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1

Detection of apoptotic cells using propidium iodide staining.  

PubMed

Flow cytometry assays are often used to detect apoptotic cells in in vitro cultures. Depending on the experimental model, these assays can also be useful in evaluating apoptosis in vivo. In this protocol, we describe a propidium iodide (PI) flow cytometry assay to evaluate B-cell lymphomas that have undergone apoptosis in vivo. B-cell lymphoma cells are injected into recipient mice and, on tumor formation, the mice are treated with the apoptosis inducer vorinostat (a histone deacetylase inhibitor). Tumor samples collected from the lymph nodes and/or the spleen are used to prepare a single-cell suspension that is exposed to a hypotonic solution containing the fluorochrome PI. The DNA content of the cells, now labeled with PI, is analyzed by flow cytometry. Nuclear DNA content is lost during apoptosis, resulting in a hypodiploid (or sub-G1) DNA profile during flow cytometry. In contrast, healthy cells display a sharp diploid DNA profile. PMID:25368311

Newbold, Andrea; Martin, Ben P; Cullinane, Carleen; Bots, Michael

2014-01-01

2

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...

3

Sodium chloride affects propidium monoazide action to distinguish viable cells.  

PubMed

Propidium monoazide (PMA) is a DNA-intercalating agent used to selectively detect DNA from viable cells by polymerase chain reaction (PCR). Here, we report that high concentrations (>5%) of sodium chloride (NaCl) prevents PMA from inhibiting DNA amplification from dead cells. Moreover, Halobacterium salinarum was unable to maintain cell integrity in solutions containing less than 15% NaCl, indicating that extreme halophilic microorganisms may not resist the concentration range in which PMA fully acts. We conclude that NaCl, but not pH, directly affects the efficiency of PMA treatment, limiting its use for cell viability assessment of halophiles and in hypersaline samples. PMID:22728959

Barth, Valdir C; Cattani, Fernanda; Ferreira, Carlos A S; de Oliveira, Sílvia D

2012-09-15

4

Phototoxicity of some novel porphyrin hybrids against the human leukemic cell line TF1  

Microsoft Academic Search

Photodynamic induced cytotoxicity by porphyrin-DNA cross linker\\/intercalator hybrid diads and triads has been studied on the human leukemic cell line TF-1. Cells were incubated for 1 to 4 h with these new photosensitizers and irradiated with white light. Cell survival was assessed by the propidium iodide staining, using flow cytometry analysis. A comparison of the dark and light cell survival

A. Viola; P. Mannoni; M. Chanon; M. Julliard; G. Mehta; B. G. Maiya; S. Muthusamy; T. Sambaiah

1997-01-01

5

Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells  

Microsoft Academic Search

The differentiation between live and dead bacterial cells presents an important challenge in many microbiological applications. Due to the persistence of DNA in the environment after cells have lost viability, DNA-based detection methods cannot differentiate whether positive signals originate from live or dead bacterial targets. We present here a novel chemical, propidium monoazide (PMA), that (like propidium iodide) is highly

Andreas Nocker; Ching-Ying Cheung; Anne K. Camper

2006-01-01

6

Rapid propidium monoazide PCR assay for the exclusive detection of viable Enterobacteriaceae cells in pasteurized milk.  

PubMed

Pasteurized milk is a complex food and contains numerous PCR inhibitors and can often contain high levels of dead Enterobacteriaceae cells, depending on the condition of food sanitation. Usually, propidium monoazide (PMA) or ethidium monoazide PCR techniques decrease the number of dead bacteria by up to 3.5 log to the associated dead bacteria with no treatment. However, this difference could be insufficient to completely inhibit DNA amplification in the PCR from 10(6) cells of dead Enterobacteriaceae bacteria/mL, potentially contaminated in pasteurized milk. Actually, such potentially high levels of dead Enterobacteriaceae cells in milk has prevented milk researchers from applying PMA- or ethidium monoazide PCR to the assay of viable Enterobacteriaceae cells in milk. We, therefore, developed a rapid PMA real-time PCR whose minimum levels of detection were 1.5 log cfu/PCR for Cronobacter muytjensii and Escherichia coli, and 2.5 log cfu/PCR for Salmonella enteritidis without DNA purification in milk matrices. The PMA real-time PCR allowed us to specifically detect viable Enterobacteriaceae cells (5-10 cfu/mL) in pasteurized milk (20 mL) within 7.5h of total testing time, following the hygienic guidelines for pasteurized milk in the United States and European Union. The long DNA amplification (mainly 2,451 bp) of the 16S-23S rRNA gene was completely suppressed in highly contaminated dead Enterobacteriaceae cells (7.5 log cfu of Cronobacter muytjensii) in 20 mL of pasteurized milk by 23-?M PMA treatment. Although the contamination of the PCR reaction with 5% milk usually causes great inhibition, our method led to the successful elongation of PCR from viable Enterobacteriaceae cells still in the pasteurized milk matrices finally corresponding to 2 to 4 mL of milk PCR inhibitors without a DNA purification step. To comply with current customer demands for chilled pasteurized milk at the most excellent possible quality, our new technique could enable laboratory persons in a factory to conduct rapid milk coliform testing before shipping from a factory. PMID:22720921

Soejima, T; Minami, J; Iwatsuki, K

2012-07-01

7

AICAR inhibits proliferation and induced S-phase arrest, and promotes apoptosis in CaSki cells  

Microsoft Academic Search

Aim:The aim of the present study was to determine the effect of 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) on proliferation, cell cycle, and apoptosis in the human epithelial cervical cancer cell line CaSki cells.Methods:Cell count and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay were used to determine cell proliferation and viability. Hoechst 33258 staining was conducted to distinguish the apoptotic cells. Cell cycle and Annexin-V\\/propidium iodide staining were

Tong-ju Guan; Feng-jin Qin; Jian-hai Du; Li Geng; You-yi Zhang; Min Li

2007-01-01

8

Study on norcantharidin-induced apoptosis in SMMC-7721 cells through mitochondrial pathways  

Microsoft Academic Search

Objective  To investigate the mechanism of norcantharidin (NCTD)-induced SMMC-7721 hepatoma cell apoptosis.\\u000a \\u000a \\u000a \\u000a Methods  SMMC-7721 cell growth inhibition was measured by the MTT method. Apoptosis was detected by Annexin V\\/propidium iodide staining.\\u000a The mitochondrial membrane potential was measured by flow cytometry. Western blot analysis was used to evaluate the level\\u000a of cytochrome c, caspase-3, AIF, Bcl-2 and Bax expression.\\u000a \\u000a \\u000a \\u000a \\u000a Results  NCTD inhibited SMMC-7721 cell

Xian-qian Li; Shi-he Shao; Gui-lian Fu; Xiao-hong Han; Hong Gao

2010-01-01

9

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

PubMed

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

Bae, Sungwoo; Wuertz, Stefan

2009-11-01

10

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

PubMed Central

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 ?g/ml) and PMA (51.10 ?g/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

Seinige, Diana; Krischek, Carsten; Klein, Gunter

2014-01-01

11

Comparative analysis and limitations of ethidium monoazide and propidium monoazide treatments for the differentiation of viable and nonviable campylobacter cells.  

PubMed

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 (R(2)) values of 0.99 (EMA) and 0.98 (PMA) between Campylobacter counts determined by qPCR and culture-based enumeration. EMA (10 ?g/ml) and PMA (51.10 ?g/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 (>10(4)) 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

Seinige, Diana; Krischek, Carsten; Klein, Günter; Kehrenberg, Corinna

2014-04-01

12

Interactions of the plasma needle with cells in culture  

NASA Astrophysics Data System (ADS)

A non-thermal atmospheric plasma source (plasma needle) has been developed. This plasma operates at room temperature, low voltages and power levels, so it can be applied for fine treatment of organic material. In this work the impact of the plasma needle on living cells is explored. For this purpose CHO-K1 (Chinese hamster ovary) cells in culture have been plasma-treated and their responses have been recorded by means of propidium iodide staining. Plasma treatment at low to intermediate power levels leads to damage of the DNA in the cell nucleus, which causes cell death. Characteristic features are high precision of plasma action (influenced cells are strictly localized) and induction of cell death without destroying the cell integrity. Possibilities of using plasma treatment for removal of unwanted cells (e.g. cancer cells) will be investigated.

Stoffels, E.; Broers, J. L. V.; Kunts, S.; Cornelis, R. A. A.; Caubet, V.; Ramaekers, F. C. S.

2002-10-01

13

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

PubMed

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

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

2014-03-01

14

Flow cytometric determination of radiation-induced chromosome damage and its correlation with cell survival  

SciTech Connect

Chinese hamster M3-1 cells were irradiated with several doses of x rays or ..cap alpha.. particles from /sup 238/Pu. Propidium iodide-stained chromosome suspensions were prepared at different times after irradiation; cells were also assayed for survival. The DNA histograms of these chromosomes showed increased background counts with increased doses of radiation. This increase in background was cell-cycle dependent and was correlated with cell survival. The correlation between radiation-induced chromosome damage and cell survival was the same for X rays and ..cap alpha.. particles. Data are presented which indicate that flow cytometric analysis of chromosomes of irradiated cell populations can be a useful adjunct to classical cytogenic analysis of irradiation-induced chromosomal damage by virtue of its ability to express and measure chromosomal damage not seen by classical cytogenic methods.

Welleweerd, J.; Wilder, M.E.; Carpenter, S.G.; Raju, M.R.

1984-07-01

15

Quantitative Detection of Viable Bifidobacterium bifidum BF-1 Cells in Human Feces by Using Propidium Monoazide and Strain-Specific Primers  

PubMed Central

We developed a PCR-based method to detect and quantify viable Bifidobacterium bifidum BF-1 cells in human feces. This method (PMA-qPCR) uses propidium monoazide (PMA) to distinguish viable from dead cells and quantitative PCR using a BF-1-specific primer set designed from the results of randomly amplified polymorphic DNA analysis. During long-term culture (10 days), the number of viable BF-1 cells detected by counting the number of CFU on modified MRS agar, by measuring the ATP contents converted to CFU, and by using PMA-qPCR decreased from about 1010 to 106 cells/ml; in contrast, the total number of (viable and dead) BF-1 cells detected by counting 4?,6-diamidino-2-phenylindolee (DAPI)-stained cells and by using qPCR without PMA and reverse transcription-qPCR remained constant. The number of viable BF-1 cells in fecal samples detected by using PMA-qPCR was highly and significantly correlated with the number of viable BF-1 cells added to the fecal samples, within the range of 105.3 to 1010.3 cells/g feces (wet weight) (r > 0.99, P < 0.001). After 12 healthy subjects ingested 1010.3 to 1011.0 CFU of BF-1 in a fermented milk product daily for 28 days, 104.5 ± 1.5 (mean ± standard deviation [SD]) BF-1 CFU/g was detected in fecal samples by using strain-specific selective agar; in contrast, 106.2 ± 0.4 viable BF-1 cells/g were detected by using PMA-qPCR, and a total of 107.6 ± 0.7 BF-1 cells/g were detected by using qPCR without PMA. Thus, the number of viable BF-1 cells detected by PMA-qPCR was about 50 times higher (P < 0.01) than that detected by the culture-dependent method. We conclude that strain-specific PMA-qPCR can be used to quickly and accurately evaluate viable BF-1 in feces. PMID:23354719

Fujimoto, Junji

2013-01-01

16

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

PubMed Central

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

2013-01-01

17

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

PubMed Central

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

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

2014-01-01

18

Cell cycle effects of CB30865, a lipophilic quinazoline-based analogue of the antifolate thymidylate synthase inhibitor ICI 198583 with an undefined mechanism of action.  

PubMed

CB30865 (p-[N-(7-bromo-3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl+ ++)-N-(prop-2-ynyl)amino]-N-(3-pyridylmethyl)benzamide) is a quinazoline-based pyridine-containing compound that emerged from a programme aimed at the development of thymidylate synthase (TS) inhibitors as anticancer agents. Its structure is based on the antifolate ICI 198583, but with a pyridine ring replacing the glutamate. Despite its structure, CB30865 does not act in vitro via inhibition of TS or, apparently, other known folate-dependent pathways, and extensive mechanistic studies suggest that it acts via a novel locus with respect to conventional antitumour agents. However, CB30865 is highly potent against a variety of human tumour cell lines (e.g., 50%-inhibitory concentration [IC50] values in the 1-10 nM range). Thus, the cell cycle effects of CB30865 were investigated. DNA histogram analysis of W1L2 human lymphoblastoid, L1210 murine leukaemia, and CH1 human ovarian cells (propidium iodide staining) has demonstrated that CB30865 does not cause a phase-specific arrest at concentrations that have been shown to inhibit colony formation. This is unexpected for an anticancer agent. In W1L2 cells, using bromodeoxyuridine (BrdUrd) labelling and bivariate Hoechst/ propidium iodide staining, it was revealed that 0.003-0.15 microM CB30865 (1-50 x 72 h IC50) caused cells to arrest in all phases of the cell cycle simultaneously after 20-24 h exposure. This effect was also observed in CH1 and L1210 cells, though the arrest was at slightly different times. Thus, using this technique, it has been demonstrated that CB30865 induces an unusual and delayed cell cycle arrest, which provides further evidence for a novel locus of action for this compound. PMID:9725559

Skelton, L A; Ormerod, M G; Titley, J C; Jackman, A L

1998-09-01

19

Neutrophil Adhesion to Vascular Prosthetic Surfaces Triggers Nonapoptotic Cell Death  

PubMed Central

Objective To test the hypothesis that neutrophil adhesion to expanded polytetrafluoroethylene (ePTFE) and Dacron triggers cell death. Summary Background Data Vascular prosthetic infections are intransigent clinical dilemmas associated with excessive rates of death and complications. Impaired neutrophil function has been implicated in the infection of implanted cardiovascular devices. ePTFE and Dacron are potent neutrophil stimuli able to elicit activation responses such as reactive oxygen species production independent of exogenous/soluble agonists. Reactive oxygen species that are released into the medium when neutrophils are challenged by soluble agonists are known to cause self-destruction. The authors therefore sought to examine whether neutrophil adhesion to prosthetic graft materials decreases neutrophil viability by means of reactive oxygen species production. Methods Neutrophils were adhered to surfaces for up to 6 hours. Cell viability was monitored with propidium iodide staining and lactate dehydrogenase release. Results Within 6 hours of adhesion to ePTFE and Dacron, respectively, 59% ± 11% and 44% ± 5% (n = 7) of the neutrophils were stained by propidium iodide. Indistinguishable results were obtained with plasma-coated ePTFE and Dacron. In contrast, less than 2% of the neutrophils adherent to fibrinogen-, immunoglobin-, or fetal bovine serum-coated polystyrene surfaces for 6 hours were positive for propidium iodide. The increase in membrane permeability to propidium iodide was accompanied by a two- to threefold increase in lactate dehydrogenase release. Pretreatment of neutrophils with N-acetyl-L-cysteine, cytochalasin D, or cyclosporin A significantly reduced the number of propidium iodide-positive ePTFE and Dacron adherent neutrophils. Conclusions Neutrophil adhesion to ePTFE and Dacron triggers a rapid nonapoptotic cell death. The effect of ePTFE and Dacron on neutrophil viability appears to be caused by reactive oxygen species production. The premature death of graft-adherent neutrophils provides a novel explanation of the defect in neutrophil bacterial killing associated with vascular prosthetic grafts. PMID:10749621

Nadzam, Geoffrey S.; De La Cruz, Carolyn; Greco, Ralph S.; Haimovich, Beatrice

2000-01-01

20

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

PubMed

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

Dermol, Janja; Miklav?i?, Damijan

2014-12-01

21

Pleiotropic effects of cadmium in mesangial cells.  

PubMed

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 microM CdCl(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(2+)/calmodulin-dependent protein kinase II (CaMK-II). In the present study we show that low level exposure of mesangial cells to Cd(2+) (0.5 microM) 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. PMID:19233221

Xiao, Weiqun; Liu, Ying; Templeton, Douglas M

2009-08-01

22

Pleiotropic effects of cadmium in mesangial cells  

SciTech Connect

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.

Xiao Weiqun; Liu Ying [University of Toronto, Laboratory Medicine and Pathobiology, 1 King's College Circle , Toronto, Ont., M5S 1A8 (Canada); Templeton, Douglas M. [University of Toronto, Laboratory Medicine and Pathobiology, 1 King's College Circle , Toronto, Ont., M5S 1A8 (Canada)], E-mail: doug.templeton@utoronto.ca

2009-08-01

23

Multinucleation and cell dysfunction induced by amorphous silica nanoparticles in an L-02 human hepatic cell line  

PubMed Central

Silica nanoparticles (SNPs) are one of the most important nanomaterials, and have been widely used in a variety of fields. Therefore, their effects on human health and the environment have been addressed in a number of studies. In this work, the effects of amorphous SNPs were investigated with regard to multinucleation in L-02 human hepatic cells. Our results show that L-02 cells had an abnormally high incidence of multinucleation upon exposure to silica, that increased in a dose-dependent manner. Propidium iodide staining showed that multinucleated cells were arrested in G2/M phase of the cell cycle. Increased multinucleation in L-02 cells was associated with increased generation of cellular reactive oxygen species and mitochondrial damage on flow cytometry and confocal microscopy, which might have led to failure of cytokinesis in these cells. Further, SNPs inhibited cell growth and induced apoptosis in exposed cells. Taken together, our findings demonstrate that multinucleation in L-02 human hepatic cells might be a failure to undergo cytokinesis or cell fusion in response to SNPs, and the increase in cellular reactive oxygen species could be responsible for the apoptosis seen in both mononuclear cells and multinucleated cells. PMID:24092974

Wang, Wen; Li, Yang; Liu, Xiaomei; Jin, Minghua; Du, Haiying; Liu, Ying; Huang, Peili; Zhou, Xianqing; Yuan, Lan; Sun, Zhiwei

2013-01-01

24

Decitabine inhibits the cell growth of cholangiocarcinoma in cultured cell lines and mouse xenografts  

PubMed Central

Decitabine (DAC), an inhibitor of DNA methyltransferase, demonstrates antitumor activities in various types of cancer. However, its therapeutic potential for cholangiocarcinoma (CCA), one of the most aggressive gastrointestinal malignancies, remains to be explored. The present study investigated the antiproliferative effects of DAC on CCA cells in vitro and in vivo. Human CCA cell lines, TFK-1 and QBC939, were used as models to investigate DAC on the cell growth and proliferation of CCA. Cell proliferation was evaluated by Cell Counting Kit-8 assay combined with clonogenic survival assay. Flow cytometry, Hoechst 33342/propidium iodide staining and green fluorescent protein-tagged MAP-LC3 detection were applied to determine cell cycle progression, apoptosis and autophagy. Nude mice with TFK-1 xenografts were evaluated for tumor growth following DAC treatment. DAC was observed to significantly suppress the proliferation of cultured TFK-1 and QBC939 cells, accompanied with enhanced apoptosis, autophagy and cell cycle arrest at G2/M phase. In TFK-1 mouse xenografts, DAC retarded the tumor growth and increased the survival of CCA tumor-bearing mice. PMID:25295073

WANG, BING; LI, HONGBO; YANG, RUI; ZHOU, SHUNCHANG; ZOU, SHENGQUAN

2014-01-01

25

HPC viability measurement: trypan blue versus acridine orange and propidium iodide  

Microsoft Academic Search

BACKGROUND: A reliable, validated method for rapidly determining HPC viability is essential for clinical cell en- gineering. STUDY DESIGN AND METHODS: A fluorometric cell viability assay using acridine orange and propidium io- dide (AO\\/PI) was compared to the current standard, trypan blue (TB) exclusion. Viable cells stained with AO\\/ PI fluoresce green under darkfield fluorescence micros- copy, while nonviable cells

K. Mascotti; J. McCullough; S. R. Burger

2000-01-01

26

Therapeutic and radiosensitizing effects of armillaridin on human esophageal cancer cells.  

PubMed

Background. Armillaridin (AM) is isolated from Armillaria mellea. We examined the anticancer activity and radiosensitizing effect on human esophageal cancer cells. Methods. Human squamous cell carcinoma (CE81T/VGH and TE-2) and adenocarcinoma (BE-3 and SKGT-4) cell lines were cultured. The MTT assay was used for cell viability. The cell cycle was analyzed using propidium iodide staining. Mitochondrial transmembrane potential was measured by DiOC6(3) staining. The colony formation assay was performed for estimation of the radiation surviving fraction. Human CE81T/VGH xenografts were established for evaluation of therapeutic activity in vivo. Results. AM inhibited the viability of four human esophageal cancer cell lines with an estimated concentration of 50% inhibition (IC50) which was 3.4-6.9??M. AM induced a hypoploid cell population and morphological alterations typical of apoptosis in cells. This apoptosis induction was accompanied by a reduction of mitochondrial transmembrane potential. AM accumulated cell cycle at G2/M phase and enhanced the radiosensitivity in CE81T/VGH cells. In vivo, AM inhibited the growth of CE81T/VGH xenografts without significant impact on body weight and white blood cell counts. Conclusion. Armillaridin could inhibit growth and enhance radiosensitivity of human esophageal cancer cells. There might be potential to integrate AM with radiotherapy for esophageal cancer treatment. PMID:23864890

Chi, Chih-Wen; Chen, Chien-Chih; Chen, Yu-Jen

2013-01-01

27

Therapeutic and Radiosensitizing Effects of Armillaridin on Human Esophageal Cancer Cells  

PubMed Central

Background. Armillaridin (AM) is isolated from Armillaria mellea. We examined the anticancer activity and radiosensitizing effect on human esophageal cancer cells. Methods. Human squamous cell carcinoma (CE81T/VGH and TE-2) and adenocarcinoma (BE-3 and SKGT-4) cell lines were cultured. The MTT assay was used for cell viability. The cell cycle was analyzed using propidium iodide staining. Mitochondrial transmembrane potential was measured by DiOC6(3) staining. The colony formation assay was performed for estimation of the radiation surviving fraction. Human CE81T/VGH xenografts were established for evaluation of therapeutic activity in vivo. Results. AM inhibited the viability of four human esophageal cancer cell lines with an estimated concentration of 50% inhibition (IC50) which was 3.4–6.9??M. AM induced a hypoploid cell population and morphological alterations typical of apoptosis in cells. This apoptosis induction was accompanied by a reduction of mitochondrial transmembrane potential. AM accumulated cell cycle at G2/M phase and enhanced the radiosensitivity in CE81T/VGH cells. In vivo, AM inhibited the growth of CE81T/VGH xenografts without significant impact on body weight and white blood cell counts. Conclusion. Armillaridin could inhibit growth and enhance radiosensitivity of human esophageal cancer cells. There might be potential to integrate AM with radiotherapy for esophageal cancer treatment. PMID:23864890

Chi, Chih-Wen; Chen, Chien-Chih; Chen, Yu-Jen

2013-01-01

28

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

PubMed Central

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

2014-01-01

29

Interference of Notch 2 inhibits the progression of gliomas and induces cell apoptosis by induction of the cell cycle at the G0/G1 phase.  

PubMed

Glioblastoma is the most common type of malignant brain tumor with a poor prognosis. The Notch signaling pathway is often aberrantly activated in glioma cells. In order to determine the expression of Notch 2 and to evaluate its possible prognostic value in malignant glioblastoma, specimens from 32 patients and 20 controls were analyzed using immunohistochemical staining and reverse transcription quantitative polymerase chain reaction. The expression of Notch 2 in the glioma tissues was significantly higher compared with that in the normal brain tissues (P<0.01). Subsequently, endogenous Notch 2 interference was effectively performed by specific small hairpin (sh)RNA in the glioma cancer cell line U251. The results from an MTT assay and from Annexin V?fluorescein isothiocyanate/propidium iodide staining indicated that interference of Notch 2 significantly inhibited the proliferation and induced the apoptosis of U251 cells. In addition, the cell cycle was analyzed using flow cytometry and the results revealed that Notch 2 shRNA induced cell cycle arrest at the G0/G1 phase in U251 cells. Additionally, proteins associated with the cell cycle and cell proliferation were detected using western blot analysis. The data demonstrated that the expression of P21, cyclin D and phosphorylated retinoblastoma was significantly inhibited in the Notch 2 shRNA?transfected U251 cells. The results of the present study provide further insights into the effects of Notch 2 and a molecular reference for brain tumor therapy. PMID:25338527

Yu, Hui-Ping; Qi, Song-Tao; Feng, Wen-Feng; Zhang, Guo-Zhong; Zhang, He-Ping; Tian, Jin-Jun

2015-01-01

30

Effects of nano-second electrical pulses (nsPEFs) on cell cycle progression and susceptibility at various phases  

NASA Astrophysics Data System (ADS)

Exposure to nano-second pulsed electrical fields (nsPEFs) has been shown to cause poration of external and internal cell membranes, DNA damage, and blebbing of the plasma membrane. Recovery from nsPEF exposure is likely dependent on multiple factors, including exposure parameters, length of time between pulses, and extent of cellular damage. As cells progress through the cell cycle, variations in DNA and nucleus structure, cytoskeletal arrangement, and elasticity of cell membrane could cause nsPEFs to affect cells differently during different cell cycle phases. To better understand the impact of nsPEF on cell cycle, we investigated CHO cell cycle progression following varying intensities of nsPEFexposures. Cell populations were examined post exposure (10 ns pulse trains at 100, 150, or 200kV/cm) by analysis of DNA content via propidium iodide staining and flow cytometric analysis to determine cell cycle phase. Populations exhibited arrest in G2/M phase, but not in G1 phase at 1h post-exposure that increased in severity and duration with increasing exposure dose. Recovery from arrest was complete after 12h, and populations did not exhibit an increase in apoptosis as a result of exposure. Post exposure arrest in G2/M phase may indicate that nsPEF-induced damage is not significant to cause G1 arrest or that mitotic checkpoints are more important regulators of cell cycle progression after nsPEF exposure.

Mahlke, Megan A.; Thompson, Gary; Estlack, Larry; Navara, Christopher; Ibey, Bennett L.

2013-02-01

31

Use of Propidium Monoazide for Live\\/Dead Distinction in Microbial Ecology  

Microsoft Academic Search

Received 22 December 2006\\/Accepted 14 June 2007 One of the prerequisites of making ecological conclusions derived from genetic fingerprints is that bacterial community profiles reflect the live portion of the sample of interest. Propidium monoazide is a membrane- impermeant dye that selectively penetrates cells with compromised membranes, which can be considered dead. Once inside the cells, PMA intercalates into the

Andreas Nocker; Priscilla Sossa-Fernandez; Mark D. Burr; Anne K. Camper

2007-01-01

32

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

PubMed

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

Kathiravan, Govindarajan; Sureban, Sripathi M

2009-12-01

33

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

PubMed Central

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

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

2013-01-01

34

The impact of retinoic acid treatment on the sensitivity of neuroblastoma cells to fenretinide.  

PubMed

Despite the successful introduction of 13-cis retinoic acid (13cisRA) therapy for the treatment of neuroblastoma, approximately 50% patients do not respond or experience relapse. A retinoid analogue, fenretinide [N-(4-hydroxyphenyl) retinamide; 4-HPR] can induce apoptosis in neuroblastoma cell lines and could have clinical use after therapy with 13cisRA. However, there are important questions concerning potential retinoid drug interactions which need to be addressed. The aim of this study was to investigate the influence of retinoic acid pre-treatment on fenretinide-induced apoptosis and fenretinide metabolism in neuroblastoma cell lines. Apoptosis was measured by flow cytometry of propidium iodide-stained neuroblastoma cells and a live-cell imaging assay. Intracellular fenretinide metabolism was determined by HPLC analysis. Pre-treatment of neuroblastoma cell lines with retinoic acid (RA) resulted in a significant decrease in the apoptotic response to fenretinide in three of the four lines tested. Comparison between responsive and non-responsive cell lines suggested that RA sensitivity was required to promote fenretinide resistance, and that this was mediated by up-regulation of Bcl-2 and the inhibition of pro-apoptotic fenretinide signalling pathways. Induction of the oxidative metabolism of fenretinide after RA pre-treatment did not significantly impact on intracellular parent drug levels and is unlikely to explain the decreased apoptotic response observed. The interaction between RA and fenretinide could have important implications for the scheduling of fenretinide in therapeutic protocols for neuroblastoma. PMID:21964808

Armstrong, Jane L; Martin, Shaun; Illingworth, Nicola A; Jamieson, David; Neilson, Abbie; Lovat, Penny E; Redfern, Chris P F; Veal, Gareth J

2012-01-01

35

Cytotoxic Activities of Physalis minima L. Chloroform Extract on Human Lung Adenocarcinoma NCI-H23 Cell Lines by Induction of Apoptosis  

PubMed Central

Physalis minima L. is reputed for having anticancer property. In this study, the chloroform extract of this plant exhibited remarkable cytotoxic activities on NCI-H23 (human lung adenocarcinoma) cell line at dose- and time-dependent manners (after 24, 48 and 72?h of incubation). Analysis of cell-death mechanism demonstrated that the extract exerted apoptotic programed cell death in NCI-H23 cells with typical DNA fragmentation, which is a biochemical hallmark of apoptosis. Morphological observation using transmission electron microscope (TEM) also displayed apoptotic characteristics in the treated cells, including clumping and margination of chromatins, followed by convolution of the nuclear and budding of the cells to produce membrane-bound apoptotic bodies. Different stages of apoptotic programed cell death as well as phosphatidylserine externalization were confirmed using annexin V and propidium iodide staining. Furthermore, acute exposure to the extract produced a significant regulation of c-myc, caspase-3 and p53?mRNA expression in this cell line. Due to its apoptotic effect on NCI-H23 cells, it is strongly suggested that the extract could be further developed as an anticancer drug. PMID:19541726

Leong, Ooi Kheng; Muhammad, Tengku Sifzizul Tengku; Sulaiman, Shaida Fariza

2011-01-01

36

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

PubMed

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

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

2013-01-01

37

Phototoxicity of some novel porphyrin hybrids against the human leukemic cell line TF-1.  

PubMed

Photodynamic induced cytotoxicity by porphyrin-DNA cross linker/intercalator hybrid diads and triads has been studied on the human leukemic cell line TF-1. Cells were incubated for 1 to 4 h with these new photosensitizers and irradiated with white light. Cell survival was assessed by the propidium iodide staining, using flow cytometry analysis. A comparison of the dark and light cell survival factor values suggests that irradiation has a significant effect on the toxicity at low concentrations for the porphyrin-chlorambucil diad and to a lesser extent at high concentrations for the porphyrin-acridone diad, the porphyrin-acridine diad and the porphyrin-cholic acid-chlorambucil triad. While the intrinsic antileukemic (via DNA cross-linking) activity of the chlorambucil moiety and the structural details may be responsible for the photoenhancement of the toxicity, the presence of acridine or acridone which are avid intercalators of DNA, is responsible for a similar effect seen for diads. PMID:9372615

Viola, A; Mannoni, P; Chanon, M; Julliard, M; Mehta, G; Maiya, B G; Muthusamy, S; Sambaiah, T

1997-10-01

38

Zinc- or cadmium-pre-induced metallothionein protects human central nervous system cells and astrocytes from radiation-induced apoptosis.  

PubMed

We have shown the protection of human central nervous system (CNS) cultures by zinc (Zn) or cadmium (Cd)-pre-induced metallothionein (MT) synthesis from radiation-induced cytotoxicity (lactate dehydrogenase (LDH) release and neuronal dendritic injury). The present study is to further define the types of cell death induced by different dose levels of radiation and investigate the effect of MT induction (by Zn or Cd) on radiation-induced apoptosis in primary human CNS and astrocyte cultures. Apoptosis was detected by fragmented DNA electrophoresis, TUNEL technique, and propidium iodide staining. Expression of MT protein was examined by immunofluorescent staining. Results showed that exposure of primary human CNS cultures to 15 and 30 Gy gamma-radiation predominantly induced apoptotic cell death, while exposure to 60 Gy gamma-radiation predominantly induced necrotic cell death. Normal primary human CNS cultures showed weak MT staining, while primary human CNS cultures exposed to Zn or Cd showed intense MT staining. The induced apoptotic cell death by exposure to 30 Gy gamma-radiation increased to a maximum level at 12 and 24 h, and was reduced significantly by Zn or Cd pre-induced MT. Using primary human astrocytes, the induction of MT protein by Zn or Cd was further confirmed. The enhanced MT expression also afforded a significant protection from 30 Gy gamma-ray-induced apoptosis in the primary human astrocytes. These results suggest that MT protected human CNS cells from apoptosis following ionizing radiation, probably through its antioxidant property. PMID:14687759

Cai, Lu; Iskander, Sammy; Cherian, M George; Hammond, Robert R

2004-02-01

39

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

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.

Chou, C.-T. [Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, 813, Taiwan (China); Department of Biological Sciences, National Sun Yat-sen University, 804, Taiwan (China); He Shiping [Department of Biological Sciences, National Sun Yat-sen University, 804, Taiwan (China); Jan, C.-R. [Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, 813, Taiwan (China)]. E-mail: crjan@isca.vghks.gov.tw

2007-02-01

40

DNA alteration and programmed cell death during ageing of sunflower seed  

PubMed Central

Sunflower (Helianthus annuus L.) seed viability is affected by moisture content (MC) during ageing and is related to accumulation of hydrogen peroxide and changes in energy metabolism. The aim of the present work was to investigate the effect of ageing on DNA alteration events by RAPD (random amplification of polymorphic DNA) analysis and to determine whether loss of seed viability might correspond to a controlled programmed cell death (PCD). Ageing of sunflower seeds was carried out at 35?°C for 7?d at different MCs. The higher the MC, the lower was the seed viability. RAPD analysis showed that DNA alterations occurred during ageing especially in seeds containing a high MC. In addition, PCD, as revealed by DNA fragmentation and TUNEL (terminal deoxynucleotide transferase-mediated dUTP nick-end labelling) assay, was detected in aged seeds at MCs which resulted in ?50% seed viability. At the cellular level, TUNEL assay and propidium iodide staining showed that cell death concerns all the cells of the embryonic axis. The quantification of the adenylate pool highlights mitochondrial dysfunction in aged seeds containing a high MC. The involvement of oxidative burst, mitochondria dysfunction, and PCD in seed loss of viability is proposed. PMID:21765164

El-Maarouf-Bouteau, Hayat; Mazuy, Claire; Corbineau, Francoise; Bailly, Christophe

2011-01-01

41

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

PubMed Central

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

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

2012-01-01

42

The Effect of Pioglitazone on the Alzheimer's Disease-Induced Apoptosis in Human Umbilical Vein Endothelial Cells  

PubMed Central

Background: Alzheimer's disease (AD) is a progressive neurodegenerative disease and nowadays the role of endothelial cell (EC) injury has been proposed in pathological process in AD. Peroxisome proliferator-activated receptor-? (PPAR-?) agonist has anti-inflammatory properties through activation in glial cells and improves vascular function and prevent atherosclerotic disease progression. The aim of this study is evaluation of pioglitazone effects as a drug of PPAR-? agonist on endothelial apoptosis induced by sera from AD patients. Methods: Human umbilical vein endothelial cells (HUVECs) were treated with sera from AD patients (n = 10) and sera from controls (n = 10). Apoptosis was identified by annexin V-propidium iodide staining and cell death detection kit. Apoptosis was evaluated after and before adding of 10 ?M pioglitazone on EC. Nitrite (NO2-) levels were determined in the culture supernatants. Results: Induced apoptosis by the serum of patients was inhibited markedly when pioglitazone used before treating HUVECs with the sera of AD. Also, the measurement of nitrite concentration showed significantly greater levels of dissolved NO2/NO3 metabolite in the culture media of HUVECs treated by sera of AD patients (P < 0.05), while the rate of nitric oxide significantly decreased when pioglitazone exists in culture media. Conclusion: Further studies are justified to investigate the novel role of the PPARs in the prevention of the neuronal and endothelial damage in neurological disorder and present a new therapeutic approach for Alzheimer's patients. PMID:23776725

Dehghani, Leila; Meamar, Rokhsareh; Askari, Gholamreza; Khorvash, Fariborz; Shaygannejad, Vahid; Pour, Azam Foroughi; Javanmard, Shaghayegh Haghjooy

2013-01-01

43

"Suicide" gene therapy of breast cancer cells is only cytostatic in vitro but anti-tumoral in vivo on breast MCF7-ras tumor.  

PubMed

Gene therapy with Herpes Simplex Virus thymidine kinase gene (HSV-tk) is effective in various tumor models in vitro and in vivo. We compared the efficacy of the HSV-tk gene therapy in vitro and in vivo in MCF-7 and MCF7-ras cells which form tumor in athymic mice. After viral infection, cells were treated with GCV (Ganciclovir) and live cells were counted. The in vitro treatment significantly inhibited cell growth but did not induce early and late apoptosis, measured, respectively, by annexin or by propidium iodide staining and a significant cell death. The HSV-tk/GCV treatment of MCF7-ras tumor in athymic mice showed a significant inhibition of tumor development until 60 days post-treatment. Some mice showed a complete tumor eradication without tumor regrowth after the end of treatment. In conclusion, we demonstrated that the HSV-tk/GCV system is not very efficient in vitro, but very efficient in vivo in our animal breast cancer model. PMID:15646826

Gadal, Franck; Bastias, Jorge; Wei, Ming X; Crépin, Michel

2004-01-01

44

Study on the relationship between cadmium chloride-induced adrenocortical cell of guinea pig apoptosis and stress-activated protein kinase activity.  

PubMed

Heavy metals are important environmental pollutants that affect many cellular functions. The signaling pathways that lead to apoptosis of adrenocortical cells following exposure to cadmium chloride (CdCl2) have not yet been fully clarified. We developed a primary culture of guinea pig adrenocortical cells and treated it with various concentrations of CdCl2 for different time periods. The apoptosis induced by CdCl2 was detected by fluorescein isothiocyanate-labeled annexin V and propidium iodide staining using a flow cytometer. Stress-activated protein kinase (SAPK) activities were measured by immunoprecipitation and chemiluminescence assay. After 2h of treatment, the apoptotic cell rate increased in a dose-dependent manner. The difference between the high-dose group and the control group was significant (P<0.01). The apoptosis was also found to increase in a time-dependent manner in cells treated with 50micromol/L CdCl2. Among the various treatment period groups, the difference between more than 1h-treated groups and the control group was significant (P<0.01). The SAPK activity increased with an increase in CdCl2 dose after 5min of exposure. However, after 15min of exposure to CdCl2, the SAPK activity decreased with an increase in exposure time. Our findings suggest that the SAPK signaling pathway might play an important role in CdCl2-induced apoptosis of adrenocortical cells. PMID:18585907

Min, Zhao; Xingfen, Yang; Qing, Wei; Ciyong, Lu; Tiejiang, Chen

2008-09-01

45

Intestinal permeability enhancement of levothyroxine sodium by straight chain fatty acids studied in MDCK epithelial cell line.  

PubMed

Levothyroxine sodium (T4), administered orally, is used for the treatment of hypothyroidism. T4 is a narrow therapeutic index drug with highly variable bioavailability (40-80%). The purpose of the present study was to increase the transepithelial transport of T4 using straight chain fatty acids across Madin-Darby Canine kidney (MDCK) cell line. Capric acid (C10), lauric acid (C12) and oleic acid (C18) were studied in molar ratios of 1:0.5, 1:1, 1:2 and 1:3 (T4:fatty acid). Transport of the hydrophilic marker, Lucifer yellow, was also studied. All three fatty acids proved to significantly increase T4 transport and the order of enhancement was to the effect of C12 approximately C18>C10. This Increase in transport was accompanied by reductions in transepithelial electrical resistance (TEER) values, which indicates an opening of tight junctions. Cytotoxic effects of the fatty acids were evaluated by TEER measurements, lactate dehydrogenase release, percent viability and propidium iodide staining of the cells. At the lower molar concentrations of 1:1, the fatty acids did not show any toxicity. However, C12 and C18 when added, to T4:fatty acid molar ratio of 1:2 and 1:3, respectively showed severe toxicity with irreversible damage to the cells. Hence, addition of fatty acids to T4 formulations at low concentrations can significantly improve intestinal permeability of T4 without any toxicity potentially leading to improved bioavailability. PMID:20580671

Pabla, Dimple; Akhlaghi, Fatemeh; Zia, Hossein

2010-08-11

46

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

PubMed Central

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-?B and NF-?B-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-?B. PMID:23738323

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

2013-01-01

47

Andrographolide inhibits hepatoma cells growth and affects the expression of cell cycle related proteins.  

PubMed

The present study is aimed to investigate the toxic effects of andrographolide (Andro) on hepatoma cells and elucidate its preliminary mechanisms. After cells were treated with different concentrations of Andro (0-50 micromol x L(-1)) for 24 h, cell viability was evaluated with 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay. Furthermore, after hepatoma cells (Hep3B and HepG2) were treated with different concentrations of Andro (0-30 micromol x L(-1)) for 14 d, the number of colony formation was accounted under microscope. Cell cycle related proteins such as Cdc-2, phosphorylated-Cdc-2, Cyclin B and Cyclin D1 were detected with Western blotting assay and the cell cycle was analyzed by flow cytometry using propidium iodide staining. MTT results showed that Andro induced growth inhibition of hepatoma cells in a concentration-dependent manner but had no significant effects on human normal liver L-02 cells. Andro dramatically decreased the colony formation of hepatoma cells in the concentration-dependent manner. Moreover, Andro induced a decrease of Hep3B cells at the G0-G1 phase and a concomitant accumulation of cells at G2-M phase. At the molecular level, Western blotting results showed that Andro decreased the expression of Cdc-2, phosphorylated-Cdc-2, Cyclin D1 and Cyclin B proteins in a time-dependent manner, which are all cell cycle related proteins. Taken together, the results demonstrated that Andro specifically inhibited the growth of hepatoma cells and cellular cell cycle related proteins were possibly involved in this process. PMID:20055171

Shen, Kai-Kai; Liu, Tian-Yu; Xu, Chong; Ji, Li-Li; Wang, Zheng-Tao

2009-09-01

48

Rabeprazole exhibits antiproliferative effects on human gastric cancer cell lines  

PubMed Central

Intracellular proton extrusion in gastric cancer cells has been reported to promote cancer cell survival under acidic conditions via hydrogen/potassium adenosine triphosphatase (H+/K+-ATPase). Rabeprazole is a frequently used second-generation proton pump inhibitor (PPI) that irreversibly inactivates gastric H+/K+-ATPase. Therefore, we hypothesized that rabeprazole could reduce the viability of gastric cancer cells. In the present study, four human gastric cancer cell lines and one non-cancer gastric cell line were cultured. Cell viability, the ?- and ?-subunits of H+/K+-ATPase and cellular apoptosis were analyzed by dye exclusion assay, reverse transcription-polymerase chain reaction and annexin V-fluorescein isothiocyanate/propidium iodide staining, respectively. The expression level of total extracellular signal-regulated protein kinase 1/2 (ERK 1/2) and phosphorylated-ERK protein was detected by western blot analysis. Gastric cancer cell lines were more tolerant of the acidic culture media than non-cancer cells. Administration of rabeprazole led to a marked decrease in the viability of MKN-28 cells. Exposure to rabeprazole induced significant apoptosis in AGS cells. Rabeprazole completely inhibited the phosphorylation of ERK 1/2 in the MKN-28 cells, whereas the same effect was not observed in either the KATO III or MKN-45 cells. The ERK 1/2 inhibitor, PD98059, attenuated the viability of the AGS cells. A similar antiproliferative effect was observed in the rabeprazole treatment group. In addition, PD98059 and rabeprazole were able to efficaciously inhibit the phosphorylation of ERK 1/2 in the gastric cancer cells. Therefore, it was concluded that rabeprazole can attenuate the cell viability of human gastric cancer cells through inactivation of the ERK1/2 signaling pathway. The results of the present study demonstrate that rabeprazole inhibits the viability of gastric cancer cells in vitro and may serve as a novel antineoplastic agent.

GU, MENGLI; ZHANG, YAN; ZHOU, XINXIN; MA, HAN; YAO, HANGPING; JI, FENG

2014-01-01

49

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

PubMed Central

Background Apoptotic cascades may frequently be impaired in tumor cells; therefore, the approaches to circumvent these obstacles emerge as important therapeutic modalities. Methodology/Principal Findings Our novel derivatives of chlorin e6, that is, its amide (compound 2) and boronated amide (compound 5) evoked no dark toxicity and demonstrated a significantly higher photosensitizing efficacy than chlorin e6 against transplanted aggressive tumors such as B16 melanoma and M-1 sarcoma. Compound 5 showed superior therapeutic potency. Illumination with red light of mammalian tumor cells loaded with 0.1 µM of 5 caused rapid (within the initial minutes) necrosis as determined by propidium iodide staining. The laser confocal microscopy-assisted analysis of cell death revealed the following order of events: prior to illumination, 5 accumulated in Golgi cysternae, endoplasmic reticulum and in some (but not all) lysosomes. In response to light, the reactive oxygen species burst was concomitant with the drop of mitochondrial transmembrane electric potential, the dramatic changes of mitochondrial shape and the loss of integrity of mitochondria and lysosomes. Within 3–4 min post illumination, the plasma membrane became permeable for propidium iodide. Compounds 2 and 5 were one order of magnitude more potent than chlorin e6 in photodamage of artificial liposomes monitored in a dye release assay. The latter effect depended on the content of non-saturated lipids; in liposomes consisting of saturated lipids no photodamage was detectable. The increased therapeutic efficacy of 5 compared with 2 was attributed to a striking difference in the ability of these photosensitizers to permeate through hydrophobic membrane interior as evidenced by measurements of voltage jump-induced relaxation of transmembrane current on planar lipid bilayers. Conclusions/Significance The multimembrane photodestruction and cell necrosis induced by photoactivation of 2 and 5 are directly associated with membrane permeabilization caused by lipid photodamage. PMID:20856679

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

2010-01-01

50

Sarcophine-diol, a Chemopreventive Agent of Skin Cancer, Inhibits Cell Growth and Induces Apoptosis through Extrinsic Pathway in Human Epidermoid Carcinoma A431 Cells1  

PubMed Central

Sarcophine-diol (SD), a structural modifications of sarcophine, has shown chemopreventive effects on 7,12-dimethylbenz(a)anthracene-initiated and 12-O-tetradecanoylphorbol-13-acetate-promoted skin tumor developments in mice. Tumorigenesis is associated with uncontrolled cell growth and loss of apoptosis. In the present study, the effects of SD on cell growth and apoptosis in human epidermoid carcinoma A431 cells were determined to assess whether SD could inhibit cell growth and/or induce apoptosis, thus elucidating possible mechanism of action. MTT assay was used for cell viability; bromodeoxyuridine incorporation assay was used for cell proliferation; fluorescence-activated cell sorting analysis of annexin V/propidium iodide staining and TUNEL assay were used for determining apoptotic cells; Western blot analysis was used for determining the expression of caspase-3 and colorimetric caspase activity assays were used for determination of caspase-3, -8, and -9 activity. The results showed that SD treatment at concentration of 200 to 600 µM resulted in a concentration-dependent decrease in cell viability and cell proliferation in A431 cells, which largely inhibited cell growth. Sarcophine-diol treatment induced a strong apoptosis and significantly (P < .05) increased DNA fragmentation in A431 cells. Furthermore, SD treatment significantly (P < .05) increased the activity and expression of caspase-3 through activation of upstream caspase-8 in A431 cells rather than the activation of caspase 9. Sarcophine-diol treatment is relatively much less cytotoxic in monkey kidney normal CV-1 cells. These results suggest that SD decreases cell growth and induces apoptosis through caspase-dependent extrinsic pathway in A431 cells, and this may contribute to its overall chemopreventive effects in mouse skin cancer models. PMID:19252748

Zhang, Xiaoying; Bommareddy, Ajay; Chen, Wei; Khalifa, Sherief; Kaushik, Radhey S; Fahmy, Hesham; Dwivedi, Chandradhar

2009-01-01

51

Anti-tumor activity of safranal against neuroblastoma cells  

PubMed Central

Objective: Safranal (2,6,6-trimethyl-1,3-cyclohexadiene-1-carboxaldehyde, C10H14O) is an active ingredient in the saffron, which is used in traditional medicine, and also, the biological activity of saffron in anti-cancer is in development. It has been reported to have anti-oxidant effects, but its anti-tumor effects remain uncertain. The aim of this study was to evaluate effects of safranal on anti-tumor on neuroblastoma cells. Materials and Methods: Neuroblastoma cells were cultured and exposed to safranal (0, 10, 15, 20, 50 ?g/ml). Cell proliferation was examined using the 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Apoptotic cells, cell cycle distribution, and sub-G1 fraction were analyzed using flow cytometric analysis after propidium iodide staining. Results: Safranal inhibited the growth of malignant cells in a dose-and time-dependent manner. The IC (50) values against the neuroblastoma cell line were determined as 11.1 and 23.3 ?g/ml after 24 and 48 h, respectively. Safranal induced a sub-G1 peak in the flow cytometry histogram of treated cells compared to control cells indicating that apoptotic cell death is involved in safranal toxicity. Conclusions: Our pre-clinical study demonstrated a neuroblastoma cell line to be highly sensitive to safranal-mediated growth inhibition and apoptotic cell death. Although the molecular mechanisms of safranal action are not yet clearly understood, it appears to have potential as a therapeutic agent. PMID:24991121

Samarghandian, Saeed; Shoshtari, Mohammad Ebrahim; Sargolzaei, Javad; Hossinimoghadam, Hosna; Farahzad, Jabbari Azad

2014-01-01

52

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

PubMed Central

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-?) 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

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

2011-01-01

53

Sonoporation-Induced Apoptosis and Cell Cycle Arrest: Initial Findings  

NASA Astrophysics Data System (ADS)

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.

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

2011-09-01

54

Adipocytes decrease Runx2 expression in osteoblastic cells: roles of PPAR? and adiponectin.  

PubMed

The mechanisms through which bone marrow adipocytes might influence differentiation and function of osteoblasts are not completely understood. To investigate the direct effects of bone marrow fat cells on osteoblast function, an ex vivo co-culture system was utilized comprising either primary fat cells or differentiated 3T3-L1 adipocytes and osteoblastic cells on transwells. In co-culture, both adipocytes and osteoblastic cells were differentiated into adipocytes or osteoblasts, respectively, before culturing on transwells. Co-culture with either primary fat cells or fully differentiated 3T3-L1 adipocytes significantly decreased mRNA and protein expression of runt-related transcription factor 2 (Runx2) in osteoblastic cells. An increase in mRNA and protein expression of peroxisome proliferator-activated receptor ? (PPAR?) occurred concomitantly with the reduction of Runx2 expression. Adiponectin concentration was increased in the media by co-culture. In addition, co-culture with conditioned media from fat cells increased PPAR? promoter activity and decreased Runx2 promoter activity. Knockdown of PPAR? or adiponectin receptor 1 in osteoblastic cells by siRNA prevented the down-regulation of mRNA expression of Runx2 in osteoblastic cells cultured with fully differentiated 3T3-L1 cells. Furthermore, co-transfection with PPAR? decreased Runx2 promoter activity. A marker of osteogenesis, alkaline phosphatase activity in osteoblastic cells was significantly decreased by co-culture. Annexin V/propidium iodide staining showed that co-culture did not induce apoptosis in osteoblastic cells. Thus, we conclude that adipocytes modulate key metabolic functions of osteoblasts through the release of secretory products. PPAR? plays a key role in mediating the effects of adipocytes on osteoblasts. PMID:20589837

Liu, Li-Fen; Shen, Wen-Jun; Zhang, Zhong Hua; Wang, Li Juan; Kraemer, Fredric B

2010-11-01

55

Growth inhibitory activity of cucurbitacin glucosides isolated from Citrullus colocynthis on human breast cancer cells.  

PubMed

Our aim was to study the effects of cucurbitacin glucosides extracted from Citrullus colocynthis leaves on human breast cancer cell growth. Leaves were extracted, resulting in the identification of cucurbitacin B/E glucosides. The cucurbitacin glucoside combination (1:1) inhibited growth of ER(+) MCF-7 and ER(-) MDA-MB-231 human breast cancer cell lines. Cell-cycle analysis showed that treatment with isolated cucurbitacin glucoside combination resulted in accumulation of cells at the G(2)/M phase of the cell cycle. Treated cells showed rapid reduction in the level of the key protein complex necessary to the regulation of G(2) exit and initiation of mitosis, namely the p34(CDC2)/cyclin B1 complex. cucurbitacin glucoside treatment also caused changes in the overall cell morphology from an elongated form to a round-shaped cell, which indicates that cucurbitacin treatment caused impairment of actin filament organization. This profound morphological change might also influence intracellular signaling by molecules such as PKB, resulting in inhibition in the transmission of survival signals. Reduction in PKB phosphorylation and inhibition of survivin, an anti-apoptosis family member, was observed. The treatment caused elevation in p-STAT3 and in p21(WAF), proven to be a STAT3 positive target in absence of survival signals. Cucurbitacin glucoside treatment also induced apoptosis, as measured by Annexin V/propidium iodide staining and by changes in mitochondrial membrane potential (DeltaPsi) using a fluorescent dye, JC-1. We suggest that cucurbitacin glucosides exhibit pleiotropic effects on cells, causing both cell cycle arrest and apoptosis. These results suggest that cucurbitacin glucosides might have therapeutic value against breast cancer cells. PMID:17049494

Tannin-Spitz, Tehila; Grossman, Shlomo; Dovrat, Sara; Gottlieb, Hugo E; Bergman, Margalit

2007-01-01

56

Cyanide induces different modes of death in cortical and mesencephalon cells.  

PubMed

A comparative study was conducted in rat primary cortical (CX) and mesencephalic (MC) neurons to investigate intracellular cascades activated during cyanide-induced injury and to determine the point at which the cascades diverge to produce either apoptosis or necrosis. Cyanide treatment (400 microM) for 24 h produced primarily apoptosis in CX cells, whereas the same concentration of cyanide induced predominantly necrosis in MC cells as indicated by increased propidium iodide staining and cellular lactate dehydrogenase efflux. Cyanide increased generation of cellular reactive oxygen species (ROS) in both CX and MC cells, but the rate of formation and nature of the oxidative species varied with cell type. Catalase decreased cyanide-induced ROS generation in CX but not in MC cells. Nitric oxide generation was more prominent after cyanide treatment of MC compared with CX cells. N-Methyl-D-aspartate receptors were more involved in CX apoptosis than in MC necrosis. Mitochondrial membrane potential decreased moderately in CX cells on exposure to cyanide, whereas MC cells responded with a more pronounced reduction in potential. In CX cells cyanide produced a concentration-dependent release of cytochrome c from mitochondria and increased caspase activity, whereas little change was seen in MC neurons. Thus, cyanide-induced necrosis of MC cells involved generation of excessive amounts of nitric oxide and superoxide accompanied by mitochondrial depolarization. In contrast cyanide causes a lower level of oxidative stress in CX cells, involving mainly hydrogen peroxide and superoxide, and a moderate change in mitochondrial membrane potential that lead to cytochrome c release, caspase activation, and apoptosis. PMID:12388630

Prabhakaran, K; Li, L; Borowitz, J L; Isom, G E

2002-11-01

57

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

PubMed Central

The Scutellaria species (Lamiaceae) is used as a source of flavonoids to treat a variety of diseases in traditional medicine. In spite of many reports about the cytotoxic and antitumor effects of some species of this genus, anticancer researches on one of the Iranian species S. litwinowii have not yet been conducted. The cytotoxic properties of total methanol extract of S. litwinowii and its fractions were investigated on different cancer cell lines including AGS, HeLa, MCF-7, PC12 and NIH 3T3. Meanwhile, the role of apoptosis in this toxicity was explored. The cells were cultured in DMEM medium and incubated with different concentrations of herb plant extracts. Cell viability was quantitated by MTT assay. Apoptotic cells were determined using propidium iodide staining of DNA fragmentation by flow cytometry (sub-G1 peak). Scutellaria litwinowii inhibited the growth of malignant cells in a dose-dependent manner. Among solvent fractions of S. litwinowii, the methylene chloride fraction was found to be more toxic compared to other fractions. The IC50 values of this fraction against AGS, HeLa, MCF-7 and PC12 cell lines after 24 h were determined, 121.2 ± 3.1, 40.9 ± 2.5, 115.9 ± 3.5 and 64.5 ± 3.4 ?g/ml, respectively. Scutellaria litwinowii induced a sub-G1 peak in the flow cytometry histogram of treated cells compared to control cells indicating that apoptotic cell death is involved in S. litwinowii toxicity. Scutellaria litwinowii exerts cytotoxic and proapototic effects in a variety of malignant cell lines and could be considered as a potential chemotherapeutic agent in cancer treatment. PMID:20028719

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

2011-01-01

58

Infrasound sensitizes human glioblastoma cells to cisplatin-induced apoptosis.  

PubMed

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

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

2013-11-01

59

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

PubMed

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-?B) 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-?B pathway. Celastrol down-modulated antiapoptotic proteins including Bcl-2 and survivin expression. The expression of NF-?B 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

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

2014-01-01

60

Inhibition of cell proliferation by mild hyperthermia at 43?C with Paris Saponin I in the lung adenocarcinoma cell line PC?9.  

PubMed

Rhizoma paridis is widely used for cancer therapy due to its potential involvement in the suppression of tumor growth. However, at present there is no clear explanation for the mechanism underlying the inhibitory effects of Rhizoma paridis combined with hyperthermia on tumor growth. The aim of the present study was to evaluate the effects of Paris saponin I (PSI) combined with hyperthermia on a variety of non?small cell lung cancer (NSCLC) cell lines. An MTT assay was used to determine the levels of growth inhibition. The cell cycle was analyzed using flow cytometry and cell apoptosis was analyzed with Annexin V/propidium iodide staining and the Hoechst assay. The morphology of cells during apoptosis was determined using a transmission electron microscope. The expression levels of B?cell lymphoma 2 (Bcl?2), Bcl?2?associated X protein (Bax) and caspase?3 proteins were detected using western blotting. The inhibition rates significantly increased with PSI in combination with hyperthermia at 43?C. PSI with hyperthermia at 43?C caused G2/M phase arrest and significantly induced apoptosis. The expression level of Bcl?2 decreased, while Bax expression increased following treatment with PSI with hyperthermia at 43?C. In addition, the protein expression of caspase?3 was significantly enhanced. PSI combined with hyperthermia is a potent antitumor treatment through the inhibition of proliferation of NSCLC cells and may be developed as a new antitumor therapy. PSI combined with hyperthermia significantly induced apoptosis through a multi regulatory process involving G2/M arrest and regulation of Bax, Bcl?2 and caspase?3 expression, resulting in cell death and tumor inhibition. PMID:25322761

Zhao, Pengjun; Jiang, Hao; Su, Dan; Feng, Jianguo; Ma, Shenglin; Zhu, Xinhai

2015-01-01

61

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

PubMed

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

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

2014-03-25

62

Modulation of cardiac Na+,K+-ATPase cell surface abundance by simulated ischemia-reperfusion and ouabain preconditioning  

PubMed Central

Na+,K+-ATPase and cell survival were investigated in a cellular model of ischemia-reperfusion (I/R)-induced injury and protection by ouabain-induced preconditioning (OPC). Rat neonatal cardiac myocytes were subjected to 30 min of substrate and coverslip-induced ischemia followed by 30 min of simulated reperfusion. This significantly compromised cell viability as documented by lactate dehydrogenase release and Annexin V/propidium iodide staining. Total Na+,K+-ATPase ?1- and ?3-polypeptide expression remained unchanged, but cell surface biotinylation and immunostaining studies revealed that ?1-cell surface abundance was significantly decreased. Na+,K+-ATPase-activity in crude homogenates and 86Rb+ transport in live cells were both significantly decreased by about 30% after I/R. OPC, induced by a 4-min exposure to 10 ?M ouabain that ended 8 min before the beginning of ischemia, increased cell viability in a PKC?-dependent manner. This was comparable with the protective effect of OPC previously reported in intact heart preparations. OPC prevented I/R-induced decrease of Na+,K+-ATPase activity and surface expression. This model also revealed that Na+,K+-ATPase-mediated 86Rb+ uptake was not restored to control levels in the OPC group, suggesting that the increased viability was not conferred by an increased Na+,K+-ATPase-mediated ion transport capacity at the cell membrane. Consistent with this observation, transient expression of an internalization-resistant mutant form of Na+,K+-ATPase ?1 known to have increased surface abundance without increased ion transport activity successfully reduced I/R-induced cell death. These results suggest that maintenance of Na+,K+-ATPase cell surface abundance is critical to myocyte survival after an ischemic attack and plays a role in OPC-induced protection. They further suggest that the protection conferred by increased surface expression of Na+,K+-ATPase may be independent of ion transport. PMID:23086991

Belliard, Aude; Sottejeau, Yoann; Duan, Qiming; Karabin, Jessa L.

2013-01-01

63

Molecular mechanism of epigallocatechin-3-gallate in human esophageal squamous cell carcinoma in vitro and in vivo.  

PubMed

Epigallocatechin-3-gallate (EGCG), the major polyphenol of green tea, has been shown to inhibit proliferation in various types of tumors. However, few studies concerning the role and mechanism of EGCG in esophageal squamous cell carcinoma are available. Therefore, the antitumor mechanism of EGCG needs to be investigated. The present study aimed to examine the antitumor effect of EGCG on the human esophageal squamous cell carcinoma cell lines, Eca-109 and Te-1, in vitro and in vivo. Cell viability was assessed using the MTT assay and tumor formation and growth in murine xenograft models with or without EGCG treatment. Cell cycle analysis and levels of reactive oxygen species (ROS) were detected using flow cytometry. Apoptosis was measured by Annexin/propidium iodide staining. Caspase-3 cleavage and vascular endothelial growth factor (VEGF) expression were detected using western blot analysis and immunohistochemistry in tumor cell lines and tumor xenografts, respectively. The results showed that EGCG inhibited proliferation in the Eca-109 and Te-1 cells in a time- and dose-dependent manner. Tumor cells were arrested in the G1 phase and apoptosis was accompanied by ROS production and caspase-3 cleavage. In a mouse model, EGCG significantly inhibited the growth of Eca-109 tumors by increasing the expression of cleaved-caspase-3 and decreasing VEGF protein levels. Taken together, the results suggest that EGCG inhibits proliferation and induces apoptosis through ROS production, caspase-3 activation, and a decrease in VEGF expression in vitro and in vivo. Furthermore, EGCG may have future clinical applications for novel approaches to treat esophageal squamous cell carcinoma. PMID:25333353

Liu, Lifeng; Hou, Lei; Gu, Shanzhi; Zuo, Xiaoxiao; Meng, Du; Luo, Minna; Zhang, Xiaojin; Huang, Shangke; Zhao, Xinhan

2015-01-01

64

2-Methoxyestradiol-bis-sulphamate refrains from inducing apoptosis and autophagy in a non-tumorigenic breast cell line  

PubMed Central

Background Anticancer research resulted in the discovery of a promising antimitotic metabolite, 2-methoxyestradiol. 2-Methoxyestradiol-bis-sulphamate, a bis-sulphamoylated analogue exerts antiproliferative- and antimitotic activity. Investigating the anticancer potential of 2-methoxyestradiol-bis-sulphamate requires demonstrating the influence of 2-methoxyestradiol-bis-sulphamate on non-tumorigenic cells. This project focused on the in vitro effects of 2-methoxyestradiol-bis-sulphamate on the non-tumorigenic MCF-12A breast epithelial cell line. Methods The in vitro influence of 2-methoxyestradiol-bis-sulphamate was investigated on cell cycle progression, possible induction of apoptosis and autophagy and reactive oxygen species generation. Cell cycle progression was done using flow cytometry in conjunction with ethanol fixation and propidium iodide staining. Displaying effects on the mitochondrial membrane potential was achieved utilizing flow cytometry and the MitoCapture TM Mitochondrial apoptosis detection kit. Autophagy detection was done by means of flow cytometry and anti-LC3B conjugated to DyLight 488. Reactive oxygen species generation was conducted employing flow cytometry and 2,7-dichlorofluorescein diacetate and hydroethidine. Results This study demonstrated that 2-methoxyestradiol-bis-sulphamate did not affect cell cycle progression or reactive oxygen species in a statistically significant manner in the non-tumorigenic MCF-12A cell line. In addition, 2-methoxyestradiol-bis-sulphamate did not statistically significantly induce apoptosis or autophagy. Conclusion Reports indicate that 2-methoxyestradiol-bis-sulphamate induces apoptosis and autophagy in several tumorigenic cell lines. The anticancer ability of 2-methoxyestradiol-bis-sulphamate is due to its antimitotic activity. However, this study demonstrates the promising notion that 2-methoxyestradiol-bis-sulphamate does not affect the non-tumorigenic MCF-12A cells. This project contributes to the embedded scientific knowledge regarding the differential death mechanisms used by 2-methoxyestradiol-bis-sulphamate on tumorigenic and non-tumorigenic cell lines. PMID:22905730

2012-01-01

65

Tanacetum polycephalum (L.) Schultz-Bip. induces mitochondrial-mediated apoptosis and inhibits migration and invasion in MCF7 cells.  

PubMed

Tanacetum polycephalum (L.) Schultz-Bip (Mokhaleseh) has been traditionally used in the treatment of headaches, migraines, hyperlipidemia and diabetes. The present study aimed to evaluate its anticancer properties and possible mechanism of action using MCF7 as an in vitro model. T. polycephalum leaves were extracted using hexane, chloroform and methanol solvents and the cytotoxicity was evaluated using the MTT assay. Detection of the early apoptotic cells was investigated using acridine orange/propidium iodide staining. An Annexin-V-FITC assay was carried out to observe the phosphatidylserine externalization as a marker for apoptotic cells. High content screening was applied to analyze the cell membrane permeability, nuclear condensation, mitochondrial membrane potential (MMP) and cytochrome c release. Apoptosis was confirmed by using caspase-8, caspase-9 and DNA laddering assays. In addition, Bax/Bcl-2 expressions and cell cycle arrest also have been investigated. MTT assay revealed significant cytotoxicity of T. Polycephalum hexane extract (TPHE) on MCF7 cells with the IC50 value of 6.42±0.35 µg/mL. Significant increase in chromatin condensation was also observed via fluorescence analysis. Treatment of MCF7 cells with TPHE encouraged apoptosis through reduction of MMP by down-regulation of Bcl-2 and up-regulation of Bax, triggering the cytochrome c leakage from mitochondria to the cytosol. The treated MCF7 cells significantly arrested at G1 phase. The chromatographic analysis elicited that the major active compound in this extract is 8?-hydroxy-4?,15-dihydrozaluzanin C. Taken together, the results presented in this study demonstrated that the hexane extract of T. Polycephalum inhibits the proliferation of MCF7 cells, resulting in the cell cycle arrest and apoptosis, which was explained to be through the mitochondrial pathway. PMID:24995928

Karimian, Hamed; Mohan, Syam; Moghadamtousi, Soheil Zorofchian; Fadaeinasab, Mehran; Razavi, Mahboubeh; Arya, Aditya; Kamalidehghan, Behnam; Ali, Hapipah Mohd; Noordin, Mohamad Ibrahim

2014-01-01

66

The mechanism of bifonazole-induced [Ca(2+)]i rises and non-Ca(2+)-triggered cell death in PC3 human prostate cancer cells.  

PubMed

Abstract Bifonazole is an antifungal drug widely used for treating skin diseases. The effect of bifonazole on physiology of cancer cells is unclear. The effect of bifonazole on cytosolic free Ca(2+) concentrations ([Ca(2+)]i) and viability in PC3 human prostate cancer cells was explored. The Ca(2+)-sensitive fluorescent dye, fura-2, was applied to measure [Ca(2+)]i. Bifonazole at concentrations of 5-30?µM induced a [Ca(2+)]i rise in a concentration-dependent manner. The response was reduced by 50% by removing extracellular Ca(2+). Bifonazole-evoked [Ca(2+)]i rise was not altered by nifedipine, econazole, SK&F96365 and protein kinase C activator, but was inhibited by 75% by GF109203X, a protein kinase C inhibitor. In Ca(2+)-free medium, treatment with the endoplasmic reticulum Ca(2+) pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) nearly abolished bifonazole-evoked [Ca(2+)]i rise. Conversely, treatment with bifonazole abolished BHQ-evoked [Ca(2+)]i rise. Inhibition of phospholipase C with U73122 abolished bifonazole-induced [Ca(2+)]i rise. At 30-100?µM, bifonazole decreased cell viability concentration-dependently, which was not reversed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N?,N'-tetraacetic acid/acetoxy methyl. Annexin V/propidium iodide staining data suggest that bifonazole (30-100?µM) induced apoptosis concentration-dependently. Together, in PC3 human prostate cancer cells, bifonazole induced [Ca(2+)]i rises by inducing phospholipase C- and protein kinase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) influx via non-store-operated pathways. Bifonazole induced cell death that might involve apoptosis. PMID:24849495

Cheng, Jin Shiung; Chou, Chiang-Ting; Liang, Wei-Zhe; Kuo, Chun-Chi; Shieh, Pochuen; Kuo, Daih-Huang; Jan, Chung-Ren

2014-12-01

67

Inhibition of the p38 MAPK pathway sensitizes human gastric cells to doxorubicin treatment in vitro and in vivo.  

PubMed

Gastric cancer is the second most common cause of cancer-related deaths worldwide. Doxorubicin-based chemotherapeutic regimes have been the mainstay of systemic treatment for disseminated gastric cancer for numerous years. However, the ef?cacy of doxorubicin is severely limited due to chemoresistance. Chemoresistance is a tightly regulated process, under the control of numerous signal transduction pathways. Amongst these, the mitogen-activated protein kinase (MAPK) pathway has received much attention. This study assessed whether the p38 MAPK pathway is involved in doxorubicin resistance in gastric cancer cells. Doxorubicin alone or combined with the p38 MAPK pathway inhibitor SB203580 was used to treat gastric cancer cells (SGC7901 and BGC823 lines). The effect of doxorubicin on the growth and apoptosis of gastric cancer cells in the presence or absence of SB203580 was investigated by western blot analysis, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Hoechst staining, Annexin V-FITC/propidium iodide staining followed by ?ow cytometry analysis, and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Next, the effects of doxorubicin and SB203580, on the sensitivity of BGC-823 cells were assessed in a tumor xenograft model. The results showed that the p38 MAPK inhibitor significantly increases gastric cancer cell sensitivity to doxorubicin. Doxorubicin in combination with SB203580 significantly reduced cell viability (P<0.01) and increased cell death (P<0.01), which may be associated with the inactivation of the p38 MAPK signaling pathway, followed by the induced expression of the pro-apoptotic protein Bax and a concomitant decrease in Bcl-2 expression. These findings suggest that p38 MAPK is involved in gastric cancer cell survival, and that the inhibition of p38 MAPK signaling can reduce the tolerance of gastric cancer cells to doxorubicin treatment. PMID:25270341

Tan, Wei; Yu, Hong-Gang; Luo, He-Sheng

2014-12-01

68

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

PubMed Central

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

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

2012-01-01

69

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

PubMed

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

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

2012-11-01

70

Cadmium induces apoptotic cell death in WI 38 cells via caspase-dependent Bid cleavage and calpain-mediated mitochondrial Bax cleavage by Bcl-2-independent pathway.  

PubMed

Previous reports have demonstrated that cadmium (Cd) may induce cell death via apoptosis, but the mechanism responsible for cellular death is not clear. In this study, we investigated the signaling pathways implicated in Cd-induced apoptosis in lung epithelial fibroblast (WI 38) cells. Apoptotic features were observed using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, propidium iodide staining and DNA laddering. A treatment of cadmium caused the caspase-8-dependent Bid cleavage, the release of cytochrome c (Cyt c), activation of caspase-9 and -3, and PARP cleavage. A caspase-8 specific inhibitor prevented the Bid cleavage, caspase-3 activation and cell death. Alternatively, we observed that full-length Bax was cleaved into 18-kDa fragment (p18/Bax); this was initiated after 12 h and by 36 h the full-length Bax protein was totally cleaved to the p18/Bax, which caused a drastic release of Cyt c from mitochondria. The p18/Bax was detected exclusively in the mitochondrial fraction, and it originated from mitochondrial full-length Bax, but not from the cytosol full-length Bax. Cd also induced the activation of the mitochondrial 30-kDa small subunit of calpain that was preceded by Bax cleavage. Cd induced the upregulation of Bcl-2 and the degradation of p53 protein. N-acetyl cysteine effectively inhibited the Cd-induced DeltaPsim reduction, indicating ROS acts upstream of mitochondrial membrane depolarization. Taken together, our results suggest that Cd-induced apoptosis was thought to be mediated at least two pathways; caspase-dependent Bid cleavage, and the other is calpain-mediated mitochondrial Bax cleavage. Moreover, we found that the function of Bid and Bax was not dependent of Bcl-2, and that ROS can also contribute in the Cd-induced cell death. PMID:15450950

Oh, Seon-Hee; Lee, Byung-Hoon; Lim, Sung-Chul

2004-11-01

71

Effect of the pesticide, deltamethrin, on Ca2+ signaling and apoptosis in OC2 human oral cancer cells.  

PubMed

Deltamethrin is a synthetic pyrethroid insecticide used extensively in pest control. Although deltamethrin has been shown to induce cytosolic free Ca(2+) concentration ([Ca(2+)]i) rises and apoptosis in different cancer cells, there is no information concerning the effects of deltamethrin on oral cancer. This study explored the effects of deltamethrin on [Ca(2+)]i and viability in OC2 human oral cancer cells. Deltamethrin, at concentrations of 5-10??M, increased [Ca(2+)]i in a concentration-dependent manner. The Ca(2+) signal was reduced partly by removing extracellular Ca(2+). Deltamethrin-induced [Ca(2+)]i rise was not inhibited by econazole, SK&F96365, phorbol 12-myristate 13 acetate (PMA) or GF109203X, but was inhibited by nifedipine. In Ca(2+)-free medium, 10-?M deltamethrin pretreatment inhibited the [Ca(2+)]i rise induced by the endoplasmic reticulum Ca(2+) pump inhibitor, 2,5-di-tert-butylhydroquinone (BHQ). Conversely, pretreatment with BHQ inhibited deltamethrin-induced [Ca(2+)]i rise. Inhibition of inositol 1,4,5-trisphosphate formation with phospholipase C (PLC) inhibitor U73122 did not suppress deltamethrin-induced Ca(2+) release. At concentrations between 20 and 100??M, deltamethrin killed cells in a concentration-dependent manner. The cytotoxic effect of deltamethrin was not reversed by prechelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl. Deltamethrin-induced cell death was not caused by a preceding [Ca(2+)]i rise. Annexin V/propidium iodide staining data suggest that deltamethrin (40-60??M) induced apoptosis in a concentration-dependent manner. To conclude, in OC2 cells, deltamethrin evoked a [Ca(2+)]i rise by inducing PLC-independent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry by nifedipine-sensitive Ca(2+) channels. Further, deltamethrin induced Ca(2+)-independent cell death might involve apoptosis. PMID:23829777

Chi, Chao-Chuan; Chou, Chiang-Ting; Liang, Wei-Zhe; Jan, Chung-Ren

2014-01-01

72

FoxP3 provides competitive fitness to CD4?CD25? T cells in leprosy patients via transcriptional regulation.  

PubMed

Leprosy is a chronic infectious disease caused by Mycobacterium leprae. FoxP3 have been shown to have important implications in various diseases. The present study describes the mechanism of action of FoxP3 in CD4?CD25? T cells derived from leprosy patients. Increased molecular interactions of FoxP3 with histone deacetylases 7/9 in the nucleus of CD4?CD25? T cells derived from borderline lepromatous leprosy/lepromatous leprosy (BL/LL) patients were found to be responsible for FoxP3-driven immune suppression activities during the progression of leprosy. Further, downregulation of CTLA-4 and CD25 genes in siFoxP3-treated PBMCs derived from BL/LL patients elucidated the transcription-activating nature of FoxP3. This observation was supported by direct binding of FoxP3 to the promoter region of the CTLA-4 and CD25 genes, and FoxP3's molecular interaction with histone acetyl transferases. The study also revealed that the increased expression of miR155 in CD4?CD25? cells from BL/LL governs the competitive fitness of these cells. Again, reduced Annexin V & propidium iodide staining and Nur77 expression, and concomitantly increased Ki-67 positivity suggested that CD4?CD25? cells derived from BL/LL patients are more competitively fit than those from borderline tuberculoid leprosy/tuberculoid leprosy and healthy controls. Taken together, the study shows the orchestration of FoxP3 leading to competitive fitness of Treg cells in leprosy. PMID:24214631

Kumar, Sudhir; Naqvi, Raza Ali; Ali, Riyasat; Rani, Richa; Khanna, Neena; Rao, D N

2014-02-01

73

Autophagy limits the cytotoxic effects of the AKT inhibitor AZ7328 in human bladder cancer cells  

PubMed Central

Background: Mutations that activate the PI3K/AKT/mTOR pathway are relatively common in urothelial (bladder) cancers, but how these pathway mutations affect AKT dependency is not known. We characterized the relationship between AKT pathway mutational status and sensitivity to the effects of the selective AKT kinase inhibitor AZ7328 using a panel of 12 well-characterized human bladder cancer cell lines. Methods: Sequenome DNA sequencing was performed to identify mutations in a panel of 12 urothelial cancer cell lines. Drug-induced proliferative inhibition and apoptosis were quantified using MTT assays and propidium iodide staining with FACS analyses. Protein activation via phosphorylation was measured by immunoblotting. Autophagy was measured by LC3 immunofluorescence and immunoblotting. Results: AZ7328 inhibited proliferation and AKT substrate phosphorylation in a concentration-dependent manner but had minimal effects on apoptosis. Proliferative inhibition correlated loosely with the presence of activating PIK3CA mutations and was strengthened in combination with the mTOR inhibitor rapamycin. AZ7328 induced autophagy in some of the lines, and in the cells exposed to a combination of AZ7328 and chemical autophagy inhibitors apoptosis was induced. Conclusions: The cytostatic effects of AZ7328 correlate with PIK3CA mutations and are greatly enhanced by dual pathway inhibition using an mTOR inhibitor. Furthermore, AZ7328 can interact with autophagy inhibitors to induce apoptosis in some cell lines. Overall, our results support the further evaluation of combinations of PI3K/AKT/mTOR pathway and autophagy inhibitors in pre-clinical in vivo models and ultimately in patients with PIK3CA mutant bladder cancers. PMID:22895070

Dickstein, Rian J.; Nitti, Giovanni; Dinney, Colin P.; Davies, Barry R.; Kamat, Ashish M.; McConkey, David J.

2012-01-01

74

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

PubMed Central

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

Berrington, Danielle; Lall, Namrita

2012-01-01

75

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

PubMed

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

Berrington, Danielle; Lall, Namrita

2012-01-01

76

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

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 µM) 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

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

77

Hyperglycemia alters PI3k and Akt signaling and leads to endothelial cell proliferative dysfunction  

PubMed Central

Diabetes mellitus is a major risk factor for the development of vascular complications. We hypothesized that hyperglycemia decreases endothelial cell (EC) proliferation and survival via phosphatidylinositol 3-kinase (PI3k) and Akt signaling pathways. We cultured human umbilical vein ECs (HUVEC) in 5, 20, or 40 mM d-glucose. Cells grown in 5, 20, and 40 mM mannitol served as a control for osmotic effects. We measured EC proliferation for up to 15 days. We assessed apoptosis by annexin V and propidium iodide staining and flow cytometry, analyzed cell lysates obtained on culture day 8 for total and phosphorylated PI3k and Akt by Western blot analysis, and measured Akt kinase activity using a GSK fusion protein. HUVEC proliferation was also tested in the presence of pharmacological inhibitors of PI3k-Akt (wortmannin and LY294002) and after transfection with a constitutively active Akt mutant. ECs in media containing 5 mM d-glucose (control) exhibited log-phase growth on days 7–10. d-Glucose at 20 and 40 mM significantly decreased proliferation versus control (P < 0.05 for both), whereas mannitol did not impair EC proliferation. Apoptosis increased significantly in HUVEC exposed to 40 mM d-glucose. dGlucose at 40 mM significantly decreased tyrosine-phosphorylated PI3k, threonine 308-phosphorylated-Akt, and Akt activity relative to control 5 mM d-glucose. Pharmacological inhibition of PI3k-Akt resulted in a dose-dependent decrease in EC proliferation. Transfection with a constitutively active Akt mutant protected ECs by enhancing proliferation when grown in 20 and 40 mM d-glucose. We conclude that d-glucose regulates Akt signaling through threonine phosphorylation of Akt and that hyperglycemia-impaired PI3k-Akt signaling may promote EC proliferative dysfunction in diabetes. PMID:15964918

Varma, Shubha; Lal, Brajesh K.; Zheng, Ruifang; Breslin, Jerome W.; Saito, Satoshi; Pappas, Peter J.; Hobson, Robert W.; Duran, Walter N.

2006-01-01

78

Effect of the phytoestrogen, genistein-8-C-glucoside, on Chinese hamster ovary cells in vitro.  

PubMed

Genistein-8-C-glucoside (G8CG) belongs to isoflavones, which are a subclass of flavonoids, a large group of polyphenolic compounds widely distributed in plants. A number of studies on flavonoids show their cardioprotective and antiosteoporosis properties in in vitro and in vivo models. As a phytoestrogen, genistein has recently generated interest as a potential anticancer and antiatherogenic agent. Several flavonoids are known as antioxidants and scavengers of free oxygen radicals. In the current investigation we used glycosylated genistein (genistein-8-C-glucoside) from flowers of lupine (Lupinus luteus L.). Many authors have found that the action of genistein is not so simple, although many reports conducted in vitro have demonstrated that it is cytotoxic and genotoxic. Therefore, the cytotoxic and genotoxic effects of this compound in Chinese hamster ovary cells (line CHO) were studied. A colorimetric MTT assay to assess cytotoxicity and a Comet assay for the detection of DNA damage were used. Apoptosis was determined by the Hoechst 33258/propidium iodide staining technique. We have also demonstrated antioxidant properties of G8CG. The level of reactive oxygen species generated by G8CG alone and/or H2O2 was evaluated with fluorescence probes: dichlorofluorescein-diacetate (DCFDA) by flow cytometry. The cells were exposed to various concentrations of genistein-8-C-glucoside (1-290 microM) and hydrogen peroxide (10-130 microM) and the effect of G8CG alone or in combination with H2O2 was determined. The results reveal that G8CG at concentrations higher than 10 microM significantly reduced cell viability, induced apoptosis and DNA damage. However at lower concentrations (5 and 7.5 microM), G8CG showed antioxidant properties, but had no cytotoxic or genotoxic activity. PMID:17601753

Rucinska, Agata; Kirko, Sergej; Gabryelak, Teresa

2007-11-01

79

Protection of primary glial cells by grape seed proanthocyanidin extract against nitrosative/oxidative stress.  

PubMed

Previous studies showed that proanthocyanidins provide potent protection against oxidative stress. Here we investigate the effects of grape seed proanthocyanidin extract (GSPE) as a novel natural antioxidant on the generation and fate of nitric oxide (NO) in rat primary glial cell cultures. GSPE treatment (50 mg/L) increased NO production (measured by NO(2-) assay) by stimulation of the inducible isoform of NOS. However, GSPE failed to affect the LPS/IFN-gamma-induced NO production or iNOS expression. Similar responses were found in the murine macrophage cell line RAW264.7. GSPE did not show any effect on dihydrodichlorofluorescein fluorescence (ROS marker with high sensitivity toward peroxynitrite) either in control or in LPS/IFN-gamma-induced glial cultures even in the presence of a superoxide generator (PMA). GSPE treatment alone had no effect on the basal glutathione (GSH) status in glial cultures. Whereas the microglial GSH level declined sharply after LPS/IFN-gamma treatment, the endogenous GSH pool was protected when such cultures were treated additionally with GSPE, although NO levels did not change. Glial cultures pretreated with GSPE showed higher tolerance toward application of hydrogen peroxide (H(2)O(2)) and tert-butylhydroperoxide. Furthermore, GSPE-pretreated glial cultures showed improved viability after H(2)O(2)-induced oxidative stress demonstrated by reduction in lactate dehydrogenase release or propidium iodide staining. We showed that, in addition to its antioxidative property, GSPE enhances low-level production of intracellular NO in primary rat astroglial cultures. Furthermore, GSPE pretreatment protects the microglial GSH pool during high output NO production and results in an elevation of the H(2)O(2) tolerance in astroglial cells. PMID:11292363

Roychowdhury, S; Wolf, G; Keilhoff, G; Bagchi, D; Horn, T

2001-04-01

80

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

PubMed Central

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 ?M) 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-3? (GSK-3?) 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-?? p65 translocation and production of proinflammatory cytokines (IL-6 and TNF-?). 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

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

2014-01-01

81

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

PubMed

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

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

2014-06-01

82

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

PubMed

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

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

2013-01-01

83

Atorvastatin attenuates homocysteine-induced apoptosis in human umbilical vein endothelial cells via inhibiting NADPH oxidase-related oxidative stress-triggered p38MAPK signaling  

PubMed Central

Aim: To examine the effect of atorvastatin on homocysteine (Hcy)-induced reactive oxygen species (ROS) production and apoptosis in human umbilical vein endothelial cells (HUVECs). Methods: HUVECs were cultured with Hcy (0.1?5 mmol/L) in the presence or absence of atorvastatin (1?100 ?mol//L) or various stress signaling inhibitors, including the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium (DPI, 10 ?mol/L), the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 (10 ?mol/L) and antioxidants N-acetyl cysteine (NAC, 1 mmol/L). Cell apoptosis was evaluated by Annexin V/propidium iodide staining and flow cytometry. ROS were detected by 2?,7?-dichlorodihydrofluorescein diacetate (H2DCFH-DA). NADPH oxidases were evaluated with lucigenin-enhanced chemiluminescence. Hcy-induced expression of p38MAPK protein was measured by Western blotting analysis. Results: Atorvastatin inhibited endothelial cell apoptosis induced by 1 mmol/L Hcy in a dose-dependent manner and the maximal inhibitory effect was reached at 100 ?mol/L. Atorvastatin (10 ?mol/L) significantly suppressed Hcy (1 mmol/L for 30 min) induced ROS accumulation (3.17±0.33 vs 4.34±0.31, P<0.05). Atorvastatin (10 ?mol/L) also antagonized Hcy (1 mmol/L for 30 min) induced activation of NADPH oxidase (2.57±0.49 vs 3.33±0.6, P<0.05). Furthermore, atorvastatin inhibited Hcy-induced phosphorylation of p38 MAPK (1.7±0.1 vs 2.22±0.25, P<0.05), similar effects occurred with DPI, NAC and SB203580. Conclusion: Atorvastatin may inhibit Hcy-induced ROS accumulation and endothelium cell apoptosis through an NADPH oxidase and/or p38MAPK-dependent mechanisms, all of which may contribute to atorvastatin-induced beneficial effect on endothelial function. PMID:19767766

Bao, Xiao-mei; Wu, Chun-fang; Lu, Guo-ping

2009-01-01

84

Detecting inactivated endospores in fluorescence microscopy using propidium monoazide  

NASA Astrophysics Data System (ADS)

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.

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

2012-04-01

85

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

PubMed Central

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

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

2011-01-01

86

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

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.

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. [National Public Health Institute, Kuopio (Finland). Dept. for Environmental Health

2007-03-15

87

Methylene Blue Modulates Transendothelial Migration of Peripheral Blood Cells  

PubMed Central

Vasoplegia is a severe complication after cardiac surgery. Within the last years the administration of nitric oxide synthase inhibitor methylene blue (MB) became a new therapeutic strategy. Our aim was to investigate the role of MB on transendothelial migration of circulating blood cells, the potential role of cyclic cGMP, eNOS and iNOS in this process, and the influence of MB on endothelial cell apoptosis. Human vascular endothelial cells (HuMEC-1) were treated for 30 minutes or 2 hours with different concentrations of MB. Inflammation was mimicked by LPS stimulation prior and after MB. Transmigration of PBMCs and T-Lymphocytes through the treated endothelial cells was investigated. The influence of MB upon the different subsets of PBMCs (Granulocytes, T- and B-Lymphocytes, and Monocytes) was assessed after transmigration by means of flow-cytometry. The effect of MB on cell apoptosis was evaluated using Annexin-V and Propidium Iodide stainings. Analyses of the expression of cyclic cGMP, eNOS and iNOS were performed by means of RT-PCR and Western Blot. Results were analyzed using unpaired Students T-test. Analysis of endothelial cell apoptosis by MB indicated a dose-dependent increase of apoptotic cells. We observed time- and dose-dependent effects of MB on transendothelial migration of PBMCs. The prophylactic administration of MB led to an increase of transendothelial migration of PBMCs but not Jurkat cells. Furthermore, HuMEC-1 secretion of cGMP correlated with iNOS expression after MB administration but not with eNOS expression. Expression of these molecules was reduced after MB administration at protein level. This study clearly reveals that endothelial response to MB is dose- and especially time-dependent. MB shows different effects on circulating blood cell-subtypes, and modifies the release patterns of eNOS, iNOS, and cGMP. The transendothelial migration is modulated after treatment with MB. Furthermore, MB provokes apoptosis of endothelial cells in a dose/time-dependent manner. PMID:24340007

Werner, Isabella; Guo, Fengwei; Bogert, Nicolai V.; Stock, Ulrich A.; Meybohm, Patrick; Moritz, Anton; Beiras-Fernandez, Andres

2013-01-01

88

Methylene blue modulates transendothelial migration of peripheral blood cells.  

PubMed

Vasoplegia is a severe complication after cardiac surgery. Within the last years the administration of nitric oxide synthase inhibitor methylene blue (MB) became a new therapeutic strategy. Our aim was to investigate the role of MB on transendothelial migration of circulating blood cells, the potential role of cyclic cGMP, eNOS and iNOS in this process, and the influence of MB on endothelial cell apoptosis. Human vascular endothelial cells (HuMEC-1) were treated for 30 minutes or 2 hours with different concentrations of MB. Inflammation was mimicked by LPS stimulation prior and after MB. Transmigration of PBMCs and T-Lymphocytes through the treated endothelial cells was investigated. The influence of MB upon the different subsets of PBMCs (Granulocytes, T- and B-Lymphocytes, and Monocytes) was assessed after transmigration by means of flow-cytometry. The effect of MB on cell apoptosis was evaluated using Annexin-V and Propidium Iodide stainings. Analyses of the expression of cyclic cGMP, eNOS and iNOS were performed by means of RT-PCR and Western Blot. Results were analyzed using unpaired Students T-test. Analysis of endothelial cell apoptosis by MB indicated a dose-dependent increase of apoptotic cells. We observed time- and dose-dependent effects of MB on transendothelial migration of PBMCs. The prophylactic administration of MB led to an increase of transendothelial migration of PBMCs but not Jurkat cells. Furthermore, HuMEC-1 secretion of cGMP correlated with iNOS expression after MB administration but not with eNOS expression. Expression of these molecules was reduced after MB administration at protein level. This study clearly reveals that endothelial response to MB is dose- and especially time-dependent. MB shows different effects on circulating blood cell-subtypes, and modifies the release patterns of eNOS, iNOS, and cGMP. The transendothelial migration is modulated after treatment with MB. Furthermore, MB provokes apoptosis of endothelial cells in a dose/time-dependent manner. PMID:24340007

Werner, Isabella; Guo, Fengwei; Bogert, Nicolai V; Stock, Ulrich A; Meybohm, Patrick; Moritz, Anton; Beiras-Fernandez, Andres

2013-01-01

89

Development of an Innovative 3D Cell Culture System to Study Tumour - Stroma Interactions in Non-Small Cell Lung Cancer Cells  

PubMed Central

Introduction We describe a novel 3D co-culture model using non-small cell lung cancer (NSCLC) cell lines in combination with lung fibroblasts. This model allows the investigation of tumour-stroma interactions and addresses the importance of having a more in vivo like cell culture model. Methods Automation-compatible multi-well hanging drop microtiter plates were used for the production of 3D mono- and co-cultures. In these hanging drops the two NSCLC cell lines A549 and Colo699 were cultivated either alone or co-cultured with lung fibroblasts. The viability of tumour spheroids was confirmed after five and ten days by using Annexin V/Propidium Iodide staining for flow-cytometry. Tumour fibroblast spheroid formation was characterized by scanning electron microscope (SEM), semi-thin sections, fluorescence microscope and immunohistochemistry (IHC). In addition to conventional histology, protein expression of E-Cadherin, vimentin, Ki67, fibronectin, cytokeratin 7 and ?-smooth muscle actin (?-SMA) was investigated by IHC. Results Lower viability was observed in A549 monocultures compared to co-cultures, whereas Colo699 monocultures showed better viability compared to co-cultures. Ki67 expression varied significantly between mono- and co-cultures in both tumour cell lines. An increase of vimentin and decreased E-Cadherin expression could be detected during the course of the cultivation suggesting a transition to a more mesenchymal phenotype. Furthermore, the fibroblast cell line showed an expression of ?-SMA only in co-culture with the cancer cell line A549, thereby indicating a mesenchymal to mesenchymal shift to an even more myofibroblast phenotype. Conclusion We demonstrate that our method is a promising tool for the generation of tumour spheroid co-cultures. Furthermore, these spheroids allow the investigation of tumour-stroma interactions and a better reflection of in vivo conditions of cancer cells in their microenvironment. Our method holds potential to contribute to the development of anti-cancer agents and support the search for biomarkers. PMID:24663399

Amann, Arno; Zwierzina, Marit; Gamerith, Gabriele; Bitsche, Mario; Huber, Julia M.; Vogel, Georg F.; Blumer, Michael; Koeck, Stefan; Pechriggl, Elisabeth J.; Kelm, Jens M.; Hilbe, Wolfgang; Zwierzina, Heinz

2014-01-01

90

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

PubMed Central

AIM: To investigate the effects of tectorigenin on human hepatocellular carcinoma (HCC) HepG2 cells. METHODS: Tectorigenin, one of the main components of rhizome of Iris tectorum, was prepared by simple methods, such as extraction, filtration, concentration, precipitation and recrystallization. HepG2 cells were incubated with tectorigenin at different concentrations, and their viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was detected by morphological observation of nuclear change, agarose gel electrophoresis of DNA ladder, and flow cytometry with Hoechst 33342, Annexin V-EGFP and propidium iodide staining. Generation of reactive oxygen species was quantified using DCFH-DA. Intracellular Ca2+ was monitored by Fura 2-AM. Mitochondrial membrane potential was monitored using Rhodamine 123. Release of cytochrome c from mitochondria to cytosol was detected by Western blotting. Activities of caspase-3, -8 and -9 were investigated by Caspase Activity Assay Kit. RESULTS: The viability of HepG2 cells treated by tectorigenin decreased in a concentration- and time-dependent manner. The concentration that reduced the number of viable HepG2 cells by 50% (IC50) after 12, 24 and 48 h of incubation was 35.72 mg/L, 21.19 mg/L and 11.06 mg/L, respectively. However, treatment with tectorigenin at 20 mg/L resulted in a very slight cytotoxicity to L02 cells after incubation for 12, 24 or 48 h. Tectorigenin at a concentration of 20 mg/L greatly inhibited the viability of HepG2 cells and induced the condensation of chromatin and fragmentation of nuclei. Tectorigenin induced apoptosis of HepG2 cells in a time- and dose-dependent manner. Compared with the viability rate, induction of apoptosis was the main mechanism of the anti-proliferation effect of tectorigenin in HepG2 cells. Furthermore, tectorigenin-induced apoptosis of HepG2 cells was associated with the generation of reactive oxygen species, increased intracellular [Ca2+]i, loss of mitochondrial membrane potential, translocation of cytochrome c, and activation of caspase-9 and -3. CONCLUSION: Tectorigenin induces apoptosis of HepG2 cells mainly via mitochondrial-mediated pathway, and produces a slight cytotoxicity to L02 cells. PMID:22553399

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

2012-01-01

91

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

PubMed Central

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?

Desfosses-Foucault, Emilie; Dussault-Lepage, Veronique; Le Boucher, Clementine; Savard, Patricia; LaPointe, Gisele; Roy, Denis

2012-01-01

92

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

PubMed Central

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: 50.20±1.55% vs. 0 ?g/ml: 2.62±0.19%; P<0.01). Cordycepin inhibited EA.hy926 cell migration (percentage of wound healing area, 2,000 ?g/ml: 3.45±0.29% vs. 0 ?g/ml: 85.48±0.84%; P<0.05), as well as tube formation (total length of tubular structure, 1,000 ?g/ml: 107±39 ?m vs. 0 ?g/ml: 936±56 ?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

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

2014-01-01

93

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...

94

Enalapril protects endothelial cells against induced apoptosis in Alzheimer's disease  

PubMed Central

Background: Alzheimer's disease (AD) is a progressive neurodegenerative disease in which endothelial cell (EC) can be affected. In brain, functional changes in ECs contribute to reductions in resting blood flow. Furthermore, angiotensin-converting enzyme inhibitors (ACE-I) have beneficial effects on endothelial dysfunction. This is the first study that presents direct experimental evidence associating endothelial apoptosis as a basis of AD pathogenesis and response to an ACE-I therapy. Materials and Methods: Human umbilical vein ECs (HUVECs) were treated with sera from AD patients and sera from healthy volunteers (each group, n = 10). Apoptosis was determined by annexin V–propidium iodide staining and cell death detection kit. The effect of 50 ?M enalapril on endothelial apoptosis was assessed. Nitrite (NO2?) levels were determined in the culture supernatants. Results: Enalapril suppressed the induction of apoptosis by the serum of patients only when used before treating HUVECs with the sera of AD. Mean ± SD of apoptosis induction in the control group was 6.7 ± 3.69; in the group treated with sera of AD for 24 h was 47.78 ± 0.65; in the group wherein sera from AD was added (pretreatment) after exposure of HUVECs by 50 ?M enalapril for 24 h was 26.6 ± 2.63; and in the group wherein HUVECs were exposed in the sera of AD for 24 h and then 50 ?M enalapril was added to these cells for another 24 h (post-treatment) was 56.87 ± 5.51. Also, the mean ± SD of NO2? concentration showed significantly greater levels of dissolved NO2/NO3 metabolite in the culture media of untreated HUVECs by enalapril (1.03 ± 0.06) as compared with control (0.26 ± 0.13; P < 0.05), while the rate of nitric oxide (NO) significantly decreased when enalapril was presented in culture both in the pretreatment (0.07 ± 0.003) and in the post-treatment group (0.06 ± 0.005; P < 0.05). Conclusion: It could be concluded that EC treated with sera from AD patients activates apoptosis in HUVECs; this effect was reversed by enalapril pretreatment. This can be proposed as a therapeutic approach for Alzheimer's patients. PMID:23961275

Meamar, Rokhsareh; Dehghani, Leila; Ghasemi, Majid; Saadatnia, Mohamad; Basiri, Keivan; Faradonbeh, Nazanin Alaei; Javanmard, Shaghayegh Haghjooy

2013-01-01

95

Positron emission tomographic monitoring of dual phosphatidylinositol-3-kinase and mTOR inhibition in anaplastic large cell lymphoma  

PubMed Central

Background Dual phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibition offers an attractive therapeutic strategy in anaplastic large cell lymphoma depending on oncogenic nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) signaling. We tested the efficacy of a novel dual PI3K/mTOR inhibitor, NVP-BGT226 (BGT226), in two anaplastic large cell lymphoma cell lines in vitro and in vivo and performed an early response evaluation with positron emission tomography (PET) imaging using the standard tracer, 2-deoxy-2-[18F]fluoro-D-glucose (FDG) and the thymidine analog, 3?-deoxy-3?-[18F] fluorothymidine (FLT). Methods The biological effects of BGT226 were determined in vitro in the NPM-ALK positive cell lines SU-DHL-1 and Karpas299 by 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, propidium iodide staining, and biochemical analysis of PI3K and mTOR downstream signaling. FDG-PET and FLT-PET were performed in immunodeficient mice bearing either SU-DHL-1 or Karpas299 xenografts at baseline and 7 days after initiation of treatment with BGT226. Lymphomas were removed for immunohistochemical analysis of proliferation and apoptosis to correlate PET findings with in vivo treatment effects. Results SU-DHL-1 cells showed sensitivity to BGT226 in vitro, with cell cycle arrest in G0/G1 phase and an IC50 in the low nanomolar range, in contrast with Karpas299 cells, which were mainly resistant to BGT226. In vivo, both FDG-PET and FLT-PET discriminated sensitive from resistant lymphoma, as indicated by a significant reduction of tumor-to-background ratios on day 7 in treated SU-DHL-1 lymphoma-bearing animals compared with the control group, but not in animals with Karpas299 xenografts. Imaging results correlated with a marked decrease in the proliferation marker Ki67, and a slight increase in the apoptotic marker, cleaved caspase 3, as revealed by immunostaining of explanted lymphoma tissue. Conclusion Dual PI3K/mTOR inhibition using BGT226 is effective in ALK-positive anaplastic large cell lymphoma and can be monitored with both FDG-PET and FLT-PET early on in the course of therapy. PMID:24920919

Graf, Nicolas; Li, Zhoulei; Herrmann, Ken; Weh, Daniel; Aichler, Michaela; Slawska, Jolanta; Walch, Axel; Peschel, Christian; Schwaiger, Markus; Buck, Andreas K; Dechow, Tobias; Keller, Ulrich

2014-01-01

96

Camptothecin analogs with enhanced activity against human breast cancer cells. II. Impact of the tumor pH gradient.  

PubMed

Human breast tumors often exist in an acidic and hypoxic microenvironment, which can promote resistance to radiation and chemotherapies. A tumor-selective pH gradient arises in these tumors which favors uptake and retention of drugs like camptothecin that are weak acids. We evaluated the effect of alkyl substitutions at the 7 position in seven CPTs with varying groups at the 10 position on modulation by acidic extracellular pH in three human breast cancer cell lines. Growth inhibition was assessed by propidium iodide staining of nucleic acids in human breast cancer cells cultured at either extracellular pH 6.8 or 7.4 that were (1) hormone-sensitive (MCF-7/wt), (2) hormone insensitive (MDA-MB-231), or (3) alkylator-resistant (MCF-7/4-hc). Over 10-fold pH modulation was observed in 7-halomethyl analogs of methylenedioxy-CPT and in 7-alkyl analogs of 10-amino-CPT. Of 39 analogs tested, the overall pattern of activity across breast tumor cell lines was similar with some notable exceptions. For example, 7-propyl-10-amino-CPT was modulated 16- to 20-fold by acidic extracellular pH in the MCF-7 cell lines, but only 6-fold in MDA-MB-231 cells. One mechanism that can contribute to pH modulation is enhanced cellular drug uptake and retention. In MCF-7/wt cells, uptake of 10-amino-CPT increased 4-fold, while retention increased over 10-fold at acidic extracellular pH. In addition, gene expression analysis of MCF-7/wt cells indicated that expression of a number of genes changed under acidic culture conditions, including down-regulation of the CPT efflux protein pump breast cancer resistance protein (BCRP). Interestingly, expression of topoisomerase I, the molecular target of CPT, was not affected by acidic growth conditions. These results highlight the importance of maintaining key features of tumor physiology in cell culture models used to study cancer biology and to discover and develop new anticancer drugs. While several substitutions at the 7 and 10 positions enhance potency, 7-halomethyl and 10-amino CPT analogs show selective activity at the acidic pH common to the microenvironment of most solid tumors. PMID:16001167

Adams, David J; Wahl, Miriam L; Flowers, James L; Sen, Banalata; Colvin, Michael; Dewhirst, Mark W; Manikumar, Govindarajan; Wani, Mansukh C

2006-01-01

97

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

SciTech Connect

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 ?m 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.

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

2007-11-28

98

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

PubMed

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 degrees C or held at 5 degrees C (controls) for 1 h. Polycarbonate filters (25 mm diameter, 0.8 microm 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 PMA with 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. PMID:18160156

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

2008-02-01

99

1-calcium phosphate-uracil, a synthesized pyrimidine derivative agent, has anti-proliferative, pro-apoptotic and anti-invasion effects on multiple tumor cell lines  

PubMed Central

1-calcium phosphate-uracil (1-CP-U), a synthetic pyrimidine derivative, has been documented to demonstrate a variety of different biological activities. However, the potency and mechanisms of this agent’s anti-cancer activity have not been elucidated to date. In the present study, the anti-cancer effects of 1-CP-U were examined in a range of in vitro assays. Different cell lines were treated with 1-CP-U at varied concentrations (0.7, 1.0, 1.4 ?mol/l) for indicated durations. The cell proliferation was then examined by MTT assay. The cellular apoptotic effects were detected by Hoechst 33342 and Annexin V/propidium iodide staining, while the capacity of 1-CP-U on invasion and migration were examined by cell invasion and wound healing assays. The expression of matrix metalloproteinase proteins, as well as pro- and antiapoptotic proteins was detected by western blotting analysis. The results identified that 1-CP-U was able to inhibit the viability of SKOV3, HeLa, SMMC-7721 and A549 cell lines in a dose- and time-dependent manner, while it exerted only marginal toxic effects on non-cancerous cells. The IC50 concentration of 1-CP-U for tumor cell lines was ~1.0 ?mol/l. The growth inhibition induced by 1-CP-U was accompanied by a broad spectrum of pro-apoptotic activities, in which different cell lines varied in their sensitivity to 1-CP-U. Meanwhile, the increased expression of the pro-apoptotic protein B-cell lymphoma-2 (Bcl-2)-associated X and a marked reduction of Bcl-2 levels were associated with increased 1-CP-U concentrations. Additionally, anti-migration and anti-invasion effects of 1-CP-U were evidently associated with the downregulation of matrix metalloproteinase proteins. Of note, it was observed that 1-CP-U significantly inhibited both the migration and invasion at a lower concentration, as compared with the dose required to achieve significant inhibition of apoptosis. These results indicated that 1-CP-U appeared to be a more effective inhibitor of cell migration and invasion, rather than of apoptosis. In conclusion, the present study was the first, to the best of our knowledge, to demonstrate the function of 1-CP-U in tumor proliferation, apoptosis and invasion with specific effects against cancer cells in vitro, suggesting 1-CP-U as a potential novel anticancer agent. PMID:25118659

PENG, JING; CHEN, XINLIAN; HU, QIAN; YANG, MEI; LIU, HONGQIAN; WEI, WEI; LIU, SHANLING; WANG, HE

2014-01-01

100

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

PubMed

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-?uorescein 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

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

2013-11-01

101

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

PubMed Central

Background Cratoxylum arborescens has been used traditionally in Malaysia for the treatment of various ailments. Methods ?-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-?B) 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-?B 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-?B and HSP70 signaling pathways.

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

102

Anticancer activity of a sub-fraction of dichloromethane extract of Strobilanthes crispus on human breast and prostate cancer cells in vitro  

PubMed Central

Background The leaves of Strobilanthes crispus (S. crispus) which is native to the regions of Madagascar to the Malay Archipelago, are used in folk medicine for their antidiabetic, diuretic, anticancer and blood pressure lowering properties. Crude extracts of this plant have been found to be cytotoxic to human cancer cell lines and protective against chemically-induced hepatocarcinogenesis in rats. In this study, the cytotoxicity of various sub-fractions of dichloromethane extract isolated from the leaves of S. crispus was determined and the anticancer activity of one of the bioactive sub-fractions, SC/D-F9, was further analysed in breast and prostate cancer cell lines. Methods The dichloromethane extract of S. crispus was chromatographed on silica gel by flash column chromatography. The ability of the various sub-fractions obtained to induce cell death of MCF-7, MDA-MB-231, PC-3 and DU-145 cell lines was determined using the LDH assay. The dose-response effect and the EC50 values of the active sub-fraction, SC/D-F9, were determined. Apoptosis was detected using Annexin V antibody and propidium iodide staining and analysed by fluorescence microscopy and flow cytometry, while caspase 3/7 activity was detected using FLICA caspase inhibitor and analysed by fluorescence microscopy. Results Selected sub-fractions of the dichloromethane extract induced death of MCF-7, MDA-MB-231, PC-3 and DU-145 cells. The sub-fraction SC/D-F9, consistently killed breast and prostate cancer cell lines with low EC50 values but is non-cytotoxic to the normal breast epithelial cell line, MCF-10A. SC/D-F9 displayed relatively higher cytotoxicity compared to tamoxifen, paclitaxel, docetaxel and doxorubicin. Cell death induced by SC/D-F9 occurred via apoptosis with the involvement of caspase 3 and/or 7. Conclusions A dichloromethane sub-fraction of S. crispus displayed potent anticancer activities in vitro that can be further exploited for the development of a potential therapeutic anticancer agent. PMID:20684795

2010-01-01

103

Flow cytometric estimation of nuclear DNA amount in diploid bananas ( Musa acuminata and M. balbisiana )  

Microsoft Academic Search

Cell nuclei were isolated from leaf tissues of wild banana (Musa balbisiana, M. acuminata ssp.banksii andM. acuminata ssp.errans) and of the two vegetative clones of diploid cultivar “Pisang Mas”. Relative fluorescence intensity was measured on propidium\\u000a iodide-stained nuclei by flow cytometry. Nuclei isolated fromGlycine max with known nuclear genome size were used as internal standard to determine nuclear DNA content

J. Doležel; M. Doleželová; F. J. Novák

1994-01-01

104

Sunflower ( Helianthus annuus) Leaves Contain Compounds that Reduce Nuclear Propidium Iodide Fluorescence  

Microsoft Academic Search

Sunflower leaves have unidentified compounds that interfere with propidium iodide (PI) intercalation and\\/or fluorescence. Independently prepared pea leaf nuclei show greater PI fluorescence than nuclei from pea leaves simultaneously processed (co-chopped) with sunflower leaves. Differences in fluorescence persist after mixing the PI-stained pea and the co-chopped pea\\/sunflower samples, i.e. PI staining protects the nuclei from the effects of the inhibitor.

H. James Price; George Hodnett; J. Spencer Johnston

2000-01-01

105

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

PubMed Central

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

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

2013-01-01

106

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

PubMed

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

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

2012-02-01

107

Evaluation of viable Mycobacterium avium subsp. paratuberculosis in milk using peptide-mediated separation and Propidium Monoazide qPCR.  

PubMed

The causative agent of paratuberculosis in ruminants, Mycobacterium avium subsp. paratuberculosis (MAP), although still a matter of debate, has been linked with Crohn's and other human diseases. The availability of rapid methods for assessing the viability of MAP cells in food, in particular milk, could be of great use for risk management in food safety. MAP viability is generally assessed using culture techniques that require prolonged incubation periods for the growth of MAP. To differentiate between viable and nonviable MAP cells in milk samples, this study explores the combination of two already described techniques: peptide magnetic bead separation followed by Propidium Monoazide qPCR. Using an Ordinal Multinomial Logistic Regression model to analyze the results obtained after spiking milk samples with mixtures containing different percentages of viable/dead cells, we were able to assess the probability of the viability status of MAP found in milk. This model was applied to contaminated pasteurized milk to ascertain the efficacy of heat treatment in MAP killing. The method reported herein can potentially be used for direct detection of MAP viability in milk. PMID:24860938

Ricchi, Matteo; De Cicco, Caterina; Kralik, Petr; Babak, Vladimir; Boniotti, Maria B; Savi, Roberto; Cerutti, Giulia; Cammi, Giuliana; Garbarino, Chiara; Arrigoni, Norma

2014-07-01

108

Use of propidium monoazide for the enumeration of viable Oenococcus oeni in must and wine by quantitative PCR.  

PubMed

Malolactic fermentation is an important step in winemaking, but it has to be avoided in some cases. It's carried out by lactic acid bacteria belonging mainly to the genus Oenococcus, which is known to be a slow growing bacterium. Classical microbiological methods to enumerate viable cells of Oenococcus oeni in must and wine take 7-9 days to give results. Moreover, RT-qPCR technique gives accurate quantitative results, but it requires time consuming steps of RNA extraction and reverse transcription. In the present work we developed a fast and reliable quantitative PCR (qPCR) method to enumerate cells of Oenococcus oeni, directly, in must and wine. For the first time we used a propidium monoazide treatment of samples to enumerate only Oenococcus oeni viable cells. The detection limit of the developed method is 0.33 log CFU/mL (2.14 CFU/mL) in must, and 0.69 log CFU/mL (4.90 CFU/mL) in wine, lower than that of the previously developed qPCR protocols. PMID:23628614

Vendrame, Marco; Iacumin, Lucilla; Manzano, Marisa; Comi, Giuseppe

2013-08-01

109

Nanosecond pulsed electric fields and the cell cycle  

NASA Astrophysics Data System (ADS)

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.

Mahlke, Megan A.

110

Parthenolide generates reactive oxygen species and autophagy in MDA-MB231 cells. A soluble parthenolide analogue inhibits tumour growth and metastasis in a xenograft model of breast cancer  

PubMed Central

Triple-negative breast cancers (TNBCs) are clinically aggressive forms associated with a poor prognosis. We evaluated the cytotoxic effect exerted on triple-negative MDA-MB231 breast cancer cells both by parthenolide and its soluble analogue dimethylamino parthenolide (DMAPT) and explored the underlying molecular mechanism. The drugs induced a dose- and time-dependent decrement in cell viability, which was not prevented by the caspase inhibitor z-VAD-fmk. In particular in the first hours of treatment (1–3?h), parthenolide and DMAPT strongly stimulated reactive oxygen species (ROS) generation. The drugs induced production of superoxide anion by activating NADPH oxidase. ROS generation caused depletion of thiol groups and glutathione, activation of c-Jun N-terminal kinase (JNK) and downregulation of nuclear factor kB (NF-kB). During this first phase, parthenolide and DMAPT also stimulated autophagic process, as suggested by the enhanced expression of beclin-1, the conversion of microtubule-associated protein light chain 3-I (LC3-I) to LC3-II and the increase in the number of cells positive to monodansylcadaverine. Finally, the drugs increased RIP-1 expression. This effect was accompanied by a decrement of pro-caspase 8, while its cleaved form was not detected and the expression of c-FLIPS markedly increased. Prolonging the treatment (5–20?h) ROS generation favoured dissipation of mitochondrial membrane potential and the appearance of necrotic events, as suggested by the increased number of cells positive to propidium iodide staining. The administration of DMAPT in nude mice bearing xenografts of MDA-MB231 cells resulted in a significant inhibition of tumour growth, an increment of animal survival and a marked reduction of the lung area invaded by metastasis. Immunohistochemistry data revealed that treatment with DMAPT reduced the levels of NF-kB, metalloproteinase-2 and -9 and vascular endothelial growth factor, while induced upregulation of phosphorylated JNK. Taken together, our data suggest a possible use of parthenolide for the treatment of TNBCs. PMID:24176849

D'Anneo, A; Carlisi, D; Lauricella, M; Puleio, R; Martinez, R; Di Bella, S; Di Marco, P; Emanuele, S; Di Fiore, R; Guercio, A; Vento, R; Tesoriere, G

2013-01-01

111

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

PubMed Central

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

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

2014-01-01

112

Detection of viable Escherichia coli O157:H7 in ground beef by propidium monoazide real-time PCR.  

PubMed

Escherichia coli O157:H7 associated with food has caused many serious public health problems in recent years. However, only viable cells of this pathogen can cause infections, and false-positive detection caused by dead cells can lead to unnecessary product recalls. The objective of this study was to develop and optimize a method that combines propidium monoazide (PMA) staining with real-time PCR to detect only viable cells of E. coli O157:H7 in ground beef. PMA is a DNA intercalating dye that can penetrate compromised membranes of dead cells and bind to cellular DNA, preventing its amplification via a subsequent PCR. Three strains of E. coli O157:H7 (505B, G5310 and C7927) at concentrations of 10(0) to 10(8)CFU/mL were used as live cells. Dead cells were obtained by heating cell suspensions at 85°C for 15 min. Suspensions were treated with PMA and the optimized assay was applied to artificially contaminated ground beef with two different fat contents (10% and 27%). DNA was extracted and amplified by TaqMan® real-time PCR assay targeting the uidA gene for detection of E. coli O157:H7. Plasmid pUC19 was added as an internal amplification control (IAC). A treatment of 25 ?M PMA with a 10-min light exposure on ice was sufficient to eliminate DNA from 10(8) dead E. coli O157:H7 cells/mL. The optimized assay could detect as low as 10(2) CFU/mL viable E. coli O157:H7 in pure culture and 10(5) CFU/g in ground beef, in the presence of 10(6)/mL or g of dead cells. With an 8-h enrichment, 1 CFU/g viable E. coli O157:H7 in ground beef was detectable without interference from 10(6) dead cells/g. In conclusion, the PMA real-time PCR could effectively detect viable E. coli O157:H7 without being compromised by dead cells. PMID:24291180

Liu, Yarui; Mustapha, Azlin

2014-01-17

113

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

PubMed

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

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

2014-02-01

114

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...

115

The DNA intercalators ethidium bromide and propidium iodide also bind to core histones.  

PubMed

Eukaryotic DNA is compacted in the form of chromatin, in a complex with histones and other non-histone proteins. The intimate association of DNA and histones in chromatin raises the possibility that DNA-interactive small molecules may bind to chromatin-associated proteins such as histones. Employing biophysical and biochemical techniques we have characterized the interaction of a classical intercalator, ethidium bromide (EB) and its structural analogue propidium iodide (PI) with hierarchical genomic components: long chromatin, chromatosome, core octamer and chromosomal DNA. Our studies show that EB and PI affect both chromatin structure and function, inducing chromatin compaction and disruption of the integrity of the chromatosome. Calorimetric studies and fluorescence measurements of the ligands demonstrated and characterized the association of these ligands with core histones and the intact octamer in absence of DNA. The ligands affect acetylation of histone H3 at lysine 9 and acetylation of histone H4 at lysine 5 and lysine 8 ex vivo. PI alters the post-translational modifications to a greater extent than EB. This is the first report showing the dual binding (chromosomal DNA and core histones) property of a classical intercalator, EB, and its longer analogue, PI, in the context of chromatin. PMID:24649406

Banerjee, Amrita; Majumder, Parijat; Sanyal, Sulagna; Singh, Jasdeep; Jana, Kuladip; Das, Chandrima; Dasgupta, Dipak

2014-01-01

116

Consequences of Stoichiometric Error on Nuclear DNA Content Evaluation in Coffea liberica var. dewevrei using DAPI and Propidium Iodide  

PubMed Central

The genome size of coffee trees (Coffea sp.) was assessed using flow cytometry. Nuclear DNA was stained with two dyes [4?,6?diamino?2?phenylindole dihydrochloride hydrate (DAPI) and propidium iodide (PI)]. Fluorescence in coffee tree nuclei (C?PI or C?DAPI) was compared with that of the standard, petunia (P?PI or P?DAPI). If there is no stoichiometric error, then the ratio between fluorescence of the target nuclei and that of the standard nuclei (R?PI or R?DAPI) is expected to be proportional to the genome size. Between?tree differences in target : standard fluorescence ratios were noted in Coffea liberica var. dewevrei using propidium iodide and DAPI. For both dyes, between?tree differences were due to a lack of proportionality when comparing locations of the coffee peak and the petunia peak. Intraspecific genome size variations clearly cannot explain variations in the target : standard fluorescence ratio. The origin of the lack of proportionality between target and standard fluorescences differed for the two dyes. With propidium iodide, there was a regression line convergence point, and no between?tree differences were noted in this respect, whereas there was no such convergence with DAPI. An accurate estimate of genome size can thus be obtained with PI. Implications with respect to accessibility and binding mode are discussed. PMID:12096798

NOIROT, MICHEL; BARRE, PHILIPPE; LOUARN, JACQUES; DUPERRAY, CHRISTOPHE; HAMON, SERGE

2002-01-01

117

Spermiogenesis defects in human: detection of transition proteins in semen from some infertile men.  

PubMed

Semen samples from 60 infertile men were examined by flow cytometry following propidium iodide staining. Of these, 23 samples contained young haploid cells. Transition proteins (TP1 and/or TP2) were detected in 12 of these, using immunohistochemical staining. The presence of TPs in spermatids in semen indicates inhibition in the differentiation pathway from round spermatids to spermatozoa. Cells of this type were found in semen from patients with nonobstructive azoospermia, severe to extreme cases of oligozoospermia, asthenozoospermia and teratozoospermia. PMID:18727729

Becker, S; Soffer, Y; Lewin, L M; Yogev, L; Shochat, L; Golan, R

2008-08-01

118

Effects of Caffeine and Chlorogenic Acid on Propidium Iodide Accessibility to DNA: Consequences on Genome Size Evaluation in Coffee Tree  

PubMed Central

Estimates of genome size using flow cytometry can be biased by the presence of cytosolic compounds, leading to pseudo?intraspecific variation in genome size. Two important compounds present in coffee trees—caffeine and chlorogenic acid—modify accessibility of the dye propidium iodide to Petunia DNA, a species used as internal standard in our genome size evaluation. These compounds could be responsible for intraspecific variation in genome size since their contents vary between trees. They could also be implicated in environmental variations in genome size, such as those revealed when comparing the results of evaluations carried out on different dates on several genotypes. PMID:12876189

NOIROT, M.; BARRE, P.; DUPERRAY, C.; LOUARN, J.; HAMON, S.

2003-01-01

119

Monitoring the prevalence of viable and dead cariogenic bacteria in oral specimens and in vitro biofilms by qPCR combined with propidium monoazide  

PubMed Central

Background Streptococcus mutans and Streptococcus sobrinus are associated with the development of dental caries in humans. However, previous diagnostic systems are unsuitable for monitoring viable cell numbers in oral specimens. Assessing the relationship between the numbers of viable and dead bacterial cells and oral status is important for understanding oral infectious diseases. Propidium monoazide (PMA) has been reported to penetrate dead cells following membrane damage and to cross-link DNA, thereby inhibiting DNA amplification. In the present study, we established an assay for selective analysis of two viable human cariogenic pathogens, S. mutans and S. sobrinus, using PMA combined with real-time PCR (PMA-qPCR). Results We designed species-specific primer sets for S. mutans and S. sobrinus, generated standard curves for measuring cell numbers, and evaluated the dynamic range of the assay. To determine the effectiveness of the assay, PMA was added to viable and autoclave-killed cell mixtures. PMA treatment effectively prevented DNA amplification from dead cells. No amplification of DNA from dead cells was observed in these organisms. In addition, we applied this assay to analyze viable cell numbers in oral specimens. A significant correlation was found between the number of viable S. mutans cells in saliva and that in plaque among caries-free patients, whereas no correlation was observed between saliva and carious dentin. The total and viable cell numbers in caries-positive saliva were significantly higher than those in caries-free saliva. Finally, we analyzed the usefulness of this assay for in vitro oral biofilm analysis. We applied PMA-qPCR for monitoring viable S. mutans cell numbers in vitro in planktonic cells and oral biofilm treated with hydrogen peroxide (H2O2). In planktonic cells, the number of viable cells decreased significantly with increasing H2O2 concentration, whereas only a small decrease was observed in biofilm cell numbers. Conclusions PMA-qPCR is potentially useful for quantifying viable cariogenic pathogens in oral specimens and is applicable to oral biofilm experiments. This assay will help to elucidate the relationship between the number of viable cells in oral specimens and the oral status. PMID:23848601

2013-01-01

120

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

PubMed Central

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

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

121

Advantageous direct quantification of viable closely related probiotics in petit-suisse cheeses under in vitro gastrointestinal conditions by Propidium Monoazide--qPCR.  

PubMed

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

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

122

BENA435, a new cell-permeant photoactivated green fluorescent DNA probe  

E-print Network

was used to develop a model of BENA435 intercalation between base pairs of a DNA helix. BENA435 of these reagents. For example, ethidium bromide and propidium iod- ide intercalate into dsDNA (2), whereas, propidium bromide does not penetrate into cells, and ethidium bromide intercalates into the DNA of living

Paris-Sud XI, Université de

123

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

NASA Astrophysics Data System (ADS)

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, ? 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 ?H2AX-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 ?-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-?1). TGF-?1 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 ? 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 cel

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

124

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

PubMed Central

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

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

2008-01-01

125

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

PubMed

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

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

2007-11-01

126

Vibrating microbubbles poking individual cells: Drug transfer into cells via sonoporation  

Microsoft Academic Search

Ultrasound contrast microbubbles have the ability to enhance endothelial cell permeability and thus may be used as a new way to deliver drugs. It facilitates the transfer of extracellular molecules into cells activated through ultrasound driven microbubbles. The present study is designed to correlate the relationship between microbubble induced cell deformation and enhanced cell membrane permeability. Propidium iodide (PI) was

Annemieke van Wamel; Klazina Kooiman; Miranda Harteveld; Marcia Emmer; Folkert J. ten Cate; Michel Versluis; Nico de Jong

2006-01-01

127

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

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

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

128

Lenticular mitoprotection. Part A: Monitoring mitochondrial depolarization with JC-1 and artifactual fluorescence by the glycogen synthase kinase-3? inhibitor, SB216763  

PubMed Central

Purpose Dissipation of the electrochemical gradient across the inner mitochondrial membrane results in mitochondrial membrane permeability transition (mMPT), a potential early marker for the onset of apoptosis. In this study, we demonstrate a role for glycogen synthase kinase-3? (GSK-3?) in regulating mMPT. Using direct inhibition of GSK-3? with the GSK-3? inhibitor SB216763, mitochondria may be prevented from depolarizing (hereafter referred to as mitoprotection). Cells treated with SB216763 showed an artifact of fluorescence similar to the green emission spectrum of the JC-1 dye. We demonstrate the novel use of spectral deconvolution to negate the interfering contributing fluorescence by SB216763, thus allowing an unfettered analysis of the JC-1 dye to determine the mitochondrial membrane potential. Methods Secondary cultures of virally transfected human lens epithelial cells (HLE-B3) were exposed to acute hypoxic conditions (approximately 1% O2) followed by exposure to atmospheric oxygen (approximately 21% O2). The fluorescent dye JC-1 was used to monitor the extent of mitochondrial depolarization upon exposure of inhibitor treatment relative to the control cells (mock inhibition) in atmospheric oxygen. Annexin V-fluorescein isothiocyanate/propidium iodide staining was implemented to determine cell viability. Results Treatment of HLE-B3 cells with SB216763 (12 µM), when challenged by oxidative stress, suppressed mitochondrial depolarization relative to control cells as demonstrated with JC-1 fluorescent dye analysis. Neither the control nor the SB216763-treated HLE-B3 cells tested positive with annexin V-fluorescein isothiocyanate/propidium iodide staining under the conditions of the experiment. Conclusions Inhibition of GSK-3? activity by SB216763 blocked mMPT relative to the slow but consistent depolarization observed with the control cells. We conclude that inhibition of GSK-3? activity by the GSK-3? inhibitor SB216763 provides positive protection against mitochondrial depolarization. PMID:23825920

Brooks, Morgan M.; Neelam, Sudha; Fudala, Rafal; Gryczynski, Ignacy

2013-01-01

129

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 ...

130

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)-...

131

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...

132

Quantification of Electroporative Uptake Kinetics and Electric Field Heterogeneity Effects in Cells  

E-print Network

Quantification of Electroporative Uptake Kinetics and Electric Field Heterogeneity Effects in Cells integrity indicator, propidium iodide (PI), in HL60 human leukemia cells resulting from exposure to 40 ms-time fluorescence microscopy and the development of a microcuvette: a specialized device designed for exposing cell

Sheridan, Jennifer

133

Amine reactive dyes: An effective tool to discriminate live and dead cells in polychromatic flow cytometry  

Microsoft Academic Search

Membrane-damaged cells caused by either mechanical trauma or through normal biological processes can produce artifacts in immunophenotyping analysis by flow cytometry. Dead cells can nonspecifically bind monoclonal antibody conjugates, potentially leading to erroneous conclusions, particularly when cell frequencies are low. To date, DNA intercalating dyes (Ethidium monoazaide (EMA), Propidium Iodide, 7AAD, etc.) or Annexin V have been commonly used to

Stephen P. Perfetto; Pratip K. Chattopadhyay; Laurie Lamoreaux; Richard Nguyen; David Ambrozak; Richard A. Koup; Mario Roederer

2006-01-01

134

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

NASA Astrophysics Data System (ADS)

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.

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

2013-04-01

135

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

SciTech Connect

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)

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

2013-04-30

136

Imaging bioluminescent indicators shows Ca2+ and ATP permeability thresholds in live cells attacked by complement.  

PubMed Central

A series of permeability thresholds to Ca2+ metabolites and macromolecules, occurring at different times when cells are attacked by complement, has been established by imaging HeLa cells transiently expressing a recombinant cytosolic fusion protein of firefly luciferase and aequorin (luciferase-aequorin) to measure changes in ATP and cytosolic free Ca2+. Nuclear fluorescence of propidium was used as a measure of permeability to small molecules, and luciferase activity imaged to assess lysis. The rise in cytosolic free Ca2+ observed after C9 attack preceded by at least 60 s both the increase in propidium fluorescence, measured in single cells, and the decrease in ATP monitored by luciferase light emission. These effects were dependent on the concentration of C9. At concentrations of C9 up to 4 micrograms/ml no loss of luciferase-aequorin protein was detected at the end of the experiment. Thus the membrane integrity of the cells remained intact, even though the cells were permeable to propidium. These results confirmed our earlier observations that propidium permeability in cells attacked by complement was not a reliable measure of cell death. They also show that it is vital to take account of cellular heterogeneity if the mechanisms by which cells respond to membrane pore former attack are to be correctly interpreted. Images Figure 2 PMID:9659235

Sala-Newby, G B; Taylor, K M; Badminton, M N; Rembold, C M; Campbell, A K

1998-01-01

137

Extended role of necrotic cell death after hypoxia–ischemia-induced neurodegeneration in the neonatal rat  

Microsoft Academic Search

The relative contribution of apoptosis and necrosis after neonatal hypoxia–ischemia (HI) is still a matter of debate. Here we determined the time course of necrotic cell death after neonatal HI and its relationship to caspase-3 activation and apoptotic cell death. Necrosis was evaluated by intracerebroventricular injection of propidium iodide (PI) before sacrificing the animal and processing brain sections for caspase-3

Silvia Carloni; Andrea Carnevali; Mauro Cimino; Walter Balduini

2007-01-01

138

Induction of apoptosis by diallyl disulfide through activation of caspase-3 in human leukemia HL60 cells 1 1 Abbreviations: Ac-DEVD-CHO, N-acetyl-Asp-Glu-Val-Asp-CHO (aldehyde); Ac-DEVD-AFC, N-acetyl-Asp-Glu-Val-Asp-AFC (7-amino-4-trifluoromethyl-coumaine); Ac-YVAD-CHO, N-acetyl-Tyr-Val-Ala-Asp-CHO (aldehyde); CAD, caspase-activated deoxyribonuclease; CM-H 2DCFDA, 5-(and -6)-chloromethyl-2?,7?-dichlorodihydrofluorescein diacetate; DADS, diallyl disulfide; FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; ICAD, inhibitor of caspase-activated deoxyribonuclease; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NAC, N-acetylcysteine; PARP, poly(ADP-ribose) polymerase; PI, propidium iodide; and ROI, reactive oxygen intermediate  

Microsoft Academic Search

Diallyl disulfide (DADS), a component of garlic (Allium sativum), has been known to exert potent chemopreventative activity against colon, lung, and skin cancers. However, its molecular mechanism of action is still obscure. The present study demonstrated that DADS induces apoptosis of human leukemia HL-60 cells in a concentration- and time-dependent manner with an ic50 for cell viability of less than

Kang-Beom Kwon; Su-Jin Yoo; Do-Gon Ryu; Jeong-Yeh Yang; Hye-Won Rho; Jong-Suk Kim; Jin-Woo Park; Hyung-Rho Kim; Byung-Hyun Park

2002-01-01

139

Major Changes in Chromatin Condensation Suggest the Presence of an Apoptotic Pathway in Plant Cells  

Microsoft Academic Search

A large decrease in fluorescence intensity of propidium iodide (PI)-stained nuclei is observed during senescence of plant cells. The phenomenon reflects a decrease in accessibility of DNA to this fluorochrome and is a consequence of chromatin condensation. This decrease is substantially greater than usually found in animal nuclei whose chromatin undergoes condensation, e.g., during differentiation or quiescence. Chromatin condensation was

Iona E. W. O'Brien; Brian G. Murray; Bruce C. Baguley; Bret A. M. Morris; Ian B. Ferguson

1998-01-01

140

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

NASA Astrophysics Data System (ADS)

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 ?m in diameter and 8 ?m in length with 10 ?m 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.

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

2013-09-01

141

Colorectal cancer cell growth inhibition by linoleic acid is related to fatty acid composition changes  

Microsoft Academic Search

Polyunsaturated fatty acids (PUFAs) possess anti-cancer action both in vitro and in vivo. In the present study, we detected\\u000a cell viability with methyl thiazolyl tetrazolium (MTT) assay and cell membrane permeability with propidium iodide (PI) fluorescence\\u000a dyeing, and calculated cell membrane fluidity change as fluorescence anisotropy. Fatty acid content in cells was measured\\u000a by gas chromatography\\/mass spectroscopy (GC\\/MS), and the

Xiao-feng Lu; Guo-qing He; Hai-ning Yu; Qi Ma; Sheng-rong Shen; Undurti N. Das

2010-01-01

142

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

PubMed

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

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

2014-01-01

143

Immunologic and biochemical effects of the fermented wheat germ extract Avemar.  

PubMed

Avemar (MSC) is a nontoxic fermented wheat germ extract demonstrated to have antitumor effects. Avemar has the potential to significantly improve the survival rate in patients suffering from malignant colon tumors. We studied its effects in the HT-29 human colon carcinoma cell line. Avemar had an inhibiting effect on colonies of HT-29 cells with an IC50 value of 118 microg/ml (7 days of incubation); this value could be decreased to 100 and 75 microg/ml in the presence of vitamin C. In the cell line examined, Avemar induced both necrosis and apoptosis, as demonstrated by Hoechst/propidium iodide staining. The incubation of cells with 3200 microg/ml Avemar for 24 hrs caused necrosis in 28% and the induction of apoptosis in 22% of the cells. Avemar inhibited the cell-cycle progression of HT-29 cells in the G1 phase of the cell cycle. In addition, Avemar inhibited the activity of the key enzyme of de novo DNA synthesis, ribonucleotide reductase. In addition, we determined the effects of Avemar on the activity of cyclooxygenase-1 and -2. Both enzymes were significantly inhibited by Avemar with IC50 values of 100 and 300 microg/ml, respectively. We outline new explanations for its antitumor activity, which might serve as the basis for further studies using Avemar. PMID:15673563

Illmer, Christoph; Madlener, Sibylle; Horvath, Zsuzsanna; Saiko, Philipp; Losert, Annemarie; Herbacek, Irene; Grusch, Michael; Krupitza, Georg; Fritzer-Szekeres, Monika; Szekeres, Thomas

2005-02-01

144

Effect of docosahexaenoic acid on ras post-translational processing and localization in a transgenic mouse colonic cell line  

E-print Network

GAS CHROMOTOGRAPHY. . . . 77 APPENDIX 11 COMPLEXING FATTY ACIDS TO BSA. 81 APPENDIX 12 CELL NUMBER DETERMINATION . 84 APPENDIX 13 FLUORESCEIN DIACETATE - PROPIDIUM IODINE VIABILITY STAINING 85 APPENDIX 14 COLUMN CHROMATOGRAPHY TO REMOVE NEUTRAL..., subconfluent cells were treated with 50 ItM DHA or LA complexed to fatty acid free BSA (Holub et al. 1971) for 72 h (Appendix 11). Cells treated with media only were used as a control for cell number, viability, and fatty acid incorporation. Following...

Collett, Esther Dick

2012-06-07

145

A novel cell culture model for studying differentiation and apoptosis in the mouse mammary gland  

E-print Network

. Cells (1 × 105) were stained with annexin V and propidium iodide using the Apoptosis Detection Kit and following the manufacturer’s instructions (R&D Systems, Abingdon, Oxford, UK). Cells were analyzed by flow cytometry using a Coulter EPICS XL flow... Primary research A novel cell culture model for studying differentiation and apoptosis in the mouse mammary gland Katrina E Gordon, Bert Binas*, Rachel S Chapman†, Kathreena M Kurian†, Richard W E Clarkson‡, A John Clark, E Birgitte Lane...

Gordon, Katrina E; Binas, Bert; Chapman, Rachel S; Kurian, Kathreena M; Clarkson, Richard W E; Clark, A John; Birgitte Lane, E; Watson, Christine J

2000-03-07

146

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

PubMed

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 (three curcuminoids and two turmerones) from CL, using human cancer cell lines HepG2, MCF-7 and MDA-MB-231. The immunomodulatory activities of turmerones (alpha 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 alpha-turmerone significantly inhibited proliferation of cancer cells in dose-dependent manner. The IC(50) values of these compounds in cancer cells ranged from 11.0 to 41.8 microg/ml. alpha-Turmerone induced MDA-MB-231 cells to undergo apoptosis, which was confirmed by annexin-V and 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 alpha-turmerone treated cells. Both alpha-turmerone and aromatic-turmerone showed stimulatory effects on PBMC proliferation and cytokine production. The anti-proliferative effect of alpha-turmerone and immunomodulatory activities of ar-turmerone was 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

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

147

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

PubMed Central

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

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

2013-01-01

148

Antioxidant, hepatoprotective and cytotoxic effects of icetexanes isolated from stem-bark of Premna tomentosa.  

PubMed

The study investigates the antioxidant, hepatoprotective and antiproliferative effects of novel icetexane diterpenoids (ice 1-4) isolated from hexane extract of stem bark of Premna tomentosa. A549, HT-29, MCF-7, MDA-MB-231, A431 cells were used to assess the antiproliferative activity by MTT assay. Cell death induced by apoptosis was determined by morphological assessment studies using acridine orange/ethidium bromide staining (dual staining), mitochondrial potential measurement by JC-1 staining, and cell cycle analysis by propidium iodide staining method by Muse cell analyser. Anti oxidant activity was investigated by in vitro assays such as DPPH, nitric oxide and superoxide scavenging activities. Hepatoprotective activity was determined in vitro with HepG2 cells and in vivo by tBHP induced hepatic damage mice model. Based on the in vitro cytotoxic assays and morphological assessment studies using fluorescence microscopic study (acridine orange and ethidium bromide double staining) and mitochondrial potential measurements, it was found that ice 2 and 3 possess good antiproliferative effect via mitochondrial mediated apoptosis in human lung and breast cancer cells. Results of in vitro antioxidant studies demonstrated that ice-4 has showed good antioxidant activity. The restoration of serum levels of SGOT, SGPT and ALKP, liver GSH status and reduction or inhibition of lipid peroxidation in liver of tBHP intoxicated mice after administration of ice-4 at dose of 250mg/kg indicated its potential use for hepatoprotective activity. PMID:24183951

V G M, Naidu; Atmakur, Hymavathi; Katragadda, Suresh Babu; Devabakthuni, Bhavana; Kota, Anudeep; S, Chenna Keshava Reddy; Kuncha, Madhusudana; M V P S, Vishnu Vardhan; Kulkarni, Prasad; Janaswamy, Madhusudana Rao; Sistla, Ramakrishna

2014-03-15

149

Complement-Mediated Killing of Microtumors in Vitro  

PubMed Central

Complement-mediated lysis of cancer cells growing in three-dimensional aggregates involves factors that are not associated with the killing of cells in suspension. We have used multicellular tumor spheroids established from breast carcinoma (T47D) and ovarian teratocarcinoma (PA-1) cell lines as models to study complement-mediated destruction of micrometastases and small solid tumors. We found that significant killing of microtumors treated with an antitumor antibody and a specific monoclonal antibody (YTH53.1) against the complement lysis inhibitor protectin (CD59) started to occur after a 1 to 2-hour lag phase. After an overnight incubation, the microtumors became totally infiltrated by the YTH53.1 monoclonal antibody and C1q, whereas C3 and C5b-9 penetrated as a frontier to the peripheral cell layers. A 51Cr release assay showed that during a 24-hour pulsed treatment with complement, 33% of cells in the spheroids were killed, and the average tumor volume decreased by 28%. According to propidium iodide staining, complement exposure resulted in killing and peeling off of the outermost tumor cells. PMID:9736033

Hakulinen, Juha; Meri, Seppo

1998-01-01

150

Cigarette Smoke Alters Respiratory Syncytial Virus-Induced Apoptosis and Replication  

PubMed Central

Individuals exposed to cigarette smoke have a greater number and severity of viral infections, including respiratory syncytial virus (RSV) infections, than do nonsmokers, but the cellular mechanism is unknown. Our objective was to determine the mechanism by which cigarette smoke augments viral infection. We hypothesize that cigarette smoke causes necrosis and prevents virus-induced cellular apoptosis, and that this is associated with increased inflammation and viral replication. Primary airway epithelial cells were exposed to cigarette smoke extract for 2 days, followed by 1 day of RSV exposure. Western blot detection of cleaved caspases 3 and 7 showed less apoptosis when cells were treated with cigarette smoke before viral infection. This finding was confirmed with ELISA and TUNEL detection of apoptosis. Measures of cell viability, including propidium iodide staining, ATP assay, and cell counts, indicated that cigarette smoke causes necrosis rather than virus-induced apoptosis. Using plaque assay and fluorescently-labeled RSV, we showed that although there were less live cells in the cigarette smoke–pretreated group, viral load was increased. The effect was inhibited by pretreatment of cells with N-acetylcysteine and aldehyde dehydrogenase, suggesting that the effect was primarily mediated by reactive aldehydes. Cigarette smoke causes necrosis rather than apoptosis in viral infection, resulting in increased inflammation and enhanced viral replication. PMID:19131644

Groskreutz, Dayna J.; Monick, Martha M.; Babor, Ellen C.; Nyunoya, Toru; Varga, Steven M.; Look, Dwight C.; Hunninghake, Gary W.

2009-01-01

151

UVA Irradiation of Dysplastic Keratinocytes: Oxidative Damage versus Antioxidant Defense  

PubMed Central

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

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

2012-01-01

152

Surface oxidation of UHMWPE for orthopedic use increases apoptosis and necrosis in human granulocytes.  

PubMed

Ultra high molecular weight polyethylene (UHMWPE) used in orthopedic prosthesis is often sterilized with gamma-rays and the subsequent oxidation was suggested to favor the in vivo wear. UHMWPE debries produced by wearing trigger an inflammatory response that can led to the implant failure. To explore direct effects of UHMWPE oxidation on immunocompetent cells and their possible role in the prosthesis failure, peripheral blood cells (PBCs) have been grown for 24 and 48 h onto plastic (Ct), UHMWPE (PE) and heat oxidized UHMWPE (PEOx) discs. PBCs necrosis and apoptosis were assessed in flow cytometry using propidium iodide staining. After 24 h, no statistically significant differences were observed in the amount of apoptotic and necrotic cells between Ct, PE and PEOx samples while, after 48 h, both necrotic and apoptotic cells were strongly increased in PEOx samples where also the granulocytes population appeared strongly reduced (6.3+/-1.1%) compared to PE (10.5+/-1.5%) and Ct (15.1+/-0.9%) samples. We conclude that surface oxidation may interfere with prosthetic failure and/or integration via granulocytes modulation. PMID:15348470

Renň, F; Sabbatini, M; Cannas, M

2003-03-01

153

Cytotoxicity of All-Trans-Retinal Increases Upon Photodegradation†  

PubMed Central

All-trans-retinal (AtRal) can accumulate in the retina as a result of excessive exposure to light. The purpose of this study was to compare cytotoxicity of AtRal and photodegraded AtRal (dAtRal) on cultured human retinal pigment epithelial cells in dark and upon exposure to visible light. AtRal was degraded by exposure to visible light. Cytotoxicity was monitored by imaging of cell morphology, propidium iodide staining of cells with permeable plasma membrane and measurements of reductive activity of cells. Generation of singlet oxygen photosensitized by AtRal and dAtRal was monitored by time-resolved measurements of characteristic singlet oxygen phosphorescence. Photodegradation of AtRal resulted in a decrease in absorption of visible light and accumulation of the degradation products with absorption maximum at ~330 nm. Toxicity of dAtRal was concentration-dependent and was greater during irradiation with visible light than in dark. DAtRal was more cytotoxic than AtRal both in dark and during exposure to visible light. Photochemical properties of dAtRal indicate that it may be responsible for the maximum in the action spectra of retinal photodamage recorded in animals. In conclusion, photodegradation products of AtRal may impose a significant threat to the retina and therefore their roles in retinal pathology need to be explored. PMID:22515697

Rozanowska, Malgorzata; Handzel, Kinga; Boulton, Michael E.; Rozanowski, Bartosz

2013-01-01

154

Antimicrobial Mechanism of Monocaprylate  

PubMed Central

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

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

2012-01-01

155

Bufalin Exerts Inhibitory Effects on IL-1?-Mediated Proliferation and Induces Apoptosis in Human Rheumatoid Arthritis Fibroblast-Like Synoviocytes.  

PubMed

Rheumatoid arthritis fibroblast-like synoviocytes (RAFLSs) proliferate abnormally and resist apoptosis. Bufalin inhibits cell proliferation and induces apoptosis in human cancer cells. In this study, we explored the effects of bufalin on interleukin-1beta (IL-1?)-induced proliferation and apoptosis of RAFLSs. The cell proliferation and apoptosis were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay and annexin V/propidium iodide staining, respectively. Bufalin dose-dependently inhibited IL-1?-induced RAFLS proliferation. Mechanistically, bufalin decreased the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-?B), both of which are involved in IL-1?-mediated RAFLS proliferation. Moreover, bufalin induced apoptosis and mitochondrial damage of RAFLSs, which was associated with Bcl-2 downregulation, Bax upregulation, mitochondrial cytochrome c release, and enhanced cleavages of caspase-3 and poly-(ADP-ribose) polymerase. Collectively, our results reveal that bufalin suppresses IL-1?-induced proliferation of RAFLSs through MAPK and NF-?B signaling pathways and induces RAFLS apoptosis via the mitochondria-dependent pathway. PMID:24752615

Chang, Yue-Wen; Zhao, Yong-Fang; Cao, Yue-Long; Gu, Wei; Pang, Jian; Zhan, Hong-Sheng

2014-10-01

156

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

PubMed

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

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

2015-01-01

157

Proteasome inhibition reverses hedgehog inhibitor and taxane resistance in ovarian cancer  

PubMed Central

The goal of this study was to determine whether combined targeted therapies, specifically those against the Notch, hedgehog and ubiquitin-proteasome pathways, could overcome ovarian cancer chemoresistance. Chemoresistant ovarian cancer cells were exposed to gamma-secretase inhibitors (GSI-I, Compound E) or the proteasome inhibitor bortezomib, alone and in combination with the hedgehog antagonist, LDE225. Bortezomib, alone and in combination with LDE225, was evaluated for effects on paclitaxel efficacy. Cell viability and cell cycle analysis were assessed by MTT assay and propidium iodide staining, respectively. Proteasome activity and gene expression were determined by luminescence assay and qPCR, respectively. Studies demonstrated that GSI-I, but not Compound E, inhibited proteasome activity, similar to bortezomib. Proteasome inhibition decreased hedgehog target genes (PTCH1, GLI1 and GLI2) and increased LDE225 sensitivity in vitro. Bortezomib, alone and in combination with LDE225, increased paclitaxel sensitivity through apoptosis and G2/M arrest. Expression of the multi-drug resistance gene ABCB1/MDR1 was decreased and acetylation of ?-tubulin, a marker of microtubule stabilization, was increased following bortezomib treatment. HDAC6 inhibitor tubastatin-a demonstrated that microtubule effects are associated with hedgehog inhibition and sensitization to paclitaxel and LDE225. These results suggest that proteasome inhibition, through alteration of microtubule dynamics and hedgehog signaling, can reverse taxane-mediated chemoresistance. PMID:25216523

Amm, Hope M.; Katre, Ashwini A.; Dobbin, Zachary C.; Jeong, Dae Hoon

2014-01-01

158

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

NASA Technical Reports Server (NTRS)

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.

Lawless, B. DeSales

1999-01-01

159

A new flavonoid regulates angiogenesis and reactive oxygen species production.  

PubMed

The tumor vascular system, which is critical to the survival and growth of solid tumors, has been an attractive target for anticancer research. Building on studies that show that some flavonoids have anticancer vascular effects, we developed and analyzed the flavonoid derivative R24 [3, 6-bis (2-oxiranylmethoxy)-9H-xanthen-9-one]. A CAM assay revealed that R24 disrupted neovascular formation; fewer dendrites were detected and overall dendritic length was shorter in the R24-treated chicken embryos. The antiproliferative effect of R24 was measured by MTT assay in A549 (lung cancer), AsPC-1 (pancreatic cancer), HCT-116 (colorectal cancer), and PC-3 (prostate cancer) cell lines. R24 reduced proliferation with an IC50 of 3.44, 3.59, 1.22, and 11.83 ?M, respectively. Cell-cycle analysis and Annexin-V/propidium iodide staining showed that R24 induced apoptosis. In addition, R24 regulated intracellular ROS production in a dose-dependent manner. CM-H2DCFDA staining indicated that intracellular ROS production increased with the R24 dose. In summary, we found that R24 exhibits potent antiangiogenic and antiproliferative effects, induces apoptosis, and promotes ROS production. PMID:24729227

Zhang, Mei; Liu, Chaomei; Zhang, Zhenhuan; Yang, Shanmin; Zhang, Bingrong; Yin, Liangjie; Swarts, Steven; Vidyasagar, Sadasivan; Zhang, Lurong; Okunieff, Paul

2014-01-01

160

Interplay between the Notch and PI3K/Akt pathways in high glucose-induced podocyte apoptosis.  

PubMed

Podocyte apoptosis contributes to the pathogenesis of diabetic nephropathy (DN). However, the mechanisms that mediate high glucose (HG)-induced podocyte apoptosis remain poorly understood. Conditionally immortalized mouse podocytes were cultured in HG medium. A chemical inhibitor or a specific short-hairpin RNA (shRNA) vector was used to inhibit the activation of the Notch pathway and the PI3K/Akt pathway in HG-treated podocytes. Western blotting and real-time PCR were used to evaluate the levels of Notch, PI3K/Akt, and apoptotic pathway signaling. The apoptosis rate of HG-treated podocytes was assessed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling and annexin V/propidium iodide staining. In HG-treated podocytes, PI3K/Akt pathway activation prevented podocyte apoptosis in the early stage of HG stimulation and Notch pathway-induced podocyte apoptosis in the late stage of HG stimulation. The inhibition of the Notch pathway or the activation of the PI3K/Akt pathway prevented cell apoptosis in HG-treated podocytes. These findings suggest that the Notch and PI3K/Akt pathways may mediate HG-induced podocyte apoptosis. PMID:24226527

Wang, Xiao-Mei; Yao, Min; Liu, Shu-Xia; Hao, Jun; Liu, Qing-Juan; Gao, Feng

2014-01-01

161

[Subcellular localization and resistance to Gibberella fujikuroi of AtELHYPRP2 in transgenic tobacco].  

PubMed

The subcellular localization and the resistance to fungal pathogen Gibberella fujikuroi of the protein encoded by Arabidopsis AtELHYPRP2 (EARLI1-LIKE HYBRID PROLINE-RICH PROTEIN 2, AT4G12500) were investigated using transgenic tobacco plants. The coding sequence of AtELHYPRP2 was amplified from genomic DNA of Col-0 ecotype. After restriction digestion, the PCR fragment was ligated into pCAMBIA1302 to produce a fusion expression vector, pCAMBIA1302-AtELHYPRP2-GFP. Then the recombinant plasmid was introduced into Agrobacterium tumefaciens strain LBA4404 and transgenic tobacco plants were regenerated and selected via leaf disc transformation method. RT-PCR and Western blotting analyses showed that AtELHYPRP2 expressed effectively in transgenic tobacco plants. Observation under laser confocal microscopy revealed that the green fluorescence of AtELHYPRP2-GFP fusion protein could overlap with the red fluorescence came from propidium iodide staining, indicating AtELHYPRP2 is localized to cell surface. Antimicrobial experiments exhibited that the constitutive expression of AtELHYPRP2 could enhance the resistance of tobacco to fungal pathogen G. fujikuroi and the infection sites could accumulate H2O2 obviously. The basal expression levels of PR1 and the systemic expression levels of PR1 and PR5 in transgenic tobacco plants were higher than that of the wild-type plants, suggesting AtELHYPRP2 may play a role in systemic acquired resistance. PMID:25007583

Chai, Qiuxia; Li, Benchang; Xu, Ziqin

2014-03-01

162

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

PubMed

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 (42)K 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 AgNO(3) rapidly reduced K(+) influx to 5% that of controls, and brought about pronounced and immediate increases in K(+) efflux, while higher doses of Au(3+) and Hg(2+) 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 AgNO(3), 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 NH(4)(+). Co-application, with silver, of the channel blockers Cs(+), TEA(+), or Ca(2+), 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. Ni(2+), Cd(2+), and Pb(2+), 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

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

2012-01-01

163

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

PubMed Central

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

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

2012-01-01

164

Artemisia absinthium (AA): a novel potential complementary and alternative medicine for breast cancer.  

PubMed

Natural products have become increasingly important in pharmaceutical discoveries, and traditional herbalism has been a pioneering specialty in biomedical science. The search for effective plant-derived anticancer agents has continued to gain momentum in recent years. The present study aimed to investigate the role of crude extracts of the aerial parts of Artemisia absinthium (AA) extract in modulating intracellular signaling mechanisms, in particular its ability to inhibit cell proliferation and promote apoptosis in a human breast carcinoma estrogenic-unresponsive cell line, MDA-MB-231, and an estrogenic-responsive cell line, MCF-7. Cells were incubated with various concentrations of AA, and anti-proliferative activity was assessed by MTT assays, fluorescence microscopy after propidium iodide staining, western blotting and cell cycle analysis. Cell survival assays indicated that AA was cytotoxic to both MDA-MB-231 and MCF-7 cells. The morphological features typical of nucleic staining and the accumulation of sub-G1 peak revealed that the extract triggered apoptosis. Treatment with 25 ?g/mL AA resulted in activation of caspase-7 and upregulation of Bad in MCF-7 cells, while exposure to 20 ?g/mL AA induced upregulation of Bcl-2 protein in a time-dependent response in MDA-MB-231 cells. Both MEK1/2 and ERK1/2 was inactivated in both cell lines after AA treatment in a time-dependent manner. These results suggest that AA-induced anti-proliferative effects on human breast cancer cells could possibly trigger apoptosis in both cell lines through the modulation of Bcl-2 family proteins and the MEK/ERK pathway. This might lead to its possible development as a therapeutic agent for breast cancer following further investigations. PMID:22311047

Shafi, Gowhar; Hasan, Tarique N; Syed, Naveed Ahmed; Al-Hazzani, Amal A; Alshatwi, Ali A; Jyothi, A; Munshi, Anjana

2012-07-01

165

Steroidal glycosides with antiproliferative activities from Digitalis trojana.  

PubMed

The phytochemical investigation of Digitalis trojana led to the isolation of two cardiac glycosides (1, 2), one pregnane glycoside (3), three furostanol type saponins (4-6), along with three cleroindicins (7-9), four phenylethanoid glycosides (10-13), two flavonoids (14, 15) and two phenolic acid derivatives (16, 17). The structure elucidation of the isolates was carried out by NMR experiments as well as ESI-MS. The cytotoxic activity of compounds 1-13 against a small panel of cancer cell lines, namely MCF-7, T98G, HT-29, PC-3, A375 and SH-SY5Y, was investigated. Compounds 1-6 showed antiproliferative activity against human breast MCF-7 and colon HT-29 cancer cell lines with IC50 values ranging from 8.3 to 50??M. In order to understand the mechanism involved in the cell death, the active compounds were tested as pro-apoptotic agents using propidium iodide staining by flow cytometry method. No significant increase was observed in the apoptosis of the MCF-7 and HT-29 cancer cells. Moreover, the effects of the active compounds on cell proliferation were assessed on the same cancer cell lines by cell cycle analysis of DNA content using flow cytometry. No significative changes were observed in the cell cycle of MCF-7, while significant changes in G2 /M cell cycle phase of HT-29 cells were observed after treatment with digitalin (1), cariensoside (3) and 22-O-methylparvispinoside B (6) at 10??M. PMID:23722601

Kirmizibekmez, Hasan; Masullo, Milena; Festa, Michela; Capasso, Anna; Piacente, Sonia

2014-04-01

166

Cells  

NSDL National Science Digital Library

Students use websites to review about cells and cell processes. The Cell Look inside a cell The Virtual Cell Another inside view of a cell. Click on the worksheet. Cells of the body Look inside cells of the body Cells Flash cards Practice cell parts with functions. Cell Concentration Play concentration matching game. Cell Differentiation Movie Watch how cells change as an organism develops. Cell Organelle Table Review Cell Organelles Inside a Cell Look Inside a Cell Nobel Prize Educational Games Play games while learning about ...

Mcnees, Mrs.

2010-09-28

167

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

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 ?-cell death have been reported. The aim of this study was to clarify the molecular mechanisms by which 1,25(OH)2D3 contributes to ?-cell protection against cytokine-induced ?-cell dysfunction and death. Human and mouse islets were exposed to IL-1? and interferon-? in the presence or absence of 1,25(OH)2D3. Effects on insulin secretion and ?-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-?B activity was tested, whereas effects on secreted chemokines/cytokines were confirmed by ELISA and migration studies. Cytokine exposure caused a significant increase in ?-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 ?-cell function/phenotype. In conclusion, these findings demonstrate a direct protective effect of 1,25(OH)2D3 against inflammation-induced ?-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

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

168

Brain-derived neurotrophic factor, but not neurotrophin-3, prevents ischaemia-induced neuronal cell death in organotypic rat hippocampal slice cultures  

Microsoft Academic Search

We have investigated the neuroprotective actions of neurotrophins in a model of ischaemia using slice cultures. Ischaemia was induced in organotypic hippocampal cultures by simultaneous oxygen and glucose deprivation. Cell death was assessed 24 h later by propidium iodide fluorescence. Pre- but not post-ischaemic addition of brain-derived neurotrophic factor (BDNF) produced a concentration-dependent reduction in neuronal damage. Neurotrophin-3 was not

A. K. Pringle; L. E. Sundstrom; G. J. C. Wilde; L. R. Williams; F. lannotti

1996-01-01

169

A simple way to identify non-viable cells within living plant tissue using confocal microscopy  

E-print Network

not stained. Non-viable cells are stained selectively The root cap, consisting of columella and lateral root cap, gation zone of the root (Figure 2) [6]. Cells at the end of the lateral root cap die, thus exposing the root epidermis [6]. The enhancer-trap line... Figure 2 SYTOX orange staining of non-viable lateral root cap cells. (A) to (D) Enhancer-trap line Q0171 expressing GFP (green) in the columella (c) and lateral root cap (lrc). (A), (C) Overlay projection image of Q0171 stained with propidium iodide...

Truernit, Elisabeth; Haseloff, Jim

2008-06-23

170

Flow cytometric viability assessment of lactic acid bacteria starter cultures produced by fluidized bed drying.  

PubMed

For starter culture production, fluidized bed drying is an efficient and cost-effective alternative to the most frequently used freeze drying method. However, fluidized bed drying also poses damaging or lethal stress to bacteria. Therefore, investigation of impact of process variables and conditions on viability of starter cultures produced by fluidized bed drying is of major interest. Viability of bacteria is most frequently assessed by plate counting. While reproductive growth of cells can be characterized by the number of colony-forming units, it cannot provide the number of viable-but-nonculturable cells. However, in starter cultures, these cells still contribute to the fermentation during food production. In this study, flow cytometry was applied to assess viability of Lactobacillus plantarum starter cultures by membrane integrity analysis using SYBR®Green I and propidium iodide staining. The enumeration method established allowed for rapid, precise and sensitive determination of viable cell concentration, and was used to investigate effects of fluidized bed drying and storage on viability of L. plantarum. Drying caused substantial membrane damage on cells, most likely due to dehydration and oxidative stress. Nevertheless, high bacterial survival rates were obtained, and granulates contained in the average 2.7?×?10(9) viable cells/g. Furthermore, increased temperatures reduced viability of bacteria during storage. Differences in results of flow cytometry and plate counting suggested an occurrence of viable-but-nonculturable cells during storage. Overall, flow cytometric viability assessment is highly feasible for rapid routine in-process control in production of L. plantarum starter cultures, produced by fluidized bed drying. PMID:24584512

Bensch, Gerald; Rüger, Marc; Wassermann, Magdalena; Weinholz, Susann; Reichl, Udo; Cordes, Christiana

2014-06-01

171

VSV-MP gene therapy strategy inhibits tumor growth in nude mice model of human lung adenocarcinoma.  

PubMed

Vesicular stomatitis virus (VSV) matrix protein (MP) can induce in vitro apoptosis of tumor cells in the absence of other viral components. Here, the antitumor activity of VSV-MP against lung adenocarcinoma was investigated in vivo. A pVAX-plasmid DNA encoding VSV-MP and control empty vectors (pVAX) were constructed and wrapped-up with liposome. A549 and Spc-A1 human lung adenocarcinoma cells were transfected with liposomal-VSV-MP (Lip-MP) or Lip-pVAX and then examined for cell viability or apoptosis using Hoechst/propidium iodide staining by flow cytometry, and further demonstrated by caspase/poly ADP-ribose polymerase (PARP) cleavage analysis. For the in vivo study, A549 and Spc-A1 lung carcinoma models in nude mice were established and randomly assigned into three groups to receive eight 2-weekly intravenous administrations of medium alone as control, Lip-pVAX or Lip-MP, respectively. Subsequently, Lip-MP significantly reduced tumor growth and prolonged the survival of tumor-bearing mice compared with Lip-pVAX and control agents (P<0.05), with much higher apoptosis index of both in vivo and in vitro tumor cells, respectively (P<0.05). In addition, in vivo antitumoral effect was associated with natural killer-(NK) cell congregation without evidence of toxicity. These observations suggest that systemically delivering Lip-MP has a specific dual antitumor activity in human lung adenocarcinoma by inducing apoptosis and possibly stimulating NK-cell responses, it may provide a clue for developing new therapeutic approaches against human lung adenocarcinoma. PMID:22052213

Jing, X-M; Wen, Y-J; Shi, W; Tang, Q-Q; Li, J; Chen, X-C

2012-02-01

172

Role of the Erythropoietin Receptor in ETV6/RUNX1-Positive Acute Lymphoblastic Leukemia  

PubMed Central

Purpose We explored the mechanisms leading to the distinct overexpression of EPOR as well as the effects of EPO signaling on ETV6/RUNX1-positive acute lymphoblastic leukemias. Experimental Design ETV6/RUNX1-expressing model cell lines and leukemic cells were used for real-time PCR of EPOR expression. Proliferation, viability, and apoptosis were analyzed on cells exposed to EPO, prednisone, or inhibitors of EPOR pathways by [3H]thymidine incorporation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and Annexin V/propidium iodide staining. Western blot analysis was done to detect activation of signaling proteins. Serum EPO levels and sequences of the EPOR (n = 53) as well as hemoglobin levels were taken from children with acute lymphoblastic leukemia enrolled in Austrian protocols. Results We show here that ectopic expression of ETV6/RUNX1 induced EPOR up-regulation. Anemia, however, did not appear to influence EPOR expression on leukemic cells, although children with ETV6/RUNX1-positive leukemias had a lower median hemoglobin than controls. Exposure to EPO increased proliferation and survival of ETV6/RUNX1-positive leukemias in vitro, whereas blocking its binding site did not alter cell survival. The latter was not caused by activating mutations in the EPOR but might be triggered by constitutive activation of phosphatidylinositol 3-kinase/Akt, the major signaling pathway of EPOR in these cells. Moreover, prednisone-induced apoptosis was attenuated in the presence of EPO in this genetic subgroup. Conclusions Our data suggest that ETV6/RUNX1 leads to EPOR up-regulation and that activation by EPO might be of relevance to the biology of this leukemia subtype. Further studies are, however, needed to assess the clinical implications of its apoptosis-modulating properties. PMID:19010836

Inthal, Andrea; Krapf, Gerd; Beck, Dominik; Joas, Ruth; Kauer, Max O.; Orel, Lukas; Fuka, Gerhard; Mann, Georg; Panzer-Grumayer, E. Renate

2014-01-01

173

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

PubMed Central

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

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

2012-01-01

174

In vitro and in vivo assessment of the biocompatibility of an Mg-6Z(n) alloy in the bile.  

PubMed

There is a great clinical need for biodegradable bile duct stents. Biodegradable stents made of an Mg-6Zn alloy were investigated in both vivo animal experiment and in vitro cell experiments. During the in vivo experiments, blood biochemical tests were performed to determine serum magnesium, serum creatinine (CREA), blood urea nitro-gen (BUN), serum lipase (LPS), total bilirubin (TB) and glutamic-pyruvic transaminase (GPT) levels. Moreover, tissue samples of common bile duct (CBD), liver and kidney were taken for histological evaluation. In the in vitro experiments, primary mouse extrahepatic bile duct epithelial cells (MEBDECs) were isolated and cultured. Cytotoxicity testing was carried out using the MTT method. Flow cytometry analyses with propidium iodide staining were performed to evaluate the effect of Mg-6Zn alloy extracts on cell cycle. The in vivo experiments revealed no significant differences (P > 0.05) in serum magnesium, CREA, BUN, LPS, TB or GPT before and after the operation. Based on the HE results, hepatocytes, bile duct epithelial cells, renal glomerulus and renal tubule tissues did not present significant necrosis. In the in vitro experiments, the cell relative growth rate curve did not change significantly from 20 to 40 % extracts. In vitro experiments showed that 20-40 % Mg-6Zn extracts are bio-safe for MEBDECs. In vivo experiments showed that Mg-6Zn stents did not affect several important bio-chemical parameters or, harm the function or morphology of the CBD, kidney, pancreas and liver. Our data suggested that this Mg-6Zn alloy is a safe biocompatible material for CBD. PMID:24243223

Chen, Yigang; Yan, Jun; Zhao, Changli; Zhang, Shaoxiang; Yu, Song; Wang, Zigang; Wang, Xiaohu; Zhang, Xiaonong; Zheng, Qi

2014-02-01

175

Flow cytometric quantification of radiation responses of murine peritoneal cells  

SciTech Connect

Methods have been developed to distinguish subpopulations of murine peritoneal cells, and these were applied to the measurement of early changes in peritoneal cells after irradiation. The ratio of the two major subpopulations in the peritoneal fluid, lymphocytes and macrophages, was measured rapidly by means of cell volume distribution analysis as well as by hypotonic propidium iodide (PI) staining. After irradiation, dose and time dependent changes were noted in the cell volume distributions: a rapid loss of peritoneal lymphocytes, and an increase in the mean cell volume of macrophages. The hypotonic PI staining characteristics of the peritoneal cells showed two or three distinctive G/sub 1/ peaks. The ratio of the areas of these peaks was also found to be dependent of the radiation dose and the time after irradiation. These results demonstrate that these two parameters may be used to monitor changes induced by irradiation (biological dosimetry), and to sort different peritoneal subpopulations.

Tokita, N.; Raju, M.R.

1982-01-01

176

Curcumin analogue T83 exhibits potent antitumor activity and induces radiosensitivity through inactivation of Jab1 in nasopharyngeal carcinoma  

PubMed Central

Background Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus–associated malignancy that is most common in East Asia, Africa, and Alaska. Radiotherapy is the main treatment option; unfortunately, disease response to concurrent radiotherapy and chemotherapy varies among patients with NPC, and in many cases, NPC becomes resistant to radiotherapy. Our previous studies indicated that Jab1/CSN5 was overexpressed and plays a role in the pathogenesis and radiotherapy resistance in NPC. Therefore, it is important to seek for innovative therapeutics targeting Jab1/CSN5 for NPC. In this study, we explored the antitumor effect of a curcumin analogue T83 in NPC, and found T83 exhibits antitumor activity and induces radiosensitivity through inactivation of Jab1 in NPC. Methods NPC cell viability and proliferation were detected by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation assays. Cell cycle distribution was detected with use of flow cytometry. Apoptosis was examined by using the Annexin V/propidium iodide staining assay and cleavage poly(ADP-ribose polymerase (PARP) and cleavage caspase-3 expression. Jab1 expression was examined by Western blotting. Results A growth inhibitory effect was observed with T83 treatment in a dose- and time-dependent manner. T83 significantly induced G2/M arrest and apoptosis in NPC. In addition, T83 inhibited Jab1 expression and sensitized NPC cells to radiotherapy. Conclusion Our data indicate that T83 exhibits potent inhibitory activity in NPC cells and induces radiotherapy sensitivity. Thus, T83 has translational potential as a chemopreventive or therapeutic agent for NPC. PMID:23815987

2013-01-01

177

Time course and mechanism of hippocampal neuronal death in an in vitro model of status epilepticus: Role of NMDA receptor activation and NMDA dependent calcium entry  

PubMed Central

The hippocampus is especially vulnerable to seizure-induced damage and excitotoxic neuronal injury. This study examined the time course of neuronal death in relationship to seizure duration and the pharmacological mechanisms underlying seizure-induced cell death using low magnesium (Mg2+) induced continuous high frequency epileptiform discharges (in vitro status epilepticus) in hippocampal neuronal cultures. Neuronal death was assessed using cell morphology and Fluorescein diacetate-Propidium iodide staining. Effects of low Mg2+ and various receptor antagonists on spike frequency were assessed using patch clamp electrophysiology. We observed a linear and time-dependent increase in neuronal death with increasing durations of status epilepticus. This cell death was dependent upon extracellular calcium that entered primarily through the N-methyl-D-aspartate (NMDA) glutamate receptor channel subtype. Neuronal death was significantly decreased by co-incubation with the NMDA receptor antagonists and was also inhibited by reduction of extracellular calcium (Ca2+) during status epilepticus. In contrast, neuronal death from in vitro status epilepticus was not significantly prevented by inhibition of other glutamate receptor subtypes or voltage-gated Ca2+ channels. Interestingly this NMDA-Ca2+ dependent neuronal death was much more gradual in onset compared to cell death from excitotoxic glutamate exposure. The results provide evidence that in vitro status epilepticus results in increased activation of the NMDA-Ca2+ transduction pathway leading to neuronal death in a time dependent fashion. The results also indicate that there is a significant window of opportunity during the initial time of continuous seizure activity to be able to intervene, protect neurons and decrease the high morbidity and mortality associated with status epilepticus. PMID:18289526

Deshpande, Laxmikant S.; Lou, Jeffrey K.; Mian, Ali; Blair, Robert E.; Sombati, Sompong; Attkisson, Elisa; DeLorenzo, Robert J.

2008-01-01

178

Anti-amoebic properties of a Malaysian marine sponge Aaptos sp. on Acanthamoeba castellanii.  

PubMed

Crude methanol extracts of a marine sponge, Aaptos aaptos, collected from three different localities namely Kapas, Perhentian and Redang Islands, Terengganu, Malaysia, were tested in vitro on a pathogenic Acanthamoeba castellanii (IMR isolate) to examine their anti-amoebic potential. The examination of anti-Acanthamoebic activity of the extracts was conducted in 24 well plates for 72 h at 30 °C. All extracts possessed anti-amoebic activity with their IC(50) values ranging from 0.615 to 0.876 mg/mL. The effect of the methanol extracts on the surface morphology of A. castellanii was analysed under scanning electron microscopy. The ability of the extracts to disrupt the amoeba cell membrane was indicated by extensive cell's blebbing, changes in the surface morphology, reduced in cell size and with cystic appearance of extract-treated Acanthamoeba. Number of acanthapodia and food cup was also reduced in this Acanthamoeba. Morphological criteria of apoptosis in Acanthamoeba following treatment with the sponge's extracts was determined by acridine orange-propidium iodide staining and observed by fluorescence microscopy. By this technique, apoptotic and necrotic cells can be visualized and quantified. The genotoxic potential of the methanol extracts was performed by the alkaline comet assay. All methanol extracts used were significantly induced DNA damage compared to untreated Acanthamoeba by having high percentage of scores 1, 2, and 3 of the DNA damage. Results from cytotoxicity and genotoxicity studies carried out in the present study suggest that all methanol extracts of A. aaptos have anti-amoebic properties against A. castellanii. PMID:22805843

Nakisah, M A; Ida Muryany, M Y; Fatimah, H; Nor Fadilah, R; Zalilawati, M R; Khamsah, S; Habsah, M

2012-03-01

179

Proliferation of chondrocytes on a 3-d modelled macroporous poly(hydroxyethyl methacrylate)-gelatin cryogel.  

PubMed

Tissue-engineering constructs should be designed to mimic the native tissue environment for cells, the scaffold matching to stiffness and strength of the tissues while maintaining an interconnected porous network and a reasonable porosity. This study presents a new single-step protocol for synthesis of a poly(hydroxyethyl methacrylate)-poly(ethylene glycol) diacrylate-gelatin (HPG) macroporous polymeric scaffold with well-controlled porous structure and good mechanical strength. The pore size of these matrices lies in the range of 30 to 100 ?m with an average pore diameter of 80 ?m and with an interconnected pore structure as analyzed by scanning electron microscopy. Further, interconnectivity was also confirmed by high solvent uptake capacity, as the cryogel reached its equilibrium within 2 min. The gels also showed substantial mechanical integrity, i.e., the average compressive modulus was 32.73 ± 2.36 kPa at 15% compression of their original length. The degree of weight loss of these cryogels was found to be approx. 88% within 8 weeks of incubation in PBS (pH 7.4) at 37°C. Physio-chemically optimized cryogel was further evaluated for in vitro growth and proliferation of isolated primary goat chondrocytes up to 3 weeks. The cell adherence on cryogel was examined by SEM analysis, while cell-matrix interaction was examined by 4-6-diamidino-2-phenylindole and propidium iodide staining. Furthermore, the cell compatibility and proliferation was evaluated using the MTT assay. Increase in total cellular metabolic activity was observed as shown by continuous increase in glycosaminoglycan and collagen contents with time. Collagen type-I and type-II gene expression analysed for over 3 weeks by RT-PCR showed the prominent expression of collagen type-II. These results suggest the use of synthesised cryogel scaffold as a matrix for chondrocyte attachment and proliferation in 3-D environment and as a delivery system in cartilage-tissue engineering. PMID:20843432

Singh, Deepti; Tripathi, Anuj; Nayak, Vijayashree; Kumar, Ashok

2011-01-01

180

Cells  

NSDL National Science Digital Library

Today you have the opportunity to explore the cell in a 3D fashion and learn more about its organelles. The following three links will help you understand the structure of the cell and organelles more clearly and help you understand their functions as well. Enjoy! Cells Alive Inside a Cell Virtual Cell When you have completed viewing the websites, please draw a picture of a animal cell or plant cell with its labeled parts. ...

Aird, Mrs.

2006-11-15

181

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

PubMed

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. PMID:1381171

Deng, M Y; Cliver, D O

1992-08-01

182

DNA Damage Response and Apoptosis  

PubMed Central

A number of methods have been developed to examine the morphologic, biochemical, and molecular changes that happen during the DNA damage response that may ultimately lead to death of cells through various mechanisms that include apoptosis. When cells are exposed to ionizing radiation or chemical DNA-damaging agents, double-stranded DNA breaks (DSB) are generated that rapidly result in the phosphorylation of histone variant H2AX. Because phosphorylation of H2AX at Ser 139 correlates well with each DSB, phospho-H2AX is a sensitive marker to used to examine the DNA damage and its repair. Apoptotic cells are characterized on the basis of their reduced DNA content and morphologic changes, including nuclear condensation, which can be detected by flow cytometry (sub-G1 DNA content), trypan blue, or Hoechst staining. The appearance of phosphatidylserine on the plasma membrane with annexin V–fluorochrome conjugates indicates the changes in plasma membrane composition and function. By combining it with propidium iodide staining, this method can also be used to distinguish early versus late apoptotic or necrotic events. The activation of caspases is another well-known biochemical marker of apoptosis. Finally, the Bcl-2 family of proteins and the mitochondria that play a critical role in DNA damage-induced apoptosis can be examined by translocation of Bax and cytochrome c in and out of mitochondria. In this chapter, we discuss the most commonly used techniques used in our laboratory for determining the DNA damage response leading to apoptosis. PMID:18603118

Plesca, Dragos; Mazumder, Suparna; Almasan, Alexandru

2010-01-01

183

Amine- and carboxyl- quantum dots affect membrane integrity of bacterium Cupriavidus metallidurans CH34.  

PubMed

The present study examines the interaction of amine- and carboxyl- PEG core/shell quantum dots (QDs) with metal resistant bacterium Cupriavidus metallidurans CH34. The evolution of the number of QDs, their hydrodynamic radius, diffusion coefficients, and single particle fluorescence were characterized before and during the contact with bacterium by fluorescence correlation spectroscopy (FCS). The obtained results showed that at nanomolar concentrations the amine- and carboxyl-PEG-QDs with average hydrodynamic radiuses of 16.4 and 13.5 nm, form stable dispersions in the absence and presence of 15 mgC L(-1) HA. The decrease of the number of fluorescent particles in the bacterial medium, determined by FCS, together with the increase of the fluorescence of bacterial cells over the background, found by flow cytometry (FCM), demonstrated the association of QDs to C. metallidurans. Furthermore, QDs enhanced the level of the reactive oxygen species in the bacterial cells and augmented the percentage of the cells with damaged and leaky membranes as probed by FCM in combination with 5-(and-6)-carboxy-27'-dichlorodihydrofluorescein diacetate and propidium iodide stains. No difference in the behavior of amine- and carboxyl-PEG-QDs was found, suggesting that different functional groups in the surface coating have no effect on bacterium-QD interactions under the studied conditions. The presence of HA does not affect the hydrodynamic characteristics of the functionalized QDs, but prevented the damage to the bacterial membrane. The slight decrease in the bacterial growth found after exposure of C. metallidurans to these QDs was attributed to the nanoparticles themselves rather the cadmium, zinc, or selenium ions released from the QDs. PMID:19673316

Slaveykova, Vera I; Startchev, Konstantin; Roberts, Joanna

2009-07-01

184

The Antimicrobial Mechanism of Action of Epsilon-Poly-l-Lysine.  

PubMed

Epsilon-poly-l-lysine (?-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 ?-PL's mechanism of action using Escherichia coli and Listeria innocua as model organisms. We examined ?-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 ?-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 ?-PL's binding efficiency. ?-PL removed the lipopolysaccharide layer and affected cell morphology of E. coli, while L. innocua underwent minor morphological changes. Propidium iodide staining showed that ?-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 ?-PL relied on negative charges on the membrane. Suspended membrane vesicles were disrupted by ?-PL, and a detergent-like disruption of E. coli membrane was confirmed by atomic force microscopy imaging of supported lipid bilayers. We hypothesize that ?-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

Hyldgaard, Morten; Mygind, Tina; Vad, Brian S; Stenvang, Marcel; Otzen, Daniel E; Meyer, Rikke L

2014-12-15

185

T-2 toxin induced skin inflammation and cutaneous injury in mice.  

PubMed

T-2 toxin is one of the most toxic among several trichothecenes involved in both human and animal poisoning cases. We investigated the biochemical and histological alterations behind inflammation and cutaneous injury caused by T-2 toxin. Swiss albino mice were exposed to T-2 toxin topically at doses of 0.5, 1 and 2 LD50 (2.97, 5.94 and 11.88 mg/kg respectively) and observed till 3, 24 and 72 h. Topical application of T-2 toxin resulted in skin oxidative stress in terms of increased reactive oxygen species generation, lipid peroxidation and neutrophil mediated myeloperoxidase activity. The histological alterations include degenerative changes like vacuolation, ballooning of basal keratinocytes and infiltration of inflammatory cells in dermis. The mRNA levels of skin pro-inflammatory cytokines TNF-?, IL-6, and IL-1? showed significant up regulation. Anti-inflammatory cytokines IL-10 showed significant up regulation at 24h whereas IL-4 showed down regulation for all the doses and time points. Gelatin zymography and immunoblot analysis of matrix metalloproteinases (MMP)-9 and 2 indicated MMP activation and their role in degenerative skin histological changes. Time dependent increase in inducible nitric oxide synthase levels was seen. Immunoblot analysis revealed significant increase in the levels of phosphorylated p38 mitogen activated protein kinase (MAPK). Flow cytometry analysis of propidium iodide stained epidermal cells showed increase in sub-G1 population at all the doses and time points indicating apoptosis. In summary, T-2 toxin induced skin inflammation and cutaneous injury is mediated through oxidative stress, activation of myeloperoxidase, MMP activity, increase in inflammatory cytokines, activation of p38 MAPK and apoptosis of epidermal cells leading to degenerative skin histological changes. PMID:22960706

Agrawal, Mona; Yadav, Preeti; Lomash, Vinay; Bhaskar, A S B; Lakshmana Rao, P V

2012-12-16

186

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

PubMed Central

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 TQ’s 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

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

2013-01-01

187

Role of 14-3-3? in poor prognosis and in radiation and drug resistance of human pancreatic cancers  

PubMed Central

Background Pancreatic cancer is the fourth leading cause of death in the US. Unlike other solid tumors such as testicular cancer which are now curable, more than 90% of pancreatic cancer patients die due to lack of response to therapy. Recently, the level of 14-3-3? mRNA was found to be increased in pancreatic cancers and this increased expression may contribute to the failure in treatment of pancreatic cancers. In the present study, we tested this hypothesis. Methods Western blot analysis was used to determine 14-3-3? protein level in fresh frozen tissues and was correlated to clinical outcome. A stable cell line expressing 14-3-3? was established and the effect of 14-3-3? over-expression on cellular response to radiation and anticancer drugs were tested using SRB assay and clonogenic assays. Cell cycle distribution and apoptosis analyses were performed using propidium iodide staining and PARP cleavage assays. Results We found that 14-3-3? protein level was increased significantly in about 71% (17 of 24) of human pancreatic cancer tissues and that the 14-3-3? protein level in cancers correlated with lymph node metastasis and poor prognosis. Furthermore, we demonstrated that over-expression of 14-3-3? in a pancreatic cancer cell line caused resistance to ?-irradiation as well as anticancer drugs by causing resistance to treatment-induced apoptosis and G2/M arrest. Conclusion The increased level of 14-3-3? protein likely contributes to the poor clinical outcome of human pancreatic cancers by causing resistance to radiation and anticancer drugs. Thus, 14-3-3? may serve as a prognosis marker predicting survival of pancreatic cancer patients and guide the clinical treatment of these patients. PMID:21040574

2010-01-01

188

Induction of apoptosis and apoptotic mediators in Balb/C splenic lymphocytes by dietary n-3 and n-6 fatty acids.  

PubMed

The present study was designed to investigate the effect of dietary n-6 and n-3 polyunsaturated fatty acids (PUFA) on anti-CD3 and anti-Fas antibody-induced apoptosis and its mediators in mouse spleen cells. Nutritionally adequate semipurified diets containing either 5% w/w corn oil (n-6 PUFA) or fish oil (n-3 PUFA) were fed to weanling female Balb/C mice, and 24 wk later mice were sacrificed. In n-3 PUFA-fed mice, serum and splenocyte lipid peroxides were increased by 20 and 28.3% respectively, compared to n-6 PUFA-fed mice. Further, serum vitamin E levels were decreased by 50% in the n-3 PUFA-fed group, whereas higher anti-Fas- and anti-CD3-induced apoptosis (65 and 66%) and necrosis (17 and 25%), compared to the n-6 PUFA-fed group, were found when measured with Annexin V and propidium iodide staining, respectively. In addition, decreased Bcl-2 and increased Fas-ligand (Fas-L) also were observed in the n-3 PUFA-fed group compared to the n-6 PUFA-fed group. No difference in the ratio of splenocyte subsets nor their Fas expression was observed between the n-3 PUFA-fed and n-6 PUFA-fed groups, whereas decreased proliferation of splenocytes was found in n-3 PUFA-fed mice compared to n-6 PUFA-fed mice. In conclusion, our results indicate that dietary n-3 PUFA induces higher apoptosis by increasing the generation of lipid peroxides and elevating Fas-L expression along with decreasing Bcl-2 expression. A reduced proliferative response of immune cells also was observed in n-3 PUFA-fed mice. PMID:10574656

Avula, C P; Zaman, A K; Lawrence, R; Fernandes, G

1999-09-01

189

MiR-99a may serve as a potential oncogene in pediatric myeloid leukemia  

PubMed Central

Background Leukemia is the most common malignant proliferative disease in children. Our previous study found that miR-99a was up-regulated in pediatric primary AML using microRNA expression profiles. Up to date, although there is a certain number of reports on microRNA expression features and functions in pediatric acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), the expression and function of miR-99a in these diseases remain to be investigated. Methods qRT-PCR was performed to measure the expression level of miR-99a in 88 samples including 68 pediatric acute myeloid leukemia patients, 8 chronic myeloid leukemia patients and 12 pediatric controls. MTT assay, apoptosis assay, dual-luciferase reporter transfection assay and western blot analysis were used to investigate the function of miR-99a. Results MiR-99a was highly expressed in pediatric-onset AML (M1-M5) and CML, while significantly lowly expressed during complete remission of these diseases. MTT assay indicated that the proliferations of K562 and HL60 cells were significantly promoted by miR-99a, and apoptosis assessment by Annexin V/propidium iodide staining demonstrated that the apoptosis of these cells was inhibited by miR-99a. Additionally, dual-luciferase reporter transfection assay and western blot analysis indicated that miR-99a may target CTDSPL and TRIB2, which are two tumor suppressor genes. Conclusions This study revealed that miR-99a may play a potential oncogenic role in pediatric myeloid leukemia including AML and CML via regulating tumor suppressors CTDSPL and TRIB2, suggesting that these two leukemias might share some common biological pathways involved in the generation and development of disease and miR-99a could be a common therapeutic target for myeloid leukemias treatment. PMID:24191888

2013-01-01

190

Inhibition of experimental tobacco carcinogen induced head and neck carcinogenesis.  

PubMed

Oral cancer models have attempted to demonstrate inhibition of oral carcinogenesis. These models used synthetic carcinogens, lacked a specific mechanism of activity or used non-physiologic doses for carcinogen or inhibitor. To correct these problems the tobacco and environmental carcinogen, dibenzo[a,l]pyrene (DB[a,l]P) (0.25%, 0.010 microM/application) was painted on the tongue and/or vitamin E acid succinate (VE(as)) (0.43 I.U./0.136 (microM/treatment) administered by gavages to Syrian hamsters (14 animals per group) using physiologic low doses, 5X/week. Oral cytology supplied keratinocytes after 1, 10, or 25 weeks of treatment. Cells were analyzed by flow cytometry/laser scanning cytometry. Initiation (1-6 weeks) was suppressed by reducing DNA damage (oxidation lesions: 8-oxo-dG), and repair (comet, fpg, OGG1, NTH1). Reduction in promotion (6-10 weeks) was identified by depressed proliferation (cell cycle, bromodeoxyuridine incorporation (BrdU)) and aneuploidy (propidium iodide stain). p53 and apoptosis expressions were increased (Sub G(1), mitochondrion activation: Apo 2.7, and nucleosomal formation: mebstain (TUNEL)). VE(as) administration reduced dysplasia (10 weeks) and oral cancer formation at 25 (0/7 vs. 5/7 DB[a,l]P) and 30 weeks (3/7 vs. 6/7 DB[a,l]P). Inhibition of oral carcinogenesis by VE(as) involved reversal of several cellular events that contribute towards oral cancer. PMID:15063390

Schwartz, Joel; Baker, Vikki; Larios, Eric; Desai, Dhimant; Amin, Shantu

2004-07-01

191

Nutrient reserves may allow for genome size increase: evidence from comparison of geophytes and their sister non-geophytic relatives  

PubMed Central

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

Vesely, Pavel; Bures, Petr; Smarda, Petr

2013-01-01

192

Apigenin impairs oral squamous cell carcinoma growth in vitro inducing cell cycle arrest and apoptosis.  

PubMed

In the present study, we investigated the effect of apigenin, a flavonoid widely present in fruits and vegetables, on a tongue oral cancer-derived cell line (SCC-25) and on a keratinocyte cell line (HaCaT), with the aim of unveiling its antiproliferative mechanisms. The effect of apigenin on cell growth was evaluated by MTT assay, while apoptosis was investigated by phosphatidyl serine membrane translocation and cell cycle distribution by propidium iodide DNA staining through flow cytometry. In addition the expression of cyclins and cyclin-dependent kinases was evaluated by western blotting. A reduction of apigenin-induced cell growth was found in both cell lines, although SCC-25 cells were significantly more sensitive than the immortalized keratinocytes, HaCaT. Moreover, apigenin induced apoptosis and modulated the cell cycle in SCC-25 cells. Apigenin treatment resulted in cell cycle arrest at both G0/G1 and G2/M checkpoints, while western blot analysis revealed the decreased expression of cyclin D1 and E, and inactivation of CDK1 upon apigenin treatment. These results demonstrate the anticancer potential of apigenin in an oral squamous cell carcinoma cell line, suggesting that it may be a very promising chemopreventive agent due to its cancer cell cytotoxic activity and its ability to act as a cell cycle modulating agent at multiple levels. PMID:23969487

Maggioni, Daniele; Garavello, Werner; Rigolio, Roberta; Pignataro, Lorenzo; Gaini, Renato; Nicolini, Gabriella

2013-11-01

193

Duhuo Jisheng decoction treatment inhibits the sodium nitroprussiate?induced apoptosis of chondrocytes through the mitochondrial?dependent signaling pathway.  

PubMed

Chondrocyte apoptosis activated by the mitochondrial?dependent signaling pathway plays a crucial role in the cartilage degeneration of osteoarthritis. Duhuo Jisheng decoction (DHJSD), a herbal formula from traditional Chinese medicine, has been widely used for treating osteoarthritis (OA). However, the molecular mechanisms behind the therapeutic effect of DHJSD remain to be elucidated. In the present study, the effects of DHJSD on the mitochondrial?dependent signaling pathway in sodium nitroprussiate (SNP)?induced chondrocyte apoptosis were investigated. Chondrocytes, from the knee articular cartilage of Sprague Dawley rats, were identified by type II collagen immunohistochemistry. The chondrocytes, stimulated with or without SNP to induce apoptosis, were treated by DHJSD for various concentrations and times. The viability of SNP?induced chondrocytes treated with DHJSD was enhanced compared to SNP?induced chondrocytes in a dose? and time?dependent manner, as assessed by the MTT assay. The apoptosis of SNP?induced chondrocytes treated by DHJSD was significantly decreased compared to SNP?induced chondrocyte, as shown by 4',6-diamidino?2?phenylindole and Annexin V/propidium iodide staining. The mitochondrial membrane potential (??m) of SNP?induced chondrocytes treated by DHJSD was significantly decreased compared to SNP?induced chondrocyte, as shown by JC?1 staining. To understand the mechanism, the mRNA and protein levels of Bax, B?cell lymphoma 2 (Bcl?2), caspase?9 and caspase?3 were detected by reverse transcription?polymerase chain reaction and western blot analysis, respectively. In SNP?induced chondrocyte treated by DHJSD, the Bcl?2 expression was increased, whereas the expression of Bax, caspase?9 and caspase?3 was decreased compared to SNP?induced chondrocyte. Taken together, these results indicated that DHJSD inhibits the apoptosis of SNP?induced chondrocyte by the mitochondrial?dependent apoptotic pathway, and this may partly explain its therapeutic efficacy for OA. PMID:25339266

Liu, Fayuan; Liu, Guozhong; Liang, Wenna; Ye, Hongzhi; Weng, Xiaping; Lin, Pingdong; Li, Huiting; Chen, Jiashou; Liu, Xianxiang; Li, Xihai

2014-12-01

194

Acute Ethanol Causes Hepatic Mitochondrial Depolarization in Mice: Role of Ethanol Metabolism  

PubMed Central

Background/Aims An increase of ethanol metabolism and hepatic mitochondrial respiration occurs in vivo after a single binge of alcohol. Here, our aim was to determine how ethanol intake affects hepatic mitochondrial polarization status in vivo in relation to ethanol metabolism and steatosis. Methods Hepatic mitochondrial polarization, permeability transition (MPT), and reduce pyridine nucleotides, and steatosis in mice were monitored by intravital confocal/multiphoton microscopy of the fluorescence of rhodamine 123 (Rh123), calcein, NAD(P)H, and BODIPY493/503, respectively, after gavage with ethanol (1–6 g/kg). Results Mitochondria depolarized in an all-or-nothing fashion in individual hepatocytes as early as 1 h after alcohol. Depolarization was dose- and time-dependent, peaked after 6 to 12 h and maximally affected 94% of hepatocytes. This mitochondrial depolarization was not due to onset of the MPT. After 24 h, mitochondria of most hepatocytes recovered normal polarization and were indistinguishable from untreated after 7 days. Cell death monitored by propidium iodide staining, histology and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was low throughout. After alcohol, mitochondrial NAD(P)H autofluorescence increased and decreased, respectively, in hepatocytes with polarized and depolarized mitochondria. Ethanol also caused steatosis mainly in hepatocytes with depolarized mitochondria. Depolarization was linked to ethanol metabolism, since deficiency of alcohol dehydrogenase and cytochrome-P450 2E1 (CYP2E1), the major ethanol-metabolizing enzymes, decreased mitochondrial depolarization by ?70% and ?20%, respectively. Activation of aldehyde dehydrogenase decreased depolarization, whereas inhibition of aldehyde dehydrogenase enhanced depolarization. Activation of aldehyde dehydrogenase also markedly decreased steatosis. Conclusions Acute ethanol causes reversible hepatic mitochondrial depolarization in vivo that may contribute to steatosis and increased mitochondrial respiration. Onset of this mitochondrial depolarization is linked, at least in part, to metabolism of ethanol to acetaldehyde. PMID:24618581

Zhong, Zhi; Ramshesh, Venkat K.; Rehman, Hasibur; Liu, Qinlong; Theruvath, Tom P.; Krishnasamy, Yasodha; Lemasters, John J.

2014-01-01

195

Antibiofilm efficacy of silver nanoparticles against biofilm of extended spectrum ?-lactamase isolates of Escherichia coli and Klebsiella pneumoniae  

NASA Astrophysics Data System (ADS)

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 ?-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 ?g/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.

Ansari, Mohammad Azam; Khan, Haris M.; Khan, Aijaz A.; Cameotra, Swaranjit Singh; Pal, Ruchita

2014-10-01

196

Effect of the probe geometry on dynamics of cavitation  

NASA Astrophysics Data System (ADS)

Cavitation bubbles accompany explosive evaporation of water after pulsed energy deposition during endosurgery. Bubbles collapsing at the time of an endo-probe produce a powerful and damaging water jet propagating forward in the axial direction of the probe. We demonstrate that formation of this flow and associated tissue damage can be prevented by application of the concave probes that slow the propagation of the back boundary of the bubble. A similar effect can be achieved by positioning an obstacle to the flow, such as a ring or a pick tip in a close proximity to the back, side or front of the tip. Dependence of the flow dynamics on geometry of the probe was studied using fast flash photography and particle velocimetry. With a flat tip a maximal jet velocity of 80 m/s is achieved at a pulse energy of 0.12 mJ, while with an optimized concave probe the jet is completely stopped. The maximal distance between the probe and the tissue at which cells were affected by the water jet was measured using choriallantoic membrane of a chick embryo and Propidium Iodide staining. Changing the tip geometry from flat or convex to an optimized concave shape resulted in reduction of the damage distance by a factor of 4 with pulse energies varying from 0.02 to 0.75 mJ. Elimination of the water jet dramatically improves precision and safety of the pulsed endosurgery reducing the axial damage zone to a size of the cavitation bubble at its maximal expansion.

Palanker, Daniel V.; Vankov, Alexander; Miller, Jason

2002-06-01

197

In vitro and in vivo evaluation of various carbonyl compounds against cyanide toxicity with particular reference to alpha-ketoglutaric acid.  

PubMed

Cyanide is a rapidly acting neurotoxin that necessitates immediate, vigorous therapy. The commonly used treatment regimen for cyanide includes the intravenous administration of sodium nitrite (SN) and sodium thiosulphate (STS). Due to many limitations of these antidotes, a search for more effective, safer molecules continues. Cyanide is known to react with carbonyl compounds to form the cyanohydrin complex. The present study addresses the efficacy of several carbonyl compounds and their metabolites or nutrients with alpha-ketoglutaric acid (A-KG), citric acid, succinic acid, maleic acid, malic acid, fumaric and oxaloacetic acid, glucose, sucrose, fructose, mannitol, sorbitol, dihydroxyacetone, and glyoxal (5 or 10 mM; -10 min) against toxicity of potassium cyanide (KCN; 10 mM) in rat thymocytes in vitro. Six hours after KCN, cell viability measured by MTT assay and crystal violet dye exclusion revealed maximum cytoprotection by A-KG, followed by oxaloacetic acid. A-KG also resolved the leakage of intracellular lactate dehydrogenase, loss in nuclear integrity (propidium iodide staining), and altered mitochondrial membrane potential (rhodamine 123 assay) as a result of cyanide toxicity. Protection Index (ratio of LD(50) of KCN in protected and unprotected animals; PI) of all the compounds (oral; 1.0 g/kg; -10 min) determined in male mice, revealed that maximum protection was afforded by A-KG (7.6 PI), followed by oxaloacetic acid (6.4 PI). Comparative evaluation of various salts of A-KG alone or with STS (intraperitoneal; 1.0 g/kg; -15 min) showed that maximum protection was conferred by disodium anhydrous salt of A-KG, which also significantly prevented the inhibition of brain cytochrome oxidase caused by 0.75 LD(50) KCN. This study indicates the potential of A-KG as alternative cyanide antidote. PMID:18161514

Bhattacharya, Rahul; Tulsawani, Rajkumar

2008-01-01

198

The carboxyl-terminal domain of inducible Hsp70 protects from ischemic injury in vivo and in vitro.  

PubMed

Heat shock protein (Hsp)70 can suppress both necrosis and apoptosis induced by various injuries in vivo and in vitro. However, the relative importance of different functions and binding partners of Hsp70 in ischemic protection is unknown. To explore this question, we tested the ability of Hsp70-K71E, an adenosine triphosphate (ATP)ase-deficient point mutant, and Hsp70-381-640, a deletion mutant lacking the ATPase domain and encoding the carboxyl-terminal portion, to protect against ischemia-like injury in vivo and in vitro. Heat shock protein 70-wild type (-WT), -K71E, -381-640, and control vector plasmid LXSN were expressed in primary murine astrocyte cultures. Astrocytes overexpressing Hsp70-WT, -K71E, or -381-640 were all significantly protected from 4 h combined oxygen-glucose deprivation and 24 h reperfusion when assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay or propidium iodide staining and cell counting (P < 0.05). Brains of rats were transfected with plasmids encoding Hsp70-WT, -K71E, -381-640, or LXSN 24 h before 2 h middle cerebral artery occlusion followed by 24 h reperfusion. Animals that overexpressed either of the mutant proteins or Hsp70-WT had significantly better neurological scores and smaller infarcts than control animals. Protection by both mutants was associated with reduced protein aggregation, as assessed by ubiquitin immunohistochemistry and reduced nuclear translocation of apoptosis-inducing factor. The results show that the carboxyl-terminal portion of Hsp70 is sufficient for neuroprotection. This indicates that neither the ability to fold denatured proteins nor interactions with cochaperones or other proteins that bind the amino-terminal half of Hsp70 are essential to ischemic protection. PMID:16292251

Sun, Yunjuan; Ouyang, Yi-Bing; Xu, Lijun; Chow, Ari Man-Yi; Anderson, Robin; Hecker, James G; Giffard, Rona G

2006-07-01

199

Quantification of propidium iodide delivery using millisecond electric pulses: Experiments  

E-print Network

, NJ 08854, USA b Department of Biomedical Engineering, Rutgers, The State University of New Jersey of Mechanical and Aerospace Engineering, Rutgers, The State University of New Jersey, 98 Brett Road, Piscataway involving two major aspects: (I) permeabilization of the membrane [22�24], and (II) transport of species

Shreiber, David I.

200

Manumycin induces apoptosis in prostate cancer cells  

PubMed Central

Background Manumycin exhibits an antitumor effect in a variety of cancer cell lines, including prostate cancer cell lines (DU145 and PC-3). Our previous studies demonstrated that manumycin induced the apoptosis of anaplastic thyroid cancer cells and leukemia cells via the intrinsic apoptosis pathway. In the current study, we further evaluated the effect of manumycin in two prostate cancer cell lines (LNCaP and 22Rv1), and here we elucidate some of the underlying mechanisms. Materials and methods The cell viability of prostate cancer cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after treatment with manumycin for 48 hours. Apoptosis was detected by flow cytometry using annexin V and propidium iodide. The expressions of B-cell lymphoma (Bcl)-2 family members and the activations of caspase-9 and caspase-3 were detected by Western blotting. Results Manumycin treatment resulted in significant decreases in the viabilities of the two prostate cancer cell lines in a dose-dependent manner through apoptosis, and this apoptosis involved caspase-9 activation. A specific inhibitor of caspase-9 protected cells from caspase-3 activation, apoptosis, and cytotoxicity induced by manumycin. We also found that manumycin downregulated Bcl-2 expression and upregulated Bax expression. Conclusion Our data suggest that manumycin induces apoptosis in prostate cancer cells through regulation of the Bcl-2 family involving caspase-9 activation. These results suggest that manumycin may be beneficial for the treatment of prostate cancer. PMID:24899815

Li, Jing-Gao; She, Miao-Rong; Lu, Ci-Yong; Wei, Shan-Shan; Xia, Ping-Fang; Lu, Ze-Sheng; Peng, Qi

2014-01-01

201

Cells  

NSDL National Science Digital Library

In this unit, students look at the components of cells and their functions and discover the controversy behind stem cell research. The first lesson focuses on the difference between prokaryotic and eukaryotic cells. In the second lesson, students learn about the basics of cellular respiration. They also learn about the application of cellular respiration to engineering and bioremediation. The third lesson continues students' education on cells in the human body and how (and why) engineers are involved in the research of stem cell behavior.

Integrated Teaching And Learning Program

202

Bax is not involved in the resveratrol-induced apoptosis in human lung adenocarcinoma cells  

NASA Astrophysics Data System (ADS)

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.

Zhang, Wei-wei; Wang, Zhi-ping; Chen, Tong-sheng

2010-02-01

203

Iodinated contrast media cause direct tubular cell damage, leading to oxidative stress, low nitric oxide, and impairment of tubuloglomerular feedback.  

PubMed

Iodinated contrast media (CM) have adverse effects that may result in contrast-induced acute kidney injury. Oxidative stress is believed to play a role in CM-induced kidney injury. We test the hypothesis that oxidative stress and reduced nitric oxide in tubules are consequences of CM-induced direct cell damage and that increased local oxidative stress may increase tubuloglomerular feedback. Rat thick ascending limbs (TAL) were isolated and perfused. Superoxide and nitric oxide were quantified using fluorescence techniques. Cell death rate was estimated using propidium iodide and trypan blue. The function of macula densa and tubuloglomerular feedback responsiveness were measured in isolated, perfused juxtaglomerular apparatuses (JGA) of rabbits. The expression of genes related to oxidative stress and the activity of superoxide dismutase (SOD) were investigated in the renal medulla of rats that received CM. CM increased superoxide concentration and reduced nitric oxide bioavailability in TAL. Propidium iodide fluorescence and trypan blue uptake increased more in CM-perfused TAL than in controls, indicating increased rate of cell death. There were no marked acute changes in the expression of genes related to oxidative stress in medullary segments of Henle's loop. SOD activity did not differ between CM and control groups. The tubuloglomerular feedback in isolated JGA was increased by CM. Tubular cell damage and accompanying oxidative stress in our model are consequences of CM-induced direct cell damage, which also modifies the tubulovascular interaction at the macula densa, and may therefore contribute to disturbances of renal perfusion and filtration. PMID:24431205

Liu, Zhi Zhao; Schmerbach, Kristin; Lu, Yuan; Perlewitz, Andrea; Nikitina, Tatiana; Cantow, Kathleen; Seeliger, Erdmann; Persson, Pontus B; Patzak, Andreas; Liu, Ruisheng; Sendeski, Mauricio M

2014-04-15

204

Conducting and permeable states of cell membrane submitted to high voltage pulses: Mathematical and numerical studies validated by the experiments.  

PubMed

The aim of this paper is to present a new model of in vitro cell electropermeabilization, which describes separately the conducting state and the permeable state of the membrane submitted to high voltage pulses. We first derive the model based on the experimental observations and we present the numerical methods to solve the non-linear partial differential equations. We then present numerical simulations that corroborate qualitatively the experimental data dealing with the uptake of propidium iodide (PI) after millipulses. This tends to justify the validity of our modeling. Forthcoming work will be to calibrate the parameters of the model for quantitative description of the uptake. PMID:25010659

Legučbe, M; Silve, A; Mir, L M; Poignard, C

2014-11-01

205

Selective suicide gene therapy of colon cancer exploiting the urokinase plasminogen activator receptor promoter.  

PubMed

Colon cancer is the third and fourth most prevalent cancer among Iranian men and women, respectively. Suicide gene therapy is one of the alternative therapeutic modalities for cancer. The application of specific promoters for therapeutic genes should decrease the adverse effects of this modality. The combined aims of this study were to design a specific suicide gene therapy construct for colon cancer and study its effect in distinct representatives of transformed and nontransformed cells. The KRAS oncogene signaling pathway is one of the most important signaling pathways activated in colon cancer; therefore, we inserted the urokinase plasminogen activator receptor (uPAR; PLAUR gene) promoter as one of the upregulated promoters by this pathway upstream of a suicide gene (thymidine kinase [TK]) and a reporter gene (beta-galactosidase, beta-gal [LacZ]). This promoter is a natural combination of different motifs responsive to the RAS signaling pathway, such as the transcription factors AP1 (FOS/JUN), SP1, SP3, and AP2alpha, and nuclear factor kappa B (NFkappaB). The reporter plasmid under the control of the uPAR promoter (PUCUPARLacZ) had the ability to express beta-gal in colon cancer cells (human colon adenocarcinoma [SW480] and human colorectal carcinoma [HCT116] cell lines), while it could not express beta-gal in nontransformed human umbilical vein endothelial cells (HUVEC) and normal colon cells. After confirming the ability of pUCUPARTK (suicide plasmid) to express TK in SW480 and HCT116 cells by real-time PCR, cytotoxicity assays showed that pUCUPARTK decreased the viability of these cells in the presence of ganciclovir 20 and 40 microg/mL (and higher), respectively. Although M30 CytoDEATH antibody could not detect a significant rate of apoptosis induced by ganciclovir in pUCUPARTK-transfected HCT116 cells, the percentage of stained cells was marked in comparison with untreated cells. While this antibody could detect apoptosis in HCT116 cell line transfected with positive control plasmid, it could not detect apoptosis in SW480 cells transfected with the same positive control. This discrepancy could be attributed to the different mechanisms of TK/ganciclovir-induced apoptosis in tumor protein p53 (TP53)-expressing (HCT116) and -deficient (SW480) cells. Annexin-propidium iodide staining could detect apoptosis in treated, pUCUPARTK-transfected SW480 and HCT116 cells. This study showed that the uPAR promoter can be considered as a suitable candidate for specific suicide gene therapy of colon cancer and probably other cancers in which the RAS signaling pathway is involved in their carcinogenesis process. PMID:20199127

Teimoori-Toolabi, Ladan; Azadmanesh, Kayhan; Amanzadeh, Amir; Zeinali, Sirous

2010-04-01

206

Synergistic Interactions between HDAC and Sirtuin Inhibitors in Human Leukemia Cells  

PubMed Central

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

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

207

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

PubMed

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 ([Ca(2+)](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/cm(2) for 5 days) condition in a microchannel environment. The cells were exposed to a short US tone burst (1.25 MHz, 8 ?s duration, 0.24 MPa) in the presence of Definity™ microbubbles to facilitate sonoporation. Membrane disruption was assessed by propidium iodide (PI) and changes in [Ca(2+)](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 [Ca(2+)](i) compared to statically cultured cells. The maximum increases of PI uptake and [Ca(2+)](i) were also significantly lower in the shear stress cultured cells. In addition, the extent of [Ca(2+)](i) waves in shear cultured HUVECs was reduced compared to the statically cultured cells. PMID:20863503

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

2011-01-01

208

Sapodilla plum (Achras sapota) induces apoptosis in cancer cell lines and inhibits tumor progression in mice.  

PubMed

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

Srivastava, Mrinal; Hegde, Mahesh; Chiruvella, Kishore K; Koroth, Jinsha; Bhattacharya, Souvari; Choudhary, Bibha; Raghavan, Sathees C

2014-01-01

209

Localized micro-scale disruption of cells using laser-generated focused ultrasound.  

PubMed

We utilize laser-generated focused ultrasound (LGFU) to create targeted mechanical disturbance on a few cells. The LGFU is transmitted through an optoacoustic lens that converts laser pulses into focused ultrasound. The tight focusing (<100 µm) and high peak pressure of the LGFU produces cavitational disturbances at a localized spot with micro-jetting and secondary shock-waves arising from micro-bubble collapse. We demonstrate that LGFU can be used as a non-contact, non-ionizing, high-precision tool to selectively detach a single cell from its culture substrate. Furthermore, we explore the possibility of biomolecule delivery in a small population of cells targeted by LGFU at pressure amplitudes below and above the cavitation threshold. We experimentally confirm that cavitational disruption is required for delivery of propidium iodide, a membrane-impermeable nucleic acid-binding dye, into cells. PMID:23420806

Baac, Hyoung Won; Frampton, John; Ok, Jong G; Takayama, Shuichi; Guo, L Jay

2013-12-01

210

Comparison of the effects of photon versus carbon ion irradiation when combined with chemotherapy in vitro  

PubMed Central

Background Characterization of combination effects of chemotherapy drugs with carbon ions in comparison to photons in vitro. Methods The human colon adenocarcinoma cell line WiDr was tested for combinations with camptothecin, cisplatin, gemcitabine and paclitaxel. In addition three other human tumour cell lines (A549: lung, LN-229: glioblastoma, PANC-1: pancreas) were tested for the combination with camptothecin. Cells were irradiated with photon doses of 2, 4, 6 and 8 Gy or carbon ion doses of 0.5, 1, 2 and 3 Gy. Cell survival was assessed using the clonogenic growth assay. Treatment dependent changes in cell cycle distribution (up to 12 hours post-treatment) were measured by FACS analysis after propidium-iodide staining. Apoptosis was monitored for up to 36 hours post-treatment by Nicoletti-assay (with qualitative verification using DAPI staining). Results All cell lines exhibited the well-known increase of killing efficacy per unit dose of carbon ion exposure, with relative biological efficiencies at 10% survival (RBE10) ranging from 2.3 to 3.7 for the different cell lines. In combination with chemotherapy additive toxicity was the prevailing effect. Only in combination with gemcitabine or cisplatin (WiDr) or camptothecin (all cell lines) the photon sensitivity was slightly enhanced, whereas purely independent toxicities were found with the carbon ion irradiation, in all cases. Radiation-induced cell cycle changes displayed the generally observed dose-dependent G2-arrest with little effect on S-phase fraction for all cell lines for photons and for carbon ions. Only paclitaxel showed a significant induction of apoptosis in WiDr cell line but independent of the used radiation quality. Conclusions Combined effects of different chemotherapeutics with photons or with carbon ions do neither display qualitative nor substantial quantitative differences. Small radiosensitizing effects, when observed with photons are decreased with carbon ions. The data support the idea that a radiochemotherapy with common drugs and carbon ion irradiation might be as feasible as respective photon-based protocols. The present data serve as an important radiobiological basis for further combination experiments, as well as clinical studies on combination treatments. PMID:24192264

2013-01-01

211

Gold nanoparticle sensitize radiotherapy of prostate cancer cells by regulation of the cell cycle  

NASA Astrophysics Data System (ADS)

Glucose-capped gold nanoparticles (Glu-GNPs) have been used to improve cellular targeting and radio-sensitization. In this study, we explored the mechanism of Glu-GNP enhanced radiation sensitivity in radiation-resistant human prostate cancer cells. Cell survival and proliferation were measured using MTT and clonogenic assay. Flow cytometry with staining by propidium iodide (PI) was performed to study the cell cycle changes induced by Glu-GNPs, and western blotting was used to determine the expression of p53 and cyclin proteins that correlated to cell cycle regulation. With 2 Gy of ortho-voltage irradiation, Glu-GNP showed a 1.5-2.0 fold enhancement in growth inhibition when compared to x-rays alone. Comparing the cell cycle change, Glu-GNPs induced acceleration in the G0/G1 phase and accumulation of cells in the G2/M phase at 29.8% versus 18.4% for controls at 24 h. G2/M arrest was accompanied by decreased expression of p53 and cyclin A, and increased expression of cyclin B1 and cyclin E. In conclusion, Glu-GNPs trigger activation of the CDK kinases leading to cell cycle acceleration in the G0/G1 phase and accumulation in the G2/M phase. This activation is accompanied by a striking sensitization to ionizing radiation, which may have clinical implications.

Roa, Wilson; Zhang, Xiaojing; Guo, Linghong; Shaw, Andrew; Hu, Xiuying; Xiong, Yeping; Gulavita, Sunil; Patel, Samir; Sun, Xuejun; Chen, Jie; Moore, Ronald; Xing, James Z.

2009-09-01

212

Induction of cell death by graphene in Arabidopsis thaliana (Columbia ecotype) T87 cell suspensions.  

PubMed

The toxicity of graphene on suspensions of Arabidopsis thaliana (Columbia ecotype) T87 cells was investigated by examining the morphology, mitochondrial dysfunction, reactive oxygen species generation (ROS), and translocation of graphene as the toxicological endpoints. The cells were grown in Jouanneau and Péaud-Lenoel (JPL) media and exposed to graphene at concentrations 0-80 mg/L. Morphological changes were observed by scanning electron microscope and the adverse effects such as fragmented nuclei, membrane damage, mitochondrial dysfunction was observed with fluorescence microscopy by staining with Hoechst 33342/propidium iodide and succinate dehydrogenase (mitochondrial bioenergetic enzyme). Analysis of intracellular ROS by 2',7'-dichlorofluorescein diacetate demonstrated that graphene induced a 3.3-fold increase in ROS, suggesting that ROS are key mediators in the cell death signaling pathway. Transmission electron microscopy verified the translocation of graphene into cells and an endocytosis-like structure was observed which suggested graphene entering into the cells by endocytosis. In conclusion, our results show that graphene induced cell death in T87 cells through mitochondrial damage mediated by ROS. PMID:23892171

Begum, Parvin; Fugetsu, Bunshi

2013-09-15

213

Human Wharton's jelly stem cells, its conditioned medium and cell-free lysate inhibit the growth of human lymphoma cells.  

PubMed

Several groups have reported that primitive mesenchymal stem cells from the gelatinous matrix of the Wharton's jelly of the human umbilical cord (hWJSCs) possess tumoricidal properties and inhibit the growth of solid tumours such as human mammary carcinoma, ovarian carcinoma and osteosarcoma. This unique characteristic led to the hypothesis that hWJSCs serve as a natural defence against migrating cancer cells from mother to fetus thus explaining why tumorigenesis in the fetus is rare. However, it is not known whether non-solid malignant hematopoietic cells are also inhibited by hWJSCs and what the exact tumoricidal mechanisms are. We therefore evaluated the influence of hWJSCs and its extracts on Burkitt's lymphoma cells. Cell proliferation (BrdU and Ki67+), viability (MTT) and cell death (Annexin V-Propidium iodide and live/dead) assays showed significant inhibition of lymphoma cell growth after 48 h exposure to hWJSCs or its extracts compared to controls. Increased cell death was observed at sub-G1 and S and decreased proliferation at G2/M phases of the mitotic cycle. Superoxide dismutase and hydrogen peroxide activity were significantly increased and glutathione peroxidase significantly decreased in treated lymphoma cells. Time lapse imaging and confocal z-stack images showed yellow fluorescent in situ hybridization (FISH) signals of lymphoma cell Y chromosomes within the cytoplasm of female red labelled hWJSCs. We hypothesize that the growth of lymphoma cells is inhibited by the molecules secreted by hWJSCs that use oxidative stress pathways to induce cell death followed by engulfment of the apoptotic remains of the lymphoma cells by the hWJSCs. PMID:24789672

Lin, Hao Daniel; Fong, Chui Yee; Biswas, Arijit; Choolani, Mahesh; Bongso, Ariff

2014-08-01

214

Glioblastoma cells deficient in DNA-dependent protein kinase are resistant to cell death.  

PubMed

DNA-dependent protein kinase (DNA-PK), a nuclear serine/threonine kinase, is responsible for the DNA double-strand break repair. Cells lacking or with dysfunctional DNA-PK are often associated with mis-repair, chromosome aberrations, and complex exchanges, all of which are known to contribute to the development of human cancers including glioblastoma. Two human glioblastoma cell lines were used in the experiment, M059J cells lacking the catalytic subunit of DNA-PK, and their isogenic but DNA-PK proficient counterpart, M059K. We found that M059K cells were much more sensitive to staurosporine (STS) treatment than M059J cells, as demonstrated by MTT assay, TUNEL detection, and annexin-V and propidium iodide (PI) staining. A possible mechanism responsible for the different sensitivity in these two cell lines was explored by the examination of Bcl-2, Bax, Bak, and Fas. The cell death stimulus increased anti-apoptotic Bcl-2 and decreased pro-apoptotic Bcl-2 members (Bak and Bax) and Fas in glioblastoma cells deficient in DNA-PK. Activation of DNA-PK is known to promote cell death of human tumor cells via modulation of p53, which can down-regulate the anti-apoptotic Bcl-2 member proteins, induce pro-apoptotic Bcl-2 family members and promote a Bax-Bak interaction. Our experiment also demonstrated that the mode of glioblastoma cell death induced by STS consisted of both apoptosis and necrosis and the percentage of cell death in both modes was similar in glioblastoma cell lines either lacking DNA-PK or containing intact DNA-PK. Taken together, our findings suggest that DNA-PK has a positive role in the regulation of apoptosis in human glioblastomas. The aberrant expression of Bcl-2 family members and Fas was, at least in part, responsible for decreased sensitivity of DNA-PK deficient glioblastoma cells to cell death stimuli. PMID:15493013

Chen, George G; Sin, Fanny L F; Leung, Billy C S; Ng, Ho K; Poon, Wai S

2005-04-01

215

Assessing carbon-encapsulated iron nanoparticles cytotoxicity in Lewis lung carcinoma cells.  

PubMed

Carbon-encapsulated iron nanoparticles (CEINs) have been considered as attractive candidates for several biomedical applications. In the present study, we synthesized CEINs (the mean diameter 40-80?nm) using a carbon arc route, and the as-synthesized CEINs were characterized (scanning and transmission electron microscopy, dynamic light scattering, turbidimetry, Zeta potential) and further tested as raw and purified nanomaterials containing the carbon surface modified with acidic groups. For cytotoxicity evaluation, we applied a battery of different methods (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, lactate dehydrogenase, calcein AM/propidium iodide, annexin V/propidium iodide, JC-1, cell cycle assay, Zeta potential, TEM and inductively coupled plasma mass spectrometry) to address the strategic cytotoxic endpoints of Lewis lung carcinoma cells due to CEIN (0.0001-100?µg?ml(-1) ) exposures in vitro. Our studies evidence that incubation of Lewis lung carcinoma cells with CEINs is accompanied in substantial changes of zeta potential in cells and these effects may result in different internalization profiles. The results show that CEINs increased the mitochondrial and cell membrane cytotoxicity; however, the raw CEIN material (Fe@C/Fe) produced higher toxicities than the rest of the CEINs studied to data. The study showed that non-modified CEINs (Fe@C/Fe and Fe@C) elevated some pro-apoptotic events to a greater extent compared to that of the surface-modified CEINs (Fe@C-COOH and Fe@C-(CH2 )2 COOH). They also diminished the mitochondrial membrane potentials. In contrast to non-modified CEINs, the surface-functionalized nanoparticles caused the concentration- and time-dependent arrest of the S phase in cells. Taken all together, our results shed new light on the rational design of CEINs, as their geometry, hydrodynamic and, in particular, surface characteristics are important features in selecting CEINs as future nanomaterials for nanomedicine applications. PMID:24474239

Grudzinski, Ireneusz P; Bystrzejewski, Michal; Cywinska, Monika A; Kosmider, Anita; Poplawska, Magdalena; Cieszanowski, Andrzej; Fijalek, Zbigniew; Ostrowska, Agnieszka; Parzonko, Andrzej

2014-04-01

216

Effects of dna-dependent protein kinase inhibition by NU7026 on dna repair and cell survival in irradiated gastric cancer cell line N87  

PubMed Central

Repair of radiation-induced dna double-strand breaks is a key mechanism in cancer cell radio-resistance. The synthesized compound NU7026 specifically inhibits dna-dependent protein kinase (dna-pk) within the non-homologous end-joining repair mechanism. Earlier studies demonstrated increased radiosensitivity in dna-pk deficient cells compared with wild-type cells. In chronic leukemia cells, NU7026 appears to enhance the cytotoxic effect of chlorambucil. The radio-modifying effects of NU7026 on cell survival, cell cycle, apoptosis, and dna double-strand break repair have yet to be studied in gastric cancer cells. Methods The gastric cancer cell line N87 was treated with 0 Gy or 4 Gy in the presence of NU7026 at a dose range of 0–20 ?mol/L. Clonogenic assays were used to assess cell survival after treatment. Cell-cycle distribution was analyzed using propidium iodide with fluorescence-activated cell sorting. Apoptosis was detected using annexin-V and propidium iodide with fluorescence-activated cell sorting. The ?H2AX assay was used to measure dna double-strand breaks. Results Statistically significant increases in G2/M arrest were observed in N87 cells treated with radiation and NU7026 compared with those treated with radiation alone (p = 0.0004). Combined treatment also led to an increase in apoptosis (p = 0.01). At 24 hours, the ?H2AX analysis revealed more dna double-strand breaks in N87 cells treated with radiation and NU7026 than in those treated with radiation alone (p = 0.04). Clonogenic assays demonstrated declining cell survival as both the radiation and the NU7026 dose increased. The dose enhancement factor at 0.1 survival fraction was 1.28 when N87 cells were treated with 4 Gy radiation and 5 ?mol/L NU7026. Conclusions In gastric cancer cells, NU7026 appears to enhance the cytotoxic effect of irradiation as assessed by clonogenic assays. This increased cytotoxicity might be the result of an increase in dna double-strand breaks resulting in G2/M cell arrest and possibly higher levels of apoptosis. PMID:24764698

Niazi, M.T.; Mok, G.; Heravi, M.; Lee, L.; Vuong, T.; Aloyz, R.; Panasci, L.; Muanza, T.

2014-01-01

217

Ingenol mebutate: induced cell death patterns in normal and cancer epithelial cells.  

PubMed

We investigated the proposed necrotic mechanism of ingenol mebutate, a natural compound with anti-cancer properties in human keratinocytes, the human squamous cell carcinoma cell line HSC-5, and HeLa cervix carcinoma cells. Topical application of a clinical dose of ingenol mebutate 0.05% (1.15 mM) gel to human reconstituted full-thickness skin equivalents strongly reduced epidermal, but not dermal viability. Ingenol mebutate showed cytotoxic potency between 200-300 M on normal and cancer cells. When keratinocytes were induced to differentiate, they became significantly less sensitive to ingenol mebutate and half-maximal induction of cell death required more than 300 M ingenol mebutate. Cytotoxic concentrations of ingenol mebutate caused rupture of the mitochondrial network within minutes paralleled by cytosolic calcium release in all cells. Subsequently, plasma membrane integrity was lost as seen by propidium uptake into the cells. This was in sharp contrast to lysis of cells with low concentrations of the detergent Triton X-100 that permeabilized the plasma membrane within minutes without affecting organelle morphology. Buffering of intracellular calcium and inhibition of the mitochondrial permeability transition pore reduced the cytotoxic effect of ingenol mebutate in cancer cells, but not in normal keratinocytes. However, these inhibitors could not prevent cell death subsequent to prolonged incubation. Our findings reveal that ingenol mebutate does not mediate cytotoxicity by a simple lytic, necrotic mechanism, but activates distinct processes involving multiple cell organelles in a cell-type and differentiation-dependent manner. These data improve our understanding of ingenol mebutate-target cell interactions and offer new insights relevant to the removal of aberrant cells in human skin. PMID:23134983

Stahlhut, Martin; Bertelsen, Malene; Hoyer-Hansen, Maria; Svendsen, Nannette; Eriksson, Andre H; Lord, Janet M; Scheel-Toellner, Dagmar; Young, Stephen P; Zibert, John R

2012-10-01

218

Hoechst fluorescence intensity can be used to separate viable bromodeoxyuridine-labeled cells from viable non-bromodeoxyuridine-labeled cells  

NASA Technical Reports Server (NTRS)

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.

Mozdziak, P. E.; Pulvermacher, P. M.; Schultz, E.; Schell, K.

2000-01-01

219

Inhibition of VDAC1 prevents Ca(2+)-mediated oxidative stress and apoptosis induced by 5-aminolevulinic acid mediated sonodynamic therapy in THP-1 macrophages.  

PubMed

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

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

220

Rapid count of microbial cells in dialysate.  

PubMed

An apparatus for the non-culture method (NCM) of microbial cell count was formerly developed and named a bioplorer. The bioplorer NCM is based on the double staining of cells with 4', 6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) and the automatic analysis of their fluorescent microscopic images. Viable cells can be stained with DAPI, while dead cells can be stained with DAPI and PI. In this study, the bioplorer NCM has been applied to the dialysate. The viable and dead cells in dialysate could be counted within 20 min. The detection limit expressed by log(10)[cells/100 mL] was 2.0. When cell-spiked dialysate samples containing prescribed number of Bacillus subtilis cells were assayed, the numbers of cells determined by the bioplorer NCM (N(VIA)(NCM)) and a conventional culture method (CM) on R2A medium (N(VIA)(R2A-CM)) were similar in the range of 2.6-4.6 within the 95% confidence interval (NCM-CM equivalent range). When test solutions sampled from a practical facility in a hospital were assayed, N(VIA)(NCM) was greater than, but comparable to, N(VIA)(R2A-CM). The endotoxin (ET) in the test samples were assayed as well using a test kit for limulus amoebocyte lysate assay. The results of microbial cells and ET concentration indicated that the dialysate supplying line was clean and well maintained. The bioplorer NCM can determine if the microbial contamination of dialysate supplying facilities is greater than 2.6 (398 cells/100 mL). PMID:17845395

Shimakita, Tomonori; Yamamoto, Hidenori; Naramura, Tomotaka; Fujimori, Akira; Ide, Takao; Tashiro, Yoshikazu; Saito, Mikako; Matsuoka, Hideaki

2007-10-01

221

Effects of esterified lactoferrin and lactoferrin on control of postharvest blue mold of apple fruit and their possible mechanisms of action.  

PubMed

The effects of esterified lactoferrin (ELF) and lactoferrin (LF) on blue mold caused by Penicillium expansum in apple fruit stored at 25 °C were investigated. Both ELF and LF provided an effective control and strongly inhibited spore germination and germ tube elongation of P. expansum in vitro. Assessment by propidium iodide staining combined with fluorescent microscopy revealed that the plasma membrane of P. expansum spores was damaged more seriously by ELF than by LF treatment, and the leakage of protein and sugar was higher from ELF-treated mycelia. Interestingly, ELF treatment induced a significant increase in the activities of chitinase, ?-1,3-glucanase, and peroxidase in apple fruit, whereas both LF treatment and the control showed no obvious difference. These findings indicated that the effects of ELF on blue mold in apple fruit might be associated with the direct fungitoxic property against the pathogens and the elicitation of defense-related enzymes in fruit. PMID:22663181

Wang, Jie; Shi, Xu-Gen; Wang, Hong-Yan; Xia, Xiao-Ming; Wang, Kai-Yun

2012-06-27

222

Interactions with DCAF1 and DDB1 in the CRL4 E3 ubiquitin ligase are required for Vpr-mediated G2 arrest  

PubMed Central

Background HIV-1 Vpr-mediated G2 cell cycle arrest is dependent on the interaction of Vpr with an E3 ubiquitin ligase that contains damage-specific DNA binding protein 1 (DDB1), Cullin 4A (Cul4A), DDB1 and Cul4-associated factor 1 (DCAF1), and Rbx1. Vpr is thought to associate directly with DCAF1 in the E3 ubiquitin ligase complex although the exact interaction pattern of the proteins in the complex is not completely defined. The Vpr of SIVagm induces G2 arrest of cognate African Green Monkey (AGM) cells but not human cells. The molecular mechanism by which SIVagm Vpr exhibits its species-specific function remained unknown. Methods Physical interaction of proteins in the E3 ubiquitin ligase complex was assessed by co-immunoprecipitation followed by western blotting. In addition, co-localization of the proteins in cells was investigated by confocal microscopy. The cell cycle was analyzed by propidium iodide staining and flow cytometry. DNA damage response elicited by Vpr was evaluated by detecting phosphorylation of H2AX, a marker for DNA damage response. Results We show that RNAi knock-down of DCAF1 prevented the co-immunoprecipitation of DDB1 with HIV-1 Vpr while DDB1 knock-down did not influence the binding of Vpr to DCAF1. HIV-1 Vpr mutants with a L64P or a R90K mutation maintained the ability to associate with DCAF1 but did not appear to be in a complex with DDB1. SIVagm Vpr associated with AGM DCAF1 and DDB1 while, in human cells, it binds to human DCAF1 but hardly binds to human DDB1, resulting in the reduced activation of H2AX. Conclusions The identification of Vpr mutants which associate with DCAF1 but only poorly with DDB1 suggests that DCAF1 is necessary but the simple binding of Vpr to DCAF1 is not sufficient for the Vpr association with DDB1-containing E3 ligase complex. Vpr may interact both with DCAF1 and DDB1 in the E3 ligase complex. Alternatively, the interaction of Vpr and DCAF1 may induce a conformational change in DCAF1 or Vpr that promotes the interaction with DDB1. The ability of SIVagm Vpr to associate with DDB1, but not DCAF1, can explain the species-specificity of SIVagm Vpr-mediated G2 arrest. PMID:24912982

2014-01-01

223

Detection of irradiated quail meat by using DNA comet assay and evaluation of comets by image analysis  

NASA Astrophysics Data System (ADS)

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.

Erel, Yakup; Yazici, Nizamettin; Özvatan, Sumer; Ercin, Demet; Cetinkaya, Nurcan

2009-09-01

224

Cryopreservation of Mycobacterium tuberculosis Complex Cells  

PubMed Central

Successful long-term preservation of Mycobacterium tuberculosis cells is important for sample transport, research, biobanking, and the development of new drugs, vaccines, biomarkers, and diagnostics. In this report, Mycobacterium bovis bacillus Calmette-Guérin and M. tuberculosis H37Ra were used as models of M. tuberculosis complex strains to study cryopreservation of M. tuberculosis complex cells in diverse sample matrices at different cooling rates. Cells were cryopreserved in diverse sample matrices, namely, phosphate-buffered saline (PBS), Middlebrook 7H9 medium with or without added glycerol, and human sputum. The efficacy of cryopreservation was quantified by microbiological culture and microscopy with BacLight LIVE/DEAD staining. In all sample matrices examined, the microbiological culture results showed that the cooling rate was the most critical factor influencing cell viability. Slow cooling (a few degrees Celsius per minute) resulted in much higher M. tuberculosis complex recovery rates than rapid cooling (direct immersion in liquid nitrogen) (P < 0.05). Among the three defined cryopreservation media (PBS, 7H9, and 7H9 plus glycerol), there was no significant differential effect on viability (P = 0.06 to 0.87). Preincubation of thawed M. tuberculosis complex cells in 7H9 broth for 20 h before culture on solid Middlebrook 7H10 plates did not help the recovery of the cells from cryoinjury (P = 0.14 to 0.71). The BacLight LIVE/DEAD staining kit, based on Syto 9 and propidium iodide (PI), was also applied to assess cell envelope integrity after cryopreservation. Using the kit, similar percentages of “live” cells with intact envelopes were observed for samples cryopreserved under different conditions, which was inconsistent with the microbiological culture results. This implies that suboptimal cryopreservation might not cause severe damage to the cell wall and/or membrane but instead cause intracellular injury, which leads to the loss of cell viability. PMID:22933596

Shu, Zhiquan; Weigel, Kris M.; Soelberg, Scott D.; Lakey, Annie; Cangelosi, Gerard A.; Lee, Kyong-Hoon

2012-01-01

225

Traditional Chinese medicinal formula Si-Wu-Tang prevents oxidative damage by activating Nrf2-mediated detoxifying/antioxidant genes  

PubMed Central

Background Induction of Nrf2-mediated detoxifying/antioxidant genes has been recognized as an effective strategy for cancer chemoprevention. Si-Wu-Tang (SWT), comprising the combination of four herbs, Paeoniae, Angelicae, Chuanxiong and Rehmanniae, is one of the most popular traditional oriental medicines for women’s diseases. The purpose of this study is to determine the effects of SWT on Nrf2 pathway in vitro and in vivo and to identify the active component(s). Results Cell viability and apoptosis were analyzed in the non-cancerous breast epithelial cell line MCF-10A after H2O2 treatment in the presence or absence of SWT using the Sulphorhodamine B assay, Annexin-V/Propidium iodide staining and flow cytometry. SWT strongly reduced H2O2 -induced cytotoxicity and apoptosis in MCF-10A cells. Expression of Nrf2 and Nrf2-regulated genes HMOX1 (heme oxygenase 1) and SLC7A11 (xCT) was evaluated by quantitative RT-PCR, Western Blot and immunocytochemistry. SWT strongly induced Nrf2-regulated genes at mRNA and protein levels and increased the nuclear translocation of Nrf2 in MCF-10A cells. The in vivo pharmacodynamic effect of SWT was evaluated in healthy female Sprague–Dawley rats. Short-term oral administration of SWT (1,000 mg/kg per day for six consecutive days) to rats resulted in an increased expression of Nrf2-regulated genes Hmox1 and Slc7A11 in the liver detected by quantitative RT-PCR. Among nine compounds that have been identified previously in the SWT products, z-liguistilide was discovered as the main component responsible for the effect of Nrf2 activation using the antioxidant response element-luciferase reporter gene assay. Z-liguistilide was confirmed with a high potency to induce Nrf2-regulated genes and Nrf2 nuclear translocation. Conclusions Our results demonstrated that SWT and its component z-liguistilide are able to activate the Nrf2 pathway in non-cancerous cells and organs in vitro and in vivo, suggesting that SWT might be an orally effective and nontoxic agent for cancer chemoprevention. PMID:24507416

2014-01-01

226

Annexin V assay-proven anti-apoptotic effect of ascorbic acid 2-glucoside after cold ischemia/reperfusion injury in rat liver transplantation.  

PubMed

Controversy exists over whether the predominant cell death of hepatocytes is due to apoptosis or necrosis after ischemia/reperfusion injury. In this study we investigated the predominant cell death of hepatocytes after cold ischemia/reperfusion injury using the Annexin V-based assay, and evaluated the anti-apoptotic effect of ascorbic acid 2-glucoside (AA-2G) added to the University of Wisconsin solution (UW solution) in rat liver transplantation. The retrieved liver was preserved in 4 UW solution for 24 h, and then transplanted orthotopically to the syngeneic Wistar recipient. The animals were divided into 2 groups, a control group (n=10), in which liver grafts were preserved in UW solution (4), and an AA-2G group (n=10), in which liver grafts were preserved in UW solution (4) with AA-2G (100 ug/ml). The serum AST level 4 h after reperfusion in the control group was significantly suppressed in the AA-2G group, and the bile production of the liver graft in the AA-2G group was well recovered. The mean survival time in the AA-2G group was significantly improved compared with that in the control group. Annexin-V and Propidium iodide staining 4 h after reperfusion showed a significantly higher percentage of viable hepatocytes in the AA-2G group compared with the control group (93.4 +/- 2.0 vs. 80.3 +- 2.1%, P<0.05). In the control group, the main cell death of hepatocytes was apoptosis (early apoptosis: 10.0 +- 4.7%, late apoptosis: 6.4 +/- 1.7%). The addition of AA-2G to the UW solution significantly inhibited both early and late apoptotic cell death 4 h after reperfusion (early apoptosis: 0.98 +/- 0.88%, late apoptosis: 2.2 +/- 1.1%). The expression of caspase 9 in the immunostaining of the liver graft was suppressed in the AA-2G group compared with in the control group. Our study using the Annexin V-based assay provided evidence that the predominant cell death of hepatocytes was apoptosis after 24 h cold ischemia/reperfusion injury in rat liver transplantation. The addition of AA-2G to the UW solution attenuated 24 h cold ischemia/reperfusion injury by inhibiting the apoptosis of hepatocytes. PMID:14679398

Liu, Jie; Yagi, Takahito; Sadamori, Hiroshi; Matsukawa, Hiroyoshi; Sun, Dong-Sheng; Mitsuoka, Naoshi; Yamamura, Masao; Matsuoka, Junji; Jin, Zaishun; Yamamoto, Itaru; Tanaka, Noriaki

2003-10-01

227

Cytotoxicity of Voriconazole on Cultured Human Corneal Endothelial Cells?  

PubMed Central

The purpose of the present study was to evaluate the toxicity of voriconazole on cultured human corneal endothelial cells (HCECs). HCECs were cultured and exposed to various concentrations of voriconazole (5.0 to 1,000 ?g/ml). Cell viability was measured using a Cell Counting Kit-8 (CCK-8) and live/dead viability/cytotoxicity assays. Cell damage was assessed using phase-contrast microscopy after 24 h of exposure to voriconazole. To analyze the effect of voriconazole on the intercellular barrier, immunolocalization of zonula occludens 1 (ZO1) was performed. A flow cytometric assay was performed to evaluate the apoptotic and necrotic effects of voriconazole on HCECs. Cytotoxicity tests demonstrated the dose-dependent toxic effect of voriconazole on HCECs. Voriconazole concentrations of ?100 ?g/ml led to a significant reduction in cell viability. The morphological characteristics of HCECs also changed in a dose-dependent manner. Increasing concentrations of voriconazole resulted in fading staining for ZO1. Higher concentrations of voriconazole resulted in an increased number of propidium iodide (PI)-positive cells, indicating activation of the proapoptotic pathway. In conclusion, voriconazole may have a dose-dependent toxic effect on cultured HCECs. The results of this study suggest that although voriconazole concentrations of up to 50 ?g/ml do not decrease cell viability, intracameral voriconazole concentrations of ?100 ?g/ml may increase the risk of corneal endothelial damage. PMID:21768517

Han, Sang Beom; Shin, Young Joo; Hyon, Joon Young; Wee, Won Ryang

2011-01-01

228

Phthalocyanine-mediated photodynamic therapy induces cell death and a G /G{sub 1} cell cycle arrest in cervical cancer cells  

SciTech Connect

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.

Haywood-Small, S.L. [Centre for Photobiology and Photodynamic Therapy, School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT (United Kingdom)]. E-mail: s.l.hankin@sheffield.ac.uk; Vernon, D.I. [Centre for Photobiology and Photodynamic Therapy, School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT (United Kingdom); Griffiths, J. [Centre for Photobiology and Photodynamic Therapy, Department of Color Chemistry, University of Leeds, Leeds LS2 9JT (United Kingdom); Schofield, J. [Centre for Photobiology and Photodynamic Therapy, Department of Color Chemistry, University of Leeds, Leeds LS2 9JT (United Kingdom); Brown, S.B. [Centre for Photobiology and Photodynamic Therapy, School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT (United Kingdom)

2006-01-13

229

Mitochondria toxin-induced acute cochlear cell death indicates cellular activity-correlated energy consumption.  

PubMed

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

Zou, Jing; Zhang, Ya; Zhang, Weikai; Poe, Dennis; Zhai, Suoqiang; Yang, Shiming; Pyykkö, Ilmari

2013-09-01

230

Systemic administration of fluoro-gold for the histological assessment of vascular structure, integrity and damage.  

PubMed

Fluoro-Gold (F-G) has been used extensively as a fluorescent retrograde neuronal-track tracer in the past. We now report that intraperitoneal administration of 10 to 30 mg/ kg of F-G from 30 min to 7 days prior to sacrifice labels vascular endothelial cells of the brain, choroid plexus and meninges and can be used to assess vascular integrity and damage. F-G vascular labeling co-localized with rat endothelial cell antigen (RECA-1) in the membrane. F-G also intensely labeled the nuclei of the endothelial cells, and co-localized with propidium iodide staining of these nuclei. As well, the administration of F-G during neurotoxic insults produced by amphetamine, kainic acid or "penetrating" wound to the brain can detect where vascular leakage/ hemorrhage has occurred. Histological methods to detect F-G labeled brain vasculature were performed in the same manner as that used for fluorescent visualization of neuronal elements labeled with F-G after perfusion fixation and coronal sectioning (15 to 40 µm) of the brain. This in vivo F-G labeling of endothelial cells and their nuclei yields a clear picture of the integrity of the vasculature and can be used to detect changes in structure. Vascular leaks after "penetrating" wounds through the cortex and striatum, hyperthermic amphetamine exposure or excitotoxic kainate exposure were detected by F-G in the extracellular space and via parenchymal F-G subsequently labeling the terminals and neurons adjacent to the lesioned or damaged vasculature. Further studies are necessary to determine the extent of the leakage necessary to detect vasculature damage. Visualization of the F-G labeling of vasculature structure and leakage is compatible with standard fluorescent immuno-labeling methods used to detect the presence and distribution of a protein in histological sections. This method should be directly applicable to studying brain vascular damage that occurs in the progression of Alzheimer's disease, diabetes and for monitoring the brain vascular changes during development. PMID:24274907

Bowyer, John F; Tranter, Karen M; Sarkar, Sumit; Raymick, James; Hanig, Joseph P; Schmued, Larry C

2014-02-01

231

Conserved Cysteine-Rich Domain of Paramyxovirus Simian Virus 5 V Protein Plays an Important Role in Blocking Apoptosis  

PubMed Central

The paramyxovirus family includes many well-known human and animal pathogens as well as emerging viruses such as Hendra virus and Nipah virus. The V protein of simian virus 5 (SV5), a prototype of the paramyxoviruses, contains a cysteine-rich C-terminal domain which is conserved among all paramyxovirus V proteins. The V protein can block both interferon (IFN) signaling by causing degradation of STAT1 and IFN production by blocking IRF-3 nuclear import. Previously, it was reported that recombinant SV5 lacking the C terminus of the V protein (rSV5V?C) induces a severe cytopathic effect (CPE) in tissue culture whereas wild-type (wt) SV5 infection does not induce CPE. In this study, the nature of the CPE and the mechanism of the induction of CPE were investigated. Through the use of DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling, and propidium iodide staining assays, it was shown that rSV5V?C induced apoptosis. Expression of wt V protein prevented apoptosis induced by rSV5V?C, suggesting that the V protein has an antiapoptotic function. Interestingly, rSV5V?C induced apoptosis in U3A cells (a STAT1-deficient cell line) and in the presence of neutralizing antibody against IFN, suggesting that the induction of apoptosis by rSV5V?C was independent of IFN and IFN-signaling pathways. Apoptosis induced by rSV5V?C was blocked by a general caspase inhibitor, Z-VAD-FMK, but not by specific inhibitors against caspases 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 13, suggesting that rSV5V?C-induced apoptosis can occur in a caspase 12-dependent manner. Endoplasmic reticulum stress can lead to activation of caspase 12; compared to the results seen with mock and wt SV5 infection, rSV5V?C infection induced ER stress, as demonstrated by increased expression levels of known ER stress indicators GRP 78, GRP 94, and GADD153. These data suggest that rSV5V?C can trigger cell death by inducing ER stress. PMID:15113888

Sun, Minghao; Rothermel, Terri A.; Shuman, Laurie; Aligo, Jason A.; Xu, Shibo; Lin, Yuan; Lamb, Robert A.; He, Biao

2004-01-01

232

Olive Oil Polyphenols Differentially Inhibit Smooth Muscle Cell Proliferation through a G1/S Cell Cycle Block Regulated by ERK1/2  

PubMed Central

We hypothesized that polyphenols contained in olive oil play a role in reducing the risk of atherosclerosis. The aim of this study was to determine if the polyphenols in olive oil, oleuropein (Ole), hydroxytyrosol (HT), and tyrosol (Tyr) could inhibit smooth muscle cell (SMC) proliferation through its influence on cell cycle regulation. Bovine vascular SMC were cultured in the presence of Ole, HT, or Tyr at concentration of 1, 10, or 100 ?mol/L. On days 1, 3, and 5, numbers of cells were counted. Cell cycle analysis was performed by flow cytometry on day 1 after SMC were stained with propidium iodide. Cell populations grown in the presence of Ole or HT at 100 ?mol/L concentration were significantly inhibited after 5 days of exposure. Tyr had a similar tendency but it did not attain significance. Cell cycle analysis revealed that 66% of cells were in G1 phase in Ole group, compared with 48% in control group. To examine the cell cycle block between G1 and S phases, we performed Western blotting and found that ERK1/2 activation was inhibited by Ole or HT. We conclude that olive oil polyphenols could inhibit SMC proliferation through a cell cycle block between G1 and S phases which may be regulated by ERK1/2. These results demonstrate a mechanism by which olive oil consumption may be atheroprotective by inhibiting SMC proliferation. PMID:23730132

Abe, Rei; Beckett, Joel; Abe, Ryuzo; Nixon, Alexander; Rochier, Adrienne; Yamashita, Norio; Sumpio, Bauer

2012-01-01

233

Silencing of periostin inhibits nicotine-mediated tumor cell growth and epithelial-mesenchymal transition in lung cancer cells.  

PubMed

Nicotine has been found to induce the proliferation of lung cancer cells through tumor invasion and to confer resistance to apoptosis. Periostin is abnormally highly expressed in lung cancer and is correlated with angiogenesis, invasion and metastasis. Here, we investigated the roles of periostin in the lung cancer cell proliferation, drug resistance, invasion and epithelial-mesenchymal transition (EMT) induced by nicotine. The periostin gene was silenced using small interfering RNA (siRNA) in A549 non-small cell lung cancer (NSCLC) cells. The cells were transfected with control or periostin siRNA plasmids. Periostin mRNA was evaluated by quantitative reverse transcription-polymerase chain reaction (RT-PCR). Cell proliferation was detected using the MTT assay and cell apoptosis was detected by Annexin V-FITC and propidium iodide (PI) double staining. Tumor invasion was detected by the Boyden chamber invasion assay. Western blotting was performed to detect the expression of the EMT marker Snail. Our results revealed that stably periostin-silenced cells were acquired by G418 screening, and the periostin mRNA expression levels of which were decreased by nearly 80%. Periostin-silenced A549 cells exhibited reduced cell proliferation, elevated sensitivity to chemotherapy with cisplatin, decreased cell invasion and Snail expression (P<0.05). Nicotine upregulated the periostin protein levels in the A549 cells and this upregulation was not blocked by the generalized nicotinic acetylcholine receptor (nAChR) antagonist, hexamethonium. In conclusion, periostin is one of the targets regulated by nicotine in lung cancer cells and is involved in the cancer cell growth, drug resistance, invasion and EMT induced by nicotine. PMID:23314871

Wu, Shu-Qiang; Lv, Ya-Er; Lin, Bai-Hua; Luo, Li-Min; Lv, Shi-Liang; Bi, Ai-Hong; Jia, Yong-Shi

2013-03-01

234

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

PubMed

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-? (TNF-?) 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

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

235

Cross-linked actin networks (CLANs) in bovine trabecular meshwork cells.  

PubMed

A cytoskeletal feature of human trabecular meshwork (HTM) cells in vitro and ex vivo is the presence of cross-linked actin networks (CLANs) that are abundant in a proportion of TM cells exposed to dexamethasone (DEX) and also in cells from glaucoma patients. We wished to determine whether CLANs were present in the bovine trabecular meshwork (BTM), whether they were similarly induced by dexamethasone and whether the structures were comparable to CLANs in HTM cells. Cultures of HTM and BTM cells and ex vivo dissections of BTM tissue were stained with phalloidin (F-actin) and propidium iodide (nuclei) and imaged by confocal microscopy, thereafter being subjected to image analysis. Some CLAN-like structures were identified in ex vivo BTM tissue cultured with and without DEX. However we found that BTM cells in culture produced abundant CLANs when exposed to DEX; comparable to the best response from HTM cells. The CLANs were of similar dimensions and morphology to those found in human cells and they had a similar half life of 2 or 3 days following the removal of DEX. This work demonstrates that BTM cells provide a suitable model for future investigations of CLAN formation and function. BTM cultures are sufficiently hardy to thrive in low serum and serum-free conditions so we were able to show that aqueous humor stimulates CLAN formation in the target cells. Future research is directed at identifying the aqueous component(s) responsible for CLAN production. PMID:19540832

Wade, N C; Grierson, I; O'Reilly, S; Hoare, M J; Cracknell, K P B; Paraoan, L I; Brotchie, D; Clark, A F

2009-11-01

236

Microfluidic device for stem cell differentiation and localized electroporation of postmitotic neurons.  

PubMed

New techniques to deliver 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

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

2014-12-01

237

Fluopsin C induces oncosis of human breast adenocarcinoma cells  

PubMed Central

Aim: Fluopsin C, an antibiotic isolated from Pseudomonas jinanesis, has shown antitumor effects on several cancer cell lines. In the current study, the oncotic cell death induced by fluopsin C was investigated in human breast adenocarcinoma cells in vitro. Methods: Human breast adenocarcinoma cell lines MCF-7 and MD-MBA-231 were used. The cytotoxicity was evaluated using MTT assay. Time-lapse microscopy and transmission electron microscopy were used to observe the morphological changes. Cell membrane integrity was assessed with propidium iodide (PI) uptake and lactate dehydrogenase (LDH) assay. Flow cytometry was used to measure reactive oxygen species (ROS) level and mitochondrial membrane potential (??m). A multimode microplate reader was used to analyze the intracellular ATP level. The changes in cytoskeletal system were investigated with Western blotting and immunostaining. Results: Fluopsin C (0.5-8 ?mol/L) reduced the cell viability in dose- and time-dependent manners. Its IC50 values in MCF-7 and MD-MBA-231 cells at 24 h were 0.9 and 1.03 ?mol/L, respectively. Fluopsin C (2 ?mol/L) induced oncosis in both the breast adenocarcinoma cells characterized by membrane blebbing and swelling, which was blocked by pretreatment with the pan-caspase inhibitor Z-VAD-fmk. In MCF-7 cells, fluopsin C caused PI uptake into the cells, significantly increased LDH release, induced cytoskeletal system degradation and ROS accumulation, decreased the intracellular ATP level and ??m. Noticeably, fluopsin C exerted comparable cytotoxicity against the normal human hepatocytes (HL7702) and human mammary epithelial cells with the IC50 values at 24 h of 2.7 and 2.4 ?mol/L, respectively. Conclusion: Oncotic cell death was involved in the anticancer effects of fluopsin C on human breast adenocarcinoma cells in vitro. The hepatoxicity of fluopsin C should not be ignored. PMID:23708552

Ma, Li-sha; Jiang, Chang-you; Cui, Min; Lu, Rong; Liu, Shan-shan; Zheng, Bei-bei; Li, Lin; Li, Xia

2013-01-01

238

Effect of tissue factor knockdown on the growth, invasion, chemoresistance and apoptosis of human gastric cancer cells  

PubMed Central

This study aimed to explore the role of tissue factor (TF) and evaluate its antitumor effects in the biological processes of gastric cancer cells using the application of RNA interference technology to silence TF in the SGC7901 gastric cancer cell line. Specific small interfering RNA (siRNA) designed for targeting human TF was transfected into SGC7901 cells. The expression levels of TF in the cells were detected by reverse transcription-polymerase chain reaction. Cell proliferation and chemosensitivity were measured by Cell Counting Kit-8. The metastatic potential of the SGC7901 cells was determined by Transwell experiments and wound-healing assays. Cell apoptosis was assessed by Annexin V-fluorescein isothiocyanate/propidium iodide double-staining method. The expression levels of TF mRNA were significantly reduced by the TF-siRNA in the SGC7901 cells, resulting in the suppression of cell proliferation, chemoresistance and invasion, and subsequently the induction of cell apoptosis. TF knockdown with siRNA inhibits the growth, invasion and chemoresistance and enhances the apoptosis of SGC7901 cells, providing a potential approach for gene therapy against human gastric cancer. PMID:24940442

YU, YONG-JIANG; HOU, XU-DONG; LI, YU-MIN

2014-01-01

239

Foodborne Cereulide Causes Beta-Cell Dysfunction and Apoptosis  

PubMed Central

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

Vangoitsenhoven, Roman; Rondas, Dieter; Crevecoeur, 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

240

Polyamine analog TBP inhibits proliferation of human K562 chronic myelogenous leukemia cells by induced apoptosis  

PubMed Central

The aim of the present study was to investigate the effects of the novel polyamine analog tetrabutyl propanediamine (TBP) on the growth of K562 chronic myelogenous leukemia (CML) cells and the underlying mechanism of these effects. MTT was used for the analysis of cell proliferation and flow cytometry was performed to analyze cell cycle distribution. DNA fragmentation analysis and Annexin V/propidium iodide double staining were used to identify apoptotic cells. The activity of the key enzymes in polyamine catabolism was detected using chemiluminescence. TBP can induce apoptosis and significantly inhibit K562 cell proliferation in a time- and dose-dependent manner. TBP treatment significantly induced the enzyme activity of spermine oxidase and acetylpolyamine oxidase in K562 cells, and also enhanced the inhibitory effect of the antitumor drug doxorubicin on K562 cell proliferation. As a novel polyamine analog, TBP significantly inhibited proliferation and induced apoptosis in K562 cells by upregulating the activity of the key enzymes in the polyamine catabolic pathways. TBP also increased the sensitivity of the K562 cells to the antitumor drug doxorubicin. These data indicate an important potential value of TBP for clinical therapy of human CML.

WANG, QING; WANG, YAN-LIN; WANG, KAI; YANG, JIAN-LIN; CAO, CHUN-YU

2015-01-01

241

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

PubMed Central

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

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

2013-01-01

242

Electrical conduction along endothelial cell tubes from mouse feed arteries: confounding actions of glycyrrhetinic acid derivatives  

PubMed Central

BACKGROUND AND PURPOSE Electrical conduction along endothelium of resistance vessels has not been determined independently of the influence of smooth muscle, surrounding tissue or blood. Two interrelated hypotheses were tested: (i) Intercellular conduction of electrical signals is manifest in endothelial cell (EC) tubes; and (ii) Inhibitors of gap junction channels (GJCs) have confounding actions on EC electrical and Ca2+ signalling. EXPERIMENTAL APPROACH Intact EC tubes were isolated from abdominal muscle feed (superior epigastric) arteries of C57BL/6 mice. Hyperpolarization was initiated with indirect (ACh) and direct (NS309) stimulation of intermediate- and small-conductance Ca2+-activated K+ channels (IKCa/SKCa). Remote membrane potential (Vm) responses to intracellular current injection defined the length constant (?) for electrical conduction. Dye coupling was evaluated following intracellular microinjection of propidium iodide. Intracellular Ca2+ dynamics were determined using Fura-2 photometry. Carbenoxolone (CBX) or ?-glycyrrhetinic acid (?GA) was used to investigate the role of GJCs. KEY RESULTS Steady-state Vm of ECs was ?25 mV. ACh and NS309 hyperpolarized ECs by ?40 and ?60 mV respectively. Electrical conduction decayed monoexponentially with distance (??1.4 mm). Propidium iodide injected into one EC spread into surrounding ECs. CBX or ?GA inhibited dye transfer, electrical conduction and EC hyperpolarization reversibly. Both agents elevated resting Ca2+ while ?GA inhibited responses to ACh. CONCLUSIONS AND IMPLICATIONS Individual cells were effectively coupled to each other within EC tubes. Inhibiting GJCs with glycyrrhetinic acid derivatives blocked hyperpolarization mediated by IKCa/SKCa channels, regardless of Ca2+ signalling, obviating use of these agents in distinguishing key determinants of electrical conduction along the endothelium. PMID:22168386

Behringer, Erik J; Socha, Matthew J; Polo-Parada, Luis; Segal, Steven S

2012-01-01

243

Highlighting a need to distinguish cell cycle signatures from cellular responses to chemotherapeutics in SR-FTIR spectroscopy.  

PubMed

Previous research has seen difficulties in establishing clear discrimination by principal component analysis (PCA) between drug-treated cells analysed by single point SR-FTIR spectroscopy, relative to multisampling cell monolayers by conventional FTIR. It is suggested that the issue arises due to signal mixing between cellular-response signatures and cell cycle phase contributions in individual cells. Consequently, chemometric distinction of cell spectra treated with multiple drugs is difficult even with supervised methods. In an effort to separate cell cycle chemistry from cellular response chemistry in the spectra, renal carcinoma cells were stained with propidium iodide and fluorescent-activated cell sorted (FACS) after exposure to a number of chemotherapeutic compounds; 5-fluorouracil (5FU) and a set of novel gold-based experimental compounds. The cell spectra were analysed separately by PCA in G(1), S or G(2)/M phase. The mode of action of established drug 5FU, known to disrupt S phase, was confirmed by FACS analysis. The chemical signature of 5FU-treated cells discriminated against both the control and gold-compound (KF0101)-treated cell spectra, suggesting a different mode of action due to a difference in cellular response. PMID:23095763

Hughes, C; Brown, M D; Ball, F J; Monjardez, G; Clarke, N W; Flower, K R; Gardner, P

2012-12-21

244

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

PubMed

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

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

245

Inhibitor of differentiation 1 (Id1) expression attenuates the degree of TiO2-induced cytotoxicity in H1299 non-small cell lung cancer cells.  

PubMed

The inhibitor of differentiation (Id) family of genes, which encodes negative regulators of basic helix-loop-helix transcription factors, has been implicated in diverse cellular processes such as proliferation, apoptosis, differentiation, and migration. However, the specific role of Id1 in titanium dioxide (TiO2)-induced lung injury has not been investigated. In the present study, we investigated whether TiO2 induces apoptosis in H1299 lung cancer cells and by which pathways. Based on the results of the LDH assay, dual staining with Annexin V-FITC and propidium iodide (PI), and RT-PCR analysis of apoptosis-related gene expression, TiO2 caused a dose- and time-dependent decrease in cell viability and appeared to involve both necrosis and apoptosis. Furthermore, Id1 expression was significantly reduced in TiO2-treated cells compared with control cells. To further investigate the functional role of Id1, cells were transduced with a recombinant adenovirus expressing Id1, and the effects on sensitivity to TiO2 were analyzed. Id1 overexpression led to the enhancement of cellular proliferation and reduced the sensitivity of H1299 cells to TiO2. Our results indicate that Id1 expression attenuates the degree of TiO2-induced cytotoxicity in lung cells. PMID:19486931

Lee, Young Sook; Yoon, Seokjoo; Yoon, Hea Jin; Lee, Kyuhong; Yoon, Hyoun Kyoung; Lee, Jeung-Hoon; Song, Chang Woo

2009-09-28

246

Basic apoptotic and necrotic cell death in human liver carcinoma (HepG2 ) cells induced by synthetic azamacrocycle.  

PubMed

Treatment of diseases with synthetic materials has been an aspiration of mankind since the dawn of human development. In this research, three complex compounds of azamacrocycle (TD1, TD2, and TD3) were synthesized, and experiments were conducted to determine whether their toxicity to human liver carcinoma (HepG2 ) cells is associated with apoptotic and/or necrotic cell death. Cell survival was determined by MTT assay. Apoptosis and necrosis were measured by annexin V FITC/PI assay using the flow cytometry and by propidium iodide (PI) assay using the cellometer vision. HepG2 cells were treated with different concentrations of azamacrocycles for 48 h. Results from MTT assay indicated that all the three azamacrocycles significantly (p < 0.05) reduce cell viability in a dose-dependent manner, showing 48 h-LD50 values of about 37.97, 33.60, and 19.29 ?M, for TD3, TD1 and TD2, respectively. Among the three compounds tested, TD2 showed the most pronounced cytotoxic activity against HepG2 cells, being about twofold more potent than TD3. The order of toxicity was TD2 > TD1 > TD3. Because TD2 exerted the most cytotoxic activity against HepG2 cells, it was used in the subsequent apoptosis and necrosis-related experiments. The flow cytometry assessment showed a strong dose-response relationship with regard to TD2 exposure and annexin V/PI positive cells. PI assay data indicated that TD2 exposure increased the proportion of fluorescence positive cells. Overall, our results indicate that azamacrocycle toxicity to HepG2 cells is associated with apoptotic and necrotic cell death resulting from phosphatidylserine externalization and loss of membrane integrity. PMID:22644747

Yedjou, Clement G; Saeed, Musabbir A; Hossain, Md Alamgir; Dorsey, Waneene; Yu, Hongtao; Tchounwou, Paul B

2014-06-01

247

Inhibitory effects of capsaicin on hepatic stellate cells and liver fibrosis.  

PubMed

Hepatic stellate cells (HSCs) play an important role in the process of liver fibrosis. In this study, we investigated the inhibitory effects of capsaicin on HSCs and liver fibrosis. Cultured HSCs were incubated with various concentrations of capsaicin. Cell proliferation was examined using a cell counting kit. Production of hydrogen peroxide was determined using a 2',7'-dichlorofluorescin diacetate (DCFH-DA) assay. The mRNA and protein expression of target genes was analyzed by reverse transcription PCR and Western blot analysis, respectively. Cell apoptosis was evaluated by annexin V-FITC and propidium iodide (PI) costaining followed by flow cytometric analysis. A CCl4 rat liver fibrosis model was used to assess in vivo effects of capsaicin by histological examination and measurement of liver fibrosis markers, including hydroxyproline content, serum type III collagen, and hyaluronic acid (HA) levels. Our results show that capsaicin dose-dependently inhibited cell proliferation, suppressed cell activation, and decreased hydrogen peroxide production in cultured HSCs. Capsaicin reduced the mRNA levels of tissue inhibitors of metalloproteinase 1 (TIMP-1) and transforming growth factor-?1 (TGF-?1) in HSCs. Moreover, capsaicin-induced cell apoptosis was associated with increased expression of Bax, cytochrome c (cyt c), and caspase-3, but reduced levels of Bcl-2. The animal studies further revealed that capsaicin efficiently reduced the extent of liver fibrosis, inhibited HSC proliferation, and promoted cell apoptosis. Our findings suggest that capsaicin might inhibit fibrogenesis by inhibiting the activities of HSCs. PMID:25289759

Yu, Fu-Xiang; Teng, Yin-Yan; Zhu, Qian-Dong; Zhang, Qi-Yu; Tang, Yin-He

2014-10-01

248

Cysteine: A Novel Neural Inducer for Rat Bone Marrow Mesenchymal Stem Cells  

PubMed Central

Objective Mesenchymal stem cells (MSCs) can differentiate into various cell types. Since cysteine has structural similarities to neuronal inducers ?-mercaptoethanol and glutathione, we examined its effect on neural induction of rat bone marrow MSCs. Materials and Methods In this experimental study, cells were treated in a medium containing 1mM cysteine for 24 hours prior to treatment with neuron inducing medium containing 10 mM cysteine for 1, 2 and 3 hours. Cell viability and morphology were assessed by 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay and, Hoechst, propidium iodide and acridine orange staining respectively. Expression of nestin and ?-Tubulin III genes, as neural cell-specific markers, was studied reverse transcription polymerase chain reaction (RT-PCR). The data was statistically analyzed using One-Way ANOVA and Tukey’s test and p<0.05 was considered significant. Results After 3 hours of treatment, neuron like morphology with a considerable expression of nestin and ?-Tubulin III genes was apparent. The mean cell viability was not significantly different at 1, 2 and 3 hours following induction, compared with the control cells. Conclusion Cysteine can induce neural features in rat bone marrow MSCs without reducing cell viability. Therefore, it can be considered as a safer alternative to toxic neural inducer agents such as ?-mercaptoethanol. PMID:24567936

Soleimani Mehranjani, Malek; Chian, Milad Falahat

2014-01-01

249

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

PubMed Central

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 ?g/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 ?g/ml in the culture medium, SWCNTs stimulated plant cells to grow out trichome clusters on their surface, with more than 50 ?g/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.

2011-01-01

250

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

NASA Astrophysics Data System (ADS)

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 ?g/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 ?g/ml in the culture medium, SWCNTs stimulated plant cells to grow out trichome clusters on their surface, with more than 50 ?g/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.

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

2011-12-01

251

Arsenic Trioxide Modulates DNA Synthesis and Apoptosis in Lung Carcinoma Cells  

PubMed Central

Arsenic trioxide, the trade name Trisenox, is a drug used to treat acute promyleocytic leukemia (APL). Studies have demonstrated that arsenic trioxide slows cancer cells growth. Although arsenic influences numerous signal-transduction pathways, cell-cycle progression, and/or apoptosis, its apoptotic mechanisms are complex and not entirely delineated. The primary objective of this research was to evaluate the effects of arsenic trioxide on DNA synthesis and to determine whether arsenic-induced apoptosis is mediated via caspase activation, p38 mitogen–activated protein kinase (MAPK), and cell cycle arrest. To achieve this goal, lung cancer cells (A549) were exposed to various concentrations (0, 2, 4, 6, 8, and 10 ?g/mL) of arsenic trioxide for 48 h. The effect of arsenic trioxide on DNA synthesis was determined by the [3H]thymidine incorporation assay. Apoptosis was determined by the caspase-3 fluorescein isothiocyanate (FITC) assay, p38 MAP kinase activity was determined by an immunoblot assay, and cell-cycle analysis was evaluated by the propidium iodide assay. The [3H]thymidine-incorporation assay revealed a dose-related cytotoxic response at high levels of exposure. Furthermore, arsenic trioxide modulated caspase 3 activity and induced p38 MAP kinase activation in A549 cells. However, cell-cycle studies showed no statistically significant differences in DNA content at subG1 check point between control and arsenic trioxide treated cells. PMID:20632473

Walker, Alice M.; Stevens, Jacqueline J.; Ndebele, Kenneth; Tchounwou, Paul B.

2010-01-01

252

Plumbagin induces the apoptosis of human tongue carcinoma cells through the mitochondria-mediated pathway  

PubMed Central

Background Plumbagin, a quinonoid constituent isolated from the root of Plumbago zeylanica L., has been proven to possess anti-tumor activity both in vitro and in vivo. However, its anti-tumor properties for human tongue carcinoma have not been reported. This study aimed to investigate the inhibitory effect and the underlying mechanism of plumbagin on the growth of human tongue carcinoma cells. Material/Methods Cell proliferation ability was detected by EdU incorporation assay and colony formation assay. Cell-cycle distribution was determined by flow cytometric analysis using propidium iodide (PI) staining. Cellular apoptosis was then evaluated by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Western blotting was applied to assay the expression of Bax and Bcl-2. Results Plumbagin inhibited the growth and proliferation of Tca8113 cells in vitro in a concentration- and time-dependent manner. The cell cycles of plumbagin-treated Tca8113 cells were arrested at the G2/M phase. Cells treated with plumbagin presented the characteristic morphological changes of apoptosis. The ratio of Bax/Bcl-2 was raised by plumbagin in a concentration-dependent manner. Conclusions These results indicate that plumbagin induces the apoptosis of Tca8113 cells through mitochondria-mediated pathway. PMID:23982457

Qiu, Jia-xuan; He, Yuan-qiao; Wang, Yong; Xu, Ru-liang; Qin, You; Shen, Xiang; Zhou, Shu-Feng; Mao, Zong-fu

2013-01-01

253

Polysaccharide of Boschniakia rossica induces apoptosis on laryngeal carcinoma Hep2 cells.  

PubMed

The aim of this study was to explore the anti-tumor potential of a polysaccharide isolated from Boschniakia rossica (BRP) in Hep2 human larynx squamous carcinoma cells. High performance size-exclusion chromatography analysis showed that BRP was a homogeneous polysaccharide and had a molecular weight of 22 kDa. Total carbohydrate content in BRP was determined to be 96.9%, without the presence of protein and nucleic acid. BRP suppressed the proliferation of Hep2 cells in a time- and dose-dependent manner. Cell cycle analysis revealed that exposure to BRP (200 ?g/ml) caused a G0/G1 cell cycle arrest in Hep2 cells. Moreover, treatment with BRP at 100-400 ?g/ml for 24h induced a significant apoptosis Hep2 cells compared to untreated control cells, as determined by flow cytometry with annexin-V/propidium iodide double staining. Additionally, BRP treatment promoted the cleavage of pro-caspase-3, pro-caspase-8, and pro-caspase-9, coupled with increased expression of death receptor DR5 and Bax and reduced expression of Bcl-2. Taken together, our data demonstrate that BRP shows potent anti-tumor activity in human larynx squamous carcinoma, largely through induction of G0/G1 cell cycle arrest and apoptosis. Activation of both mitochondria-mediated and death receptor-mediated apoptosis pathways is involved in the cytotoxicity of BRP. PMID:24334128

Wang, Zhenghui; Lu, Chuangxin; Wu, Caiqin; Xu, Min; Kou, Xiaohui; Kong, Demin; Jing, Gangli

2014-02-15

254

Effects of a novel cyclic RGD peptidomimetic on cell proliferation, migration and angiogenic activity in human endothelial cells  

PubMed Central

Background Cyclic RGD peptidomimetics containing a bifunctional diketopiperazine scaffold are a novel class of high-affinity ligands for the integrins ?V?3 and ?V?5. Since integrins are a promising target for the modulation of normal and pathological angiogenesis, the present study aimed at characterizing the ability of the RGD peptidomimetic cyclo[DKP-RGD] 1 proliferation, migration and network formation in human umbilical vein endothelial cells (HUVEC). Methods Cell viability was assessed by flow cytometry and annexin V (ANX)/propidium iodide (PI) staining. Cell proliferation was evaluated by the ELISA measurement of bromodeoxyuridine (BrdU) incorporation. Network formation by HUVEC cultured in Matrigel-coated plates was evaluated by optical microscopy and image analysis. Integrin subunit mRNA expression was assessed by real time-PCR and Akt phosphorylation by western blot analysis. Results Cyclo[DKP-RGD] 1 does not affect cell viability and proliferation either in resting conditions or in the presence of the pro-angiogenic growth factors VEGF, EGF, FGF, and IGF-I. Addition of cyclo[DKP-RGD] 1 however significantly decreased network formation induced by pro-angiogenic growth factors or by IL-8. Cyclo[DKP-RGD] 1 did not affect mRNA levels of ?V, ?3 or ?5 integrin subunits, however it significantly reduced the phosphorylation of Akt. Conclusions Cyclo[DKP-RGD] 1 can be a potential modulator of angiogenesis induced by different growth factors, possibly devoid of the adverse effects of cytotoxic RGD peptidomimetic analogues. PMID:25053992

2014-01-01

255

Blockade of adenosine A2A receptors prevents interleukin-1?-induced exacerbation of neuronal toxicity through a p38 mitogen-activated protein kinase pathway  

PubMed Central

Background and purpose Blockade of adenosine A2A receptors (A2AR) affords robust neuroprotection in a number of brain conditions, although the mechanisms are still unknown. A likely candidate mechanism for this neuroprotection is the control of neuroinflammation, which contributes to the amplification of neurodegeneration, mainly through the abnormal release of pro-inflammatory cytokines such as interleukin(IL)-1?. We investigated whether A2AR controls the signaling of IL-1? and its deleterious effects in cultured hippocampal neurons. Methods Hippocampal neuronal cultures were treated with IL-1? and/or glutamate in the presence or absence of the selective A2AR antagonist, SCH58261 (50 nmol/l). The effect of SCH58261 on the IL-1?-induced phosphorylation of the mitogen-activated protein kinases (MAPKs) c-Jun N-terminal kinase (JNK) and p38 was evaluated by western blotting and immunocytochemistry. The effect of SCH58261 on glutamate-induced neurodegeneration in the presence or absence of IL-1? was evaluated by nucleic acid and by propidium iodide staining, and by lactate dehydrogenase assay. Finally, the effect of A2AR blockade on glutamate-induced intracellular calcium, in the presence or absence of IL-1?, was studied using single-cell calcium imaging. Results IL-1? (10 to 100 ng/ml) enhanced both JNK and p38 phosphorylation, and these effects were prevented by the IL-1 type 1 receptor antagonist IL-1Ra (5 ?g/ml), in accordance with the neuronal localization of IL-1 type 1 receptors, including pre-synaptically and post-synaptically. At 100 ng/ml, IL-1? failed to affect neuronal viability but exacerbated the neurotoxicity induced by treatment with 100 ?mol/l glutamate for 25 minutes (evaluated after 24 hours). It is likely that this resulted from the ability of IL-1? to enhance glutamate-induced calcium entry and late calcium deregulation, both of which were unaffected by IL-1? alone. The selective A2AR antagonist, SCH58261 (50 nmol/l), prevented both the IL-1?-induced phosphorylation of JNK and p38, as well as the IL-1?-induced deregulation of calcium and the consequent enhanced neurotoxicity, whereas it had no effect on glutamate actions. Conclusions These results prompt the hypothesis that the neuroprotection afforded by A2AR blockade might result from this particular ability of A2AR to control IL-1?-induced exacerbation of excitotoxic neuronal damage, through the control of MAPK activation and late calcium deregulation. PMID:22901528

2012-01-01

256

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

PubMed

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

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

257

Long-term in vitro toxicity models: comparisons between a flow-cell bioreactor, a static-cell bioreactor and static cell cultures.  

PubMed

In vitro long-term toxicity testing is becoming an important issue in the field of toxicology, and there is a need to develop new model systems that mimic human chronic exposure and its effects. The aim of this work was to test two long-term in vitro toxicity systems which are available, a flow-cell bioreactor (Tecnomouse, Integra, Wallisellen, Switzerland) and a static cell bioreactor system (CELLine CL 6-well, Integra), and to compare them with the use of conventional cell culture flasks. A human cell line, Int 407, was exposed to cadmium chloride (CdCl(2); 10-(7-)10-(8)M) for 4 weeks. Cell numbers and cell viabilities were determined by the trypan blue (TB) exclusion assay and from exclusion of propidium iodide (PI) as determined by flow cytometry; and cell viability and metabolic activity were determined by the MTT assay. In addition, total protein determination and cadmium uptake measurements were performed. The results obtained with TB and PI exclusion did not show clear differences in cell viability with increasing CdCl(2) concentration. However, in the static cell-culture systems, an increase in MTT reduction was found at low concentrations of CdCl(2). Expression of heat-shock protein (Hsp27 and Hsp70) increased differently, depending on the CdCl(2) concentration applied and the system used. In summary, of the two bioreactors, the CELLine CL 6-well bioreactor was shown to be the more efficient system for performing long-term cytotoxicity studies. It is easy to handle, it permits the assessment of several endpoints, and sufficient replicates can be made available. PMID:12405880

Pazos, Patricia; Fortaner, Salvador; Prieto, Pilar

2002-01-01

258

Crocus sativus L. (saffron) stigma aqueous extract induces apoptosis in alveolar human lung cancer cells through caspase-dependent pathways activation.  

PubMed

Worldwide, lung cancer is the most common form of cancer. Saffron has been used in folk medicine for centuries. We investigated the potential of saffron to induce cytotoxic and apoptotic effects in lung cancer cells (A549). We also examined the caspase-dependent pathways activation of saffron-induced apoptosis against the A549 cells. A549 cells were incubated with different concentrations of saffron extract; then cell morphological changes, cell viability, and apoptosis were determined by the normal invertmicroscope, MTT assay, Annexin V and propidium iodide, and flow cytometric analysis, respectively. Activated caspases were detected by treatment of saffron in lung cancer cells using fluorescein-labeled inhibitors of polycaspases. The proliferation of the A549 cells were decreased after treatment with saffron in a dose- and time-dependent manner. The percentage of apoptotic cells increased with saffron concentrations. Saffron induced morphological changes, decreased percentage of viable cells, and induced apoptosis. Saffron could induce apoptosis in the A549 cells and activate caspase pathways. The levels of caspases involved in saffron-induced apoptosis in the A549 cells indicating caspase-dependent pathway were induced by saffron. The anticancer activity of the aqueous extract of saffron could be attributed partly to its inhibition of the cell proliferation and induction of apoptosis in cancer cells through caspase-dependent pathways activation. PMID:24288678

Samarghandian, Saeed; Borji, Abasalt; Farahmand, Seyed Kazem; Afshari, Reza; Davoodi, Saeideh

2013-01-01

259

Crocus sativus L. (Saffron) Stigma Aqueous Extract Induces Apoptosis in Alveolar Human Lung Cancer Cells through Caspase-Dependent Pathways Activation  

PubMed Central

Worldwide, lung cancer is the most common form of cancer. Saffron has been used in folk medicine for centuries. We investigated the potential of saffron to induce cytotoxic and apoptotic effects in lung cancer cells (A549). We also examined the caspase-dependent pathways activation of saffron-induced apoptosis against the A549 cells. A549 cells were incubated with different concentrations of saffron extract; then cell morphological changes, cell viability, and apoptosis were determined by the normal invertmicroscope, MTT assay, Annexin V and propidium iodide, and flow cytometric analysis, respectively. Activated caspases were detected by treatment of saffron in lung cancer cells using fluorescein-labeled inhibitors of polycaspases. The proliferation of the A549 cells were decreased after treatment with saffron in a dose- and time-dependent manner. The percentage of apoptotic cells increased with saffron concentrations. Saffron induced morphological changes, decreased percentage of viable cells, and induced apoptosis. Saffron could induce apoptosis in the A549 cells and activate caspase pathways. The levels of caspases involved in saffron-induced apoptosis in the A549 cells indicating caspase-dependent pathway were induced by saffron. The anticancer activity of the aqueous extract of saffron could be attributed partly to its inhibition of the cell proliferation and induction of apoptosis in cancer cells through caspase-dependent pathways activation. PMID:24288678

Samarghandian, Saeed; Borji, Abasalt; Farahmand, Seyed Kazem; Afshari, Reza; Davoodi, Saeideh

2013-01-01

260

Puromycin aminonucleoside induces glomerular epithelial cell apoptosis.  

PubMed

Glomerular epithelial cell (GEC) injury has been considered to play an important role in puromycin aminonucleoside (PAN)-induced nephrosis. We studied the effect of PAN on rat as well as human GEC apoptosis. Morphogic evaluation of GEC apoptosis and necrosis was carried out by staining with H-33342 and propidium iodide. GEC apoptosis was further confirmed by DNA fragmentation assay (by both agarose gel electrophoresis and end-labeling). To determine the dose- and time-response effect of PAN, GECs were treated with variable concentrations of PAN (10 to 500 microg/ml) for variable time periods (6 to 48 h). To determine the role of gene synthesis, we studied the effect of actinomycin D (a transcriptional inhibitor) on PAN-induced GEC apoptosis. To determine the role of free radicals, we evaluated the effect of superoxide dismutase (SOD), dimethylthiourea (DMTU), and catalase on PAN-induced GEC apoptosis. PAN induced GEC apoptosis in a dose- and time-dependent manner. PAN at a high concentration (PAN, 100 microg/ml) also induced a moderate degree of GEC necrosis. In DNA fragmentation assays PAN-treated GECs showed the classic ladder pattern. PAN-induced GEC apoptosis was partly attenuated with free radical scavengers, such as SOD, DMTU, and catalase. In addition, actinomycin D attenuated PAN-induced GEC apoptosis. PAN induces GEC apoptosis, which may be mediated through the generation of reactive oxygen species. PMID:11170791

Sanwal, V; Pandya, M; Bhaskaran, M; Franki, N; Reddy, K; Ding, G; Kapasi, A; Valderrama, E; Singhal, P C

2001-02-01

261

Tualang honey induces apoptosis and disrupts the mitochondrial membrane potential of human breast and cervical cancer cell lines.  

PubMed

Honey is reported to contain various compounds such as phenols, vitamins and antioxidants. The present study investigates the anticancer potential of Tualang honey (Agromas) (TH) in human breast (MCF-7 and MDA-MB-231) and cervical (HeLa) cancer cell lines; as well as in the normal breast epithelial cell line, MCF-10A. The cells were treated with increasing doses of TH (1-10%) for up to 72 h. Increase in lactate dehydrogenase (LDH) leakage from the cell membranes indicates that TH is cytotoxic to all three cancer cells with effective concentrations (EC(50)) of 2.4-2.8%. TH is however, not cytotoxic to the MCF-10A cells. Reactivity with annexin V fluorescence antibody and propidium iodide as analysed by flow cytometry and fluorescence microscopy shows that apoptosis occurred in these cancer cells. TH also reduced the mitochondrial membrane potential (??(m)) in the cancer cell lines after 24h of treatment. The activation of caspase-3/7 and -9 was observed in all TH-treated cancer cells indicating the involvement of mitochondrial apoptotic pathway. This study shows that TH has significant anticancer activity against human breast and cervical cancer cell lines. PMID:21167897

Fauzi, Agustine Nengsih; Norazmi, Mohd Nor; Yaacob, Nik Soriani

2011-04-01

262

Magnetic nanoparticles sensitize MCF-7 breast cancer cells to doxorubicin-induced apoptosis  

PubMed Central

Background Resistance of breast cancer cells to the available chemotherapeutics is a major obstacle to successful treatment. Recent studies have shown that magnetic nanoparticles might have significant application in different medical fields including cancer treatment. The goal of this study is to verify the ability of magnetic nanoparticles to sensitize cancer cells to the clinically available chemotherapy. Methods The role of iron oxide nanoparticles, static magnetic field, or a combination in the enhancement of the apoptotic potential of doxorubicin against the resistant breast cancer cells, MCF-7 was evaluated using the MTT assay and the propidium iodide method. Results In the present study, results revealed that pre-incubation of MCF-7 cells with iron oxide nanoparticles before the addition of doxorubicin did not enhance doxorubicin-induced growth inhibition. Pre-incubation of MCF-7 cells with iron oxide nanoparticles followed by a static magnetic field exposure significantly (P?cell death. Conclusions These results might point to the importance of combining magnetic nanoparticles with a static magnetic field in treatment of doxorubicin-refractory breast cancer cells. PMID:22533492

2012-01-01

263

Cell death along single microfluidic channel after freeze-thaw treatments  

PubMed Central

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

Li, Yuhui; Wang, Fen; Wang, Hao

2010-01-01

264

Zedoary oil (Ezhu You) inhibits proliferation of AGS cells  

PubMed Central

Background Zedoary (Curcumae Rhizoma, Ezhu), a Chinese medicinal herb, has been reported to show anticancer activity. This study aims to investigate the effect of zedoary oil (Ezhu You) on the proliferation of AGS cells which is one gastric cancer cell line. Methods The main ingredients of the herb were detected by GC-MS for herbal quality control. Cell viability was measured by MTT assay and cell proliferation was investigated by immunocytochemical staining for proliferating cell nuclear antigen (PCNA) protein. In addition, the cell cycle distributions were detected by flow cytometry with propidium iodine (PI) staining and the apoptosis rates were evaluated by flow cytometry with annexin V/PI double-staining. The morphological changes associated with apoptosis were observed by Hoechst 33342/PI double-staining. Protein expression was determined by western blot analysis. Results The main ingredients of the herb, including curzerene (26.45%), eucalyptol (12.04%), curcumol (9.04%), pyridine (7.97%), germacrone (7.89%), ?-elemene (7.36%), ?-elemene (4.11%) and 28 other ingredients, including curdione, were consistent with the chemical profiles of zedoary. Zedoary oil significantly decreased the cell viability of AGS cells (P?cells (P?cells. At low concentrations (?60 ?g/mL), zedoary oil was less inhibitory toward normal gastric epithelial cells than gastric cancer cell lines. In AGS cells, zedoary oil inhibited cell proliferation in a dose- and time-dependent manner, with decreased PCNA protein expression in the zedoary oil-treated cells, and arrested the cell cycle at S, G2/M and G0/G1 stages after treatment for 6–48 h. At concentrations of 30, 60 and 90 ?g/mL, which resulted in significant inhibition of proliferation and cell cycle arrest, zedoary oil induced cell apoptosis. In addition, Hoechst 33342/PI double-staining confirmed the morphological characteristics of cell apoptosis at 24 h. Zedoary oil upregulated the ratio of Bax/Bcl-2 protein expression (P?cell proliferation through cell cycle arrest and cell apoptosis promotion, which were related to Bax/Bcl-2 protein expression. PMID:23805830

2013-01-01

265

Targeted microbubble mediated sonoporation of endothelial cells in vivo.  

PubMed

Ultrasound contrast agents as drug-delivery systems are an emerging field. Recently, we reported that targeted microbubbles are able to sonoporate endothelial cells in vitro. In this study, we investigated whether targeted microbubbles can also induce sonoporation of endothelial cells in vivo, thereby making it possible to combine molecular imaging and drug delivery. Live chicken embryos were chosen as the in vivo model. ?vß3-targeted microbubbles attached to the vessel wall of the chicken embryo were insonified at 1 MHz at 150 kPa (1 × 10 000 cycles) and at 200 kPa (1 × 1000 cycles) peak negative acoustic pressure. Sonoporation was studied by intravital microscopy using the model drug propidium iodide (PI). Endothelial cell PI uptake was observed in 48% of microbubble-vessel-wall complexes at 150 kPa (n = 140) and in 33% at 200 kPa (n = 140). Efficiency of PI uptake depended on the local targeted microbubble concentration and increased up to 80% for clusters of 10 to 16 targeted microbubbles. Ultrasound or targeted microbubbles alone did not induce PI uptake. This intravital microscopy study reveals that sonoporation can be visualized and induced in vivo using targeted microbubbles. PMID:25265175

Skachkov, Ilya; Luan, Ying; van der Steen, Antonius F W; de Jong, Nico; Kooiman, Klazina

2014-10-01

266

Xiao Jin Wan, a traditional Chinese herbal formula, inhibits proliferation via arresting cell cycle progression at the G2/M phase and promoting apoptosis via activating the mitochondrial?dependent pathway in U-2OS human osteosarcoma cells.  

PubMed

Xiao Jin Wan (XJW) is a well-known traditional Chinese folk-medicine, which is commonly used for the treatment of various types of diseases including cancers. However, the mechanism of the anticancer activity of XJW against U-2OS human osteosarcoma cells, have not yet been reported. In the present study, we investigated the cellular effects of the XJW on the U-2OS human osteosarcoma cell line. Our results showed that XJW induced cell morphological changes, reduced cell viability in a dose- and time-dependent manner and arrested in the G2/M phase of the cell cycle suggesting that XJW inhibited the proliferation of U-2OS cells. Hoechst 33258 staining and Annexin V/propidium iodide double staining exhibited the typical nuclear features of apoptosis and increased the proportion of apoptotic Annexin V-positive cells in a dose-dependent manner, respectively. In addition, XJW treatment caused loss of plasma membrane asymmetry, collapse of mitochondrial membrane potential, activation of caspase-9 and caspase-3, and increase of the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2. Taken together, the results indicate that the U-2OS cell growth inhibitory activity of XJW was due to cell cycle arrested and mitochondrial-mediated apoptosis, which may partly explain the anticancer activity of Xiao Jin Wan. PMID:23354363

Wu, Guangwen; Chu, Jianfeng; Huang, Zhengrong; Ye, Jinxia; Chen, Panyu; Zheng, Chunsong; Li, Xihai; Liu, Xianxiang; Wu, Mingxia

2013-03-01

267

The alteration of protein profile of Walker 256 carinosarcoma cells during the apoptotic process induced by ultrasound.  

PubMed

The objective of this study was to investigate the alteration of the protein profile in cells after sonication and to identify the key proteins involved in the process of cell apoptosis. Walker 256 carinosarcoma cells were exposed to focused ultrasound (US) at the intensity of 2.0, 7.0, 10.2, 14.2 and 17.0 W/cm2 (I(spta)) for 10 min in vitro and the morphologic and functional changes of the cells were detected by hematoxylin & eosin staining and flow cytometry, with double staining of fluorescein isothiocyanate (FITC)-labeled Annexin V/propidium iodide (PI). The protein compositions in the cells after sonication were detected by 2-D SDS polyacrylamide gel electrophoresis. Our results showed that apoptosis of Walker 256 carinosarcoma cells could be induced by US. The percentage of early apoptosis and secondary necrosis increased with increasing intensity of US irradiation. Comparing with the protein patterns of cells before sonication, it was found that around 420 new protein spots were present in the gel after sonication. Among them, Hsp60 and Bcl-2 like protein 13 were found to be involved in the process of cell apoptosis and US-induced apoptosis of the cells was probably performed through the pathway of promoting the activation of caspase-3. PMID:15653239

Tian, Zhong-Min; Wan, Ming-Xi; Lu, Ming-Zhu; Wang, Xiao-Dong; Wang, Liang

2005-01-01

268

Two-color cytofluorometry and cellular properties of the urokinase receptor associated with a human metastatic carcinomatous cell line  

SciTech Connect

Purified human urokinase was labeled with either fluorescein isothiocyanate or iodine-125 and used as a probe for binding to the human metastatic carcinomatous cell line, Detroit 562. Cytofluorometry showed that the ligand bound preferentially to cells that had been exposed to acidic pH. The binding was competitive and decreased after mild tryptic digestion. The bound ligand could be removed by restoration of the cells to a low pH. Therefore, the cells had specific binding sites. The bound urokinase was involved in the breakdown of fibrin. Two-color cytofluorometric maps were constructed by counterstaining with propidium iodide. Results suggested that there were different cell populations that had different numbers of receptors and amounts of DNA. We cloned cells and found that single clones had homogeneous levels of receptors with different dissociation constants (from 10(-13) to 10(-11) mol/mg protein) for different clones. Cells of one clone, C5, which had high levels of receptor production, moved characteristically on a glass substratum coated with gold particles and reacted with wheat germ agglutinin, but not with concanavalin A. The receptors were found together with adhesion proteins at the sites where the cells adhered to the substrate. These results and the data obtained by zymography of the cellular proteins suggested that the urokinase-type plasminogen activators were bound to the receptors. The membrane-associated activator may stimulate local proteolysis, facilitating the migration of the tumor cell across the substrate.

Takahashi, K.; Gojobori, T.; Tanifuji, M. (Shimane Medical Univ., Izumo (Japan))

1991-02-01

269

Human Peripheral Blood Mononuclear Cells Cultured in Normal and Hyperglycemic Media in Simulated Microgravity Using NASA Bioreactors  

NASA Technical Reports Server (NTRS)

We sought answers to several questions this summer at NASA Johnson Space Center. Initial studies involved the in vitro culture of human peripheral blood mononuclear in cells in different conditioned culture media. Several human cancer clones were similarly studied to determine responses to aberrant glycosylation by the argon laser. The cells were grown at unit gravity in flasks and in simulated microgravity using NASA bioreactors. The cells in each instance were analyzed by flow cytometry. Cell cycle analysis was acquired by staining nuclear DNA with propidium iodide. Responses to the laser stimulation was measured by observing autofluorescence emitted in the green and red spectra after stimulation. Extent of glycosylation correlated with the intensity of the laser stimulated auto-fluorescence. Our particular study was to detect and monitor aberrant glycosylation and its role in etiopathogenesis. Comparisons were made between cells known to be neoplastic and normal cell controls using the same Laser Induced Autofluorescence technique. Studies were begun after extensive literature searches on using the antigen presenting potential of dendritic cells to induce proliferation of antigen specific cytotoxic T-cells. The Sendai virus served as the antigen. Our goal is to generate sufficient numbers of such cells in the simulated microgravity environment for use in autologous transplants of virally infected individuals including those positive for hepatitis and HIV.

Lawless, DeSales

2003-01-01

270

Vernonia amygdalina--Induced Growth Arrest and Apoptosis of Breast Cancer (MCF-7) Cells  

PubMed Central

Breast cancer is the second leading cause of cancer-related deaths of women in the United States. Fortunately, the mortality rate from breast cancer has decreased in recent years due to an increased emphasis on early detection and more effective treatments. Although great advancements have been made in the treatment and control of cancer progression, significant deficiencies and room for improvement remain. The central objective of this research was to further determine the in vitro mechanisms of Vernonia amygdalina (VA) leaf extracts as an anticancer candidate for the treatment of breast cancer. To achieve our objective, MCF-7 cells were treated with different concentrations of VA for 24 hand 48 h. Cell viability, live and dead cells were determined by the means of trypan blue exclusion test. Live and dead cells were further evaluated by propidium iodine (PI) assay using the Cellometer Vision. Cell apoptosis was measured by flow cytometry assessment using annexin V/PI kit. Data obtained from the trypan blue test demonstrated that VA treatment reduces cell viability in a concentration- and time-dependent manner. Result of the PI assay showed a gradual increase in the population of necrotic cells (fluorescence positive cells) in VA-treated cells compared to the control cells (fluorescence negative cells). Treatment of these cancer cells (MCF-7) for 48 h at concentrations ranging from 250 ?g/mL to 1000 ?g/mL caused early signs of apoptosis resulting from phosphatidylserine externalization as judged by annexin V assay. We observed a strong concentration-response relationship with regard to VA exposure and annexin V/PI positive cells. In summary, our finding demonstrates that VA-induced cytotoxicity and apoptosis in MCF-7 cells involve phosphatidylserine externalization accompanied by secondary necrotic cell death. With previous findings in our laboratory, the data generated in the present study confirms that VA is a valuable botanical therapeutic agent for the treatment of breast cancer. PMID:24353904

Yedjou, Clement G.; Izevbigie, Ernest B.; Tchounwou, Paul B.

2013-01-01

271

Bax Translocation Mediated Mitochondrial Apoptosis and Caspase Dependent Photosensitizing Effect of Ficus religiosa on Cancer Cells  

PubMed Central

The main aim of the present work was to investigate the potential effect of acetone extract of Ficus religosa leaf (FAE) in multiple apoptosis signalling in human breast cancer cells. FAE treatment significantly induced dose and time dependent, irreversible inhibition of breast cancer cell growth with moderate toxicity to normal breast epithelial cells. This observation was validated using Sulforhodamine B assay. Cell cycle analysis by Flow cytometry showed cell cycle arrest in G1 phase and induction of sub-G0 peak. FAE induced chromatin condensation and displayed an increase in apoptotic population in Annexin V-FITC/PI (Fluorescein isothiocyanate/Propidium iodide) double staining. FAE stimulated the loss of mitochondrial membrane potential in multiple breast cancer cell lines when compared to normal diploid cells. To understand the role of Bax in FAE induced apoptosis, we employed a sensitive cell based platform of MCF-7 cells expressing Bax-EGFP. Bax translocation to mitochondria was accompanied by the disruption of mitochondrial membrane potential and marked elevation in LEHDase activity (Caspase 9). Consistent with this data, FAE induced Caspase activation as evidenced by ratio change in FRET Caspase sensor expressing MCF-7 cell line and cleavage of prominent Caspases and PARP. Interestingly, FAE accelerated cell death in a mitochondrial dependent manner in continuous live cell imaging mode indicating its possible photosensitizing effect. Intracellular generation of reactive oxygen species (ROS) by FAE played a critical role in mediating apoptotic cell death and photosensitizing activity. FAE induced dose and time dependent inhibition of cancer cell growth which was associated with Bax translocation and mitochondria mediated apoptosis with the activation of Caspase 9 dependent Caspase cascade. FAE also possessed strong photosensitizing effect on cancer cell line that was mediated through rapid mitochondrial transmembrane potential loss and partial Caspase activation involving generation of intracellular ROS. PMID:22792212

Thankayyan R, Santhosh Kumar; Sithul, Hima; Sreeharshan, Sreeja

2012-01-01

272

Bax translocation mediated mitochondrial apoptosis and caspase dependent photosensitizing effect of Ficus religiosa on cancer cells.  

PubMed

The main aim of the present work was to investigate the potential effect of acetone extract of Ficus religosa leaf (FAE) in multiple apoptosis signalling in human breast cancer cells. FAE treatment significantly induced dose and time dependent, irreversible inhibition of breast cancer cell growth with moderate toxicity to normal breast epithelial cells. This observation was validated using Sulforhodamine B assay. Cell cycle analysis by Flow cytometry showed cell cycle arrest in G1 phase and induction of sub-G0 peak. FAE induced chromatin condensation and displayed an increase in apoptotic population in Annexin V-FITC/PI (Fluorescein isothiocyanate/Propidium iodide) double staining. FAE stimulated the loss of mitochondrial membrane potential in multiple breast cancer cell lines when compared to normal diploid cells. To understand the role of Bax in FAE induced apoptosis, we employed a sensitive cell based platform of MCF-7 cells expressing Bax-EGFP. Bax translocation to mitochondria was accompanied by the disruption of mitochondrial membrane potential and marked elevation in LEHDase activity (Caspase 9). Consistent with this data, FAE induced Caspase activation as evidenced by ratio change in FRET Caspase sensor expressing MCF-7 cell line and cleavage of prominent Caspases and PARP. Interestingly, FAE accelerated cell death in a mitochondrial dependent manner in continuous live cell imaging mode indicating its possible photosensitizing effect. Intracellular generation of reactive oxygen species (ROS) by FAE played a critical role in mediating apoptotic cell death and photosensitizing activity. FAE induced dose and time dependent inhibition of cancer cell growth which was associated with Bax translocation and mitochondria mediated apoptosis with the activation of Caspase 9 dependent Caspase cascade. FAE also possessed strong photosensitizing effect on cancer cell line that was mediated through rapid mitochondrial transmembrane potential loss and partial Caspase activation involving generation of intracellular ROS. PMID:22792212

Haneef, Jazir; Parvathy, Muraleedharan; M, Parvathy; Thankayyan R, Santhosh Kumar; Sithul, Hima; Sreeharshan, Sreeja

2012-01-01

273

The role of local renin-angiotensin system on high glucose-induced cell toxicity, apoptosis and reactive oxygen species production in PC12 cells  

PubMed Central

Objective(s): Hyperglycemia, oxidative stress and apoptosis have key roles in pathogenesis of diabetic neuropathy. There are local renin-angiotensin systems (RASs) in different tissues such as neural tissue. Local RASs are involved in physiological and pathophysiological processes such as inflammation, proliferation and apoptosis. This study aimed to investigate the role of local renin-angiotensin system on high glucose-induced cell toxicity, apoptosis and reactive oxygen species (ROS) production in PC12 cells, as a cell model of diabetic neuropathy. Materials and Methods: PC12 cells were exposed to a high glucose concentration (27 mg/ml), captopril (ACE inhibitor), telmisartan and losartan (AT1 antagonists), and also PD123319 (AT2 antagonist) were administered before and after induction of high glucose toxicity. Then cell viability was assessed by MTT assay and apoptotic cells and intracellular ROS production were detected by annexin V-propidium iodide and DCFDA, respectively, using flow cytometry. Results: High glucose concentration decreased cell viability, and increased apoptotic cells. Intracellular ROS production was also increased. In PC12 cells pretreatment and treatment by the drugs showed a significant improvement in cell viability and reduced apoptosis in captopril, telmisartan and PD123319 but only captopril and telmisartan were able to reduce ROS production. Losrtan significantly lowered ROS but didn't show any improvements in cell viability and apoptotic cells. Conclusion: The results of the present study showed that RAS inhibitors reduced cell toxicity and apoptosis and ROS production was induced by high glucose. It may be suggested that local RAS has a role in high glucose toxicity.

Shahveisi, Kaveh; Mousavi, Seyed Hadi; Hosseini, Mahmoud; Rad, Abolfazl Khajavi; Jalali, Seyed Amir; Rajaei, Ziba; Sadeghnia, Hamid Reza; Hadjzadeh, Mousa-Al-Reza

2014-01-01

274

Adenosine induces cell cycle arrest and apoptosis via cyclinD1/Cdk4 and Bcl-2/Bax pathways in human ovarian cancer cell line OVCAR-3.  

PubMed

Adenosine is a regulatory molecule with widespread physiological effects in almost every cells and acts as a potent regulator of cell growth. Adenosine has been shown to inhibit cell growth and induce apoptosis in the several cancer cells via caspase activation and Bcl-2/Bax pathway. The present study was designed to understand the mechanism underlying adenosine-induced apoptosis in the OVCAR-3 human ovarian cancer cells. MTT viability, BrdU and cell counting assays were used to study the cell proliferation effect of adenosine in presence of adenosine deaminase inhibitor and the nucleoside transporter inhibitor. Cell cycle analysis, propidium iodide and annexin V staining, caspase-3 activity assay, cyclinD1, Cdk4, Bcl-2 and Bax protein expressions were assessed to detect apoptosis. Adenosine significantly inhibited cell proliferation in a concentration-dependent manner in OVCAR-3 cell line. Adenosine induced cell cycle arrest in G0/G1 phase via Cdk4/cyclinD1-mediated pathway. Adenosine induced apoptosis, which was determined by Annexin V-FITC staining and increased sub-G1 population. Moreover, down-regulation of Bcl-2 protein expression, up-regulation of Bax protein expression and activation of caspase-3 were observed in response to adenosine treatment. The results of this study suggest that extracellular adenosine induced G1 cell cycle arrest and apoptosis in ovarian cancer cells via cyclinD1/ Cdk4 and Bcl-2/Bax pathways and caspase-3 activation. These data might suggest that adenosine could be used as an agent for the treatment of ovarian cancer. PMID:23345014

Shirali, Saeid; Aghaei, Mahmoud; Shabani, Mahdi; Fathi, Mojtaba; Sohrabi, Majid; Moeinifard, Marzieh

2013-04-01

275

The effect of Amaryllidaceae alkaloids haemanthamine and haemanthidine on cell cycle progression and apoptosis in p53-negative human leukemic Jurkat cells.  

PubMed

Plants from the Amaryllidaceae family have been shown to be a promising source of biologically active natural compounds of which some selected are currently in pre-clinical development. Regardless of interesting pioneer works, little is known about Amaryllidaceae alkaloids that have shown promising anti-cancer activities. The crinane group of the Amaryllidaceae, including haemanthamine and haemanthidine, was amongst the first of these compounds to exhibit an interesting cytotoxic potential against cancer cell lines. However, the mechanism of cytotoxic and anti-proliferative activity is not yet entirely clear. The primary objectives of the current study were to investigate the effects of haemanthamine and haemanthidine on the induction of apoptosis and the cell cycle regulatory pathway in p53-null Jurkat cells. Results indicate that haemanthamine and haemanthidine treatment decreases cell viability and mitochondrial membrane potential, leads to a decline in the percentage of cells in the S phase of the cell cycle, induces apoptosis detected by Annexin V staining and increases caspase activity. Dose dependent apoptosis was cross verified by fluorescence and bright field microscopy through Annexin V/propidium iodine staining and morphological changes which characteristically attend programmed cell death. The apoptotic effect of haemanthamine and haemanthidine on leukemia cells is more pronounced than that of gamma radiation. Contrary to gamma radiation, Jurkat cells do not completely halt the cell cycle 24h upon haemanthamine and haemanthidine exposure. Both Amaryllidaceae alkaloids accumulate cells preferentially at G1 and G2 stages of the cell cycle with increased p16 expression and Chk1 Ser345 phosphorylation. Concerning the pro-apoptotic effect, haemanthidine was more active than haemanthamine in the Jurkat leukemia cell line. PMID:24182986

Havelek, Radim; Seifrtova, Martina; Kralovec, Karel; Bruckova, Lenka; Cahlikova, Lucie; Dalecka, Marketa; Vavrova, Jirina; Rezacova, Martina; Opletal, Lubomir; Bilkova, Zuzana

2014-03-15

276

Relationship between Membrane Damage and Cell Death in Pressure-Treated Escherichia coli Cells: Differences between Exponential- and Stationary-Phase Cells and Variation among Strains  

PubMed Central

The relationship between membrane damage and loss of viability following pressure treatment was examined in Escherichia coli strains C9490, H1071, and NCTC 8003. These strains showed high, medium, and low resistance to pressure, respectively, in stationary phase but similar resistance to pressure in exponential phase. Loss of membrane integrity was measured as loss of osmotic responsiveness or as increased uptake of the fluorescent dye propidium iodide. In exponential-phase cells, loss of viability was correlated with a permanent loss of membrane integrity in all strains, whereas in stationary-phase cells, a more complicated picture emerged in which cell membranes became leaky during pressure treatment but resealed to a greater or lesser extent following decompression. Strain H1071 displayed a very unusual pressure response in stationary phase in which survival decreased to a minimum at 300 MPa but then increased at 400 to 500 MPa before decreasing again. Membranes were unable to reseal after treatment at 300 MPa but could do so after treatment at higher pressures. Membrane damage in this strain was thus typical of exponential-phase cells under low-pressure conditions but of stationary-phase cells under higher-pressure conditions. Heat shock treatment of strain H1071 cells increased pressure resistance under low-pressure conditions and also allowed membrane damage to reseal. Growth in the presence of IPTG (isopropyl-?-d-thiogalactopyranoside) increased resistance under high-pressure conditions. The mechanisms of inactivation may thus differ at high and low pressures. These studies support the view that membrane damage is an important event in the inactivation of bacteria by high pressure, but the nature of membrane damage and its relation to cell death may differ between species and phases of growth. PMID:10877775

Pagan, Rafael; Mackey, Bernard

2000-01-01

277

Cell disruption using a different methodology for proteomics analysis of Trypanosoma cruzi strains.  

PubMed

We have developed a cell disruption method to produce a protein extract using Trypanosoma cruzi cells based on a straightforward hypoosmotic lysis protocol. The procedure consists of three steps: incubation of the cells in a hypoosmotic lysis buffer, sonication in a water bath, and centrifugation. The final protein extract was designated TcS12. The stages of cell disruption at different incubation times were monitored by differential interference contrast microscopy. After 30min of incubation in lysis buffer at 4°C, the T. cruzi epimastigote forms changed from slender to round-shaped parasites. Nevertheless, cell disruption took place following sonication of the sample for 30min. The efficiency of the methodology was also validated by flow cytometry, which resulted in 72% of propidium iodide (PI)-labeled cells. To estimate the protein extraction yield and the differential protein expression, the proteomics profile of four T. cruzi strains (CL-Brener, Dm28c, Y, and 4167) were analyzed by liquid chromatography tandem mass spectrometry (LCMS/MS) on a SYNAPT HDMS system using the label-free MS(E) approach. ProteinLynx Global Server (version 2.5) with Expression(E) analysis identified a total of 1153 proteins and revealed 428 differentially expressed proteins among the strains. Gene ontology analysis showed that not only cytosolic proteins but also nuclear and organellar ones were present in the extract. PMID:24291641

Silva Galdino, Tainah; Menna-Barreto, Rubem Figueiredo Sadok; Britto, Constança; Samudio, Franklyn; Brandăo, Adeilton; Kalume, Dário Eluan

2014-03-01

278

Cordycepin induces S phase arrest and apoptosis in human gallbladder cancer cells.  

PubMed

Gallbladder cancer is the most common malignant tumor of the biliary tract, and this condition has a rather dismal prognosis, with an extremely low five-year survival rate. To improve the outcome of unresectable and recurrent gallbladder cancer, it is necessary to develop new effective treatments and drugs. The purpose of the present study was to evaluate the effects of cordycepin on human gallbladder cells and uncover the molecular mechanisms responsible for these effects. The Cell Counting Kit-8 (CCK-8) and colony formation assays revealed that cordycepin affected the viability and proliferation of human gallbladder cancer cells in a dose- and time-dependent manner. Flow cytometric analysis showed that cordycepin induced S phase arrest in human gallbladder cancer cell lines(NOZ and GBC-SD cells). Cordycepin-induced apoptosis was observed using an Annexin V/propidium iodide (PI) double-staining assay, and the mitochondrial membrane potential (??m) decreased in a dose-dependent manner. Additionally, western blot analysis revealed the upregulation of cleaved-caspase-3, cleaved-caspase-9, cleaved-PARP and Bax and the downregulation of Bcl-2, cyclin A and Cdk-2 in cordycepin-treated cells. Moreover, cordycepin inhibited tumor growth in nude mice bearing NOZ tumors. Our results indicate that this drug may represent an effective treatment for gallbladder carcinoma. PMID:25090123

Wang, Xu-An; Xiang, Shan-Shan; Li, Huai-Feng; Wu, Xiang-Song; Li, Mao-Lan; Shu, Yi-Jun; Zhang, Fei; Cao, Yang; Ye, Yuan-Yuan; Bao, Run-Fa; Weng, Hao; Wu, Wen-Guang; Mu, Jia-Sheng; Hu, Yun-Ping; Jiang, Lin; Tan, Zhu-Jun; Lu, Wei; Wang, Ping; Liu, Ying-Bin

2014-01-01

279

Bipolar nanosecond electric pulses are less efficient at electropermeabilization and killing cells than monopolar pulses.  

PubMed

Multiple studies have shown that bipolar (BP) electric pulses in the microsecond range are more effective at permeabilizing cells while maintaining similar cell survival rates as compared to monopolar (MP) pulse equivalents. In this paper, we investigated whether the same advantage existed for BP nanosecond-pulsed electric fields (nsPEF) as compared to MP nsPEF. To study permeabilization effectiveness, MP or BP pulses were delivered to single Chinese hamster ovary (CHO) cells and the response of three dyes, Calcium Green-1, propidium iodide (PI), and FM1-43, was measured by confocal microscopy. Results show that BP pulses were less effective at increasing intracellular calcium concentration or PI uptake and cause less membrane reorganization (FM1-43) than MP pulses. Twenty-four hour survival was measured in three cell lines (Jurkat, U937, CHO) and over ten times more BP pulses were required to induce death as compared to MP pulses of similar magnitude and duration. Flow cytometry analysis of CHO cells after exposure (at 15 min) revealed that to achieve positive FITC-Annexin V and PI expression, ten times more BP pulses were required than MP pulses. Overall, unlike longer pulse exposures, BP nsPEF exposures proved far less effective at both membrane permeabilization and cell killing than MP nsPEF. PMID:24332942

Ibey, Bennett L; Ullery, Jody C; Pakhomova, Olga N; Roth, Caleb C; Semenov, Iurii; Beier, Hope T; Tarango, Melissa; Xiao, Shu; Schoenbach, Karl H; Pakhomov, Andrei G

2014-01-10

280

Magnetic-field-assisted photothermal therapy of cancer cells using Fe-doped carbon nanoparticles  

NASA Astrophysics Data System (ADS)

Photothermal therapy with assistance of nanoparticles offers a solution for the destruction of cancer cells without significant collateral damage to otherwise healthy cells. However, minimizing the required number of injected nanoparticles is a major challenge. Here, we introduce the use of magnetic carbon nanoparticles (MCNPs), localizing them in a desired region by applying an external magnetic-field, and irradiating the targeted cancer cells with a near-infrared laser beam. The MCNPs were prepared in benzene, using an electric plasma discharge, generated in the cavitation field of an ultrasonic horn. The CNPs were made ferromagnetic by use of Fe-electrodes to dope the CNPs, as confirmed by magnetometry. Transmission electron microscopy measurements showed the size distribution of these MCNPs to be in the range of 5 to 10 nm. For photothermal irradiation, a tunable continuous wave Ti: Sapphire laser beam was weakly focused on to the cell monolayer under an inverted fluorescence microscope. The response of different cell types to photothermal irradiation was investigated. Cell death in the presence of both MCNPs and laser beam was confirmed by morphological changes and propidium iodide fluorescence inclusion assay. The results of our study suggest that MCNP based photothermal therapy is a promising approach to remotely guide photothermal therapy.

Gu, Ling; Vardarajan, Vijaylakshmi; Koymen, Ali R.; Mohanty, Samarendra K.

2012-01-01

281

Gemcitabine combined with gum mastic causes potent growth inhibition and apoptosis of pancreatic cancer cells  

PubMed Central

Aim: To investigate the antiproliferative and apoptotic effects of gemcitabine combined with gum mastic and the underlying mechanisms in human pancreatic cancer cell lines. Methods: Cell proliferation and apoptosis were examined using the methyl thiazolyl tetrazolium (MTT) assay and propidium iodine staining, respectively. The expression of Bcl-2, Bax, NF-?B p65 subunit, and I?B? protein was measured using Western blotting. Results: Gemcitabine 0.01?100 ?g/mL inhibited cell proliferation and induced apoptosis in both pancreatic cancer BxPC-3 and COLO 357 cells. Gum mastic 40 ?g/mL significantly potentiated the antiproliferative and apoptotic effects of gemcitabine 10 ?g/mL after 72-h treatment. When cells were treated with gemcitabine in combination with gum mastic, the I?B? level was increased, whereas NF-?B activation was blocked; the expression of Bax protein was substantially increased, but Bcl-2 protein was down-regulated. Conclusion: Gemcitabine combined with gum mastic causes potent apoptosis in pancreatic cancer cells. The combination may be an effective therapeutic strategy for pancreatic cancer. PMID:20523344

Huang, Xin-yu; Wang, Hong-cheng; Yuan, Zhou; Li, Ang; He, Mei-lan; Ai, Kai-xing; Zheng, Qi; Qin, Huan-long

2010-01-01

282

Rapid separation and counting of viable microbial cells in food by nonculture method with bioplorer, a focusing-free microscopic apparatus with a novel cell separation unit.  

PubMed

A nonculture method utilizing a novel apparatus, the bioplorer, was developed. The bioplorer is composed of an efficient cell separation unit, a focusing-free microscopic device, and an image analysis program. A meat or vegetable suspension is poured into the cell separation funnel, and insoluble matter in the sample suspension is trapped by prefilters. Microbial cells passing through the two prefilters are then trapped by the membrane filter (pore size, 0.4 microm). Trapped cells are double-stained with 4',6'-diamidino-2-phenylindole and propidium iodide, and the membrane filter is removed and set on the focusing-free microscope. A fluorescent image is then recorded. Total numbers of viable and dead cells on the membrane filter can thus be determined automatically. One assay can be performed within 10 min, which is much faster than the culture method. The results obtained with both the nonculture method and the culture method for meat and vegetable samples were highly correlated (r = 0.953 to 0.998). This method is feasible for the practical purpose of food safety control. PMID:16416915

Shimakita, Tomonori; Tashiro, Yoshikazu; Katsuya, Akira; Saito, Mikako; Matsuoka, Hideaki

2006-01-01

283

Characterization of the cellular response during apoptosis induction in cadmium-treated Hep G2 human hepatoma cells.  

PubMed

Cadmium is a toxic transition heavy metal of continuing occupational and environmental concern, with a wide variety of adverse effects on regulation of gene expression and cellular signal transduction pathways. Injury to cells by cadmium leads to a complex series of events that can culminate in the death of the cell. It has been reported that cadmium induces apoptosis in many cell lines. However, the morphological characteristics leading to apoptosis or subsequent regeneration in cells exposed to cadmium have not been clarified. We evaluated whether human hepatoma cells maintained in culture undergo apoptosis when exposed to cadmium. Cytotoxic activity of cadmium on Hep G2 cells determined using 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide assay. A DNA ladder assay was performed by electrophoresis. Cell cycle analysis was quantified by flow cytometry. Nuclear morphology was studied by fluorescence microscopy after staining with propidium iodide and Hoechst 33342. Morphologic alterations in culture hepatocytes treated with CdCl2 were observed by transmission electron microscopy. We have demonstrated that apoptosis is a major mode of elimination of damaged HepG2 cells in cadmium toxicity and it precedes necrosis. PMID:14645995

Aydin, Hikmet Hakan; Celik, Handan Ak; Deveci, Remziye; Terzioglu, Ender; Karacali, Sabire; Mete, Nihal; Akarca, Ulus; Batur, Yücel

2003-11-01

284

The histone deacetylase inhibitor SAHA acts in synergism with fenretinide and doxorubicin to control growth of rhabdoid tumor cells  

PubMed Central

Background Rhabdoid tumors are highly aggressive malignancies affecting infants and very young children. In many instances these tumors are resistant to conventional type chemotherapy necessitating alternative approaches. Methods Proliferation assays (MTT), apoptosis (propidium iodide/annexin V) and cell cycle analysis (DAPI), RNA expression microarrays and western blots were used to identify synergism of the HDAC (histone deacetylase) inhibitor SAHA with fenretinide, tamoxifen and doxorubicin in rhabdoidtumor cell lines. Results HDAC1 and HDAC2 are overexpressed in primary rhabdoid tumors and rhabdoid tumor cell lines. Targeting HDACs in rhabdoid tumors induces cell cycle arrest and apoptosis. On the other hand HDAC inhibition induces deregulated gene programs (MYCC-, RB program and the stem cell program) in rhabdoid tumors. These programs are in general associated with cell cycle progression. Targeting these activated pro-proliferative genes by combined approaches of HDAC-inhibitors plus fenretinide, which inhibits cyclinD1, exhibit strong synergistic effects on induction of apoptosis. Furthermore, HDAC inhibition sensitizes rhabdoid tumor cell lines to cell death induced by chemotherapy. Conclusion Our data demonstrate that HDAC inhibitor treatment in combination with fenretinide or conventional chemotherapy is a promising tool for the treatment of chemoresistant rhabdoid tumors. PMID:23764045

2013-01-01

285

Bioactivity of the Murex Homeopathic Remedy and of Extracts from an Australian Muricid Mollusc against Human Cancer Cells  

PubMed Central

Marine molluscs from the family Muricidae are the source of a homeopathic remedy Murex, which is used to treat a range of conditions, including cancer. The aim of this study was to evaluate the in vitro bioactivity of egg mass extracts of the Australian muricid Dicathais orbita, in comparison to the Murex remedy, against human carcinoma and lymphoma cells. Liquid chromatography coupled with mass spectrometry (LC-MS) was used to characterize the chemical composition of the extracts and homeopathic remedy, focusing on biologically active brominated indoles. The MTS (tetrazolium salt) colorimetric assay was used to determine effects on cell viability, while necrosis and apoptosis induction were investigated using flow cytometry (propidium iodide and Annexin-V staining, resp.). Cells were treated with varying concentrations (1–0.01?mg/mL) of crude and semi-purified extracts or preparations (dilute 1?M and concentrated 4?mg/mL) from the Murex remedy (4?h). The Murex remedy showed little biological activity against the majority of cell lines tested. In contrast, the D. orbita egg extracts significantly decreased cell viability in the majority of carcinoma cell lines. Flow cytometry revealed these extracts induce necrosis in HT29 colorectal cancer cells, whereas apoptosis was induced in Jurkat cells. These findings highlight the biomedical potential of Muricidae extracts in the development of a natural therapy for the treatment of neoplastic tumors and lymphomas. PMID:19491143

Benkendorff, Kirsten; McIver, Cassandra M.; Abbott, Catherine A.

2011-01-01

286

1-Nitropyrene (1-NP) induces apoptosis and apparently a non-apoptotic programmed cell death (paraptosis) in Hepa1c1c7 cells.  

PubMed

Mechanistic studies of nitro-PAHs (polycyclic aromatic hydrocarbons) of interest might help elucidate which chemical characteristics are most important in eliciting toxic effects. 1-Nitropyrene (1-NP) is the predominant nitrated PAH emitted in diesel exhaust. 1-NP-exposed Hepa1c1c7 cells exhibited marked changes in cellular morphology, decreased proliferation and different forms of cell death. A dramatic increase in cytoplasmic vacuolization was observed already after 6 h of exposure and the cells started to round up at 12 h. The rate of cell proliferation was markedly reduced at 24 h and apoptotic as well as propidium iodide (PI)-positive cells appeared. Electron microscopic examination revealed that the vacuolization was partly due to mitochondria swelling. The caspase inhibitor Z-VAD-FMK inhibited only the apoptotic cell death and Nec-1 (an inhibitor of necroptosis) exhibited no inhibitory effects on either cell death or vacuolization. In contrast, cycloheximide markedly reduced both the number of apoptotic and PI-positive cells as well as the cytoplasmic vacuolization, suggesting that 1-NP induced paraptotic cell death. All the MAPKs; ERK1/2, p38 and JNK, appear to be involved in the death process since marked activation was observed upon 1-NP exposure, and their inhibitors partly reduced the induced cell death. The ERK1/2 inhibitor PD 98057 completely blocked the induced vacuolization, whereas the other MAPKs inhibitors only had minor effects on this process. These findings suggest that 1-NP may cause apoptosis and paraptosis. In contrast, the corresponding amine (1-aminopyrene) elicited only minor apoptotic and necrotic cell death, and cells with characteristics typical of paraptosis were absent. PMID:18417179

Asare, Nana; Landvik, Nina E; Lagadic-Gossmann, Dominique; Rissel, Mary; Tekpli, Xavier; Ask, Kjetil; Lĺg, Marit; Holme, Jřrn A

2008-07-15

287

Antibacterial Compounds of Canadian Honeys Target Bacterial Cell Wall Inducing Phenotype Changes, Growth Inhibition and Cell Lysis That Resemble Action of ?-Lactam Antibiotics  

PubMed Central

Honeys show a desirable broad spectrum activity against Gram-positive and negative bacteria making antibacterial activity an intrinsic property of honey and a desirable source for new drug development. The cellular targets and underlying mechanism of action of honey antibacterial compounds remain largely unknown. To facilitate the target discovery, we employed a method of phenotypic profiling by directly comparing morphological changes in Escherichia coli induced by honeys to that of ampicillin, the cell wall-active ?-lactam of known mechanism of action. Firstly, we demonstrated the purity of tested honeys from potential ?-lactam contaminations using quantitative LC-ESI-MS. Exposure of log-phase E. coli to honey or ampicillin resulted in time- and concentration-dependent changes in bacterial cell shape with the appearance of filamentous phenotypes at sub-inhibitory concentrations and spheroplasts at the MBC. Cell wall destruction by both agents, clearly visible on microscopic micrographs, was accompanied by increased permeability of the lipopolysaccharide outer membrane as indicated by fluorescence-activated cell sorting (FACS). More than 90% E. coli exposed to honey or ampicillin became permeable to propidium iodide. Consistently with the FACS results, both honey-treated and ampicillin-treated E. coli cells released lipopolysaccharide endotoxins at comparable levels, which were significantly higher than controls (p<0.0001). E. coli cells transformed with the ampicillin-resistance gene (?–lactamase) remained sensitive to honey, displayed the same level of cytotoxicity, cell shape changes and endotoxin release as ampicillin-sensitive cells. As expected, ?–lactamase protected the host cell from antibacterial action of ampicillin. Thus, both honey and ampicillin induced similar structural changes to the cell wall and LPS and that this ability underlies antibacterial activities of both agents. Since the cell wall is critical for cell growth and survival, honey active compounds would be highly applicable for therapeutic purposes while differences in the mode of action between honey and ampicillin may provide clinical advantage in eradicating ?-lactam-resistant pathogens. PMID:25191847

Brudzynski, Katrina; Sjaarda, Calvin

2014-01-01

288

Enhanced detection of fluorescence quenching in labeled cells  

DOEpatents

A method is provided for quantifying BrdU labeled DNA in cells. The BrdU is substituted onto the DNA and the DNA is stained with a first fluorochrome having a fluorescence which is quenchable by BrdU. The first fluorochrome is preferably a thymidine base halogen analogue, such as a Hoechst fluorochrome. The DNA is then stained with a second fluorochrome having a fluorescence which is substantially uneffected by BrdU. The second fluorochrome may be selected from the group consisting of mithramycin, chromomycin A3, olivomycin, propidium iodide and ethidium bromine. The fluorescence from the first and second fluorochromes is then measured to obtain first and second output signals, respectively. The first output signal is subtracted from the second output signal to obtain a difference signal which is functionally related to the quantity of BrdU incorporated into DNA. The technique is particularly useful for quantifying the synthesis of DNA during the S-phase of the cell cycle. 2 figs.

Crissman, H.A.; Steinkamp, J.A.

1987-11-30

289

Multivariate analysis of apoptotic markers versus cell cycle phase in living human cancer cells by microfluidic cytometry  

PubMed Central

Measurement of apoptotic markers in tumors can be directly correlated with the cell cycle phase using flow cytometry (FCM). The conventional DNA content analysis requires cell permeabilization to stain nuclei with fluorescent probes such as propidium iodide or use of a costly UV-excitation line for Hoechst 33342 probe. The access to FCM is also still limited to centralized core facilities due to its inherent high costs and complex operation. This work describes development and proof-of-concept validation of a portable and user-friendly microfluidic flow cytometer (?FCM) that can perform multivariate real time analysis on live cells using sampling volumes as small as 10 microliters. The ?FCM system employs disposable microfluidic cartridges fabricated using injection molding in poly(methylmethacrylate) transparent thermoplastic. Furthermore, the dedicated and miniaturized electronic hardware interface enables up to six parameter detection using a combination of spatially separated solid-state 473 (10 mW) and 640 nm (20 mW) lasers and x-y stage for rapid laser alignment adjustment. We provide new evidence that a simple 2D flow focusing on a chip is sufficient to measure cellular DNA content in live tumor cells using a far-red DNA probe DRAQ5. The feasibility of using the ?FCM system for a dose-response profiling of investigational anti-cancer agents on human hematopoietic cancer cells is also demonstrated. The data show that ?FCM can provide a viable novel alternative to conventional FCM for multiparameter detection of caspase activation and dissipation of mitochondrial inner membrane potential (??m) in relation to DNA content (cell cycle phase) in live tumor cells. PMID:24386542

Akagi, Jin; Skommer, Joanna; Matuszek, Anna; Takeda, Kazuo; Fujimura, Yuu; Khoshmanesh, Khashayar; Kalantar-Zadeh, Kourosh; Mitchell, Arnan; Errington, Rachel; Smith, Paul J; Darzynkiewicz, Zbigniew; Wlodkowic, Donald

2013-01-01

290

Multivariate analysis of apoptotic markers versus cell cycle phase in living human cancer cells by microfluidic cytometry  

NASA Astrophysics Data System (ADS)

Measurement of apoptotic markers in tumors can be directly correlated with the cell cycle phase using flow cytometry (FCM). The conventional DNA content analysis requires cell permeabilization to stain nuclei with fluorescent probes such as propidium iodide or use of a costly UV-excitation line for Hoechst 33342 probe. The access to FCM is also still limited to centralized core facilities due to its inherent high costs and complex operation. This work describes development and proof-of-concept validation of a portable and user-friendly microfluidic flow cytometer (?FCM) that can perform multivariate real time analysis on live cells using sampling volumes as small as 10 microliters. The ?FCM system employs disposable microfluidic cartridges fabricated using injection molding in poly(methylmethacrylate) transparent thermoplastic. Furthermore, the dedicated and miniaturized electronic hardware interface enables up to six parameter detection using a combination of spatially separated solid-state 473 (10 mW) and 640 nm (20 mW) lasers and x-y stage for rapid laser alignment adjustment. We provide new evidence that a simple 2D flow focusing on a chip is sufficient to measure cellular DNA content in live tumor cells using a far-red DNA probe DRAQ5. The feasibility of using the ?FCM system for a dose-response profiling of investigational anti-cancer agents on human hematopoietic cancer cells is also demonstrated. The data show that ?FCM can provide a viable novel alternative to conventional FCM for multiparameter detection of caspase activation and dissipation of mitochondrial inner membrane potential (??m) in relation to DNA content (cell cycle phase) in live tumor cells.

Akagi, Jin; Skommer, Joanna; Matuszek, Anna; Takeda, Kazuo; Fujimura, Yuu; Khoshmanesh, Khashayar; Kalantar-Zadeh, Kourosh; Mitchell, Arnan; Errington, Rachel; Smith, Paul J.; Darzynkiewicz, Zbigniew; Wlodkowic, Donald

2013-03-01

291

Assessment of cytotoxic properties of safranal and nanoliposomal safranal in various cancer cell lines.  

PubMed

Saffron (Crocus sativus) is a widely used food additive used for its color and taste. It has been reported that saffron possesses significant in vivo and in vitro anti-tumor activity. In the present study, anti-tumor effects of safranal, the major aromatic compound in saffron, and its liposomal form were investigated. The role of apoptosis has also been explored in this toxicity. HeLa, MCF7 and L929 cell lines were cultured and exposed to safranal (0.01-3?mM) or liposomal safranal (0.04-0.32?mM). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay was performed to assess cytotoxicity. Apoptosis was evaluated by staining cells with propidium iodide and quantifying sub-Gl peak by flow cytometry. MTT assay revealed a significant and concentration-dependent cytotoxic effect of safranal on HeLa and MCF7 cell lines. Liposomal safranal showed enhanced effect compared to the safranal solution, as compared by their IC50 concentrations. Flow cytometry results revealed induction of apoptosis by safranal. It might be concluded that safranal could be involved in saffron-induced cell death in HeLa and MCF7 cells. Liposome encapsulation improved anti-tumor effect of safranal. Safranal and particularly its liposomal form could be investigated as promising chemotherapeutic agents in cancer treatment. PMID:23494763

Malaekeh-Nikouei, Bizhan; Mousavi, Seyed Hadi; Shahsavand, Shabnam; Mehri, Soghra; Nassirli, Horiyeh; Moallem, Seyed Adel

2013-12-01

292

Characterization of zinc-induced neuronal death in primary cultures of rat cerebellar granule cells.  

PubMed

Although zinc is essential for the activity of numerous biological systems, and zinc deficiency has been associated with various pathologies, this metal can also exert direct neurotoxic action. In primary cultures of rat cerebellar granule neurons, a brief, 15- to 30-min exposure to zinc (100-500 microM) resulted in concentration-dependent delayed neuronal death. The toxicity of zinc depended on the maturity of the neuronal cultures-it was not apparent prior to Day 5 and it reached a plateau at about 9-10 days in vitro. We assayed cell injury by measuring mitochondrial functioning (MTT assay) and cell death with the trypan blue exclusion assay. Apoptosis was assayed by the morphological appearance of cells following fluorescence staining with propidium iodide and by the in situ TUNEL technique. Mitochondrial injury was an early result of zinc treatment. Actinomycin D, an inhibitor of macromolecular synthesis, attenuated delayed cell death. The calcium channel blockers nimodipine and amlodipine reduced both mitochondrial injury and cell death; the blockade of ionotropic glutamate receptors with MK-801 or CNQX was ineffective. These results suggest that calcium channel-blocker-sensitive mitochondrial injury and DNA damage are operative in the protein-synthesis-dependent neurotoxicity of zinc. An imbalance of zinc homeostasis might play a role in the pathophysiology of apoptosis-associated neurodegenerative disorders. PMID:9225750

Manev, H; Kharlamov, E; Uz, T; Mason, R P; Cagnoli, C M

1997-07-01

293

Galangin induces apoptosis of hepatocellular carcinoma cells via the mitochondrial pathway  

PubMed Central

AIM: To investigate the mechanism by which galangin, a polyphenolic compound derived from medicinal herbs, induces apoptosis of hepatocellular carcinoma (HCC) cells. METHODS: The 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay was used to measure cell viability. Apoptosis was evaluated by in situ uptake of propidium iodide and Hoechst 33258 and was then detected by fluorescence microscopy. Protein expressions were detected by Western blotting. To confirm the apoptotic pathway mediated by galangin, cells were transfected by bcl-2 gene to overexpress Bcl-2 or siRNA to down-regulate Bcl-2 expression. RESULTS: Galangin (46.25-370.0 ?mol/L) exerted an anti-proliferative effect, induced apoptosis, and decreased mitochondrial membrane potential in a dose and time-dependent manner. Treatment with galangin induced apoptosis by translocating the pro-apoptotic protein Bax to the mitochondria, which released apoptosis-inducing factor and cytochrome c into the cytosol. Overexpression of Bcl-2 attenuated galangin-induced HepG2 cell apoptosis, while decreasing Bcl-2 expression enhanced galangin-induced cell apoptosis. CONCLUSION: Our data suggests that galangin mediates apoptosis through a mitochondrial pathway, and may be a potential chemotherapeutic drug for the treatment of HCC. PMID:20632439

Zhang, Hai-Tao; Luo, Hui; Wu, Jun; Lan, Liu-Bo; Fan, Da-Hua; Zhu, Kai-Dan; Chen, Xiao-Yi; Wen, Min; Liu, Hui-Ming

2010-01-01

294

Stable overexpression of DNA fragmentation factor in T-47D cells: sensitization of breast cancer cells to apoptosis in response to acetazolamide and sulfabenzamide.  

PubMed

Alterations in expression of the DFF40 gene have been reported in some cancers. This study is an in vitro study of the therapeutic effects of gene transfer that lead to elevation in DFF40 expression within T-47D cells in the presence of sulfonamide drugs. In this study, we have constructed a eukaryotic expression vector for DFF40 and transfected it into T-47D cancer cells. We used real time RT-PCR to detect the expression of DFF40 and the MTT assay to determine effects of the sulfonamide drugs acetazolamide, sulfabenzamide, sulfathiazole and sulfacetamide on cell viability in the presence of increased and normal DFF40 levels. Cell cycle distribution was assessed by propidium iodide (PI) staining and the rates of apoptosis by annexin V/PI staining. The DNA laddering analysis was employed to evaluate apoptosis. We observed that overexpression of DFF40 was only effective in decreasing viability in cells incubated with acetazolamide and sulfabenzamide. There was enhanced apoptosis in these groups, particularly with acetazolamide. The cell cycle distribution analysis showed that in the presence of sulfonamide drugs there were no substantial changes in empty-vector or DFF40-transfected cells, except for those cells treated with sulfabenzamide or sulfathiazole. There was no DNA laddering in cells that expressed the empty vector when incubated with sulfonamide drugs. In contrast, we observed DNA laddering in cells that expressed DFF40 in the presence of acetazolamide. Our results have demonstrated that combinatorial use of some sulfonamides such as acetazolamide along with increased expression of DFF40 can potently kill tumor cells via apoptosis and may be beneficial for treatment of some chemoresistant cancers. PMID:25086620

Bagheri, Fatemeh; Safarian, Shahrokh; Eslaminejad, Mohamadreza Baghaban; Sheibani, Nader

2014-11-01

295

Silver nanoparticles exert a long-lasting antiproliferative effect on human keratinocyte HaCaT cell line.  

PubMed

For their antibacterial activity, silver nanoparticles (Ag NPs) are largely used in various commercially available products designed to come in direct contact with the skin. In this study we investigated the effects of Ag NPs on skin using the human-derived keratinocyte HaCaT cell line model. Ag NPs caused a concentration- and time-dependent decrease of cell viability, with IC(50) values of 6.8 ± 1.3 ?M (MTT assay) and 12 ± 1.2 ?M (SRB assay) after 7 days of contact. A 24h treatment, followed by a 6 day recovery period in Ag NPs-free medium, reduced cell viability with almost the same potency (IC(50)s of 15.3 ± 4.6 and 35 ± 20 ?M, MTT and SRB assays, respectively). Under these conditions, no evidence of induction of necrotic events (propidium iodide assay) was found. Apocynin, NADPH-oxidase inhibitor, or N(G)-monomethyl-L-argynine, nitric oxide synthase inhibitor, did not prevent NPs-induced reduction of cell viability. TEM analysis of cells exposed to NPs for 24h revealed alteration of nuclear morphology but only a marginal presence of individual NPs inside the cells. These results demonstrate that on HaCaT keratinocytes a relatively short time of contact with Ag NPs causes a long-lasting inhibition of cell growth, not associated with consistent Ag NPs internalization. PMID:21501681

Zanette, Caterina; Pelin, Marco; Crosera, Matteo; Adami, Gianpiero; Bovenzi, Massimo; Larese, Francesca Filon; Florio, Chiara

2011-08-01

296

Overexpression of Human Arginine Decarboxylase Rescues Human Mesenchymal Stem Cells against H2O2 Toxicity through Cell Survival Protein Activation  

PubMed Central

In this study, we explored the potentiality of human arginine decarboxylase (ADC) to enhance the survival of mesenchymal stem cells (MSCs) against unfavorable milieu of host tissues as the low survival of MSCs is the issue in cell transplantation therapy. To address this, human MSCs overexpressing human ADC were treated with H2O2 and the resultant intracellular events were examined. First, we examined whether human ADC is overexpressed in human MSCs. Then, we investigated cell survival or death related events. We found that the overexpression of human ADC increases formazan production and reduces caspase 3 activation and the numbers of FITC, hoechst, or propidium iodide positive cells in human MSCs exposed to H2O2. To elucidate the factors underlying these phenomena, AKT, CREB, and BDNF were examined. We found that the overexpression of human ADC phosphorylates AKT and CREB and increases BDNF level in human MSCs exposed to H2O2. The changes of these proteins are possibly relevant to the elevation of agmatine. Collectively, our data demonstrate that the overexpression of human ADC stimulates pro-survival factors to protect human MSCs against H2O2 toxicity. In conclusion, the present findings support that ADC can enhance the survival of MSCs against hostile environment of host tissues. PMID:23487582

Seo, Su Kyoung; Yang, Wonsuk; Park, Yu Mi; Lee, Won Taek; Park, Kyung Ah

2013-01-01

297

Cryptosporidium parvum infection of Caco-2 cell monolayers induces an apical monolayer defect, selectively increases transmonolayer permeability, and causes epithelial cell death.  

PubMed Central

Caco-2 cells were grown on permeable filters and infected with Cryptosporidium parvum. Infection rates exceeded 50% of target cells with a sufficient inoculum dose of parasites. Infection induced a dose- and time-dependent fall in transmonolayer resistance, which was closely related to both the inoculum dose and the number of parasites detected by immunofluorescence. Caco-2a, MDBK, and MDBK subclone F5D2 evidenced similar declines in resistance when grown and infected under similar circumstances. Caco-2 monolayers became permeable to molecules of < or = 1,000 Da but continued to remain impermeable to exogenously added, or endogenously produced, proteins of > or = 1,881 Da. We found that infected monolayers released up to 50% of the total cellular lactase dehydrogenase into apical media, but not basal media, and that the vital dye propidium iodide avidly stained infected cells, and often parasites, when added to the apical reservoir. Cryptosporidium infection of Caco-2 monolayers increases transmonolayer permeability, induces an apical cellular and monolayer defect, and causes cell death. Images PMID:7927716

Griffiths, J K; Moore, R; Dooley, S; Keusch, G T; Tzipori, S

1994-01-01

298

Inhibition of Ca2+-independent phospholipase A2 decreases prostate cancer cell growth by p53-dependent and independent mechanisms.  

PubMed

The mechanisms by which Ca(2+)-independent phospholipase A(2) (iPLA(2)) mediates cell growth in p53-positive LNCaP and p53-negative PC-3 prostate cancer cell lines were studied. Exposure of cells to the iPLA(2) selective inhibitor bromoenol lactone (BEL; 0-20 microM) induced concentration- and time-dependent decreases in cell growth based on 3-(4, dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide staining and cell number. Decreased cell growth was not caused by cell death as BEL exposure did not alter nuclear morphology or increase annexin V (apoptotic cell marker) or propidium iodide (necrotic cell marker) staining after 48 h. Decreased growth correlated to a G(1)/G(0) arrest in LNCaP cells and aG(2)/M arrest in PC-3 cells. In LNCaP cells, G(1) arrest was preceded by time- (0-48 h) and concentration-dependent (0-10 microM) increases in the expression of the tumor suppressor protein p53 and the cyclin-dependent kinase inhibitor p21. Increases in p53 expression preceded increases in p21 expression by 8 h. In LNCaP cells, BEL treatment decreased the expression of the p53 antagonist Mdm2, while increasing Akt phosphorylation. BEL treatment also increased Akt phosphorylation in PC-3 cells, but Mdm2 was not detected. The ability of BEL to increase Akt phosphorylation was inhibited by the phosphoinositide 3-kinase inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one]. BEL treatment also decreased agonist-induced activation of the epidermal growth factor receptor. These data suggest that inhibition of iPLA(2) decreases prostate cancer cell growth by p53-dependent and independent mechanisms. Furthermore, alterations in Mdm2 and epidermal growth factor receptor activation following BEL exposure suggest novel roles for iPLA(2) in prostate cancer cell signaling. PMID:18441250

Sun, Bin; Zhang, Xiaoling; Talathi, Sonia; Cummings, Brian S

2008-07-01

299

DNA fragmentation and apoptosis induced by safranal in human prostate cancer cell line  

PubMed Central

Objectives: Apoptosis, an important mechanism that contributes to cell growth reduction, is reported to be induced by Crocus sativus (Saffron) in different cancer types. However, limited effort has been made to correlate these effects to the active ingredients of saffron. The present study was designed to elucidate cytotoxic and apoptosis induction by safranal, the major coloring compound in saffron, in a human prostate cancer cell line (PC-3). Materials and Methods: PC-3 and human fetal lung fibroblast (MRC-5) cells were cultured and exposed to safranal (5, 10, 15, and 20 ?g/ml). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to assess cytotoxicity. DNA fragmentation was assessed by gel electrophoresis. Cells were incubated with different concentrations of safranal, and cell morphologic changes and apoptosis were determined by the normal inverted microscope, Annexin V, and propidium iodide, followed by flow cytometric analysis, respectively. Results: MTT assay revealed a remarkable and concentration-dependent cytotoxic effect of safranal on PC-3 cells in comparison with non-malignant cell line. The morphologic alterations of the cells confirmed the MTT results. The IC50 values against PC-3 cells were found to be 13.0 ? 0.07 and 6.4 ? 0.09 ?g/ml at 48 and 72 h, respectively. Safranal induced an early and late apoptosis in the flow cytometry histogram of treated cells, indicating apoptosis is involved in this toxicity. DNA analysis revealed typical ladders as early as 48 and 72 h after treatment, indicative of apoptosis. Conclusions: Our preclinical study demonstrated a prostate cancer cell line to be highly sensitive to safranal-mediated growth inhibition and apoptotic cell death. Although the molecular mechanisms of safranal action are not clearly understood, it appears to have potential as a therapeutic agent. PMID:24082436

Samarghandian, Saeed; Shabestari, Mahmoud M

2013-01-01

300

13-Acetoxysarcocrassolide Induces Apoptosis on Human Gastric Carcinoma Cells Through Mitochondria-Related Apoptotic Pathways: p38/JNK Activation and PI3K/AKT Suppression  

PubMed Central

13-acetoxysarcocrassolide (13-AC), an active compound isolated from cultured Formosa soft coral Sarcophyton crassocaule, was found to possess anti-proliferative and apoptosis-inducing activities against AGS (human gastric adenocarcinoma cells) gastric carcinoma cells. The anti-tumor effects of 13-AC were determined by MTT assay, colony formation assessment, cell wound-healing assay, TUNEL/4,6-Diamidino-2-phenylindole (DAPI) staining, Annexin V-fluorescein isothiocyanate/propidium iodide (PI) staining and flow cytometry. 13-AC inhibited the growth and migration of gastric carcinoma cells in a dose-dependent manner and induced both early and late apoptosis as assessed by flow cytometer analysis. 13-AC-induced apoptosis was confirmed through observation of a change in ??m, up-regulated expression levels of Bax and Bad proteins, down-regulated expression levels of Bcl-2, Bcl-xl and Mcl-1 proteins, and the activation of caspase-3, caspase-9, p38 and JNK. Furthermore, inhibition of p38 and JNK activity by pretreatment with SB03580 (a p38-specific inhibitor) and SP600125 (a JNK-specific inhibitor) led to rescue of the cell cytotoxicity of 13-AC-treated AGS cells, indicating that the p38 and the JNK pathways are also involved in the 13-AC-induced cell apoptosis. Together, these results suggest that 13-AC induces cell apoptosis against gastric cancer cells through triggering of the mitochondrial-dependent apoptotic pathway as well as activation of the p38 and JNK pathways. PMID:25342459

Su, Ching-Chyuan; Chen, Jeff Yi-Fu; Din, Zhong-Hao; Su, Jui-Hsin; Yang, Zih-Yan; Chen, Yi-Jen; Wang, Robert Y.L.; Wu, Yu-Jen

2014-01-01

301

The distinct role of guanine nucleotide exchange factor Vav1 in Bcl-2 transcription and apoptosis inhibition in Jurkat leukemia T cells  

PubMed Central

Aim: To investigate a novel function of proto-oncogene Vav1 in the apoptosis of human leukemia Jurkat cells. Methods: Jurkat cells, Jurkat-derived vav1-null cells (J.Vav1) and Vav1-reconstituted J.WT cells were treated with a Fas agonist antibody, IgM clone CH11. Apoptosis was determined using propidium iodide (PI) staining, Annexin-V staining, DNA fragmentation, cleavage of caspase 3/caspase 8, and poly (ADP-ribose) polymerase (PARP). Mitochondria transmembrane potential (??m) was measured using DiOC6(3) staining. Transcription and expression of the Bcl-2 family of proteins were evaluated using semi-quantitative RT-PCR and Western blot, respectively. Bcl-2 promoter activity was analyzed using luciferase reporter assays. Results: Cells lacking Vav1 were more sensitive to Fas-mediated apoptosis than Jurkat and J.WT cells. J.Vav1 cells lost mitochondria transmembrane potential (??m) more rapidly upon Fas induction. These phenotypes could be rescued by re-expression of Vav1 in J.Vav1 cells. The expression of Vav1 increased the transcription of pro-survival Bcl-2. The guanine nucleotide exchange activity of Vav1 was required for enhancing Bcl-2 promoter activity, and the Vav1 downstream substrate, small GTPase Rac2, was likely involved in the control of Bcl-2 expression. Conclusion: Vav1 protects Jurkat cells from Fas-mediated apoptosis by promoting Bcl-2 transcription through its GEF activity. PMID:21151158

Yin, Jie; Wan, Ya-juan; Li, Shi-yang; Du, Ming-juan; Zhang, Cui-zhu; Zhou, Xing-long; Cao, You-jia

2011-01-01

302

13-Acetoxysarcocrassolide Induces Apoptosis on Human Gastric Carcinoma Cells Through Mitochondria-Related Apoptotic Pathways: p38/JNK Activation and PI3K/AKT Suppression.  

PubMed

13-acetoxysarcocrassolide (13-AC), an active compound isolated from cultured Formosa soft coral Sarcophyton crassocaule, was found to possess anti-proliferative and apoptosis-inducing activities against AGS (human gastric adenocarcinoma cells) gastric carcinoma cells. The anti-tumor effects of 13-AC were determined by MTT assay, colony formation assessment, cell wound-healing assay, TUNEL/4,6-Diamidino-2-phenylindole (DAPI) staining, Annexin V-fluorescein isothiocyanate/propidium iodide (PI) staining and flow cytometry. 13-AC inhibited the growth and migration of gastric carcinoma cells in a dose-dependent manner and induced both early and late apoptosis as assessed by flow cytometer analysis. 13-AC-induced apoptosis was confirmed through observation of a change in ??m, up-regulated expression levels of Bax and Bad proteins, down-regulated expression levels of Bcl-2, Bcl-xl and Mcl-1 proteins, and the activation of caspase-3, caspase-9, p38 and JNK. Furthermore, inhibition of p38 and JNK activity by pretreatment with SB03580 (a p38-specific inhibitor) and SP600125 (a JNK-specific inhibitor) led to rescue of the cell cytotoxicity of 13-AC-treated AGS cells, indicating that the p38 and the JNK pathways are also involved in the 13-AC-induced cell apoptosis. Together, these results suggest that 13-AC induces cell apoptosis against gastric cancer cells through triggering of the mitochondrial-dependent apoptotic pathway as well as activation of the p38 and JNK pathways. PMID:25342459

Su, Ching-Chyuan; Chen, Jeff Yi-Fu; Din, Zhong-Hao; Su, Jui-Hsin; Yang, Zih-Yan; Chen, Yi-Jen; Wang, Robert Y L; Wu, Yu-Jen

2014-01-01

303

Responses of Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus to Simulated Food Processing Treatments, Determined Using Fluorescence-Activated Cell Sorting and Plate Counting?  

PubMed Central

Three common food pathogenic microorganisms were exposed to treatments simulating those used in food processing. Treated cell suspensions were then analyzed for reduction in growth by plate counting. Flow cytometry (FCM) and fluorescence-activated cell sorting (FACS) were carried out on treated cells stained for membrane integrity (Syto 9/propidium iodide) or the presence of membrane potential [DiOC2(3)]. For each microbial species, representative cells from various subpopulations detected by FCM were sorted onto selective and nonselective agar and evaluated for growth and recovery rates. In general, treatments giving rise to the highest reductions in counts also had the greatest effects on cell membrane integrity and membrane potential. Overall, treatments that impacted cell membrane permeability did not necessarily have a comparable effect on membrane potential. In addition, some bacterial species with extensively damaged membranes, as detected by FCM, appeared to be able to replicate and grow after sorting. Growth of sorted cells from various subpopulations was not always reflected in plate counts, and in some cases the staining protocol may have rendered cells unculturable. Optimized FCM protocols generated a greater insight into the extent of the heterogeneous bacterial population responses to food control measures than did plate counts. This study underlined the requirement to use FACS to relate various cytometric profiles generated by various staining protocols with the ability of cells to grow on microbial agar plates. Such information is a prerequisite for more-widespread adoption of FCM as a routine microbiological analytical technique. PMID:21602370

Kennedy, Deirdre; Cronin, Ultan P.; Wilkinson, Martin G.

2011-01-01

304

Comet assay, cloning assay, and light and electron microscopy on one preselected cell  

NASA Astrophysics Data System (ADS)

In order to perform long-term studies up to one week on a preselected single cell after micromanipulation (e.g. UVA and NIR microbeam exposure) in comparison with non-treated neighbor cells (control cells) we applied a variety of single cell diagnostic techniques and developed a special comet assay for single preselected cells. For that purpose adherent cells were grown in low concentrations and maintained in special sterile centimeter-sized glass cell chambers. After preselection, a single cell was marked by means of diamond-produced circles on the outer cell chamber window. During exposure to microbeams, NADH-attributed autofluorescence of the chosen cell was detected by fluorescence imaging and spectroscopy. In addition, cell morphology was video-monitored (formation of pseudopodia, membrane blebbing,...). Maintaining the microchamber in the incubator, the irradiated cell was examined 24 h later for cell division (clone formation) and modifications in autofluorescence and morphology (including daughter cells). In the case that no division occurred the vitality of the light-exposed cell and of the control cells were probed by intranuclear propidium iodide accumulation. After fixation, either electron microscopy or single cell gel electrophoresis (comet assay) was performed. To monitor comet formation indicating photoinduced DNA damage in the preselected single cell in comparison with the non-exposed neighbor cells the chamber was filled with low-melting gel and lysis solution and exposed to an electric field. In contrast to the conventional comet assay, where only randomly chosen cells of a suspension are investigated, the novel optimized electrophoresis technique should enhance the possibilities of DNA damage detection to a true single (preselected) cell level. The single cell techniques applied to UVA microexposed Chinese hamster ovary cells (364 nm, 1 mW, 3.5 W/cm2) revealed significant cell damage for J/cm2 fluences such as modifications of intracellular redox state, impaired cell division, formation of giant cells and cell shrinking, swelling of mitochondria and loss of cristae as well as DNA damage.

Koenig, Karsten; Oehring, Hartmut; Halbhuber, Karl-Juergen; Fiedler, Ursula; Bauer, Eckhard; Greulich, Karl-Otto

1998-01-01

305

Comet assay, cloning assay, and light and electron microscopy on one preselected cell  

NASA Astrophysics Data System (ADS)

In order to perform long-term studies up to one week on a preselected single cell after micromanipulation (e.g. UVA and NIR microbeam exposure) in comparison with non-treated neighbor cells (control cells) we applied a variety of single cell diagnostic techniques and developed a special comet assay for single preselected cells. For that purpose adherent cells were grown in low concentrations and maintained in special sterile centimeter-sized glass cell chambers. After preselection, a single cell was marked by means of diamond-produced circles on the outer cell chamber window. During exposure to microbeams, NADH-attributed autofluorescence of the chosen cell was detected by fluorescence imaging and spectroscopy. In addition, cell morphology was video-monitored (formation of pseudopodia, membrane blebbing,...). Maintaining the microchamber in the incubator, the irradiated cell was examined 24 h later for cell division (clone formation) and modifications in autofluorescence and morphology (including daughter cells). In the case that no division occurred the vitality of the light-exposed cell and of the control cells were probed by intranuclear propidium iodide accumulation. After fixation, either electron microscopy or single cell gel electrophoresis (comet assay) was performed. To monitor comet formation indicating photoinduced DNA damage in the preselected single cell in comparison with the non-exposed neighbor cells the chamber was filled with low-melting gel and lysis solution and exposed to an electric field. In contrast to the conventional comet assay, where only randomly chosen cells of a suspension are investigated, the novel optimized electrophoresis technique should enhance the possibilities of DNA damage detection to a true single (preselected) cell level. The single cell techniques applied to UVA microexposed Chinese hamster ovary cells (364 nm, 1 mW, 3.5 W/cm2) revealed significant cell damage for J/cm2 fluences such as modifications of intracellular redox state, impaired cell division, formation of giant cells and cell shrinking, swelling of mitochondria and loss of cristae as well as DNA damage.

Koenig, Karsten; Oehring, H.; Halbhuber, Karl-Juergen; Fiedler, Ursula; Bauer, Eckhard; Greulich, Karl O.

1997-12-01

306

Endothelial cell Blood cells  

E-print Network

Endothelial cell HSCs Blood cells Blood vessel Haemogenic endothelial cell Figure 1 | Relationship between endothelial cells and blood cells. Endothelial cells line the inside of blood vessels. During mouse embryonic development, a subset of these cells, known as haemogenic endothelial cells, seems

Wilmers, Chris

307

Millimeter wave treatment induces apoptosis via activation of the mitochondrial-dependent pathway in human osteosarcoma cells.  

PubMed

Millimeter wave (MW) is an electromagnetic wave with a wavelength between 1 and 10 mm and a frequency of 30-300 GHz that causes multiple biological effects and has been used as a major component in physiotherapies for the clinical treatment of various types of diseases including cancers. However, the precise molecular mechanism of the anticancer activity of millimeter wave remains to be elucidated. In the present study, we investigated the cellular effects of the MW in the U-2OS human osteosarcoma cell line. Our results showed that MW induced cell morphological changes and reduced cell viability in a dose- and time-dependent manner suggesting that MW inhibited the growth of U-2OS cells as demonstrated. Hoechst 33258 staining and Annexin V/propidium iodide double staining exhibited the typical nuclear features of apoptosis and increased the proportion of apoptotic Annexin V-positive cells in a dose-dependent manner, respectively. In addition, MW treatment caused loss of plasma membrane asymmetry, release of cytochrome c, collapse of mitochondrial membrane potential, activation of caspase-9 and -3, and increase of the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2. Taken together, the results indicate that the U-2OS cell growth inhibitory activity of MW was due to mitochondrial-mediated apoptosis, which may partly explain the anticancer activity of millimeter wave treatment. PMID:22246399

Wu, Guangwen; Chen, Xuzheng; Peng, Jun; Cai, Qiaoyan; Ye, Jinxia; Xu, Huifeng; Zheng, Chunsong; Li, Xihai; Ye, Hongzhi; Liu, Xianxiang

2012-05-01

308

L-carvone induces p53, caspase 3 mediated apoptosis and inhibits the migration of breast cancer cell lines.  

PubMed

A wide variety of natural compounds exists that possesses significant cytotoxic as well as chemopreventive activity through induction of apoptosis in cancer cells. The antiproliferative and apoptotic effect of L-carvone, an active component of spearmint (Mentha spicata) was studied on breast cancer (MCF 7 and MDA MB 231) and normal (MCF 10A) cell lines, and insight into its mechanism of action was attained. L-carvone inhibited proliferation of MCF 7 (IC50 1.2 mM) and MDA MB 231 cells (IC50 1.0 mM) and inhibited the migration of breast cancer cell lines. L-carvone induced apoptosis as observed by nuclei fragmentation and the presence of apoptotic bodies in DAPI, AnnexinV/propidium iodide, and TUNEL assays. L-carvone exposure arrested MCF 7 cells in S phase of the cell cycle. DNA damage caused by L-carvone was apparent from the increased tail moment in COMET assay, which could be induced by an increase in ROS that was measured using a fluorescence probe. Glutathione levels were also increased. The increased level of p53, Bad, cleaved caspase 3, and cleaved PARP explained p53 and caspase-mediated apoptosis. PMID:24611509

Patel, Pinaki B; Thakkar, Vasudev R

2014-01-01

309

Apoptosis Induction of Salvia chorassanica Root Extract on Human Cervical Cancer Cell Line  

PubMed Central

Salvia chorassanica Bunge is one of the Iranian endemic species of Salvia. There is not any reported literature on S. chorassanica. This study was designed to examine the in-vitro anti-proliferative and proapoptotic effects of the methanol extract of S. chorassanica and its fractions on HeLa cell line. Cells were cultured in EX-CELL®, an animal free medium specially designed for HeLa cell line and incubated with different concentrations of plant extracts. Cell viability was quantified by MTS assay. Apoptotic cells were determined using propidium iodide (PI) staining of DNA fragmentation by flow cytometry (sub-G1 peak). Activity of caspase -3, -8 and -9 was measured by the caspase colorimetric kit assay. S. chorassanica inhibited the growth of malignant cells and the CH2Cl2 fraction was determined as the most cytotoxic fraction in comparison with other fractions. The calculated IC50 values for methanol extract, n-hexane, CH2Cl2 and EtOAc fractions were 8.841, 5.45, 2.38, and 58.03 ?g/mL, respectively. S. chorassanica induced a sub-G1 peak in the flow cytometry histogram of treated cells compared to control cells indicating that the cytotoxic mechanism is characterized by apoptosis induction. The activity of caspase-3 and 8 proteins in treated HeLa cells was significantly higher than that of the control while caspase-9 activity did not change significantly. Based on the result obtained from our study, the apoptosis pathway involved in S. chorassanica-induced cell death may be through the extrinsic pathway and it can be a novel promising candidate in the treatment of cancer. PMID:24250574

Parsaee, Heydar; Asili, Javad; Mousavi, Seyed Hadi; Soofi, Hojjat; Emami, Seyed Ahmad; Tayarani-Najaran, Zahra

2013-01-01

310

Interactions of human bone cells with diamond-like carbon polymer hybrid coatings.  

PubMed

Diamond-like carbon (DLC) coatings produced using the plasma-accelerating filtered pulsed arc discharge (FPAD) method display excellent adherence to the substrate and improve its corrosion resistance. This article reports the interactions of human osteoblastic cells with DLC and two DLC polymer hybrid (DLC-p-h) coatings deposited on smooth, matt and rough silicon wafers by the FPAD method. The DLC-p-h materials were DLC-polytetrafluoroethylene hybrid (DLC-PTFE-h) and DLC-polydimethylsiloxane hybrid (DLC-PDMS-h) coatings. The biocompatibility of the coatings was assayed by using mesenchymal stem cells, primary osteoblasts and Saos-2 cells. Human mesenchymal stem cells proliferated when cultured on DLC and DLC-PTFE-h, but their numbers diminished on DLC-PDMS-h. In all three cell types studied, phalloidin-TRITC staining disclosed cell-type organization typical of an actin cytoskeleton on DLC and DLC-PTFE-h, but minimal and disorganized stress fibers on cells cultured on DLC-PDMS-h. The microtubular cytoskeleton was similarly disorganized on DLC-PDMS-h. Cells on DLC-PDMS-h developed a peculiar form of membrane damage, with nuclear staining by propidium iodide associated with granular calcein staining of the cytoplasm. Active caspase-3 labeling was only seen in cells cultured on DLC-PDMS-h, indicating that these cells undergo apoptosis induced by defective cell adhesion. Results suggest that DLC-PDMS-h coatings might be useful in orthopedic applications where an implant or implant-facet should be protected against bone overgrowth while DLC and DLC-PTFE-h coatings might improve osseointegration. PMID:20197124

Calzado-Martín, Alicia; Saldańa, Laura; Korhonen, Hannu; Soininen, Antti; Kinnari, Teemu J; Gómez-Barrena, Enrique; Tiainen, Veli-Matti; Lappalainen, Reijo; Munuera, Luis; Konttinen, Yrjö T; Vilaboa, Nuria

2010-08-01

311

BNIP3 upregulation by ERK and JNK mediates cadmium-induced necrosis in neuronal cells.  

PubMed

Cadmium (Cd) is a toxic heavy metal that may cause neurological disorders. We studied the mechanism underlying Cd-mediated cell death in neuronal cells. In Cd-induced neurotoxicity, caspase-3 was only modestly activated, and accordingly, zVAD-fmk, a pan-caspase inhibitor, partially attenuated cell death. However, pretreatment with Necrox-2 or Necrox-5, two novel necrosis inhibitors, suppressed cell death more markedly compared with pretreatment with zVAD-fmk. Moreover, the necrosis inhibitors did not prevent cleavage of caspase-3. These results indicate that caspase-independent necrosis is more prevalent in Cd-induced neurotoxicity. Bcl-2 and adenovirus E1B-19 kDa-interacting protein 3 (BNIP3) has been reported to be related to caspase-independent cell death. Cd treatment caused a dramatic upregulation of BNIP3 mRNA and protein levels in vitro and in vivo. Furthermore, knockdown of BNIP3 greatly inhibited Cd-induced cell death. Importantly, BNIP3 RNAi decreased lactate dehydrogenase release and the percentage of propidium iodide-positive cells, two markers of necrotic cell death due to rupture of the cell membrane, whereas it had no effect on activation of caspase-3 in Cd-treated cells. These data suggest that BNIP3 mediates caspase-independent necrosis, but not apoptosis. Moreover, our results indicate that induction of BNIP3 by Cd may not be related to HIF-1 which is generally regarded as a mediator responsible for BNIP3 expression. Finally, we show that mitogen-activated protein kinases (MAPKs) are activated by Cd in vitro and in vivo; ERK and JNK promote BNIP3 upregulation and subsequent necrosis. Taken together, our results suggest BNIP3, upregulated by activation of ERK and JNK, mediates Cd-induced necrosis in neuronal cells. PMID:24824807

Wang, Bin; Xiao, Jia-Li; Ling, Yi-Hui; Meng, Xiao-Jing; Wu, Bing; Yang, Xin-Yi; Zou, Fei

2014-08-01

312

Modulation of doxorubicin cytotoxicity by resveratrol in a human breast cancer cell line  

PubMed Central

Background Breast cancer is the most common cancer in the Arab world and it ranked first among Saudi females. Doxorubicin (DOX), an anthracycline antibiotic is one of the most effective anticancer agents used to treat breast cancer. chronic cardiotoxicity is a major limiting factor of the use of doxorubicin. Therefore, our study was designed to assess the role of a natural product resveratrol (RSVL) on sensitization of human breast cancer cells (MCF-7) to the action of DOX in an attempt to minimize doxorubicin effective dose and thereby its side effects. Methods Human breast cancer cell line MCF-7, was used in this study. Cytotoxic activity of DOX was determined using (sulforhodamine) SRB method. Apoptotic cells were quantified after treatment by annexin V-FITC- propidium iodide (PI) double staining using flow-cytometer. Cell cycle disturbance and doxorubicin uptake were determined after RSVL or DOX treatment. Results Treatment of MCF-7 cells with 15 ?g/ml RSVL either simultaneously or 24 h before DOX increased the cytotoxicity of DOX, with IC50 were 0.056 and 0.035 ?g/ml, respectively compared to DOX alone IC50 (0.417 ?g/ml). Moreover, flow cytometric analysis of the MCF-7 cells treated simultaneously with DOX (0.5 ?g/ml) and RSVL showed enhanced arrest of the cells in G0 (80%). On the other hand, when RSVL is given 24 h before DOX although there was more increased in the cytotoxic effect of DOX against the growth of the cells, however, there was decreased in percentage arrest of cells in G0, less inhibition of DOX-induced apoptosis and reduced DOX cellular uptake into the cells. Conclusion RSVL treatment increased the cytotoxic activity of DOX against the growth of human breast cancer cells when given either simultaneously or 24 h before DOX. PMID:23153194

2012-01-01

313

The antiproliferative effects of ouabain and everolimus on adrenocortical tumor cells.  

PubMed

Ouabain is a cardiotonic steroid obtained from Strophanthus. Recently its role as antiproliferative agent has been investigated in tumor cells. Everolimus is a derivative of rapamycin and acts as a signal transduction inhibitor. Adrenocortical carcinoma is a rare cancer, with poor prognosis. This research focuses on antineoplastic properties of ouabain and its association with everolimus. We analyzed the effects of drugs on cells by MTT assay, by [(3)H] thymidine assay, by Wright's staining, by homogeneous caspases assay, by flow cytometry analysis and by Western blot analysis on H295R and SW13 cells and on primary adrenocortical tumor cells. Ouabain induced cell viability reduction in SW13, H295R and 5 primary adrenocortical tumor cells. Combination of ouabain with everolimus produced a stronger cytotoxic effect on cell proliferation and viability. Marked morphological changes were observed in both SW13 and H295R cell lines after ouabain treatment, with an increase in necrosis. Cell cycle distribution was altered by ouabain in SW13. Analysis of apoptosis demonstrated an increase in caspase activity, clearly evident for SW13 at 72h. FACS analysis by Annexin V-FITC kit and propidium iodide confirmed an increased level of necrosis at higher concentrations. Western blot analysis showed that PI3k/Akt signaling pathway was modified after ouabain treatments in SW13. Ouabain exerts antiproliferative effects on SW13 and H295R cell lines and on primary adrenocortical tumor cells. These data suggest that ouabain or ouabain derivatives may be potential anticancer agents. PMID:24153038

Pezzani, Raffaele; Rubin, Beatrice; Redaelli, Marco; Radu, Claudia; Barollo, Susi; Cicala, Maria Verena; Salvŕ, Monica; Mian, Caterina; Mucignat-Caretta, Carla; Simioni, Paolo; Iacobone, Maurizio; Mantero, Franco

2014-01-01

314

Rumex L. species induce apoptosis in 1301, EOL-1 and H-9 cell lines.  

PubMed

The Rumex L. (dock) species for many centuries have been used in medical treatment, through their adstringent, spasmolitic or cholagogic action. In the present study, the in vitro screening of cytotoxic activities of ethanol extract from roots, leaves and fruits of six Rumex species: R. acetosa L., R. acetosella L., R. confertus Willd., R. crispus L., R. hydrolapathum Huds. and R. obtusifolius L. were performed. We found remarkable cytotoxic activities on leukemic 1301 and EOL-1 cell lines and T cell line at concentration dependent manners. Cytotoxic activity was determined in two ways: trypan blue test and annexin-V FITC and propidium iodide assay. Received IC50 values of investigated extracts on 1301, EOL-1 and H-9 cell lines ranged from 0.22, 0.17 and 0.04 to 2.56, 1.91 and 1.83 mg/mL, respectively. Analysis of morphological changes demonstrated that the extract exerted cell-death via apoptosis. The overall activities of Rumex species support the traditional use of the extract from the fruits of R. confertus, R. crispus, R. hydrolapathum and R. obtusifolius in the treatment of cancer. PMID:22594263

Wegiera, Magdalena; Smolarz, Helena D; Bogucka-Kocka, Anna

2012-01-01

315

Endosulfan decreases cytotoxic activity of nonspecific cytotoxic cells and expression of granzyme gene in Oreochromis niloticus.  

PubMed

The effect of the organochlorinated insecticide endosulfan, on the cytotoxic activity of Nile tilapia nonspecific cytotoxic cells (NCC) was assessed. Juvenile Nile tilapia were exposed to endosulfan (7 ppb) for 96 h and splenic NCC were isolated. Flow cytometric phenotyping of NCC was based on the detection of the NCC specific membrane signaling protein NCCRP-1 by using the monoclonal antibody Mab 5C6; granzyme expression was evaluated by quantitative RT-PCR. The cytotoxic activity of sorted NCC on HL-60 tumoral cells was assessed using propidium iodide (PI) staining of DNA in HL-60 nuclei, indicating dead cells. Nile tilapia splenic NCC had the ability to kill HL-60 tumoral cells, however, the exposure to endosulfan significantly reduced, by a 65%, their cytotoxic activity when using the effector:target ratio of 40:1. Additionally, the exposure to endosulfan tended to increase the expression of NCCRP-1, which is involved in NCC antigen recognition and signaling. Moreover, it decreased the expression of the granzyme gene in exposed group as compared with non-exposed group; however significant differences between groups were not detected. In summary, the acute exposure of Nile tilapia to sublethal concentration of endosulfan induces alteration in function of NCC: significant decrease of cytotoxic activity and a tendency to lower granzyme expression, severe enough to compromise the immunity of this species. PMID:24657320

Téllez-Bańuelos, Martha Cecilia; Ortiz-Lazareno, Pablo Cesar; Jave-Suárez, Luis Felipe; Siordia-Sánchez, Victor Hugo; Bravo-Cuellar, Alejandro; Santerre, Anne; Zaitseva, Galina P

2014-05-01

316

Botulinum protease-cleaved SNARE fragments induce cytotoxicity in neuroblastoma cells  

PubMed Central

Soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) are crucial for exocytosis, trafficking, and neurite outgrowth, where vesicular SNAREs are directed toward their partner target SNAREs: synaptosomal-associated protein of 25 kDa and syntaxin. SNARE proteins are normally membrane bound, but can be cleaved and released by botulinum neurotoxins. We found that botulinum proteases types C and D can easily be transduced into endocrine cells using DNA-transfection reagents. Following administration of the C and D proteases into normally refractory Neuro2A neuroblastoma cells, the SNARE proteins were cleaved with high efficiency within hours. Remarkably, botulinum protease exposures led to cytotoxicity evidenced by spectrophotometric assays and propidium iodide penetration into the nuclei. Direct delivery of SNARE fragments into the neuroblastoma cells reduced viability similar to botulinum proteases' application. We observed synergistic cytotoxic effects of the botulinum proteases, which may be explained by the release and interaction of soluble SNARE fragments. We show for the first time that previously observed cytotoxicity of botulinum neurotoxins/C in neurons could be achieved in cells of neuroendocrine origin with implications for medical uses of botulinum preparations. PMID:24372287

Arsenault, Jason; Cuijpers, Sabine A G; Ferrari, Enrico; Niranjan, Dhevahi; Rust, Aleksander; Leese, Charlotte; O'Brien, John A; Binz, Thomas; Davletov, Bazbek

2014-01-01

317

Synthesis of solasodine glycoside derivatives and evaluation of their cytotoxic effects on human cancer cells.  

PubMed

Solasodine glycosides, such as solamargine, have been proved to be very important anti-cancer agents. In order to discover more potent cytotoxic agents and explore the preliminary structure activity relationship, a new series of solasodine glycosides 2-9 were synthesized via a transglycosylation strategy, and their cytotoxic activity against a panel of human cancer cell lines (MCF-7, KB, K562, and PC3 cells) were evaluated by MTT assays. The results indicated that compounds 2, 8, and 9 with the substitute moiety of rhamnose, 2-hydroxyethoxymethyl, and 1,3-dihydroxypropan-2-yloxy-methyl, respectively, exhibited quite strong anticancer activity. The underlying mechanism tests demonstrated that these compounds could induce apoptosis detected by DAPI staining, and Annexin V and propidium iodide binding. Cell cycle analysis indicated that the cancer cells were predominantly arrested at the G2/M phase when exposure to these compounds was examined by flow cytometry. These compounds may serve as lead candidates in the development of novel chemotherapeutics for cancer treatment. PMID:22460423

Cui, C Z; Wen, X S; Cui, M; Gao, J; Sun, B; Lou, H X

2012-02-01

318

WP 631 and Epo B synergize in SKOV-3 human ovarian cancer cells.  

PubMed

Combined therapy is one of the basic methods of treatment different types of cancer. It allows to reduce the side effects of each component while maximizing the therapeutic action. The aim of this study was to evaluate the impact of two new drugs: WP 631 (bisanthracycline) and epothilone B (Epo B), added in combination on the SKOV-3 human ovarian cancer cells. To assess the type of interaction between WP 631 and Epo B isobolografic analysis was applied based on the cytotoxicity of drugs determined by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolinum bromide) assay. Apoptotic and necrotic cell levels were measured by double staining with Hoechst 33258 and propidium iodide, Annexin V-FITC staining and by using TUNEL assay. The combination of WP 631 and Epo B is more potent than drugs added alone. The quantitative analysis indicated that the major mode of cell death induced by the combination after 72 h treatment was early apoptosis, whereas drugs administered alone generated less intensive apoptosis. The present report demonstrates for the first time that WP 631 and Epo B co-administered synergize in SKOV-3 cell line (Z(ex)/Z(th)<1). PMID:24374386

Marczak, Agnieszka; Bukowska, Barbara; Rogalska, Aneta

2014-01-01

319

Detection of live Escherichia coli O157:H7 cells by PMA-qPCR.  

PubMed

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source. PMID:24513664

Li, Baoguang; Hu, Zonglin; Elkins, Christopher A

2014-01-01

320

Validation of SYTO 9\\/Propidium Iodide Uptake for Rapid Detection of Viable but Noncultivable Legionella pneumophila  

Microsoft Academic Search

Legionella pneumophila is an ubiquitous environmental microorganism that can cause Legionnaires’ disease or Pontiac fever. As a waterborne pathogen,\\u000a it has been found to be resistant to chlorine disinfection and survive in drinking water systems, leading to potential outbreaks\\u000a of waterborne disease. In this work, the effect of different concentrations of free chlorine was studied (0.2, 0.7, and 1.2 mg\\u000a l?1),

M. S. Giăo; S. A. Wilks; N. F. Azevedo; M. J. Vieira; C. W. Keevil

2009-01-01

321

Propidium Monoazide-based Method for Identifying Phylogenetic Association of Necromass Near Hydrothermal Systems  

NASA Astrophysics Data System (ADS)

Black Smoker hydrothermal systems are geologically driven systems located near subduction zones and spreading centers associated with plate margins. The high temperature and low pH of fluids that are often associated with basalt-hosted hydrothermal systems select for unique microbial communities primarily comprised of prokaryotes capable of S and Fe cycling. High temperature fluids, where temperatures exceed 300° C, are likely to have a lethal effect on transient deep water planktonic communities and, over long temporal scales, may influence the molecular composition of pelleted necromass aggregates near the chimney system. We have developed a method for discriminative sequencing permitting intra vs. extracellular 16S rDNA sequencing to reveal community differences between biologically-relevant and necromass-associated DNA. This method has only recently been applied to marine environments and, here, we propose its use as relevant tool for studying the molecular ecology of high temperature hydrothermal systems, as physical drivers of massive transient community die offs and associated detrital 16S rDNA community shifts. Ultimately, we aim to understand the fraction of 16S rDNA communities that do not represent living taxa, or the information-containing fraction of total necromass pool, to better frame ecological hypotheses regarding environmental biogeochemical cycling in hydrothermal system environments.

Ramírez, Gustavo; Edwards, Katrina

2014-05-01

322

Cryptosporidium Propidium Monoazide-PCR, a Molecular Biology-Based Technique for Genotyping Viable Cryptosporidium Oocysts  

EPA Science Inventory

Cryptosporidium is an important waterborne protozoan parasite that can cause severe diarrhea and death in the immunocompromised. Current methods to monitor for Cryptosporidium oocysts in water are microscopy-based USEPA Methods 1622 and 1623. These methods assess total levels o...

323

Enhancing laser thermal-therapy using ultrasound-microbubbles and gold nanorods of in vitro cells.  

PubMed

Gold nanorods (GNRs) are being exploited for their absorption properties to improve thermal therapy. However, a key challenge is delivering sufficient concentration of GNRs to induce a therapeutic effect. In this study, ultrasound and microbubbles (USMBs) were used to enhance intracellular uptake of GNRs. AML-5 cells in suspension (0.6 mL) were exposed to ultrasound (1.3 and 1.7 MPa peak negative pressure) and definity microbubbles (1.7% v/v) for 1 min at varying GNR concentrations (0-2.5×10(11) per mL). Following ultrasound-microbubble treatment, cells were centrifuged twice and treated with an 810 nm laser at an average fluence rate of 3.6 W/cm(2) for 5 min. In addition, cells were incubated with GNRs for 12 h prior to laser treatment. Following the treatment, cell viability (V(PI)) was assessed using propidium iodide (PI) and flow cytometry. Cell viability decreased by ?4-folds with the combined treatment of USMB+GNR+Laser (V(PI)=17%) compared to cells incubated with GNR+Laser (V(PI)=68%). This effect depended on ultrasound pressure and GNR concentration. Higher cell death was achieved at higher GNR concentration and 1.3 MPa peak negative pressure. Cell viability decreased from 92% to 29% with increasing GNR concentration from 1×10(11) to 1.5×10(11) GNR/mL. In addition, higher temperatures were observed using a thermal camera with the combined treatment (USMB+GNR+Laser) of 59±1°C compared to 54±0.9°C for cells incubated with GNRs. The combined treatment of ultrasound-microbubble and gold nanorod laser induced thermal-therapy improved treatment response of in vitro cells. PMID:23290827

Tarapacki, Christine; Kumaradas, Carl; Karshafian, Raffi

2013-03-01

324

Nickel nanowires induced and reactive oxygen species mediated apoptosis in human pancreatic adenocarcinoma cells  

PubMed Central

Background The ability to evade apoptosis is one of the key properties of cancer. The apoptogenic effect of nickel nanowires (Ni NWs) on cancer cell lines has never been adequately addressed. Due to the unique physicochemical characteristics of Ni NWs, we envision the development of a novel anticancer therapeutics specifically for pancreatic cancer. Thus, we investigated whether Ni NWs induce ROS-mediated apoptosis in human pancreatic adenocarcinoma (Panc-1) cells. Methods In this study Ni NWs were fabricated using the electrodeposition method. Synthesized Ni NWs were physically characterized by energy dispersive X-ray analysis, UV-Vis spectroscopy of NanoDrop 2000 (UV-Vis), magnetization study, scanning electron microscopy, and transmission electron microscopy. Assessment of morphological apoptotic characteristics by phase contrast microscopy (PCM), Ni-NWs-induced apoptosis staining with ethidium bromide (EB) and acridine orange (AO) followed by fluorescence microscopy (FM) was performed. For molecular biological and biochemical characterization, Panc-1 cell culture and cytotoxic effect of Ni NWs were determined by using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Quantitative apoptosis was analyzed by flow cytometry staining with propidium iodide through cell cycle arrest and generation of ROS using 2?, 7?-dichlorofluorescein diacetate fluorescence intensity. In all experiments, Panc-1 cancer cells without any treatment were used as the negative controls. Results The intracellular uptake of Ni NWs through endocytosis by Panc-1 cells was observed by PCM. EB and AO staining of FM and MTT assay qualitatively and quantitatively confirmed the extent of apoptosis. Flow cytometric cell cycle arrest and ROS generation indicated Ni NWs as inducers of apoptotic cell death. Conclusion We investigated the role of Ni NWs as inducers of ROS-mediated apoptosis in Panc-1 cells. These results suggested that Ni NWs could be an effective apoptotic agent for Panc-1 cells and have good potential for further research into a clinical treatment selective for pancreatic cancer. PMID:21845039

Hossain, Md. Zakir; Kleve, Maurice G

2011-01-01

325

Effects of Triterpenoid Glycosides from Fresh Ginseng Berry on SW480 Human Colorectal Cancer Cell Line  

PubMed Central

Purpose The pharmacological activities, notably the anticancer properties, of bioactive constituents fromfresh American ginseng berry have not yet been well studied. In this study, we investigated the antiproliferative effects of fresh American ginseng berry extract (AGBE) and its representative triterpenoid glycosides using the human colorectal cancer cell line SW480. Materials and Methods Using high performance liquid chromatography (HPLC), the contents of 8 ginsenosides in AGBE were determined. The cell growth inhibitory effects of AGBE and three triterpenoid glycosides (ginsenosides Rb3, Re, and Rg3) were evaluated by proliferation assay and 3H-thymidine incorporation assay. Cell cycle and apoptotic effects were analyzed by using flow cytometry after staining with propidium iodide and annexin V. Results HPLC analysis data showed that AGBE has a distinct ginsenoside profile. AGBE inhibited SW480 cell growth significantly in a time-dependent (24-96 hours) and concentration-dependent (0.1-1.0 mg/mL) manner. Ginsenosides Rb3, Re, and Rg3 also possess significant antiproliferative activities on SW480 cells. 3H-thymidine incorporation assay indicated that AGBE and ginsenosides Rb3, Re, and Rg3 might inhibit the transferring and duplication of DNA in SW480 cells. Flow cytometric assay data suggested that AGBE arrested SW480 cells in S and G2/M phases, and significantly induced cell apoptosis. Conclusion AGBE and ginsenosides Rb3, Re, and Rg3 possessed significant antiproliferative effects and induced changes of morphological appearance on SW480 cells. The mechanisms of the antiproliferation of AGBE and tested ginsenosides involved could be cell cycle arrest and induction of apoptosis. PMID:21509163

Xie, Jing-Tian; Du, Guang-Jian; McEntee, Eryn; Aung, Han H.; He, Hui; Mehendale, Sangeeta R.; Wang, Chong-Zhi

2011-01-01

326

Interactions Between Ataxia Telangiectasia Mutated Kinase Inhibition, Poly(ADP-ribose) Polymerase-1 Inhibition and BRCA1 Status in Breast Cancer Cells  

PubMed Central

Background: Cells harboring BRCA1/BRCA2 mutations are hypersensitive to inhibition of poly(ADP-ribose) polymerase-1 (PARP-1). We recently showed that interference with PARP-1 activity by NU1025 is strongly cytotoxic for BRCA1-positive BT-20 cells but not BRCA1-deficient SKBr-3 cells. These unexpected observations prompted speculation that other PARP-1 inhibitor(s) may be more cytotoxic towards SKBr-3 cells. In addition, interference with the DNA damage signaling pathway via (for instance) Ataxia telangiectasia mutated (ATM) kinase inhibition may induce synthetic lethality in DNA repair-deficient breast cancer cells and pharmacological interference with ATM activity may sensitize breast cancer cells to PARP-1 inactivation. Methods: We determined drug cytotoxicity in human MCF-7 and SKBr-3 breast cancer cells using the CellTiterGLO Luminescent cell viability assay and a Tecan multi-label, multitask plate counter to measure generated luminescence. Changes in cell cycle progression were monitored by flow cytometric measurement of DNA content in cells stained with propidium iodide. Results: Unlike NU1025, AZD2461, a new PARP-1 inhibitor, markedly reduced the numbers of living MCF-7 and SKBr-3 cells. ATM kinase inhibition (CP466722) was also cytotoxic for both MCF-7 and SKBr-3 cells. Furthermore, AZD2461 enhanced the cytotoxicity of CP466722 in both cell lines by inducing apoptosis, and concurrent inhibition of ATM and PARP-1 reduced cell proliferation more strongly than either single treatment. Conclusions: Our data show that inhibition of PARP-1 by AZD2461 is synthetically lethal for NU1025-resistant MCF-7 and SKBr-3 breast cancer cells. They also indicate that DNA damage signaling is essential for survival of both SKBr-3 and MCF-7 cells, especially after inactivation of PARP-1. PMID:25337581

Wesierska-Gadek, Jozefa; Heinzl, Sarah

2014-01-01

327

Combination of BCL11A siRNA with vincristine increases the apoptosis of SUDHL6 cells  

PubMed Central

Background B cell chronic lymphocytic leukemia/lymphoma 11 A (BCL11A) is associated with human B cell malignancy initiation. Our previous study has shown that downregulation of BCL11A mRNA by small interfering RNA (siRNA) is capable of inducing apoptosis in the SUDHL6 cell line. To further explore the effects of BCL11A siRNA on the enhanced cytotoxicity of a chemotherapeutic drug, we investigated the effects of BCL11A siRNA combined with vincristine (VCR) on SUDHL6 cell proliferation and apoptosis. Methods Chemically synthesized BCL11A siRNA was transfected into SUDHL6 cells using the HiPerFect Transfection Reagent in combination with VCR. Cell proliferation was measured by the CCK8 assay. The morphology of apoptotic cells was observed with Hoechst 33258 staining. The rate of cell apoptosis was determined by annexin V-fluorescein isothiocyanate/propidium iodide double staining using fluorescence-activated cell sorting (FACS) analysis. Results After BCL11A siRNA plus VCR treatment, cell proliferation was significantly decreased in comparison with VCR or BCL11A siRNA treatment alone and negative control siRNA plus VCR treatment (P <0.05). The apoptotic rate of BCL11A siRNA plus VCR treated cells was significantly increased compared with BCL11A siRNA and VCR treatment alone and negative control siRNA plus VCR treatment (P <0.05). Conclusions The combination of BCL11A siRNA and VCR increases apoptosis in SUDHL6 cells. Our study implies that BCL11A siRNA in combination with VCR may be a useful approach for improving effective treatment for B cell lymphoma. PMID:24961604

2014-01-01

328

Mitochondrial-mediated apoptosis in lymphoma cells by the diterpenoid lactone Andrographolide, the active component of Andrographis paniculata  

PubMed Central

Purpose Andrographolide is a diterpenoid lactone isolated from Andrographis paniculata (King of Bitters), an herbal medicine used in Asia. It has been reported to have anti-inflammatory, antihypertensive, anti-viral and immune-stimulant properties. Furthermore, it has been shown to inhibit cancer cell proliferation and induce apoptosis in leukemia and solid tumor cell lines. Experimental Design We studied the Burkitt p53 mutated Ramos cell line, the mantle-cell lymphoma (MCL) line Granta, the follicular lymphoma (FL) cell line HF-1 and the diffuse large B-cell lymphoma (DLBCL) cell line SUDHL4, as well as primary cells from patients with FL, DLBCL, and MCL. Results We found that andrographolide resulted in dose- and time-dependent cell death as measured by MTT. Andrographolide significantly increased reactive oxygen species (ROS) production in all cell lines. To determine mechanism of cell death, we measured apoptosis by Annexin-V/propidium iodide (PI) in the presence and absence of the antioxidant N-acetyl-L-cysteine (NAC), the glutathione-depleting agent buthionine sulfoxamine (BSO), or caspase inhibitors. We found that apoptosis was greatly enhanced by BSO, blocked by NAC, and accompanied by PARP cleavage and activation of caspases 3, 8 and 9. We measured BAX conformational change, and mitochondrial membrane potential, and using mouse embryonic fibroblast (MEF) Bax/Bak double knockouts (MEFBax?/?/Bak?/?), we found that apoptosis was mediated through mitochondrial pathways, but dependent on caspases in both cell lines and in patient samples. Conclusions Andrographolide caused ROS-dependent apoptosis in lymphoma cell lines and in primary tumor samples, which was enhanced by depletion of GSH and inhibited by NAC or the pan-caspase inhibitor Z-VAD-FMK. Further studies of diterpenoid lactones in lymphoma are warranted. PMID:20798229

Yang, Shuo; Evens, Andrew M.; Prachand, Sheila; Singh, Amareshwar T.K; Bhalla, Savita; David, Kevin; Gordon, Leo I.

2010-01-01

329

Pinosylvin at a high concentration induces AMPK-mediated autophagy for preventing necrosis in bovine aortic endothelial cells.  

PubMed

Pinosylvin is a known functional compound of the Pinus species. Pinosylvin at low concentrations (?pmol/L) was reported to promote cell proliferation in endothelial cells. However, this study found that pinosylvin at a high concentration (100 ?mol/L) induces cell death in bovine aortic endothelial cells. Therefore, we examined how pinosylvin was associated with apoptosis, autophagy, and necrosis. Pinosylvin at a high concentration appeared to promote caspase-3 activation, nuclear condensation, and the "flip-flop" of phosphatidylserine, indicating that pinosylvin induces apoptosis. However, based on flow cytometry data obtained from double-staining with annexin V and propidium iodide, pinosylvin was shown to inhibit necrosis, a postapoptotic process. Pinosylvin induced LC3 conversion from LC3-I to LC3-II and p62 degradation, which are important indicators of autophagy. In addition, AMP-activated protein kinase (AMPK) appeared to be activated by pinosylvin, and an AMPK inhibitor was markedly shown to reduce the LC3 conversion. The inhibitory effect of an AMPK inhibitor was reversed by pinosylvin. These results suggest that pinosylvin induces autophagy via AMPK activation. Further, necrosis was found to be promoted by an autophagy inhibitor and then restored by pinosylvin, while the caspase-3 inhibitor had no effect on necrosis. These findings indicate that pinosylvin-induced autophagy blocks necrotic progress in endothelial cells. PMID:25393712

Park, Jinsun; Pyee, Jaeho; Park, Heonyong

2014-12-01

330

Cadmium induces Ca2+-dependent necrotic cell death through calpain-triggered mitochondrial depolarization and reactive oxygen species-mediated inhibition of nuclear factor-kappaB activity.  

PubMed

This study investigates the mechanism of cell death induced by cadmium (Cd) in Chinese hamster ovary (CHO) cells. Cells exposed to 4 microM Cd for 24 h did not show signs of apoptosis, such as DNA fragmentation and caspase-3 activation. The pro-apoptotic (Bax) or anti-apoptotic (Bcl-2 and Bcl-xL) protein levels in the Bcl-2 family were not altered. However, an increase in propidium iodide uptake and depletion of ATP, characteristics of necrotic cell death, were observed. Cd treatment increased the intracellular calcium (Ca2+) level. Removal of the Ca2+ by a chelator, BAPTA-AM, efficiently inhibited Cd-induced necrosis. The increased Ca2+ subsequently mediated calpain activation and intracellular ROS production. Calpains then triggered mitochondrial depolarization resulting in cell necrosis. Cyclosporin A, an inhibitor of mitochondrial permeability transition, recovered the membrane potential and reduced the necrotic effect. The generated ROS reduced basal NF-kappaB activity and led cells to necrosis. An increase of NF-kappaB activity by its activator, PMA, attenuated Cd-induced necrosis. Calpains and ROS act cooperatively in this process. The calpain inhibitor and the ROS scavenger synergistically inhibited Cd-induced necrosis. Results in this study suggest that Cd stimulates Ca2+-dependent necrosis in CHO cells through two separate pathways. It reduces mitochondrial membrane potential by activating calpain and inhibits NF-kappaB activity by increasing the ROS level. PMID:17323976

Yang, Pei-Ming; Chen, Hung-Chi; Tsai, Jia-Shiuan; Lin, Lih-Yuan

2007-03-01

331

Piscidin-1, an antimicrobial peptide from fish (hybrid striped bass morone saxatilis x M. chrysops), induces apoptotic and necrotic activity in HT1080 cells.  

PubMed

Piscidin-1, a 22-residue cationic peptide isolated from mast cells of a hybrid striped bass, has potent antimicrobial activities against both gram-positive and -negative bacteria. To date, there is no report of its antitumor activity on any tumor cell lines. In this study, we examined the antitumor activity of a synthetic piscidin-1 peptide against several human cancer cell lines using an MTS assay and soft-agar colony-formation assay. We found that a low dose of piscidin induces both apoptosis and necrosis in HT1080 cells, as shown by annexin-V/propidium iodide and acridine orange/ethidium bromide staining, and triggers a necrotic cell death pathway in a short period with high-dose treatment. The destruction of cell membranes by piscidin-1 was demonstrated by transmission electron microscopy. Furthermore, piscidin-1 also inhibits the migration of HT1080 cells in a dose-dependent manner. This study provides the first evidence of the anticancer activity of the antimicrobial peptide, piscidin-1, with potential implications for the treatment of cancer. PMID:22559967

Lin, Heng-Ju; Huang, Tsui-Chin; Muthusamy, Sasikala; Lee, Jheng-Fong; Duann, Yeh-Fang; Lin, Cheng-Hui

2012-05-01

332

A micro-Raman spectroscopic investigation of leukemic U-937 cells treated with Crotalaria agatiflora Schweinf and the isolated compound madurensine.  

PubMed

In South Africa traditional medicine plays an important role in primary health care and therefore it is very important that the medicinal use of plants is scientifically tested for toxicity and effectiveness. It was established that the ethanolic extract of the leaves of Crotalaria agatiflora, as well as the isolated compound madurensine, is moderately toxic against leukemic U-937 cells. Light microscopic investigations indicated that symptoms of cell death are induced during treatments, but flow cytometry analysis of treated cells, using annexin-V and propidium iodide, showed that apoptosis and necrosis are insignificantly induced. The Raman results suggested that protein extraction and DNA melting occur in the cells during treatment with the ethanolic extracts (IC(50) value 73.9 ?g/mL), drastically changing the molecular content of the cells. In contrast, treatment with madurensine (IC(50) value 136.5 ?g/mL), an isolated pyrrolizidine alkaloid from the ethanolic extract of the leaves, did not have the same effect. The results are also compared to that of cells treated with actinomycin D, a compound known to induce apoptosis. The investigation showed that micro-Raman spectroscopy has great promise to be used for initial screening of samples to determine the effects of different treatments on cancerous cell lines together with conventional methods. The results highlight the fact that for many natural products used for medicinal purposes, the therapeutic effect of the crude plant extract tends to be significantly more effective than the particular action of its individual constituents. PMID:22580136

le Roux, Karlien; Prinsloo, Linda C; Hussein, Ahmed A; Lall, Namrita

2012-09-01

333

Heat shock protein 70 antisense oligonucleotide inhibits cell growth and induces apoptosis in human gastric cancer cell line SGC-7901  

PubMed Central

AIM: Heat shock protein (HSP)70 is over-expressed in human gastric cancer and plays an important role in the progression of this cancer. We investigated the effects of antisense HSP70 oligomer on human gastric cancer cell line SGC-7901, and its potential role in gene therapy for this cancer. METHODS: Human gastric cancer cell line SGC-7901 was treated in vitro with various concentrations of antisense HSP70 oligonucleotides at different intervals. Growth inhibition was determined as percentage by trypan blue dye exclusion test. Extracted DNA was electrophoresed on agarose gel, and distribution of cell cycle and kinetics of apoptosis induction were analyzed by propidium iodide DNA incorporation using flow cytometry, which was also used to detect the effects of antisense oligomer pretreatment on the subsequent apoptosis induced by heat shock in SGC-7901 cells. Proteins were extracted for simultaneous measurement of HSP70 expression level by SDS-PAGE Western blotting. RESULTS: The number of viable cells decreased in a dose- and time-dependent manner, and ladder-like patterns of DNA fragments were observed in SGC-7901 cells treated with antisense HSP70 oligomers at a concentration of 10 ?mol/L for 48 h or 8 ?mol/L for 72 h, which were consistent with inter-nucleosomal DNA fragmentation. Flow cytometric analysis showed a dose- and time-dependent increase in apoptotic rate by HSP70 antisense oligomers. This response was accompanied with a decrease in the percentage of cells in the G1 and S phases of the cell cycle, suggesting inhibition of cell proliferation. In addition, flow cytometry also showed that pretreatment of SGC-7901 cells with HSP70 antisense oligomers enhanced the subsequent apoptosis induced by heat shock treatment. Western blotting demonstrated that HSP70 antisense oligomers inhibited HSP70 expression, which preceded apoptosis, and HSP70 was undetectable at the concentration of 10 ?mol/L for 48 h or 8 ?mol/L for 72 h. CONCLUSION: Antisense HSP70 oligomers can abrogate HSP70 expression in SGC-7901 cells, which may in turn induce apoptosis and inhibit cell proliferation, conversely suggesting that HSP70 is required for the proliferation and survival of human gastric cancer cells under normal conditions. PMID:15609400

Zhao, Zhi-Gang; Shen, Wen-Lu

2005-01-01

334

Macromitophagy, neutral lipids synthesis, and peroxisomal fatty acid oxidation protect yeast from "liponecrosis", a previously unknown form of programmed cell death.  

PubMed

We identified a form of cell death called "liponecrosis." It can be elicited by an exposure of the yeast Saccharomyces cerevisiae to exogenous palmitoleic acid (POA). Our data imply that liponecrosis is: (1) a programmed, regulated form of cell death rather than an accidental, unregulated cellular process and (2) an age-related form of cell death. Cells committed to liponecrotic death: (1) do not exhibit features characteristic of apoptotic cell death; (2) do not display plasma membrane rupture, a hallmark of programmed necrotic cell death; (3) akin to cells committed to necrotic cell death, exhibit an increased permeability of the plasma membrane for propidium iodide; (4) do not display excessive cytoplasmic vacuolization, a hallmark of autophagic cell death; (5) akin to cells