target tumor cells (Daudi, Raji, RPMI8226, U266, U937, and K562 cells) and CAM-stained PBMCs (E:T ratio, 12.5:1 to 50:1) or expanded NK cells (E:T ratio, 0.5 to 4:1) was observed after incubation for 1 or 4 hours. In summary, we successfully developed an effective flow cytometry-based assay for assessing the activity of NK cells in PBMCs and expanded NK cells against K562 cells and various types of hematologic cancer cells. PMID:22371909

  14. An improved flow cytometry-based natural killer cytotoxicity assay involving calcein AM staining of effector cells.

    TOXLINE Toxicology Bibliographic Information

    Jang YY; Cho D; Kim SK; Shin DJ; Park MH; Lee JJ; Shin MG; Shin JH; Suh SP; Ryang DW

    2012-01-01

    Several flow cytometric methods for measuring natural killer cell activity have been developed. Commonly used protocols involve the staining of target cells with various fluorescent dyes. However, these protocols are not applicable to certain experimental settings. Therefore, we used Calcein AM (CAM), which has been reported to be the most suitable dye for use in target cell staining protocols, as a means of developing an improved flow cytometry-based NK cytotoxicity assay involving effector cell staining. Peripheral blood mononuclear cells (PBMCs) isolated by gradient density centrifugation and expanded NK cells were used as effector cells. Cytotoxicity against K562 cells and several hematologic cancer cell lines was measured by a flow cytometry-based method using CAM and propidium iodide. The new assay was compared with a standard (51)Cr release assay (CRA) in terms of its ability to measure the cytotoxicity of NK cells in PBMCs and expanded NK cells against K562 cells. The optimal concentration of CAM for staining effector cells was 0.05 μM, and CAM fluorescence intensity in effector cells was maintained for 4 hours. CAM staining had no significant effect on NK cell activity in human PBMCs or expanded NK cells. Comparison of the CRA and this new assay using K562 cells revealed a good correlation (PBMCs, r = 0.894; expanded NK cells, r = 0.887). Distinct separation between target tumor cells (Daudi, Raji, RPMI8226, U266, U937, and K562 cells) and CAM-stained PBMCs (E:T ratio, 12.5:1 to 50:1) or expanded NK cells (E:T ratio, 0.5 to 4:1) was observed after incubation for 1 or 4 hours. In summary, we successfully developed an effective flow cytometry-based assay for assessing the activity of NK cells in PBMCs and expanded NK cells against K562 cells and various types of hematologic cancer cells.

  15. Foodborne Cereulide Causes Beta-Cell Dysfunction and Apoptosis

    PubMed Central

    Vangoitsenhoven, Roman; Rondas, Dieter; Crèvecoeur, Inne; D'Hertog, Wannes; Baatsen, Pieter; Masini, Matilde; Andjelkovic, Mirjana; Van Loco, Joris; Matthys, Christophe; Mathieu, Chantal; Overbergh, Lut; Van der Schueren, Bart

    2014-01-01

    Aims/Hypothesis To study the effects of cereulide, a food toxin often found at low concentrations in take-away meals, on beta-cell survival and function. Methods Cell death was quantified by Hoechst/Propidium Iodide in mouse (MIN6) and rat (INS-1E) beta-cell lines, whole mouse islets and control cell lines (HepG2 and COS-1). Beta-cell function was studied by glucose-stimulated insulin secretion (GSIS). Mechanisms of toxicity were evaluated in MIN6 cells by mRNA profiling, electron microscopy and mitochondrial function tests. Results 24 h exposure to 5 ng/ml cereulide rendered almost all MIN6, INS-1E and pancreatic islets apoptotic, whereas cell death did not increase in the control cell lines. In MIN6 cells and murine islets, GSIS capacity was lost following 24 h exposure to 0.5 ng/ml cereulide (P<0.05). Cereulide exposure induced markers of mitochondrial stress including Puma (p53 up-regulated modulator of apoptosis, P<0.05) and general pro-apoptotic signals as Chop (CCAAT/-enhancer-binding protein homologous protein). Mitochondria appeared swollen upon transmission electron microscopy, basal respiration rate was reduced by 52% (P<0.05) and reactive oxygen species increased by more than twofold (P<0.05) following 24 h exposure to 0.25 and 0.50 ng/ml cereulide, respectively. Conclusions/Interpretation Cereulide causes apoptotic beta-cell death at low concentrations and impairs beta-cell function at even lower concentrations, with mitochondrial dysfunction underlying these defects. Thus, exposure to cereulide even at concentrations too low to cause systemic effects appears deleterious to the beta-cell. PMID:25119564

  16. Psoralen/UV inactivation of HIV-1-infected cells for use in cytologic and immunologic procedures

    SciTech Connect

    Watson, A.J.; Klaniecki, J.; Hanson, C.V. )

    1990-04-01

    A rapid procedure for the inactivation of HIV-1-infected cells using psoralen and ultraviolet (UV) light is described. Exposure of HIV-1-infected cells to 5 micrograms/ml psoralen followed by UV irradiation (320-380 nm) for 5 minutes yields cells that are noninfectious as assessed by extended infectivity assays. The psoralen/UV inactivation procedure described is effective with cells chronically or acutely infected with HIV-1 and is unaffected by cell densities up to 12 x 10(6)/ml. At 5 micrograms/ml psoralen does little damage to cellular permeability as shown by the ability of treated cells to exclude trypan blue and propidium iodide. Psoralen/UV treatment of HIV-1-infected cells does not cause a significant decrease in the reactivity of HIV-1 core and envelope antigens or cellular antigens to monoclonal antibodies. Experiments are presented demonstrating the use of these cells for flow cytometry studies and for cell surface labeling using the lactoperoxidase {sup 125}I iodination procedure.

  17. The radiosensitizing effect of doranidazole on human colorectal cancer cells exposed to high doses of irradiation

    PubMed Central

    Zhang, Li; Gong, Aimin; Ji, Jun; Wu, Yuanyuan; Zhu, Xiaoyu; Lv, Suqing; Lv, Hongzhu; Sun, Xizhuo

    2007-01-01

    Background This paper investigates the effects of a new radiosensitizer, doranidazole, and enhancing irradiation on colorectal cancer cells. Methods The radiosensitizing effect of doranidazole was determined using colony formation and propidium iodide (PI) assays to measure cell growth inhibition and the cell killing effect of human colorectal cancer cell lines exposed to high doses of γ-ray irradiation under hypoxic conditions in vitro. Fluorescence staining and cell migration assays were also used to assess the radiosensitizing effect. Results Cell proliferation evaluated by clonogenic survival curves was significantly inhibited by 5 mmol/L doranidazole, particularly at doses ranging from 10 to 30 Gy of irradiation. The radiosensitizing effect of doranidazole on colorectal cancer cells occurs in a time- and dose-dependent manner. Doranidazole also inhibited the mobility of cell invasion and migration. Conclusion Doranidazole can enhance the killing effect and the cell growth inhibition of colorectal cancer after high-dose irradiation in a time and dose-dependent manner. PMID:17919337

  18. The Effects of Fenugreek on Radiation Induced Toxicity for Human Blood T-Cells in Radiotherapy.

    PubMed

    Tavakoli, Mohamed Bagher; Kiani, Ali; Roayaei, Mahnaz

    2015-01-01

    Many cellular damages either in normal or cancerous tissues are the outcome of molecular events affected by ionizing radiation. T-cells are the most important among immune system agents and are used for biological radiation dose measurement in recommended standard methods. The herbs with immune modulating properties may be useful to reduce the risk of the damages and subsequently the diseases. The T-cells as the most important immune cells being targeted for biological dosimetry of radiation. This study proposes a flowcytometric-method based on fluorescein isothiocyanate- and propidium iodide (PI)-labeled annexin-V to assess apoptosis in blood T-cells after irradiation in both presence and absence of fenugreek extract. T-cells peripheral blood lymphocyte isolated from blood samples of healthy individuals with no irradiated job background. The media of cultured cells was irradiated 1-h after the fenugreek extract was added. The number of apoptotic cells was assessed by annexin-V protocol and multicolor flowcytometry. An obvious variation in apoptotic cells number was observed in presence of fenugreek extract (>80%). The results suggest that fenugreek extract can potentiate the radiation induced apoptosis or radiation toxicity in blood T-cells (P < 0.05). PMID:26284174

  19. The Effects of Fenugreek on Radiation Induced Toxicity for Human Blood T-Cells in Radiotherapy

    PubMed Central

    Tavakoli, Mohamed Bagher; Kiani, Ali; Roayaei, Mahnaz

    2015-01-01

    Many cellular damages either in normal or cancerous tissues are the outcome of molecular events affected by ionizing radiation. T-cells are the most important among immune system agents and are used for biological radiation dose measurement in recommended standard methods. The herbs with immune modulating properties may be useful to reduce the risk of the damages and subsequently the diseases. The T-cells as the most important immune cells being targeted for biological dosimetry of radiation. This study proposes a flowcytometric-method based on fluorescein isothiocyanate- and propidium iodide (PI)-labeled annexin-V to assess apoptosis in blood T-cells after irradiation in both presence and absence of fenugreek extract. T-cells peripheral blood lymphocyte isolated from blood samples of healthy individuals with no irradiated job background. The media of cultured cells was irradiated 1-h after the fenugreek extract was added. The number of apoptotic cells was assessed by annexin-V protocol and multicolor flowcytometry. An obvious variation in apoptotic cells number was observed in presence of fenugreek extract (>80%). The results suggest that fenugreek extract can potentiate the radiation induced apoptosis or radiation toxicity in blood T-cells (P < 0.05). PMID:26284174

  20. Effect of stent coating alone on in vitro vascular smooth muscle cell proliferation and apoptosis.

    PubMed

    Curcio, Antonio; Torella, Daniele; Cuda, Giovanni; Coppola, Carmela; Faniello, Maria Concetta; Achille, Francesco; Russo, Viviana G; Chiariello, Massimo; Indolfi, Ciro

    2004-03-01

    Synthetic polymers, like methacrylate (MA) compounds, have been clinically introduced as inert coatings to locally deliver drugs that inhibit restenosis after stent. The aim of the present study was to evaluate the effects of MA coating alone on vascular smooth muscle cell (VSMC) growth in vitro. Stainless steel stents were coated with MA at the following doses: 0.3, 1.5, and 3 ml. Uncoated/bare metal stents were used as controls. VSMCs were cultured in dishes, and a MA-coated stent or an uncoated bare metal stent was gently added to each well. VSMC proliferation was assessed by bromodeoxyuridine (BrdU) incorporation. Apoptosis was analyzed by three distinct approaches: 1) annexin V/propidium iodide fluorescence detection; 2) DNA laddering; and 3) caspase-3 activation and PARP cleavage. MA-coated stents induced a significant decrease of BrdU incorporation compared with uncoated stents at both the low and high concentrations. In VSMCs incubated with MA-coated stents, annexin V/propidium iodide fluorescence detection showed a significant increase in apoptotic cells, which was corroborated by the typical DNA laddering. Apoptosis of VSMCs after incubation with MA-coated stents was characterized by caspase-3 activation and PARP cleavage. The MA-coated stent induced VSMC growth arrest by inducing apoptosis in a dose-dependent manner. Thus MA is not an inert platform for eluting drugs because it is biologically active per se. This effect should be taken in account when evaluating an association of this coating with antiproliferative agents for in-stent restenosis prevention. PMID:14592937

  1. Hydropropidine: A novel, cell-impermeant fluorogenic probe for detecting extracellular superoxide

    PubMed Central

    Michalski, Radoslaw; Zielonka, Jacek; Hardy, Micael; Joseph, Joy; Kalyanaraman, Balaraman

    2013-01-01

    Here we report the synthesis and characterization of a membrane-impermeant fluorogenic probe, hydropropidine (HPr+), the reduction product of propidium iodide, for detecting extracellular superoxide (O2·−). HPr+ is a positively-charged water-soluble analog of hydroethidine (HE), a fluorogenic probe commonly used for monitoring intracellular O2·−. We hypothesized that the presence of a highly localized positive charge on the nitrogen atom would impede cellular uptake of HPr+ and allow for exclusive detection of extracellular O2·−. Our results indicate that O2·− reacts with HPr+ (k = 1.2 × 104 M−1s−1) to form exclusively 2-hydroxypropidium (2-OH-Pr++) in cell-free and cell-based systems. This reaction is analogous to the reaction between HE and O2·− (Zhao H et al. Free Radic Biol Med 34:1359-68, 2003). During the course of this investigation, we also reassessed the rate constants for the reactions of O2·− with HE and its mitochondria targeted analog (Mito-HE or Mito-SOX Red®) and addressed the discrepancies between the present values and those reported previously by us. Our results indicate that the rate constant between O2·− and HPr+ is slightly higher than that of HE and O2·− and is closer to that of Mito-HE and O2·−. Similar to HE, HPr+ undergoes oxidation in the presence of various oxidants (peroxynitrite – derived radicals, Fenton’s reagent, and ferricytochrome c) forming the corresponding propidium dication (Pr++) and the dimeric products (e.g., Pr++-Pr++). In contrast to HE, there was very little intracellular uptake of HPr+. We conclude that HPr+ is a useful probe for detecting O2·− and other one-electron oxidizing species in an extracellular milieu. PMID:23051008

  2. Natural killer cells induce eosinophil activation and apoptosis.

    PubMed

    Awad, Ali; Yassine, Hanane; Barrier, Mathieu; Vorng, Han; Marquillies, Philippe; Tsicopoulos, Anne; Duez, Catherine

    2014-01-01

    Eosinophils are potent inflammatory cells with numerous immune functions, including antigen presentation and exacerbation of inflammatory responses through their capacity to release a range of largely preformed cytokines and lipid mediators. Thus, timely regulation of eosinophil activation and apoptosis is crucial to develop beneficial immune response and to avoid tissue damage and induce resolution of inflammation. Natural Killer (NK) cells have been reported to influence innate and adaptive immune responses by multiple mechanisms including cytotoxicity against other immune cells. In this study, we analyzed the effect of the interaction between NK cells and eosinophils. Co-culture experiments revealed that human NK cells could trigger autologous eosinophil activation, as shown by up-regulation of CD69 and down-regulation of CD62L, as well as degranulation, evidenced by increased CD63 surface expression, secretion of eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN). Moreover, NK cells significantly and dose dependently increased eosinophil apoptosis as shown by annexin V and propidium iodide (PI) staining. Direct contact was necessary for eosinophil degranulation and apoptosis. Increased expression of phosphorylated extracellular signal-regulated kinase (ERK) in cocultured eosinophils and inhibition of eosinophil CD63 expression by pharmacologic inhibitors suggest that MAPK and PI3K pathways are involved in NK cell-induced eosinophil degranulation. Finally, we showed that NK cells increased reactive oxygen species (ROS) expression by eosinophils in co-culture and that mitochondrial inhibitors (rotenone and antimycin) partially diminished NK cell-induced eosinophil apoptosis, suggesting the implication of mitochondrial ROS in NK cell-induced eosinophil apoptosis. Pan-caspase inhibitor (ZVAD-FMK) only slightly decreased eosinophil apoptosis in coculture. Altogether, our results suggest that NK cells regulate eosinophil functions by inducing their activation and their apoptosis. PMID:24727794

  3. Intracellular delivery of trehalose into mammalian cells by electropermeabilization.

    PubMed

    Shirakashi, R; Köstner, C M; Müller, K J; Kürschner, M; Zimmermann, U; Sukhorukov, V L

    2002-09-01

    The disaccharide trehalose is increasingly being used as a very efficient stabilizer of cells, membranes and macromolecules during cryo- and lyoconservation. Although extracellular trehalose can reduce cryo- and lyodamage to mammalian cells, the sugar is required on both sides of the plasma membrane for maximum protection efficiency. In the present study, mouse myeloma cells were loaded with the disaccharide by means of reversible electropermeabilization in isotonic trehalose-substituted medium, which contained 290 mM trehalose as the major solute. By using the membrane-impermeable fluorescent dye propidium iodide as the reporter molecule, optimum electropulsing conditions were found, at which most permeabilized cells survived and recovered (i.e., resealed) their original membrane integrity within a few minutes after electric treatment. Microscopic examination during the resealing phase revealed that electropulsed cells shrank gradually to about 60% of their original volume. The kinetics of the dye uptake and the volumetric response of cells to electropulsing were analyzed using a theoretical model that relates the observed cell volume changes to the solute transport across the transiently permeabilized cell membrane. From the best fit of the model to the experimental data, the intracellular trehalose concentration in electropulsed cells was estimated to be about 100 mM. This loading efficiency compares favorably to other methods currently used for intracellular trehalose delivery. The results presented here point toward application of the electropermeabilization technique for loading cells with membrane-impermeable bioprotectants, with far-reaching implications for cryo- and lyopreservation of rare and valuable mammalian cells and tissues. PMID:12202951

  4. Differentiated NSC-34 cells as an in vitro cell model for VX.

    PubMed

    Kanjilal, Baishali; Keyser, Brian M; Andres, Devon K; Nealley, Eric; Benton, Betty; Melber, Ashley A; Andres, Jaclynn F; Letukas, Valerie A; Clark, Offie; Ray, Radharaman

    2014-10-01

    The US military has placed major emphasis on developing therapeutics against nerve agents (NA). Current efforts are hindered by the lack of effective in vitro cellular models to aid in the preliminary screening of potential candidate drugs/antidotes. The development of an in vitro cellular model to aid in discovering new NA therapeutics would be highly beneficial. In this regard, we have examined the response of a differentiated hybrid neuronal cell line, NSC-34, to the NA VX. VX-induced apoptosis of differentiated NSC-34 cells was measured by monitoring the changes in caspase-3 and caspase-9 activity post-exposure. Differentiated NSC-34 cells showed an increase in caspase-3 activity in a manner dependent on both time (17-23 h post-exposure) and dose (10-100 nM). The maximal increase in caspase-3 activity was found to be at 20-h post-exposure. Caspase-9 activity was also measured in response to VX and was found to be elevated at all concentrations (10-100 nM) tested. VX-induced cell death was also observed by utilizing annexin V/propidium iodide flow cytometry. Finally, VX-induced caspase-3 or -9 activities were reduced with the addition of pralidoxime (2-PAM), one of the current therapeutics used against NA toxicity, and dizocilpine (MK-801). Overall the data presented here show that differentiated NSC-34 cells are sensitive to VX-induced cell death and could be a viable in vitro cell model for screening NA candidate therapeutics. PMID:25045830

  5. Inhibitory effect of ibuprofen on tumor survival and angiogenesis in gastric cancer cell.

    PubMed

    Akrami, Hassan; Aminzadeh, Saman; Fallahi, Hossein

    2015-05-01

    Numerous epidemiological studies have suggested effectiveness of long-term and regular use of non-steroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen and aspirin, in preventing and treatment of certain cancers including prostate, colon, breast, lung, and gastric cancers. We have studied the potential anti-turmeric effect of ibuprofen in adenocarcinoma gastric cell line (AGS). The effects of ibuprofen were investigated on cell proliferation, apoptosis, angiogenesis, and expression of stemness marker genes using real-time RT-PCR, DNA laddering, and tube formation assays via ECM gel and human umbilical vein endothelial cells (HUVECs). Annexin-V-FLUOS and propidium iodide (PI) were used to stain the apoptotic cells. Our findings indicate that ibuprofen at the concentrations of 100, 200, 300, 400, and 500 μM is able to reduce the cancerous characteristics of the AGS cells by inducing apoptosis, inhibition of cell proliferation, and angiogenesis. Real-time RT-PCR showed that ibuprofen altered the expression of several genes including Akt, P53, PCNA, Bax, and Bcl2 in the AGS cells. In addition, reduction in CD44 and OCT3/4 transcript levels revealed that ibuprofen reduces the stemness of the AGS cells and therefore it could be used as a potential anti-tumor drug. PMID:25542229

  6. The furano norclerodane diterpenoid disobulbin-D induces apoptosis in normal human liver L-02 cells.

    PubMed

    Ma, Min; Jiang, Zhenzhou; Ruan, Jinlan; Tan, Xinqi; Liu, Jin; Wang, Cuifen; Zha, Xiao Ming; Zhang, Luyong

    2012-09-01

    Disobulbin-D (DBD), a hepatotoxic furano norclerodane diterpenoid, was isolated by bio-guided fractionation from the rhizome of Dioscorea bulbifera L. In working toward elucidating the cellular and molecular mechanisms of DBD toxicity, we treated normal human liver cell line L-02 cells with DBD in vitro and evaluated its toxicity in terms of cell viability, morphologic changes, induction of apoptosis/necrosis, and caspase 3 activity. The viability of L-02 cells was inhibited by DBD in a concentration and time-dependent manner. Apoptosis was supported by the Annexin V and propidium iodide assay, Hoechst 33258 staining, and the occurrence of a sub-G(1) peak. DBD can cause an increase in caspase 3 activity, and pretreatment with Ac-DEVD-CHO blocked cell death and attenuated the apoptosis, showing that DBD-induced L-02 cell apoptosis is caspase 3-dependent. These results suggest that the effects of DBD on the growth of normal human liver L-02 cells may be due to its induction of cell apoptosis, which may also explain the toxicity observed in the plants containing furano clerodane diterpenoids. PMID:21211949

  7. Single Walled Carbon Nanotubes Exhibit Dual-Phase Regulation to Exposed Arabidopsis Mesophyll Cells

    PubMed Central

    2011-01-01

    Herein we are the first to report that single-walled carbon nanotubes (SWCNTs) exhibit dual-phase regulation to Arabidopsis mesophyll cells exposed to different concentration of SWCNTs. The mesophyll protoplasts were prepared by enzyme digestion, and incubated with 15, 25, 50, 100 μg/ml SWCNTs for 48 h, and then were observed by optical microscopy and transmission electron microscopy, the reactive oxygen species (ROS) generation was measured. Partial protoplasts were stained with propidium iodide and 4'-6- diamidino-2-phenylindole, partial protoplasts were incubated with fluorescein isothiocyanate-labeled SWCNTs, and observed by fluorescence microscopy. Results showed that SWCNTs could traverse both the plant cell wall and cell membrane, with less than or equal to 50 μg/ml in the culture medium, SWCNTs stimulated plant cells to grow out trichome clusters on their surface, with more than 50 μg/ml SWCNTs in the culture medium, SWCNTs exhibited obvious toxic effects to the protoplasts such as increasing generation of ROS, inducing changes of protoplast morphology, changing green leaves into yellow, and inducing protoplast cells' necrosis and apoptosis. In conclusion, single walled carbon nanotubes can get through Arabidopsis mesophyll cell wall and membrane, and exhibit dose-dependent dual-phase regulation to Arabidopsis mesophyll protoplasts such as low dose stimulating cell growth, and high dose inducing cells' ROS generation, necrosis or apoptosis.

  8. Mouse testis cell sorting according to DNA and mitochondrial changes during spermatogenesis

    SciTech Connect

    Petit, J.M.; Ratinaud, M.H.; Cordelli, E.; Spano, M.; Julien, R.

    1995-04-01

    Flow cytometry can measure variations in DNA content and chromatin structure as well as dramatic changes in the mitochondria of germ cells during maturation from spermatogonia to elongated spermatids. Using 10-N nonyl acridine orange (NAO), an inner mitochondrial membrane dye, it is easy to follow mitochondria rearrangements. Mouse testis cells stained with the DNA fluorescent probe propidium iodide (PI) and analyzed by flow cytometry can be discriminated on the basis of their ploidy levels into five main regions corresponding to elongated spermatids, round spermatids, diploid, S-phase, and tetraploid cells. The simultaneous use of PI and NAO demonstrated the presence of cells having low and high mitochondrial content in the haploid, diploid, and tetraploid compartments. Eleven sorting windows were selected from the bivariate analysis (PI/NAO) and the corresponding cells were identified by microscopic observation. Cells were also discriminated by two parameter analysis of DNA content vs. cell diameter. The definition of seven different regions allowed us to determine NAO or rhodamine 123 (Rh 123) uptakes in each compartment. We observed that the ratio (Rh 123/NAO) dramatically changed according to the progression of cell differentiation which occurs during spermatogenesis. 45 refs., 5 figs., 2 tabs.

  9. Single Walled Carbon Nanotubes Exhibit Dual-Phase Regulation to Exposed Arabidopsis Mesophyll Cells

    NASA Astrophysics Data System (ADS)

    Yuan, Hengguang; Hu, Shanglian; Huang, Peng; Song, Hua; Wang, Kan; Ruan, Jing; He, Rong; Cui, Daxiang

    2011-12-01

    Herein we are the first to report that single-walled carbon nanotubes (SWCNTs) exhibit dual-phase regulation to Arabidopsis mesophyll cells exposed to different concentration of SWCNTs. The mesophyll protoplasts were prepared by enzyme digestion, and incubated with 15, 25, 50, 100 μg/ml SWCNTs for 48 h, and then were observed by optical microscopy and transmission electron microscopy, the reactive oxygen species (ROS) generation was measured. Partial protoplasts were stained with propidium iodide and 4'-6- diamidino-2-phenylindole, partial protoplasts were incubated with fluorescein isothiocyanate-labeled SWCNTs, and observed by fluorescence microscopy. Results showed that SWCNTs could traverse both the plant cell wall and cell membrane, with less than or equal to 50 μg/ml in the culture medium, SWCNTs stimulated plant cells to grow out trichome clusters on their surface, with more than 50 μg/ml SWCNTs in the culture medium, SWCNTs exhibited obvious toxic effects to the protoplasts such as increasing generation of ROS, inducing changes of protoplast morphology, changing green leaves into yellow, and inducing protoplast cells' necrosis and apoptosis. In conclusion, single walled carbon nanotubes can get through Arabidopsis mesophyll cell wall and membrane, and exhibit dose-dependent dual-phase regulation to Arabidopsis mesophyll protoplasts such as low dose stimulating cell growth, and high dose inducing cells' ROS generation, necrosis or apoptosis.

  10. Acetonic Extract of Buxus sempervirens Induces Cell Cycle Arrest, Apoptosis and Autophagy in Breast Cancer Cells

    PubMed Central

    Ait-Mohamed, Ouardia; Battisti, Valentine; Joliot, Véronique; Fritsch, Lauriane; Pontis, Julien; Medjkane, Souhila; Redeuilh, Catherine; Lamouri, Aazdine; Fahy, Christine; Rholam, Mohamed; Atmani, Djebbar; Ait-Si-Ali, Slimane

    2011-01-01

    Plants are an invaluable source of potential new anti-cancer drugs. Here, we investigated the cytotoxic activity of the acetonic extract of Buxus sempervirens on five breast cancer cell lines, MCF7, MCF10CA1a and T47D, three aggressive triple positive breast cancer cell lines, and BT-20 and MDA-MB-435, which are triple negative breast cancer cell lines. As a control, MCF10A, a spontaneously immortalized but non-tumoral cell line has been used. The acetonic extract of Buxus sempervirens showed cytotoxic activity towards all the five studied breast cancer cell lines with an IC50 ranging from 7.74 µg/ml to 12.5 µg/ml. Most importantly, the plant extract was less toxic towards MCF10A with an IC50 of 19.24 µg/ml. Fluorescence-activated cell sorting (FACS) analysis showed that the plant extract induced cell death and cell cycle arrest in G0/G1 phase in MCF7, T47D, MCF10CA1a and BT-20 cell lines, concomitant to cyclin D1 downregulation. Application of MCF7 and MCF10CA1a respective IC50 did not show such effects on the control cell line MCF10A. Propidium iodide/Annexin V double staining revealed a pre-apoptotic cell population with extract-treated MCF10CA1a, T47D and BT-20 cells. Transmission electron microscopy analyses indicated the occurrence of autophagy in MCF7 and MCF10CA1a cell lines. Immunofluorescence and Western blot assays confirmed the processing of microtubule-associated protein LC3 in the treated cancer cells. Moreover, we have demonstrated the upregulation of Beclin-1 in these cell lines and downregulation of Survivin and p21. Also, Caspase-3 detection in treated BT-20 and T47D confirmed the occurrence of apoptosis in these cells. Our findings indicate that Buxus sempervirens extract exhibit promising anti-cancer activity by triggering both autophagic cell death and apoptosis, suggesting that this plant may contain potential anti-cancer agents for single or combinatory cancer therapy against breast cancer. PMID:21935420

  11. Possible use of quercetin, an antioxidant, for protection of cells suffering from overload of intracellular Ca2+: a model experiment.

    PubMed

    Sakanashi, Yoko; Oyama, Keisuke; Matsui, Hiroko; Oyama, Toshihisa B; Oyama, Tomohiro M; Nishimura, Yumiko; Sakai, Hitomi; Oyama, Yasuo

    2008-08-01

    Quercetin is known to protect the cells suffering from oxidative stress. The oxidative stress elevates intracellular Ca(2+) concentration, one of the phenomena responsible for cell death. Therefore, we hypothesized that quercetin would protect the cells suffering from overload of intracellular Ca(2+). To test the hypothesis, the effects of quercetin on the cells suffering from oxidative stress and intracellular Ca(2+) overload were examined by using a flow cytometer with appropriate fluorescence probes (propidium iodide, fluo-3-AM, and annexin V-FITC) and rat thymocytes. The concentrations (1-30 microM) of quercetin to protect the cells suffering from intracellular Ca(2+) overload by A23187, a calcium ionophore, were similar to those for the cells suffering from oxidative stress by H(2)O(2). The cell death respectively induced by H(2)O(2) and A23187 was significantly suppressed by removal of external Ca(2+). Furthermore, quercetin greatly delayed the process of Ca(2+)-dependent cell death although it did not significantly affect the elevation of intracellular Ca(2+) concentration by H(2)O(2) and A23187, respectively. It is concluded that quercetin can protect the cells from oxidative injury in spite of increased concentration of intracellular Ca(2+). Results suggest that quercetin is also used for protection of cells suffering from overload of intracellular Ca(2+). PMID:18586279

  12. High-throughput optical injection of mammalian cells using a non-diffracting beam in a microfluidic platform

    NASA Astrophysics Data System (ADS)

    Rendall, Helen A.; Marchington, Robert F.; Praveen, Bavishna B.; Bergmann, Gerald; Arita, Yoshihiko; Heisterkamp, Alexander; Gunn-Moore, Frank J.; Dholakia, Kishan

    2013-03-01

    Femtosecond photoporation is an optical, non-invasive method of injecting membrane impermeable substances contained within the surrounding medium into cells. The technique typically addresses individual cells in a static monolayer. While this gives excellent selectivity, it can be time consuming or impractical to treat larger samples. We build on previous work using a microfluidic platform, which allows for a suspension of cells to be dosed with femtosecond light as they flow through a microfluidic channel. A reusuable quartz chip is designed with an 's'-bend with facilitates the delivery of a 'non-diffracting' femtosecond Bessel beam along the centre of the channel. By implementing off-chip hydrodynamic focusing, cells are confined to the central region of the channel and pass along the Bessel beam core where they are photoporated. This new parallel approach allows for higher flow rates to be used compared to the previous, orthogonal, design whilst maintaining the necessary dwell time in the Bessel beam core. Optical injection of the cell membrane impermeable stain propidium iodide has been successful with two cell lines. These have yielded viable injection efficiencies of 31.0+/-9.5% Chinese hamster ovary cells (CHO-K1) and 20.4+/-4.2% human promyelocytic cells (HL60) with a cell throughput of up to 10 cells/second. This marks an order of magnitude increase compared to the previous microfluidic design.

  13. Growth inhibition and apoptosis in cancer cells induced by polyphenolic compounds of Acacia hydaspica: Involvement of multiple signal transduction pathways.

    PubMed

    Afsar, Tayyaba; Trembley, Janeen H; Salomon, Christine E; Razak, Suhail; Khan, Muhammad Rashid; Ahmed, Khalil

    2016-01-01

    Acacia hydaspica R. Parker is known for its medicinal uses in multiple ailments. In this study, we performed bioassay-guided fractionation of cytotoxic compounds from A. hydaspica and investigated their effects on growth and signaling activity in prostate and breast cancer cell lines. Four active polyphenolic compounds were identified as 7-O-galloyl catechin (GC), catechin (C), methyl gallate (MG), and catechin-3-O-gallate (CG). The four compounds inhibited prostate cancer PC-3 cell growth in a dose-dependent manner, whereas CG and MG inhibited breast cancer MDA-MB-231 cell growth. All tested compounds inhibited cell survival and colony growth in both cell lines, and there was evidence of chromatin condensation, cell shrinkage and apoptotic bodies. Further, acridine orange, ethidium bromide, propidium iodide and DAPI staining demonstrated that cell death occurred partly via apoptosis in both PC-3 and MDA-MB-231 cells. In PC-3 cells treatment repressed the expression of anti-apoptotic molecules Bcl-2, Bcl-xL and survivin, coupled with down-regulation of signaling pathways AKT, NFκB, ERK1/2 and JAK/STAT. In MDA-MB-231 cells, treatment induced reduction of CK2α, Bcl-xL, survivin and xIAP protein expression along with suppression of NFκB, JAK/STAT and PI3K pathways. Our findings suggest that certain polyphenolic compounds derived from A. hydaspica may be promising chemopreventive/therapeutic candidates against cancer. PMID:26975752

  14. Mycobacterial di-O-acyl-trehalose inhibits mitogen- and antigen-induced proliferation of murine T cells in vitro.

    PubMed

    Saavedra, R; Segura, E; Leyva, R; Esparza, L A; López-Marín, L M

    2001-11-01

    2,3-Di-O-acyl-trehalose (DAT) is a glycolipid located on the outer layer of the Mycobacterium tuberculosis cell envelope. Due to its noncovalent linkage to the mycobacterial peptidoglycan, DAT could easily interact with host cells located in the focus of infection. The aim of the present work was to study the effects of DAT on the proliferation of murine spleen cells. DAT was purified from reference strains of M. tuberculosis, or M. fortuitum as a surrogate source of the compound, by various chromatography and solvent extraction procedures and then chemically identified. Incubation of mouse spleen cells with DAT inhibited in a dose-dependent manner concanavalin A-stimulated proliferation of the cells. Experiments, including the propidium iodide exclusion test, showed that these effects were not due to death of the cells. Tracking of cell division by labeling with 5,6-carboxyfluorescein diacetate succinimidyl ester revealed that DAT reduces the rounds of cell division. Immunofluorescence with an anti-CD3 monoclonal antibody indicated that T lymphocytes were the population affected in our model. Our experiments also suggest that the extent of the suppressive activity is strongly dependent on the structural composition of the acyl moieties in DATs. Finally, the inhibitory effect was also observed on antigen-induced proliferation of mouse spleen cells specific for Toxoplasma gondii. All of these data suggest that DAT could have a role in the T-cell hyporesponsiveness observed in chronic tuberculosis. PMID:11687444

  15. Growth inhibition and apoptosis in cancer cells induced by polyphenolic compounds of Acacia hydaspica: Involvement of multiple signal transduction pathways

    PubMed Central

    Afsar, Tayyaba; Trembley, Janeen H.; Salomon, Christine E.; Razak, Suhail; Khan, Muhammad Rashid; Ahmed, Khalil

    2016-01-01

    Acacia hydaspica R. Parker is known for its medicinal uses in multiple ailments. In this study, we performed bioassay-guided fractionation of cytotoxic compounds from A. hydaspica and investigated their effects on growth and signaling activity in prostate and breast cancer cell lines. Four active polyphenolic compounds were identified as 7-O-galloyl catechin (GC), catechin (C), methyl gallate (MG), and catechin-3-O-gallate (CG). The four compounds inhibited prostate cancer PC-3 cell growth in a dose-dependent manner, whereas CG and MG inhibited breast cancer MDA-MB-231 cell growth. All tested compounds inhibited cell survival and colony growth in both cell lines, and there was evidence of chromatin condensation, cell shrinkage and apoptotic bodies. Further, acridine orange, ethidium bromide, propidium iodide and DAPI staining demonstrated that cell death occurred partly via apoptosis in both PC-3 and MDA-MB-231 cells. In PC-3 cells treatment repressed the expression of anti-apoptotic molecules Bcl-2, Bcl-xL and survivin, coupled with down-regulation of signaling pathways AKT, NFκB, ERK1/2 and JAK/STAT. In MDA-MB-231 cells, treatment induced reduction of CK2α, Bcl-xL, survivin and xIAP protein expression along with suppression of NFκB, JAK/STAT and PI3K pathways. Our findings suggest that certain polyphenolic compounds derived from A. hydaspica may be promising chemopreventive/therapeutic candidates against cancer. PMID:26975752

  16. Identification of resting cells by dual-parameter flow cytometry of statin expression and DNA content

    SciTech Connect

    Pellicciari, C.; Mangiarotti, R.; Bottone, M.G.; Danova, M.; Wang, E.

    1995-12-01

    Statin, a 57-kDa nuclear protein, has been recognized as a unique marker of quiescent (G{sub 0}) cells; specific monoclonal antibodies (MoAb) against statin have been produced and used to label resting cells in tissue sections and in cultured cells. We present an improved method for the identification of G{sub 0} cells by dual-parameter flow cytometry of statin expression and DNA content. The appropriate technical conditions were set up by using resting and cycling human fibroblasts as a model cell system. Several fixatives proved to be suitable for the immunocytochemical detection of statin; among them, 70% ethanol was selected because this fixation procedure is suitable for DNA staining with intercalating dyes and is routinely used for the immunolabeling of proliferation markers (such as proliferating cell nuclear antigen [PCNA] and Ki-67) and of bromodeoxyuridine (BrdUrd) incorporation. Following cell permeabilization with detergent, exposure to the antistatin antibody (S-44), and indirect fluorescein isothiocyanate immunolabeling, cells were counterstained for DNA with propidium iodide and analyzed by dual-parameter flow cytometry. In cells from several animal sources (rat thymocytes and C6 glioma cells, mouse 3T3 cells, and human MCF-7 cells), under different experimental conditions, the expression of statin was found to correlate inversely with that of PCNA and Ki-67, and with the BrdUrd labeling index. In dual-parameter flow scattergrams, G{sub 0} (statin positive) cells can be discriminated from the potentially cycling (statin negative) G{sub 1} cells, i.e., within a cell fraction having the same DNA content. This approach can be envisaged as a powerful tool both for monitoring changes in the resting cell fraction and for investigating the process of G{sub 0}-G{sub 1} transition in unperturbed and drug-treated cell populations. 48 refs., 5 figs., 1 tab.

  17. Nobiletin, a Polymethoxylated Flavone, Inhibits Glioma Cell Growth and Migration via Arresting Cell Cycle and Suppressing MAPK and Akt Pathways.

    PubMed

    Lien, Li-Ming; Wang, Meng-Jiy; Chen, Ray-Jade; Chiu, Hou-Chang; Wu, Jia-Lun; Shen, Ming-Yi; Chou, Duen-Suey; Sheu, Joen-Rong; Lin, Kuan-Hung; Lu, Wan-Jung

    2016-02-01

    Nobiletin, a bioactive polymethoxylated flavone (5,6,7,8,3(') ,4(') -hexamethoxyflavone), is abundant in citrus fruit peel. Although nobiletin exhibits antitumor activity against various cancer cells, the effect of nobiletin on glioma cells remains unclear. The aim of this study was to determine the effects of nobiletin on the human U87 and Hs683 glioma cell lines. Treating glioma cells with nobiletin (20-100 µm) reduced cell viability and arrested the cell cycle in the G0/G1 phase, as detected using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and propidium iodide (PI) staining, respectively; however, nobiletin did not induce cell apoptosis according to PI-annexin V double staining. Data from western blotting showed that nobiletin significantly attenuated the expression of cyclin D1, cyclin-dependent kinase 2, cyclin-dependent kinase 4, and E2 promoter-binding factor 1 (E2F1) and the phosphorylation of Akt/protein kinase B and mitogen-activated protein kinases, including p38, extracellular signal-regulated kinase, and c-Jun N-terminal kinase. Our data also showed that nobiletin inhibited glioma cell migration, as detected by both functional wound healing and transwell migration assays. Altogether, the present results suggest that nobiletin inhibits mitogen-activated protein kinase and Akt/protein kinase B pathways and downregulates positive regulators of the cell cycle, leading to subsequent suppression of glioma cell proliferation and migration. Our findings evidence that nobiletin may have potential for treating glioblastoma multiforme. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26560814

  18. Multidrug-resistant hela cells overexpressing MRP1 exhibit sensitivity to cell killing by hyperthermia: Interactions with etoposide

    SciTech Connect

    Souslova, Tatiana; Averill-Bates, Diana A. . E-mail: averill.diana@uqam.ca

    2004-12-01

    Purpose: Multidrug resistance (MDR) remains one of the primary obstacles in cancer chemotherapy and often involves overexpression of drug efflux transporters such as P-glycoprotein and multidrug resistance protein 1 (MRP1). Regional hyperthermia is undergoing clinical investigation in combination with chemotherapy or radiotherapy. This study evaluates whether hyperthermia can reverse MDR mediated by MRP1 in human cervical adenocarcinoma (HeLa) cells. Methods and materials: Cytotoxicity of hyperthermia and/or etoposide was evaluated using sulforhodamine-B in HeLa cells overexpressing MRP1 and their drug-sensitive counterparts. Glutathione, glutathione peroxidase (GPx), and glutathione S-transferase (GST) were quantified by spectrophotometry. GST isoenzymes were quantified by immunodetection. Caspase activation was evaluated by fluorometry and chromatin condensation by fluorescence microscopy using Hoechst 33258. Necrosis was determined using propidium iodide. Results: The major finding is that HeLa and HeLaMRP cells are both sensitive to cytotoxicity of hyperthermia (41-45 deg C). Hyperthermia induced activation of caspase 3 and chromatin condensation. Although total levels of cell killing were similar, there was a switch from apoptotic to necrotic cell death in MDR cells. This could be explained by decreased glutathione and GPx in MDR cells. MDR cells also contained very low levels of GST and were resistant to etoposide-induced apoptosis. Hyperthermia caused a modest increase in etoposide-induced apoptosis in HeLa and HeLaMRP cells, which required appropriate heat-drug scheduling. Conclusions: Hyperthermia could be useful in eliminating MDR cells that overexpress MRP1.

  19. Midazolam regulated caspase pathway, endoplasmic reticulum stress, autophagy, and cell cycle to induce apoptosis in MA-10 mouse Leydig tumor cells

    PubMed Central

    So, Edmund Cheung; Chen, Yung-Chia; Wang, Shu-Chun; Wu, Chia-Ching; Huang, Man-Chi; Lai, Meng-Shao; Pan, Bo-Syong; Kang, Fu-Chi; Huang, Bu-Miin

    2016-01-01

    Purpose Midazolam is widely used as a sedative and anesthetic induction agent by modulating the different GABA receptors in the central nervous system. Studies have also shown that midazolam has an anticancer effect on various tumors. In a previous study, we found that midazolam could induce MA-10 mouse Leydig tumor cell apoptosis by activating caspase cascade. However, the detailed mechanism related to the upstream and downstream pathways of the caspase cascade, such as endoplasmic reticulum (ER) stress, autophagy, and p53 pathways plus cell cycle regulation in MA-10 mouse Leydig tumor cells, remains elusive. Methods Flow cytometry assay and Western blot analyses were exploited. Results Midazolam significantly decreased cell viability but increased sub-G1 phase cell numbers in MA-10 cells (P<0.05). Annexin V/propidium iodide double staining further confirmed that midazolam induced apoptosis. In addition, expressions of Fas and Fas ligand could be detected in MA-10 cells with midazolam treatments, and Bax translocation and cytochrome c release were also involved in midazolam-induced MA-10 cell apoptosis. Moreover, the staining and expression of LC3-II proteins could be observed with midazolam treatment, implying midazolam could induce autophagy to control MA-10 cell apoptosis. Furthermore, the expressions of p-EIF2α, ATF4, ATF3, and CHOP could be induced by midazolam, indicating that midazolam could stimulate apoptosis through ER stress in MA-10 cells. Additionally, the expressions of cyclin A, cyclin B, and CDK1 could be inhibited by midazolam, and the phosphorylation of p53, P27, and P21 could be adjusted by midazolam, suggesting that midazolam could manage cell cycle through the regulation of p53 pathway to induce apoptosis in MA-10 cells. Conclusion Midazolam could induce cell apoptosis through the activation of ER stress and the regulation of cell cycle through p53 pathway with the involvement of autophagy in MA-10 mouse Leydig tumor cells. PMID:27175086

  20. Cellometer image cytometry as a complementary tool to flow cytometry for verifying gated cell populations.

    PubMed

    Kuksin, Dmitry; Kuksin, Christina Arieta; Qiu, Jean; Chan, Leo Li-Ying

    2016-06-15

    Traditionally, many cell-based assays that analyze cell populations and functionalities have been performed using flow cytometry. However, flow cytometers remain relatively expensive and require highly trained operators for routine maintenance and data analysis. Recently, an image cytometry system has been developed by Nexcelom Bioscience (Lawrence, MA, USA) for automated cell concentration and viability measurement using bright-field and fluorescent imaging methods. Image cytometry is analogous to flow cytometry in that gating operations can be performed on the cell population based on size and fluorescent intensity. In addition, the image cytometer is capable of capturing bright-field and fluorescent images, allowing for the measurement of cellular size and fluorescence intensity data. In this study, we labeled a population of cells with an enzymatic vitality stain (calcein-AM) and a cell viability dye (propidium iodide) and compared the data generated by flow and image cytometry. We report that measuring vitality and viability using the image cytometer is as effective as flow cytometric assays and allows for visual confirmation of the sample to exclude cellular debris. Image cytometry offers a direct method for performing fluorescent cell-based assays but also may be used as a complementary tool to flow cytometers for aiding the analysis of more complex samples. PMID:27033005

  1. Tirofiban counteracts endothelial cell apoptosis through the VEGF/VEGFR2/pAkt axis.

    PubMed

    Giordano, Arturo; Romano, Simona; D'Angelillo, Anna; Corcione, Nicola; Messina, Stefano; Avellino, Raffaella; Biondi-Zoccai, Giuseppe; Ferraro, Paolo; Romano, Maria Fiammetta

    2016-05-01

    Tirofiban is used in the treatment of patients with acute coronary syndrome submitted to percutaneous coronary intervention (PCI). We have, previously, shown that tirofiban stimulates VEGF expression and promotes proliferation of endothelial cells. VEGF is a well known inhibitor of endothelial cell apoptosis. TNF-α is a pro-apoptotic cytokine released in the site of a vascular injury, including balloon angioplasty. We thought to investigate whether tirofiban was able to protect endothelial cells from cell death induced by TNF-α. For this study, we used human umbilical vein endothelial cells (HUVEC). Analysis of apoptosis was performed by propidium iodide incorporation, annexin V staining and measure of active caspase 3 levels. Western blot served for a semiquantitative measure of Akt activation, VEGF, and the pro-apoptotic Bim and Bak. Our results show that TNF-α was unable to activate caspase 3 and produce cell death in the presence of tirofiban. Activation of apoptosis was preceded by upregulation of Bim and Bak that resulted decreased after addition of tirofiban. The anti-apoptosis effect of tirofiban was reproduced by VEGF and counteracted by VEGFR2 blockade and the cation chelating agent ethylene glycol tetraacetic acid (EGTA). The use of p-Akt inhibitor, BEZ235,and Akt knockdown, suggested that pAkt mediated the prosurvival effect of tirofiban. In conclusion, tirofiban protects endothelial cells from apoptosis stimulated by TNF-α, due to its ability to stimulate VEGF production. PMID:26699078

  2. Cell death along single microfluidic channel after freeze-thaw treatments.

    PubMed

    Li, Yuhui; Wang, Fen; Wang, Hao

    2010-01-01

    Cryotherapy is a prospective green method for malignant tumor treatment. At low temperature, the cell viability relates with the cooling rate, temperature threshold, freezing interface, as well as ice formation. In clinical applications, the growth of ice ball must reach a suitable size as cells could not be all killed at the ice periphery. The cell death ratio at the ice periphery is important for the control of the freezing destruction. The mechanisms of cryoinjury around the ice periphery need thorough understanding. In this paper, a primary freeze-thaw control was carried out in a cell culture microchip. A series of directional freezing processes and cell responses was tested and discussed. The temperature in the microchip was manipulated by a thermoelectric cooler. The necrotic and apoptotic cells under different cryotreatment (duration of the freezing process, freeze-thaw cycle, postculture, etc.) were stained and distinguished by propidium iodide and fluorescein isothiocyanate (FITC)-Annexin V. The location of the ice front was recorded and a cell death boundary which was different from the ice front was observed. By controlling the cooling process in a microfluidic channel, it is possible to recreate a sketch of biological effect during the process of simulated cryosurgery. PMID:20644680

  3. Gliotoxin Isolated from Marine Fungus Aspergillus sp. Induces Apoptosis of Human Cervical Cancer and Chondrosarcoma Cells

    PubMed Central

    Nguyen, Van-Tinh; Lee, Jung Suck; Qian, Zhong-Ji; Li, Yong-Xin; Kim, Kil-Nam; Heo, Soo-Jin; Jeon, You-Jin; Park, Won Sun; Choi, Il-Whan; Je, Jae-Young; Jung, Won-Kyo

    2013-01-01

    Gliotoxin, a secondary metabolite produced by marine fungus Aspergillus sp., possesses various biological activities including anticancer activity. However, the mechanism underlying gliotoxin-induced cytotoxicity on human cervical cancer (Hela) and human chondrosarcoma (SW1353) cells remains unclear. In this study, we focused on the effect of gliotoxin induction on apoptosis, the activating expressions of caspase family enzymes in the cells. Apoptotic cell levels were measured through DAPI and Annexin V/Propidium Iodide (PI) double staining analysis. The apoptotic protein expression of Bcl-2 and caspase family was detected by Western blot in Hela and SW1353 cells. Our results showed that gliotoxin treatment inhibited cell proliferation and induced significant morphological changes. Gliotoxin induced apoptosis was further confirmed by DNA fragmentation, chromatin condensation and disrupted mitochondrial membrane potential. Gliotoxin-induced activation of caspase-3, caspase-8 and caspase-9, down-regulation of Bcl-2, up-regulation of Bax and cytochromec (cyt c) release showed evidence for the gliotoxin activity on apoptosis. These findings suggest that gliotoxin isolated from marine fungus Aspergillus sp. induced apoptosis in Hela and SW1353 cells via the mitochondrial pathway followed by downstream events leading to apoptotic mode of cell death. PMID:24368570

  4. The interaction of the carbon nanoparticles with human cell plasma membrane

    NASA Astrophysics Data System (ADS)

    Overchuk, M.; Prylutska, S.; Bilyy, Rostyslav; Prylutsky, Yu.; Ritter, U.

    2013-09-01

    The study of carbon nanostructures is a highly topical branch of bionanotechnology because of their potential application in biomedicine. Carbon nanotubes (CNTs) are known for their ability to kill tumor cells causing hyperthermia shock and can be used in photothermal therapy respectively. Also chemically modified CNTs can be used for drug delivery. The needle-like shape of CNTs allows them to penetrate into the cell plasma membrane without killing the cell. C60 fullerenes are regarded as valuable nanocarriers for different hydrophobic molecules as well as potential antiviral agents or photosensitizers. In our previous studies we have demonstrated that all types of carbon nanoparticles cause externalization of phosphatidylserine (PS) from the inner to the outer layer of the cell membrane in the small local patches (points of contact), leaving the other parts of plasma membrane PS-negative. In the current work there were studied the interactions of pristine C60 fullerenes and different types of CNTs with human blood cells (erythrocytes and Jurkat T-cells