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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

2014-11-01

2

Assessment of Sarcocystis neurona sporocyst viability and differentiation between viable and nonviable sporocysts using propidium iodide stain.  

PubMed

Sarcocystis neurona has become recognized as the major causative agent of equine protozoal myeloencephalitis (EPM) in the Americas. At least 3 pathogenic species of Sarcocystis, including S. neurona, can be isolated from opossums. Methods are needed to ascertain whether these isolates are viable and capable of causing infections. In this study, the nuclear stain propidium iodide (PI) was used to differentiate between live (viable) and heat-killed (nonviable) S. neurona sporocysts. PI was excluded by live sporocysts but penetrated compromised sporocyst membrane and stained sporozoite nuclei of dead sporocysts. After live and dead sporocysts were mixed at various ratios, the number of unstained sporocysts detected after the staining procedure correlated significantly (r2 = 0.9978) with the expected numbers of live sporocysts. Sporocyst mixtures were also assayed for in vitro excystation and development in tissue cultures. The correlation between the percentage of plaques formed in tissue cultures and the percentage of expected infectious (live) sporocysts in each mixture was r2 = 0.6712. By analysis of variance, no statistically significant difference was measured between the percentage of viable sporocysts and the percentage of infectious sporocysts (P = 0.3902) in each mixture. In addition, there was evidence of a relation between PI impermeability of sporocysts and animal infectivity. These results suggest that the PI dye-exclusion technique can be a useful tool in identifying viability and potential infectivity of S. neurona sporocysts and in differentiating between viable and nonviable sporocysts. PMID:15357088

Elsheikha, Hany M; Mansfield, Linda S

2004-08-01

3

A 'fragile cell' sub-population revealed during cytometric assessment of Saccharomyces cerevisiae viability in lipid-limited alcoholic fermentation.  

PubMed

Aims:? To show that in anaerobic fermentation with limiting lipid nutrients, cell preparation impacts the viability assessment of yeast cells, and to identify the factors involved. Methods and Results:? Saccharomyces cerevisiae viability was determined using propidium iodide staining and the flow cytometry. Analyses identified intact cells, dead cells and, under certain conditions, the presence of a third subpopulation of apparently damaged cells. This intermediate population could account for up to 40% of the entire cell population. We describe, analyse and discuss the effects of different solutions for cell resuspension on the respective proportion of these three populations, in particular that of the intermediate population. We show that this intermediate cell population forms in the absence of Ca(2+) /Mg(2+) . Conclusions:? Cell preparation significantly impacts population viability assessment by FCM. The intermediate population, revealed under certain conditions, could be renamed as 'fragile cells'. For these cells, Ca(2+) and Mg(2+) reduce cell membrane permeability to PI. Significance and Impact of the Study:? This is the first study that analyses and discusses the factors influencing the formation of an intermediate population when studying viability in yeast alcoholic fermentation. With a wider application in biological research, this study provides important support to the relatively new questioning of propidium iodide staining as a universal cell death indicator. PMID:22909384

Delobel, P; Pradal, M; Blondin, B; Tesniere, C

2012-08-21

4

Propidium iodide quenches the fluorescence of TdT-incorporated FITC-labeled dUTP in apoptotic cells.  

PubMed

Apoptotic cells with frequent DNA strand breaks may be detected by tagging with directly or indirectly labeled nucleotides incorporated by the use of terminal deoxynucleotidyl transferase (TdT). Propidium iodide (PI) is typically added for the simultaneous assessment of DNA content. PI was found to quench the specific in situ FITC-fluorescence of apoptotic cells which were labeled by TdT with FITC-conjugated dUTP, biotin-dUTP followed by streptavidin-FITC, or digoxigenin-dUTP followed by FITC-labeled anti-digoxigenin antibodies as measured by flow cytometry. The effect was concentration-dependent, with 50% quenching occurring at 0.8 microg/ml, 1.5 microg/ml, and 5 microg/ml PI, respectively, at approximately 1 x 10(6) cells/ml. Spectrofluorimetry in solution revealed that 15 microg/ml PI was required to quench 50% of the fluorescence of ss FITC-labeled poly(dU)35. In contrast, the fluorescence of ds FITC-labeled poly(dU)35-poly(dA) was quenched to 50% at 3 microg/ml PI. The maximum of the fluorescence excitation spectrum of PI shifted from 490 nm to 535 nm upon binding to ds DNA as well as ss poly(dU)35, and the fluorescence yield of PI at 610 nm increased, but the binding required 10-fold higher concentrations of poly(dU)35 as compared to ds DNA. The spectroscopic properties of PI are therefore similar whether bound to poly(dU) or to double-stranded DNA, but the binding to poly(dU) is much weaker. The observed quenching in situ therefore cannot be explained by direct binding of PI to the poly(dU) tails synthesized by TdT in situ in apoptotic cells, but may rather be due to radiationless energy transfer from FITC to PI bound to double-stranded DNA close to the nicks where TdT is known to start polymerization. PMID:9845437

Stokke, T; Solberg, K; DeAngelis, P; Steen, H B

1998-12-01

5

2-methoxyjuglone induces apoptosis in HepG2 human hepatocellular carcinoma cells and exhibits in vivo antitumor activity in a H22 mouse hepatocellular carcinoma model.  

PubMed

In order to discover anticancer agents from natural sources, an ethanol-soluble extract of the root bark of Juglans cathayensis was investigated and showed cytotoxic effects against various human cancer cell lines. A subsequent phytochemical study on the EtOAc-soluble fraction determined 2-methoxyjuglone (1) as one of the main active constituents. Compound 1 was shown to be cytotoxic against HepG2 cells. Morphological features of apoptosis were observed in 1-treated HepG2 cells, including cell shrinkage, membrane blebbing, nuclear condensation, and apoptotic body formation. Cell cycle analysis with propidium iodide staining showed that 1 induced cell cycle arrest at the S phase in HepG2 cells. Flow cytometric analysis with annexin V and propidium iodide staining demonstrated that 1 induced HepG2 cell apoptotic events in a dose-dependent manner (0-8 ?g/mL). Western blot analysis of apoptosis-related proteins revealed that 1 induces HepG2 cell apoptosis through mitochondrial cytochrome c-dependent activation of the caspase-9 and caspase-3 cascade pathway (intrinsic pathway). An in vivo experiment using tumor-bearing mice showed that treatment with 1 at 0.5 and 1.0 mg/kg per day decreased the tumor mass by 56% and 67%, respectively. PMID:23597099

Yu, Heng-Yi; Zhang, Xiao-Qiong; Li, Xue-; Zeng, Fan-Bo; Ruan, Han-Li

2013-05-24

6

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

7

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

8

1-(2,6-dihydroxy-4-methoxyphenyl)-2-(4-hydroxyphenyl) ethanone-induced cell cycle arrest in G?/G? in HT-29 cells human colon adenocarcinoma cells.  

PubMed

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; Malek, Sri Nurestri Abd

2014-01-01

9

Exploratory study on the effects of biodegradable nanoparticles with drugs on malignant B cells and on a human/mouse model of Burkitt lymphoma.  

PubMed

The aim of this study was to determine if Rituximab coated Biodegradable Nanoparticles (BNPs) loaded with Chlorambucil and Hydroxychloroquine could induce apoptosis of B-Chronic Lymphocytic Leukemia (B-CLL), MEC-1 and BJAB cells in vitro and evaluate their toxic and therapeutic effects on a Human/Mouse Model of Burkitt Lymphoma at an exploratory, proof of concept scale. We found that Rituximab-Chlorambucil-Hydroxychloroquine BNPs induce a decrease in cell viability of malignant B cells in a dose-dependent manner. The mediated cytotoxicity resulted from apoptosis, and was confirmed by monitoring the B-CLL cells after Annexin V/propidium iodide staining. Additional data revealed that these BNPs were non toxic for healthy animals, and had prolonged survival in this mice model of human lymphoma. PMID:20925646

Marín, Gustavo H; Mansilla, Eduardo; Mezzaroba, Nelly; Zorzet, Sonia; Núñez, Luis; Larsen, Gustavo; Tau, José M; Maceira, Alberto; Spretz, Ruben; Mertz, Carol; Ingrao, Sabrina; Tripodo, Claudio; Tedesco, Francesco; Macor, Paolo

2010-11-01

10

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

11

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

PubMed Central

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

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

2012-01-01

12

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

13

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

PubMed Central

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

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

2013-01-01

14

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

PubMed

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

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

2005-01-01

15

Piperine impairs cell cycle progression and causes reactive oxygen species-dependent apoptosis in rectal cancer cells.  

PubMed

Piperine, an alkaloid phytochemical found in the fruit of black and long pepper plants, is reported to inhibit the growth of cancer cells; however, the mechanism of action in human cancer cells is not clear. In this study we investigated the effect of piperine on the growth of HRT-18 human rectal adenocarcinoma cells. MTT assays showed that piperine inhibited the metabolic activity of HRT-18 cells in a dose- and time-dependent fashion, suggesting a cytostatic and/or cytotoxic effect. Flow cytometric analysis of Oregon Green 488-stained and propidium iodide-stained HRT-18 cells showed that piperine inhibited cell cycle progression. Piperine also caused HRT-18 cells to die by apoptosis, as determined by Annexin-V-FLUOS staining and characteristic changes in cell morphology. Flow cytometric analysis of dihydroethidium- and 2',7'-dichlorofluorescein diacetate-stained HRT-18 cells showed increased production of reactive oxygen species in piperine-treated cells. Furthermore, the antioxidant N-acetylcysteine reduced apoptosis in cultures of piperine-treated HRT-18 cells, indicating that piperine-induced cytotoxicity was mediated at least in part by reactive oxygen species. The cytostatic and cytotoxic effects of piperine on rectal cancer cells suggest that this dietary phytochemical may be useful in cancer treatment. PMID:23063564

Yaffe, Paul B; Doucette, Carolyn D; Walsh, Mark; Hoskin, David W

2013-02-01

16

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

17

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

PubMed

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

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

2015-04-01

18

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

PubMed Central

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

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

2013-01-01

19

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

20

Activin A regulates proliferation, invasion and migration in osteosarcoma cells.  

PubMed

Activin A is a member of the TGF?? superfamily. Previous studies have demonstrated that activin A exhibited pluripotent effects in several tumours. However, the roles of activin A signaling in osteosarcoma pathogenesis have not been previously investigated. Therefore, the present study aimed to investigate the effects of activin A on osteosarcoma cell proliferation, invasion and migration. Firstly, the expression of activin A in osteosarcoma cell lines (MG63, SaOS?2 and U2OS) and a human osteoblastic cell line (hFOB1.19) was detected using reverse transcription quantitative polymerase chain reaction and western blotting. Activin A was upregulated in osteosarcoma cell lines compared with hFOB1.19 cells. To investigate the effects of activin A on osteosarcoma cell proliferation, invasion and migration, MG63 cells were generated in which activin A was either overexpressed or depleted. MTT staining, propidium iodide staining and a Transwell assay were used to analyze the cell cycle, proliferation, invasion and migration of MG63 cells, respectively. The results of the present study revealed that the abilities of proliferation, invasion and migration were suppressed in MG63 cells in which activin A was depleted, while they were enhanced in activin A-overexpressing cells. In conclusion, the results of the present study suggested that activin A may facilitate proliferation, invasion and migration of osteosarcoma cells, and it may therefore be a potential target for the treatment of osteosarcoma. PMID:25634369

Zhu, Jianwei; Liu, Fan; Wu, Quanming; Liu, Xiancheng

2015-06-01

21

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

22

Altered antioxidant system stimulates dielectric barrier discharge plasma-induced cell death for solid tumor cell treatment.  

PubMed

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

Kaushik, Nagendra K; Kaushik, Neha; Park, Daehoon; Choi, Eun H

2014-01-01

23

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

PubMed Central

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

2014-01-01

24

Study the oxidative injury of yeast cells by NADH autofluorescence  

NASA Astrophysics Data System (ADS)

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

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

2007-06-01

25

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

26

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

PubMed Central

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

2014-01-01

27

Effects of cinobufacini injection on cell proliferation and the expression of topoisomerases in human HepG?2 hepatocellular carcinoma cells.  

PubMed

The present study aimed to investigate the effects of cinobufacini injection on the proliferation and expression of topoisomerases in human HepG?2 hepatocarcinoma cells. The cells were divided into a control group and an experimental group, in which 0.105, 0.21, 0.42 mg/l cinobufacini was injected. Cell proliferation was assessed using a 3?(4,5?dimethylthiazol?2?yl) ?2,5?diphenyltetrazolium bromide assay, levels of apoptosis were detected using annexin V/propidium iodide staining and cell cycles were analyzed using flow cytometric analysis. The mRNA and protein expression levels of topoisomerase (TOPO) I and TOPO ? were determined by reverse transcription?quantitative polymerase chain reaction and western blotting, respectively. Cinobufacini injection significantly inhibited the proliferation of the HepG?2 cells (P<0.05), induced apoptosis (P<0.05) in a dose? and time?dependent manner, induced tumor cell arrest at the S phase in a dose?dependent manner, and downregulated the mRNA and protein expression levels of TOPO I and TOPO ? (P<0.05) in a dose?dependent manner. Therefore, cinobufacini was found to inhibit human HepG?2 hepatocellular carcinoma cell proliferation, and downregulation of the expression levels of TOPO I and TOPO ? may contribute to the effect on proliferation observed in the Hep?G2 cells following cinobbufacini injection. PMID:25815590

Liu, Ying; Ban, Li-Ying; Su, Xi; Gao, Shan; Liu, Ji-Wei; Cui, Xiao-Nan

2015-07-01

28

Co?culture with bone marrow stromal cells protects PC12 neuronal cells from tumor necrosis factor???induced apoptosis by inhibiting the tumor necrosis factor receptor/caspase signaling pathway.  

PubMed

Bone marrow stromal cells (BMSCs), derived from the mesoderm, have been applied in the repair and reconstruction of injured tissues. The present study was conducted to explore the effects of BMSCs on cell viability of tumor necrosis factor?? (TNF??)?stimulated PC12 cells. PC12 cells were co?cultured with BMSCs under TNF?? treatment, with normal PC12 cells as controls. Results from an MTT assay indicated that BMSCs significantly increased cell growth and proliferation of TNF???treated PC12 cells (survival rates were 56.71 and 76.86% for the positive control (PC) and co?culture group, respectively). Furthermore, Annexin V/propidium iodide staining and flow cytometric analysis demonstrated that TNF?? increased PC12?cell apoptosis from 3.49 to 40.74% in the negative control and PC group, and the apoptotic rate was significantly reduced upon co?culture with BMSCs to 16.97%. In addition, data from reverse transcription?quantitative polymerase chain reaction and western blot analyses illustrated that TNF???induced upregulation in TNF receptor (TNFR)?1 (TNFR1) and caspase?8 expression in PC12 cells were partially reversed by co?culture with BMSCs. In conclusion, the present study suggested that BMSCs protect PC12 cells against stimulation with TNF??, which is partially mediated through the TNFR/caspase signaling pathway. The results of the present study also suggested a therapeutic use of BMSCs in clinical neurodegenerative diseases. PMID:25738414

Li, Li; Wang, Jing; Tang, Ling; Yu, Xin; Sui, Yi; Zhang, Chaodong

2015-07-01

29

In vitro biocompatibility evaluation of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer in fibroblast cells.  

PubMed

Among the various biomaterials available for tissue engineering and therapeutic applications, microbial polyhydroxyalkanoates offer the most diverse range of thermal and mechanical properties. In this study, the biocompatibility of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB); containing 50 mol % of 4-hydroxybutyrate] copolymer produced by Delftia acidovorans was evaluated. The cytotoxicity, mode of cell death, and genotoxicity of P(3HB-co-4HB) extract against V79 and L929 fibroblast cells were assessed using MTT assay, acridine orange/propidium iodide staining, and alkaline comet assay, respectively. Our results demonstrate that P(3HB-co-4HB) treated on both cell lines were comparable with clinically-used Polyglactin 910, where more than 60% of viable cells were observed following 72-h treatment at 200 mg/mL. Further morphological investigation on the mode of cell death showed an increase in apoptotic cells in a time-dependent manner in both cell lines. On the other hand, P(3HB-co-4HB) at 200 mg/mL showed no genotoxic effects as determined by alkaline comet assay following 72-h treatment. In conclusion, our study indicated that P(3HB-co-4HB) compounds showed good biocompatibility in fibroblast cells suggesting that it has potential to be used for future medical applications. PMID:17120221

Siew, Ee Ling; Rajab, Nor Fadilah; Osman, Annuar Bin; Sudesh, Kumar; Inayat-Hussain, Salmaan Hussain

2007-05-01

30

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

PubMed Central

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

Tanih, Nicoline Fri; Ndip, Roland Ndip

2013-01-01

31

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

PubMed Central

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

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

1993-01-01

32

Small interfering RNA targeting 14-3-3? increases efficacy of chemotherapeutic agents in head and neck cancer cells.  

PubMed

Patients diagnosed in advanced stages of head and neck squamous cell carcinoma often show limited response to chemotherapeutic agents. Recently, we reported the overexpression of 14-3-3? protein in head and neck premalignant and cancer tissues using liquid chromatography-tandem mass spectrometry with isotopic labeling and revealed its significance as a prognostic marker using immunohistochemical analysis. In this study, we determined the potential of 14-3-3? as a therapeutic target for head and neck cancer. Small interfering RNA (siRNA) targeting 14-3-3? was used to downregulate its expression in head and neck cancer cells in culture. Cell cycle analysis showed that head and neck cancer cells transfected with siRNA targeting 14-3-3? showed G(2)-M arrest. These siRNA transfectants also showed increased cell death on treatment with any one of the following chemotherapeutic agents: cisplatin, 5-fluorouracil, paclitaxel, or doxorubicin in comparison with the no transfection controls. Flow cytometric analysis using propidium iodide staining showed increased sub-G(0) fraction in siRNA-transfected cells treated with any of these chemotherapeutic agents, suggesting cell death; in addition, Annexin V staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay revealed increased apoptosis. Taken together, our results strongly showed that downregulation of 14-3-3? expression may serve to improve the sensitivity of head and neck cancer cells to chemotherapeutic agents. PMID:20924126

Matta, Ajay; DeSouza, Leroi V; Ralhan, Ranju; Siu, K W Michael

2010-10-01

33

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

PubMed

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

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

2011-06-01

34

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

PubMed Central

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

35

Effect of Methoxychlor on Ca²? Homeostasis and Apoptosis in HA59T Human Hepatoma Cells.  

PubMed

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

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

2015-02-28

36

The mechanism of NPC-14686-induced [Ca²?]i rises and non-Ca²?-triggered cell death in MG63 human osteosarcoma cells.  

PubMed

NPC-14686 has been shown to have anti-inflammatory effect in previous studies, but the mechanisms are unclear. The effect of NPC-14686 on cytosolic Ca²? concentrations ([Ca²?]i) and viability in MG63 human osteosarcoma cells was explored. The Ca²?-sensitive fluorescent dye fura-2 was applied to measure [Ca²?]i. NPC-14686 at concentrations of 100-500 ?M induced a [Ca²?]i rise in a concentration-dependent manner. The response was reduced by 80% by removing Ca²?. NPC-14686 induced Mn²? influx leading to quenching of fura-2 fluorescence. NPC-14686-evoked Ca²? entry was suppressed by nifedipine, econazole, SK&F96365, and protein kinase C inhibitor. Inhibition of phospholipase C with U73122 abolished NPC-14686-induced [Ca²?]i rise. At 20-50 ?M, NPC-14686 decreased cell viability, which was not reversed by chelating cytosolic Ca²? with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/propidium iodide staining data suggest that NPC-14686 (30-50 ?M) induced apoptosis in a concentration-dependent manner. NPC-14686 also increased levels of reactive oxygen species. Together, in human osteosarcoma cells, NPC-14686 induced a [Ca²?]i rise by inducing phospholipase C-dependent Ca²? release from the endoplasmic reticulum and Ca²? entry via protein kinase C-sensitive store-operated Ca²? channels. NPC-14686 induced cell death that might involve apoptosis via mitochondrial pathways. PMID:24826784

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

2014-06-30

37

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

PubMed

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

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

2012-07-01

38

Intracellular pH distribution as a cell health indicator in Saccharomyces cerevisiae  

PubMed Central

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

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

2011-01-01

39

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

NASA Astrophysics Data System (ADS)

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

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

2011-05-01

40

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

41

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

PubMed

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

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

2015-07-01

42

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

PubMed

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

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

2015-02-01

43

DNA alteration and programmed cell death during ageing of sunflower seed.  

PubMed

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, Françoise; Bailly, Christophe

2011-10-01

44

Extract from Rhus verniciflua Stokes is capable of inhibiting the growth of human lymphoma cells.  

PubMed

Rhus verniciflua Stokes (RVS), used as a food additive and a traditional herbal medicine, has both antioxidant and antitumor activities which are known to be closely associated with the polyphenolic compounds that it contains. In the present study, we purified a fraction from a crude acetone extract of RVS, named RCMF (RVS chloroform-methanol fraction), and evaluated its ability to scavenge free radicals and inhibit cell growth. In addition, the active compounds responsible for the activities were identified. Results showed that RCMF contained an antioxidant potential and strongly suppressed the proliferative capability of B lymphoma cells. RCMF-mediated suppression of cell growth was verified to be apoptotic, based on the increased DNA fragmentation and low fluorescence intensity in the nuclei after propidium iodide staining, and also on the appearance of DNA laddering. Finally, EI-MS, 1H-NMR, and 13C-NMR spectra confirmed that RCMF contained flavonoid derivatives, including protocatechuic acid, fustin, fisetin, sulfuretin, and butein, suggesting that these flavonoid derivatives are the main active compounds responsible for the antioxidant and antiproliferative activities of RCMF. PMID:15234068

Lee, J-C; Lee, K-Y; Kim, J; Na, C-S; Jung, N-C; Chung, G-H; Jang, Y-S

2004-09-01

45

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

46

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

47

Cytotoxic activity of octahydropyrazin[2,1-a:5,4-a']diisoquinoline derivatives in human breast cancer cells.  

PubMed

Evaluation of the cytotoxicity of novel octahydropyrazin[2,1-a:5,4-a']diisoquinoline derivatives (1a-2c) employing a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and inhibition of [(3)H]thymidine incorporation into DNA demonstrated that these compounds were more active than etoposide and camptothecin in both MDA-MB-231 and MCF-7 human breast cancer cells. Flow cytometric analysis after Annexin V-FITC and propidium iodide staining also confirmed that apoptosis was the main response of human breast cancer cells to 1a-2c treatment. Our results suggest that apoptosis of human breast cancer cells in the presence of 1a-2c follows the mitochondrial pathway, with the decrease in mitochondrial membrane potential and activation of caspase 9, as well as by the external pathway with the significant increase in caspase 8 expression. Cytotoxic properties of compounds 1a-2c in cultured human breast cancer cells correlate to their ability to inhibit topoisomerase I/II. PMID:25060945

Lepiarczyk, Monika; Ka?u?a, Zbigniew; Bielawska, Anna; Czarnomysy, Robert; Gornowicz, Agnieszka; Bielawski, Krzysztof

2014-07-25

48

Icariin Prevents Amyloid Beta-Induced Apoptosis via the PI3K/Akt Pathway in PC-12 Cells  

PubMed Central

Icariin is a prenylated flavonol glycoside derived from the Chinese herb Epimedium sagittatum that exerts a variety of pharmacological activities and shows promise in the treatment and prevention of Alzheimer's disease. In this study, we investigated the neuroprotective effects of icariin against amyloid beta protein fragment 25–35 (A?25–35) induced neurotoxicity in cultured rat pheochromocytoma PC12 cells and explored potential underlying mechanisms. Our results showed that icariin dose-dependently increased cell viability and decreased A?25–35-induced apoptosis, as assessed by MTT assay and Annexin V/propidium iodide staining, respectively. Results of western blot analysis revealed that the selective phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 suppressed icariin-induced Akt phosphorylation, suggesting that the protective effects of icariin are associated with activation of the PI3K/Akt signaling pathway. LY294002 also blocked the icariin-induced downregulation of proapoptotic factors Bax and caspase-3 and upregulation of antiapoptotic factor Bcl-2 in A?25–35-treated PC12 cells. These findings provide further evidence for the clinical efficacy of icariin in the treatment of Alzheimer's disease. PMID:25705234

Zhang, Dongdong; Wang, Zhe; Sheng, Chenxia; Peng, Weijun; Hui, Shan; Gong, Wei; Chen, Shuai

2015-01-01

49

Icariin Prevents Amyloid Beta-Induced Apoptosis via the PI3K/Akt Pathway in PC-12 Cells.  

PubMed

Icariin is a prenylated flavonol glycoside derived from the Chinese herb Epimedium sagittatum that exerts a variety of pharmacological activities and shows promise in the treatment and prevention of Alzheimer's disease. In this study, we investigated the neuroprotective effects of icariin against amyloid beta protein fragment 25-35 (A? 25-35) induced neurotoxicity in cultured rat pheochromocytoma PC12 cells and explored potential underlying mechanisms. Our results showed that icariin dose-dependently increased cell viability and decreased A? 25-35-induced apoptosis, as assessed by MTT assay and Annexin V/propidium iodide staining, respectively. Results of western blot analysis revealed that the selective phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 suppressed icariin-induced Akt phosphorylation, suggesting that the protective effects of icariin are associated with activation of the PI3K/Akt signaling pathway. LY294002 also blocked the icariin-induced downregulation of proapoptotic factors Bax and caspase-3 and upregulation of antiapoptotic factor Bcl-2 in A? 25-35-treated PC12 cells. These findings provide further evidence for the clinical efficacy of icariin in the treatment of Alzheimer's disease. PMID:25705234

Zhang, Dongdong; Wang, Zhe; Sheng, Chenxia; Peng, Weijun; Hui, Shan; Gong, Wei; Chen, Shuai

2015-01-01

50

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

51

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. PMID:25202402

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

2014-01-01

52

Rabeprazole exhibits antiproliferative effects on human gastric cancer cell lines.  

PubMed

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. PMID:25202402

Gu, Mengli; Zhang, Yan; Zhou, Xinxin; Ma, Han; Yao, Hangping; Ji, Feng

2014-10-01

53

Heteronemin, a spongean sesterterpene, inhibits TNF alpha-induced NF-kappa B activation through proteasome inhibition and induces apoptotic cell death.  

PubMed

In this study, we investigated the biological effects of heteronemin, a marine sesterterpene isolated from the sponge Hyrtios sp. on chronic myelogenous leukemia cells. To gain further insight into the molecular mechanisms triggered by this compound, we initially performed DNA microarray profiling and determined which genes respond to heteronemin stimulation in TNFalpha-treated cells and which genes display an interaction effect between heteronemin and TNFalpha. Within the differentially regulated genes, we found that heteronemin was affecting cellular processes including cell cycle, apoptosis, mitogen-activated protein kinases (MAPKs) pathway and the nuclear factor kappaB (NF-kappaB) signaling cascade. We confirmed in silico experiments regarding NF-kappaB inhibition by reporter gene analysis, electrophoretic mobility shift analysis and I-kappaB degradation. In order to assess the underlying molecular mechanisms, we determined that heteronemin inhibits both trypsin and chymotrypsin-like proteasome activity at an IC(50) of 0.4 microM. Concomitant to the inhibition of the NF-kappaB pathway, we also observed a reduction in cellular viability. Heteronemin induces apoptosis as shown by annexin V-FITC/propidium iodide-staining, nuclear morphology analysis, pro-caspase-3, -8 and -9 and poly(ADP-ribose) polymerase (PARP) cleavage as well as truncation of Bid. Altogether, results show that this compound has potential as anti-inflammatory and anti-cancer agent. PMID:19814997

Schumacher, Marc; Cerella, Claudia; Eifes, Serge; Chateauvieux, Sébastien; Morceau, Franck; Jaspars, Marcel; Dicato, Mario; Diederich, Marc

2010-02-15

54

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

55

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

PubMed Central

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

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

2015-01-01

56

Flow microfluorometric system for screening gynecologic cytology specimens using propidium iodide-fluorescein isothiocyanate.  

PubMed

Seventy cervical cytology specimens have been screened by a xero resolution flow analyzer-sorter using propidium iodide and fluorescein isothiocyanate as fluorochromes for nucleus and cytoplasm, respectively. This system shows a 1% sensitivity for detection of abnormal cells using only crude visual data analysis. Screening of clinical specimens was performed on the instrument with a 5.8% false negative rate and a 11.8% false positive rate by comparison with routine visual cytologic evaluation of the same samples. PMID:1254927

Fowlkes, B J; Herman, C J; Cassidy, M

1976-01-01

57

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

PubMed Central

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

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

2014-01-01

58

Isoflavone content and estrogenic activity of different batches of red clover (Trifolium pratense L.) extracts: an in vitro study in MCF-7 cells.  

PubMed

The estrogenicity of different batches of red clover (Trifolium pratense L., Fabaceae; RCL) extracts and its relationship with the isoflavone content were assessed by measuring MCF-7 cell proliferation by flow cytometry and propidium iodide staining. RCL extracts were compared to estradiol (E2) and to the main RCL isoflavones biochanin A, daidzein, genistein and formononetin. Isoflavone content in the extracts was assayed by HPLC. E2 and isoflavones increased MCF-7 proliferation in a concentration-dependent fashion, with the following potency order: E2>genistein>biochanin A=daidzein>formononetin. Extracts increased MCF-7 proliferation with different potencies, which in four out of five extracts correlated with the ratios 5,7-dihydroxyisoflavones/7-hydroxyisoflavones. The efficacy of all extracts increased with decreasing genistein contents. A solution containing the main isoflavones at the average concentration of RCL extracts increased MCF-7 proliferation with higher potency and steeper concentration-response curve. The effects of E2, of RCL extracts and of the isoflavone solution were inhibited by the estrogen receptor antagonist 4-hydroxytamoxifen. Flow cytometric analysis of MCF-7 proliferation is a suitable bioassay for the estrogenicity of RCL extracts, thus expanding the characterization of individual batches beyond assessment of chemical composition and contributing to improved standardization of quality and activity. PMID:24508860

Spagnuolo, Paola; Rasini, Emanuela; Luini, Alessandra; Legnaro, Massimiliano; Luzzani, Marcello; Casareto, Enrico; Carreri, Massimiliano; Paracchini, Silvano; Marino, Franca; Cosentino, Marco

2014-04-01

59

Characterization of immunosuppressive surface coat proteins from Steinernema glaseri that selectively kill blood cells in susceptible hosts.  

PubMed

Surface coat proteins (SCPs) of entomopathogenic nematodes are implicated in the suppression/evasion of host immune responses, which is required for successful host colonization. Steinernema glaseri NC strain SCPs suppressed immune responses in oriental beetle larvae (Exomala orientalis), a susceptible host for S. glaseri, in a dosage-dependent manner, thus protecting Heterorhabditis bacteriophora from being killed in the same host. Melanization of H. bacteriophora decreased from 92+/-5% in the untreated check to 1+/-3% when protected by injection of 230ng of S. glaseri SCPs. As the SCPs dosage increased, freely moving H. bacteriophora increased from 3+/-4% in the untreated group to 57+/-15% with an SCPs dose of 940ng. At 2h and in the absence of SCPs, 8% and 11% of hemocytes of E. orientalis were stained by propidium iodide and Hoechst, respectively. When exposed to 300ng/microl SCPs, 70% and 96% were stained, respectively. At 6h, propidium iodide stained 37% and 92% of the hemocytes without and with SCPs, respectively. In contrast, more than 90% of the cells were stained by Hoechst with or without SCPs. As native proteins, two isolated S. glaseri SCPs had an immunosuppressive effect; they were each composed of 38kDa (PI=4.6) and 56kDa (PI=3.6) subunits. SCP peptides were sequenced using LC-MS/MS and the mass fingerprints obtained with MALDI-TOF-MS; there were no significant matches found in peptide databases, which suggests that the SCPs studied are novel proteins. Twelve cDNA sequences were derived based on short peptides and 7 of them had no significant match against the Caenorhabditis elegans protein database. One of the cDNA matched an unknown C. elegans protein and the remaining 4 cDNAs matched proteins of C. elegans and Brugia malayi. PMID:19428663

Li, Xinyi; Cowles, E A; Cowles, R S; Gaugler, R; Cox-Foster, D L

2009-06-01

60

Dihydroorotate dehydrogenase inhibitor A771726 (leflunomide) induces apoptosis and diminishes proliferation of multiple myeloma cells.  

PubMed

Multiple myeloma is still an incurable disease; therefore, new therapeutics are urgently needed. A771726 is the active metabolite of the immunosuppressive drug leflunomide, which is currently applied in the treatment of rheumatoid arthritis, BK virus nephropathy, and cytomegaly viremia. Here, we show that dihydroorotate dehydrogenase (DHODH) is commonly expressed in multiple myeloma cell lines and primary multiple myeloma cells. The DHODH inhibitor A771726 inhibits cell growth in common myeloma cell lines at clinically achievable concentrations in a time- and dose-dependent manner. Annexin V-FITC/propidium iodide staining revealed induction of apoptosis of multiple myeloma cell lines and primary multiple myeloma cells. The 5-bromo-2'-deoxyuridine cell proliferation assay showed that inhibition of cell growth was partly due to inhibition of multiple myeloma cell proliferation. A771726 induced G(1) cell cycle arrest via modulation of cyclin D2 and pRb expression. A771726 decreased phosphorylation of protein kinase B (Akt), p70S6K, and eukaryotic translation initiation factor 4E-binding protein-1 as shown by Western blotting experiments. Furthermore, we show that the stimulatory effect of conditioned medium of HS-5 bone marrow stromal cells on multiple myeloma cell growth is completely abrogated by A771726. In addition, synergism studies revealed synergistic and additive activity of A771726 together with the genotoxic agents melphalan, treosulfan, and doxorubicin as well as with dexamethasone and bortezomib. Taken together, we show that inhibition of DHODH by A771726/leflunomide is effective in multiple myeloma. Considering the favorable toxicity profile and the great clinical experience with leflunomide in rheumatoid arthritis, this drug represents a potential new candidate for targeted therapy in multiple myeloma. PMID:19174558

Baumann, Philipp; Mandl-Weber, Sonja; Völkl, Andreas; Adam, Christian; Bumeder, Irmgard; Oduncu, Fuat; Schmidmaier, Ralf

2009-02-01

61

H9 Induces Apoptosis via the Intrinsic Pathway in Non-Small-Cell Lung Cancer A549 Cells.  

PubMed

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

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

2015-03-28

62

Immunofluorescence analysis of cytokeratin 8/18 staining is a sensitive assay for the detection of cell apoptosis  

PubMed Central

Apoptosis is one of the major types of programmed cell death. During this process, cells experience a series of morphological and biochemical changes. Flow cytometric analysis of Annexin V staining of cell surface phosphatidylserine, in combination with a DNA-staining dye to probe the permeability of the cell membrane, is an established method for detecting apoptosis. The present study aimed to show that the immunofluorescence analysis of cytokeratin (CK) 8/18 staining may provide a new and sensitive assay for the detection of apoptotic cells. Tumor cells were treated with 20 ?M cisplatin to induce apoptosis. Following 12 and 24 h of cisplatin treatment, cells were collected and stained with 4?,6-diamidine-2?-phenylindole dihydrochloride (DAPI) and fluorescein-labeled anti-CK8/18 antibody. The apoptotic cells were subsequently examined by fluorescence microscopy. Annexin V-fluorescein isothiocyanate/propidium iodide staining followed by flow cytometric analysis confirmed that cisplatin was able to induce apoptosis in tumor cells. Immunofluorescence analysis demonstrated that apoptotic cells had a distinct CK8/18 staining pattern. In living cells, CK8/18 was uniformly distributed in the cytoplasm and cytosol; however in the apoptotic cells with a condensed and/or fragmented apoptotic nucleus (as identified by DAPI staining), fluorescein-labeled anti-CK8/18 antibody exhibited unusual punctate and/or bubbly staining in the cytosol. In the apoptotic cells that could not be identified by DAPI staining, fluorescein-labeled CK8/18 displayed polarized aggregated staining in the cytosol. These results indicate that fluorescein-conjugated CK8/18 may be a useful and sensitive indicator of cell apoptosis. PMID:25663887

DONG, QIAO-MEI; LING, CHUN; ZHAO, LI

2015-01-01

63

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

64

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

PubMed Central

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

65

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

PubMed Central

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

2015-01-01

66

Effect of theophylline and specific phosphodiesterase IV inhibition on proliferation and apoptosis of progenitor cells in bronchial asthma  

PubMed Central

Theophylline possesses anti-inflammatory activities in asthma. We examined whether theophylline and agents that modulate cyclic AMP can determine the survival and proliferation of progenitor cells. Progenitor cells from the blood of normal and asthmatic subjects were cultured for 14 days in methylcellulose with GM-CSF, stem cell factor, IL-3 and IL-5. Apoptosis was measured by flow cytometry of propidium-iodide-stained cells. A greater number of colonies with a higher proportion of cells of eosinophil lineage from asthmatics compared to normal subjects were grown. Theophylline (at 5 and 20 ?g ml?1) significantly inhibited colony formation and increased apoptotic cells in asthmatics compared to control. Salbutamol (0.1, 1, 10 ?M), dibutyryl-cAMP (0.1, 1 mM) and rolipram (0.1, 1 mM), a phosphodiesterase IV inhibitor, also dose-dependently decreased colony numbers and increased apoptosis of progenitor cells from asthmatics. There was no significant effect of theophylline, db-cAMP, salbutamol or rolipram on colony formation or the survival of progenitor cells from normal subjects. AMP did not affect the colony formation and apoptosis. Expression of Bcl-2 protein on progenitor cells of asthma was downregulated by theophylline, salbutamol, db-cAMP and rolipram. Theophylline and rolipram decreased colony formation committed to the eosinophil lineage, together with an increase in apoptosis through an inhibition of Bcl-2 expression effects that may occur through cAMP. The anti-inflammatory properties of theophylline include an inhibition of circulating progenitor cells. PMID:12684271

Wang, Chun-Hua; Lin, Horng-Chyuan; Lin, Chien-Huang; Yu, Chih-Teng; Liu, Su-Ling; Huang, Kuo-Hsiung; Chung, Kian Fan; Kuo, Han-Pin

2003-01-01

67

Ent-11?-Hydroxy-15-oxo-kaur-16-en-19-oic-acid Inhibits Growth of Human Lung Cancer A549 Cells by Arresting Cell Cycle and Triggering Apoptosis  

PubMed Central

Objective To examine the apoptotic effect of ent-11?-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F), a compound isolated from Pteris semipinnata L (PsL), in human lung cancer A549 cells. Methods A549 cells were treated with 5F (0–80 ?g/ml) for different time periods. Cytotoxicity was examined using a MTT method. Cell cycle was examined using propidium iodide staining. Apoptosis was examined using Hoechst 33258 staining, enzyme-linked immunosorbent assay (ELISA) and caspase-3 activity analysis. Expression of representative apoptosis-related proteins was evaluated by Western blot analysis. Reactive oxygen species (ROS) level was measured using standard protocols. Potential interaction of 5F with cisplatin was also examined. Results 5F inhibited the proliferation of A549 cells in a concentration- and time-dependent manner. 5F increased the accumulation of cells in sub-G1 phase and arrested the cells in the G2 phase. Exposure to 5F induced morphological changes and DNA fragmentation that are characteristic of apoptosis. The expression of p21 was increased. 5F exposure also increased Bax expression, release of cytochrome c and apoptosis inducing factor (AIF), and activation of caspase-3. 5F significantly sensitized the cells to cisplatin toxicity. Interestingly, treatment with 5F did not increase ROS, but reduced ROS production induced by cisplatin. Conclusion 5F could inhibit the proliferation of A549 cells by arresting the cells in G2 phase and by inducing mitochondrial-mediated apoptosis. PMID:23358391

Li, Li; Chen, George G; Lu, Ying-nian; Liu, Yi; Wu, Ke-feng; Gong, Xian-ling; Gou, Zhan-ping; Li, Ming-yue

2012-01-01

68

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

SciTech Connect

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

Wu Xiaoyan; Chen Peili [Department of Medicine, University of Chicago, Chicago, Illinois (United States); Sonis, Stephen T. [Division of Oral Medicine, Brigham and Women's Hospital, Boston, Massachusetts (United States); Biomodels, Watertown, Massachusetts (United States); Lingen, Mark W. [Department of Pathology, University of Chicago, Chicago, Illinois (United States); Berger, Ann [NephRx Corporation, Kalamazoo, Michigan (United States); Toback, F. Gary, E-mail: gtoback@medicine.bsd.uchicago.edu [Department of Medicine, University of Chicago, Chicago, Illinois (United States)

2012-07-01

69

Antiproliferative effects of copper(II)-polypyridyl complexes in breast cancer cells through inducing apoptosis.  

PubMed

Although cisplatin has been used for decades to treat human cancer, some toxic side effects and resistance are observed. Previous investigations have suggested copper complexes as a novel class of tumor-cell apoptosis inducers. The present study aimed to evaluate the anti-breast cancer activities of two polypyridyl-based copper(II) complexes, [Cu(tpy)(dppz)](NO3)2 (1) and [Cu(tptz)2](NO3)2 (2) (tpy = 2,2':6',2?-terpyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine, tptz = 2,4,6-tris(2-pyridyl)-1,3,5-triazine), using human breast adenocarcinoma cell line (MCF-7). The ability of the complexes to cleave supercoiled DNA in the presence and absence of external agents was also examined. The apoptotic activities of the complexes were assessed using flow cytometry, fluorescence microscope and western blotting analysis. Our results indicated the high DNA affinity and nuclease activity of complexes 1 and 2. The cleavage mechanisms between the complexes and plasmid DNA are likely to involve a singlet oxygen or singlet oxygen-like entity as the reactive oxygen species. Complexes 1 and 2 also significantly inhibited the proliferation of MCF-7 cells in a dose-dependent manner (IC50 values = 4.57 and 1.98 ?M at 24 h, respectively). Complex 2 remarkably induced MCF-7 cells to undergo apoptosis, which was demonstrated by cell morphology, annexin-V and propidium iodide staining. The caspase cascade was activated as shown by the proteolytic cleavage of caspase-3 after treatment of MCF-7 cells with complex 2. Additionally, complex 2 significantly increased the expression of the Bax-to-Bcl-2 ratio to induce apoptosis. In conclusion, these results revealed that complex 2 may be a potential and promising chemotherapeutic agent to treat breast cancer. PMID:25673217

Salimi, Mona; Abdi, Khatereh; Kandelous, Hirsa Mostafapour; Hadadzadeh, Hassan; Azadmanesh, Kayhan; Amanzadeh, Amir; Sanati, Hassan

2015-04-01

70

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

PubMed

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

Liu, Jia; Guo, Wenjie; Li, Jing; Li, Xiang; Geng, Ji; Chen, Qiuyun; Gao, Jing

2015-03-01

71

Isoflurane induces a postconditioning effect on bovine pulmonary arterial endothelial cells exposed to oxygen-glucose deprivation  

PubMed Central

Application of volatile anesthetics during the onset of reperfusion reduced ischemia-induced cardiac and brain injury (anesthetic postconditioning). This study was designed to evaluate whether volatile anesthetics induced a postconditioning effect in endothelial cells. Bovine pulmonary arterial endothelial cell (BPAEC) cultures were exposed to oxygen-glucose deprivation, a condition to simulate ischemia in vitro, for 3 h. The volatile anesthetics isoflurane and desflurane were applied during the early phase of simulated reperfusion. Cell injury was quantified by lactate dehydrogenase (LDH) release and flow cytometrical measurement after annexin V and propidium iodide staining. Oxygen-glucose deprivation and the subsequent simulated reperfusion increased LDH release and annexin V-positive staining cells, a characteristic of cell apoptosis. Posttreatment with isoflurane, but not desflurane, reduced this cell injury. This protection was apparent even when 2% isoflurane was applied at 60 min after the onset of reperfusion. The isoflurane postconditioning effect was abolished by glybenclamide, a general ATP sensitive K+ (KATP) channel blocker, 5-hydroxydecanoate, a mitochondrial KATP channel blocker, and chelerythrine, a protein kinase C inhibitor. Diazoxide, a mitochondrial KATP channel activator, applied at the onset of reperfusion also decreased oxygen-glucose deprivation-induced endothelial cell injury. This diazoxide-induced protection was abolished by chelerythrine and 5-hydroxydecanoate. We conclude that isoflurane induced a postconditioning effect in BPAEC. The effective time window of isoflurane postconditioning was from 0 to 60 min after the onset of reperfusion. This isoflurane postconditioning effect may be mediated by mitochondrial KATP channels and PKC. PKC may be downstream of mitochondrial KATP channels for this isoflurane effect. PMID:19464284

Kim, Jie Ae; Li, Liaoliao; Zuo, Zhiyi

2009-01-01

72

The effects of Mn(III) ortho N-alkylpyridyl- and diortho N,N’-imidazolylporphyrins on Oxygen and Glucose Deprivation -Induced Cell Death in Mixed Neuronal/Glial Cortical Cell Cultures  

PubMed Central

In vivo investigations have confirmed the beneficial effects of hydrophilic, cationic Mn(III) -based catalytic antioxidants in models of oxidative stress. We hypothesized the octyl porphyrin, MnTnOct-2-PyP5+, a lipophilic but equally potent antioxidant, would be more efficacious in reducing oxygen and glucose deprivation (OGD)-induced cell death. Using a cell culture model of rat mixed neuronal/glial cells, we investigated the effect of MnTnOct-2-PyP5+ on the OGD-induced cell death as compared to the effects of widely studied hydrophilic analogues MnTE-2-PyP5+ and MnTDE-2-ImP5+ P and a standard compound, dizocilpine (MK-801). Cell death was evaluated at 24 hours using lactate dehydrogenase (LDH) release, 3-(4,5-dimethyltiazol-2-yl) -2,5 diphenyltetrazolium bromide(MTT) and propidium iodide staining. At lower concentrations, all three porphyrins reduced cell death as compared to cultures exposed to OGD alone. When the cultures were exposed to MnTnOct-2-PyP5+ before OGD but not during the deprivation, it was a very efficacious compound as judged by LDH release. MnTnOct-2-PyP5+ becomes less efficacious if the exposure was prolonged. While no extensive toxicity was seen with MnTnOct-2-PyP5+ P , the effects observed, though, might have been the result of the interplay of efficacy and toxicity leading to a diminished protectiveness. PMID:19259881

WISE-FABEROWSKI, LISA; WARNER, DAVID S.; SPASJOVIC, IVAN; BATINIC-HABERLE, INES

2010-01-01

73

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

PubMed Central

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

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

2014-01-01

74

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

75

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

PubMed Central

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

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

2009-01-01

76

Propidium iodide as a probe for the study of chromatin thermal denaturation in situ.  

PubMed

The possibility of using propidium iodide, a phenanthridinic fluorochrome specific for double-stranded nucleic acids, for the study of chromatin thermal denaturation in situ has been examined. Smears of lymphocytes and hepatocyte nuclei from 15-day-old rats were fixed in acetic acid--ethanol (1:3 v/v), treated with RNAse and submitted to different protein extraction procedures, namely, incubation with pepsin, trypsin and sodium chloride. Denaturation experiments were performed in Sörensen buffer at pH 7.4 containing 10% formamide at temperatures between 27 and 95 degrees C. The samples were stained with propidium iodide and mounted in buffer or glycerol. Measurements were performed with a microfluorometer at a wavelength of 446 nm. The results indicate a higher thermostability of lymphocytes as compared to hepatocytes. The denaturation pattern suggests a certain organization complexity of chromatin, better emphasized by the derivative curves which show the presence of at least three fractions with different melting points. After protein extraction, the denaturation curves exhibit a somewhat simplified pattern, with the disappearance of the most stable peak in the derivative curves. The samples mounted in glycerine exhibit a better stability of staining with time, and an increased quantum efficiency of the fluorochrome with regard to those mounted in buffer. These data confirm the importance of protein--DNA interactions in the organization of chromatin and point to some differences, depending on the cell type and on functional activity. PMID:7298376

Barni, S; de Piceis Polver, P; Gerzeli, G; Nano, R

1981-09-01

77

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

PubMed

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; Hussein, Sharifah Salwa Syed; Lo, Jia Ye; Supriady, Hadi; Abdul Kadir, Habsah

2014-01-01

78

(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

79

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

PubMed Central

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

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

2013-01-01

80

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

PubMed Central

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

2010-01-01

81

Fluorescein isothiocynate-dextran uptake by chinese hamster ovary cells in a 1.5 MHz ultrasonic standing wave in the presence of contrast agent.  

PubMed

Uptake of fluorescein isothiocynate-dextran (FITC-dextran) by Chinese hamster ovary cells was studied after exposure to ultrasonic standing wave (USW) in presence of Optison, an ultrasound contrast agent. Confluent Chinese hamster ovary cells were harvested and suspended in phosphate-buffered saline + 0.1% bovine serum albumin containing FITC-dextran (10, 40, and 500 kDa) at 10 microM final concentration. The suspension was seeded with contrast agent (75 microL/mL) and exposed to a 1.5 MHz USW system at acoustic pressures ranging from 0.98 to 4.2 MPa. Macromolecular uptake was assessed by fluorescent microscopy and quantified by flow cytometry 10 min after exposure. FITC-dextran positive cells, as assessed by flow cytometry, were 1 +/- 0.05% and 2.58 +/- 0.27% for acoustic pressures of 1.96 and 4.2 MPa, respectively (p = 0.006). Fluorescent microscopy indicated a degree of macromolecular loading at 0.98 MPa with 46% of peripherally FITC-dextran- and/or propidium iodide-stained cells coincident with the appearance of significant frequency (f0/2 and 2 f0) emission signals. At higher pressures, high macromolecular loading with 6% peripherally stained cells at 1.96 MPa was associated with lower order emission signals and white noise. The study conclusively demonstrates macromolecular loading in an USW, a significantly higher macromolecular loading at higher pressures and indicates potential of emission signals for a feedback loop to control the acoustic power outputs and fine-tune the biologic effects associated with sonoporation. PMID:16464674

Khanna, Sanjay; Hudson, Benjamin; Pepper, Christopher J; Amso, Nazar N; Coakley, W Terence

2006-02-01

82

Acidic pH conditions mimicking degenerative intervertebral discs impair the survival and biological behavior of human adipose-derived mesenchymal stem cells.  

PubMed

This study was designed to examine the survival and biological behavior of adipose-derived mesenchymal stem cells (ADMSCs) under an intervertebral disc (IVD)-like acidic environment. Human ADMSCs isolated from two age groups were cultured under four different pH levels (pH 7.4, 7.1, 6.8 and 6.5) which mimicked the standard condition and the normal, mildly degenerated and severely degenerated IVD. Cell viability was measured by fluorescein isothiocyanate-Annexin-V/propidium iodide staining, and cell proliferation was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. The expression of aggrecan, collagen-I, collagen-II, matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinase-3 (TIMP-3), p53 and caspase-3 at the mRNA level was examined by realtime quantitative polymerase chain reaction, and the expression of aggrecan, collagen-I, collagen-II, MMP-2 and TIMP-3 at the protein level was measured by enzyme-linked immunosorbent assay. Acidic pH inhibited the viability and proliferation, and the expression of aggrecan, collagen-I and collagen-II of ADMSCs from both age groups. ADMSCs harvested from young and mature donors exhibited similar responses to the acidic pH, although cells from young donors appeared less sensitive to the low pH levels. The results demonstrated that acidic pH in IVD may be an important deleterious factor for ADMSC-based IVD regeneration. ADMSCs harvested from young donors may be more suitable to be utilized for the implantation into degenerated IVD, and the implantations may be more effective at an early stage of IVD degeneration when the pH of matrix acidity is higher than 6.8. PMID:22829705

Li, Hao; Liang, Chengzhen; Tao, Yiqing; Zhou, Xiaopeng; Li, Fangcai; Chen, Gang; Chen, Qi-xin

2012-07-01

83

A simple sperm nuclear vacuole assay with propidium iodide.  

PubMed

Our aim was to develop a new simple sperm nuclear vacuole assay (SNVA) with propidium iodide (PI) to determine the status of nuclear vacuole (NV) of individual spermatozoa. After PI staining, sperm nuclei were classified into the 14 categories according to both nuclear morphology and the status of NV. The incidence was 57.8% (range 28-84%) in fertile controls (n = 40), and 85.1% (range 67-99%) in men with varicocele (n = 40). In the fertile group, normal nuclear-shaped spermatozoa without NV or with one small NV located in the ante-nuclear region were significantly more in comparison with the varicocele group. In the varicocele group, abnormal nuclear-shaped spermatozoa with one large NV and with multiple NVs located in the ante-nuclear region were most frequent findings. Besides, spermatozoa with NVs in both ante- and post-nuclear regions in the varicocele group were significantly more than those in the fertile group. In both fertile and varicocele groups, normal or abnormal nuclear-shaped spermatozoa with one or more vacuoles only located in the post-nuclear region occurred sparingly. The SNVA provides a useful additional approach to identify the status of NV in human spermatozoa for diagnostic purposes. A good sperm sample would have more spermatozoa without NV or with one small NV located in the ante-nuclear region. PMID:25220411

Zhu, W-J; Li, J

2014-09-13

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

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

PubMed Central

Background Silver nanoparticles (AgNPs) possess unique physical, chemical, and biological properties. AgNPs have been increasingly used as anticancer, antiangiogenic, and antibacterial agents for the treatment of bacterial infections in open wounds as well as in ointments, bandages, and wound dressings. The present study aimed to investigate the effects of two different sizes of AgNPs (10 nm and 20 nm) in male somatic Leydig (TM3) and Sertoli (TM4) cells and spermatogonial stem cells (SSCs). Methods Here, we demonstrate a green and simple method for the synthesis of AgNPs using Bacillus cereus culture supernatants. The synthesized AgNPs were characterized using ultraviolet and visible absorption spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and transmission electron microscopy (TEM). The toxicity of the synthesized AgNPs was evaluated by the effects on cell viability, metabolic activity, oxidative stress, apoptosis, and expression of genes encoding steroidogenic and tight junction proteins. Results AgNPs inhibited the viability and proliferation of TM3 and TM4 cells in a dose- and size-dependent manner by damaging cell membranes and inducing the generation of reactive oxygen species, which in turn affected SSC growth on TM3 and TM4 as feeder cells. Small AgNPs (10 nm) were more cytotoxic than medium-sized nanoparticles (20 nm). TEM revealed the presence of AgNPs in the cell cytoplasm and nucleus, and detected mitochondrial damage and enhanced formation of autosomes and autolysosomes in the AgNP-treated cells. Flow cytometry analysis using Annexin V/propidium iodide staining showed massive cell death by apoptosis or necrosis. Real-time polymerase chain reaction and western blot analyses indicated that in TM3 and TM4 cells, AgNPs activated the p53, p38, and pErk1/2 signaling pathways and significantly downregulated the expression of genes related to testosterone synthesis (TM3) and tight junctions (TM4). Furthermore, the exposure of TM3 and TM4 cells to AgNPs inhibited proliferation and self-renewal of SSCs. Conclusion Our results suggest that AgNPs exhibit size-dependent nanoreprotoxicity in male somatic cells and SSCs, strongly suggesting that applications of AgNPs in commercial products must be carefully evaluated. Further studies of AgNPs-induced nanoreprotoxicity in animal models are required. PMID:25733828

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

2015-01-01

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

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

PubMed Central

AIM: To investigate the effect of aspirin on neuroendocrine tumor (NET) cell growth and signaling in vitro. METHODS: Human pancreatic BON1, bronchopulmonary NCI-H727 and midgut GOT1 neuroendocrine tumor cells were treated with different concentrations of aspirin (from 0.001 to 5 mmol/L), and the resulting effects on metabolic activity/cell proliferation were measured using cell proliferation assays and SYBR-DNA-labeling after 72, 144 and 216 h of incubation. The effects of aspirin on the expression and phosphorylation of several critical proteins that are involved in the most common intracellular growth factor signaling pathways (especially Akt protein kinase B) and mammalian target of rapamycin (mTOR) were determined by Western blot analyses. Propidium iodide staining and flow cytometry were used to evaluate changes in cell cycle distribution and apoptosis. Statistical analysis was performed using a 2-tailed Student’s t-test to evaluate the proliferation assays and cell cycle analyses. The results are expressed as the mean ± SD of 3 or 4 independently performed experiments. Statistical significance was set at P < 0.05. RESULTS: Treatment with aspirin suppressed the viability/proliferation of BON1, NCI-H727 and GOT1 cells in a time- and dose-dependent manner. Significant effects were observed at starting doses of 0.5-1 mmol/L and peaked at 5 mmol/L. For instance, after treatment with 1 mmol/L aspirin for 144 h, the viability of pancreatic BON1 cells decreased to 66% ± 13% (P < 0.05), the viability of bronchopulmonary NCI-H727 cells decreased to 53% ± 8% (P < 0.01) and the viability of midgut GOT1 cells decreased to 89% ± 6% (P < 0.01). These effects were associated with a decreased entry into the S phase, the induction of the cyclin-dependent kinase inhibitor p21 and reduced expression of cyclin-dependent kinase 4 and cyclin D3. Aspirin suppressed mTOR downstream signaling, evidenced by the reduced phosphorylation of the mTOR substrates 4E binding protein 1, serine/threonine kinase P70S6K and S6 ribosomal protein and inhibited glycogen synthase kinase 3 activity. We observed the (compensatory) activation of tuberous sclerosis 2, the serine/threonine specific protein kinase AKT and extracellular signal-regulated kinases. CONCLUSION: Aspirin demonstrates promising anticancer properties for NETs in vitro. Further preclinical and clinical studies are needed. PMID:25110431

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

2014-01-01

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

P25. In vitro evaluation of the anticancer activity of ganetespib in combination with standard chemotherapeutics in small cell lung cancer (SCLC) cell lines  

PubMed Central

Background Ganetespib (STA-9090) has anticancer activity due to inhibition of the chaperoning activity of HSP90 for oncogenes and other cellular proteins which support malignant growth. Especially, clinical activity as single drug was found in tumors addicted to modified kinases, such as HER2, ALK, BRAF, KIT and others. In the present work we tested the cytotoxic effects of ganetespib either alone or in combination with chemotherapeutics against a panel of small cell lung cancer cell lines. Methods Cytotoxicity was assessed using MTT assays, cell cycle effects by propidium iodide staining, HSP90 expression by flow cytometry and synergism of drug combinations was calculated employing the Chou-Talalay method. Results The chemoresistant small cell lung cancer (SCLC) cell lines GLC16, NCI-H417 and DMS153 were highly sensitive to ganetespib (IC50 <30 nM), whereas chemosensitive GLC14 and primary SCLC26A cells exhibited chemoresistance to this drug. Ganetespib showed marked synergy in combination with cisplatin/carboplatin, etoposide and doxorubicin, whereas limited cooperative effects were observed for topotecan and the cisplatin/etoposide triple combination. Determinations of HSP90 expression using a monoclonal antibody pointed to an association of the cytotoxic effects of ganetespib with cell surface expression of this target and not with total expression of this heat shock protein. Conclusions In contrast to the first-generation HSP90 inhibitors, such as geldanamycin or 17-N-allylamino-17-demethoxygeldanamycin (17AAG), ganetespib has a far more favorable safety profile and has shown clinical anticancer activity in breast cancer, non-small cell lung cancer (NSCLC), gastrointestinal stromal tumors (GIST) and various hematological malignancies. According to our results, ganetespib is not expected to be active against primary, untreated SCLCs as single drug; however, is estimated to show cytotoxicity for pretreated SCLCs, either alone or in combinations with platinum drugs, etoposide or doxorubicin. These in vitro results are compatible with inhibition of an HSP90 client protein by ganetespib which is induced in response to prior exposure of the cells to chemotherapy in vivo.

Hamilton, Gerhard; Rath, Barbara; Maritschnegg, Elisabeth; Zeillinger, Robert

2014-01-01

92

Induction of Apoptosis in Human Cancer Cells by Candidaspongiolide, a Novel Sponge Polyketide  

PubMed Central

Background Candidaspongiolide (CAN), a novel polyketide from a marine sponge, is the active component of a mixture that was found to be potently cytotoxic in the National Cancer Institute’s 60-cell-line screen. Methods Effects of CAN on U251 glioma and HCT116 colorectal cancer cells and on normal fibroblasts were assessed using radiolabeling studies to measure protein synthesis, clonogenic assays to measure cell survival, flow cytometry of annexin V– and propidium iodide–stained cells to measure apoptosis, and western blots in the presence or absence of specific inhibitors to assess accumulation and phosphorylation of potential downstream target proteins. Results CAN inhibited protein synthesis and potently induced apoptosis in both U251 and HCT116 cells, the latter in part by a caspase 12–dependent pathway. For example, 25%–30% of U251 or HCT116 cells became apoptotic after 24 hours of treatment with 100 nM CAN. CAN also rapidly induced sustained phosphorylation of eukaryotic translation initiation factor-2 (eIF2)-? at Ser51 and of the translation elongation factor eEF2 at Thr56, which could contribute to its dose-dependent inhibition of protein synthesis. Stable expression of dominant-negative eIF2? was sufficient to prevent CAN-induced eIF2? phosphorylation and induction of apoptosis but insufficient to prevent inhibition of protein synthesis. CAN induction of eIF2? phosphorylation did not occur by a classic endoplasmic reticulum stress pathway. However, an inhibitor of and small-interfering RNAs to the double-stranded RNA–dependent protein kinase PKR prevented CAN-mediated eIF2? phosphorylation and apoptosis, respectively. Although CAN inhibited protein synthesis in both cancer cells and normal human fibroblasts, it induced eIF2? phosphorylation and apoptosis only in cancer cells. Conclusions CAN triggers PKR/eIF2?/caspase 12–dependent apoptosis and inhibits protein synthesis in cancer cells but only inhibits protein synthesis in normal cells. PMID:18728285

Trisciuoglio, Daniela; Uranchimeg, Badarch; Cardellina, John H.; Meragelman, Tamara L.; Matsunaga, Shigeki; Fusetani, Nobuhiru; Del Bufalo, Donatella; Shoemaker, Robert H.

2008-01-01

93

Ring-substituted analogs of 3,3'-diindolylmethane (DIM) induce apoptosis and necrosis in androgen-dependent and -independent prostate cancer cells.  

PubMed

We recently reported that novel ring-substituted analogs of 3,3'-diindolylmethane (ring-DIMs) have anti-androgenic and growth inhibitory effects in androgen-dependent prostate cancer cells. The objectives of this study were to confirm the ability of 4,4'- and 7,7'-dibromo- and dichloro-substituted ring-DIMs to inhibit androgen-stimulated proliferation of androgen-dependent LNCaP human prostate cancer cells using a non-invasive, real-time monitoring technique. In addition, their ability to induce apoptotic and necrotic cell death in androgen-dependent as well as -independent (PC-3) prostate cancer cells was studied. Prostate cancer cells were treated with increasing concentrations of DIM and ring-DIMs (0.3-30 ?M) and effects on cell proliferation were measured in real-time using an xCELLigence cellular analysis system. Chromatin condensation and loss of membrane integrity were determined by Hoechst and propidium iodide staining, respectively. Apoptotic protein markers were measured by immunoblotting and activation of caspases determined using selective fluorogenic substrates. Intra- and extracellular concentrations of DIM and ring-DIMs were assessed by electrospray ionization tandem mass spectrometry. Ring-DIMs inhibited androgen-stimulated LNCaP cell proliferation and induced apoptosis and necrosis in LNCaP and PC-3 cells with 2-4 fold greater potencies than DIM. DIM and the ring-DIMs increased caspases -3, -8 and -9 activity, elevated expression of Fas, FasL, DR4 and DR5 protein, and induced PARP cleavage in both cell lines. The cytotoxicity of the most potent ring-DIM, 4,4'-dibromoDIM, but not the other compounds was decreased by an inhibitor of caspase -3. The 4,4'-dibromoDIM was primarily found in the extracellular medium, whereas all other compounds were present to a much larger extent in the cell. In conclusion, ring-DIMs inhibited prostate cancer cell growth and induced cell death in LNCaP and PC-3 cells with greater potencies than DIM; they also structure-dependently activated different cell death pathways suggesting that these compounds have clinical potential as chemopreventive and chemotherapeutic agents in prostate cancer, regardless of hormone-dependency. PMID:23709189

Goldberg, A A; Titorenko, V I; Beach, A; Abdelbaqi, K; Safe, S; Sanderson, J T

2014-02-01

94

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

PubMed

Positive allosteric modulation of ?7 isoform of nicotinic acetylcholine receptors (?7-nAChRs) is emerging as a promising therapeutic approach for central nervous system disorders such as schizophrenia or Alzheimer's disease. However, its effect on Ca(2+) signaling and cell viability remains controversial. This study focuses on how the type II positive allosteric modulator (PAM II) PNU120596 affects intracellular Ca(2+) signaling and cell viability. We used human SH-SY5Y neuroblastoma cells overexpressing ?7-nAChRs (?7-SH) and their control (C-SH). We monitored cytoplasmic and endoplasmic reticulum (ER) Ca(2+) with Fura-2 and the genetically encoded cameleon targeting the ER, respectively. Nicotinic inward currents were measured using patch-clamp techniques. Viability was assessed using methylthiazolyl blue tetrazolium bromide or propidium iodide staining. We observed that in the presence of a nicotinic agonist, PNU120596 (i) reduced viability of ?7-SH but not of C-SH cells; (ii) significantly increased inward nicotinic currents and cytosolic Ca(2+) concentration; (iii) released Ca(2+) from the ER by a Ca(2+) -induced Ca(2+) release mechanism only in ?7-SH cells; (iv) was cytotoxic in rat organotypic hippocampal slice cultures; and, lastly, all these effects were prevented by selective blockade of ?7-nAChRs, ryanodine receptors, or IP3 receptors. In conclusion, positive allosteric modulation of ?7-nAChRs with the PAM II PNU120596 can lead to dysregulation of ER Ca(2+) , overloading of intracellular Ca(2+) , and neuronal cell death. This study focuses on how the type II positive allosteric modulator PNU120596 (PAM II PNU12) affects intracellular Ca(2+) signaling and cell viability. Using SH-SY5Y neuroblastoma cells overexpressing ?7-nAChRs (?7-SH) and their control (C-SH), we find that PAM of ?7-nAChRs with PNU120596: (i) increases inward calcium current (ICa ) and cytosolic Ca(2+) concentration ([Ca(2+) ]cyt ); (ii) releases Ca(2+) from the ER ([Ca(2+) ]ER ) by a Ca(2+) -induced Ca(2+) release mechanism; and (iv) reduces cell viability. These findings were corroborated in rat hippocampal organotypic cultures. [Ca(2+) ]cyt , cytosolic Ca(2+) concentration; [Ca(2+) ]ER , endoplasmic reticulum Ca(2+) concentration; ?7 nAChR, ?7 isoform of nicotinic acetylcholine receptors; ?7-SH, SH-SY5Y stably overexpressing ?7 nAChRs cells; C-SH, control SH-SY5Y cells; Nic, nicotine; PNU12, PNU120596. PMID:25650007

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

2015-05-01

95

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

PubMed Central

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

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

2013-01-01

96

Transcriptional regulation of kinases downstream of the T cell receptor: another immunomodulatory mechanism of glucocorticoids  

PubMed Central

Background Glucocorticoids affect peripheral immune responses, including modulation of T-cell activation, differentiation, and apoptosis. The quantity and quality of T-cell receptor (TCR)-triggered intracellular signals modulate T-cell function. Thus, glucocorticoids may affect T cells by interfering with the TCR signaling cascade. The purpose of the study was to search for glucocorticoid-modulated kinases downstream of the TCR. Methods Gene modulation in lymphoid cells either treated with glucocorticoids or from glucocorticoid-treated mice was studied using a RNase protection assay, real-time PCR, and western blotting. The sensitivity of genetically modified thymocytes to glucocorticoid-induced apoptosis was studied by performing hypotonic propidium iodide staining and flow cytometry. The Student’s t-test was employed for statistical evaluation. Results We found that transcription of Itk, a non-receptor tyrosine kinase of the Tec family, was up-regulated in a mouse T-cell hybridoma by the synthetic glucocorticoid dexamethasone. In contrast, dexamethasone down-regulated the expression of Txk, a Tec kinase that functions redundantly with Itk, and Lck, the Src kinase immediately downstream of the TCR. We investigated the expression of Itk, Txk, and Lck in thymocytes and mature lymphocytes following in vitro and in vivo dexamethasone treatment at different time points and doses. Kinase expression was differentially modulated and followed distinct kinetics. Itk was up-regulated in all cell types and conditions tested. Txk was strongly up-regulated in mature lymphocytes but only weakly up-regulated or non-modulated in thymocytes in vitro or in vivo, respectively. Conversely, Lck was down-regulated in thymocytes, but not modulated or up-regulated in mature lymphocytes in the different experimental conditions. This complex behaviour correlates with the presence of both positive and negative glucocorticoid responsive elements (GRE and nGRE, respectively) in the Itk, Txk and Lck genes. To investigate the function associated with Itk up-regulation, dexamethasone-induced apoptosis of thymocytes from Itk-deficient mice was evaluated. Our results demonstrated that Itk deficiency causes increased sensitivity to dexamethasone but not to other pro-apoptotic stimuli. Conclusions Modulation of Itk, Txk, and Lck in thymocytes and mature lymphocytes is another mechanism by which glucocorticoids modulate T-cell activation and differentiation. Itk up-regulation plays a protective role in dexamethasone-treated thymocytes. PMID:24993777

2014-01-01

97

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

98

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

PubMed Central

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

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

2015-01-01

99

Coloration de cellules de levures au Propidium Iodide pour analyse au FACS.  

E-print Network

Coloration de cellules de levures au Propidium Iodide pour analyse au FACS. But : Voir le pourcentage de cellules dans chacune des phases du cycle cellulaire. Matériel : - FACS buffer - Éthanol 70.0 20 ml ddH2O 380 ml Méthode : 1. Récolter 1 ml de cellules (OD600 voulue) dans un Falcon 12 ml. Spin 1

Abou Elela, Sherif

100

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

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. PMID:25395836

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

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

PubMed Central

Background Development of tumour resistance to chemotherapeutic drugs and concerns over their toxic effects has led to the increased use of medicinal herbs or natural products by cancer patients. Strobilanthes crispus is a traditional remedy for many ailments including cancer. Its purported anticancer effects have led to the commercialization of the plant leaves as medicinal herbal tea, although the scientific basis for its use has not been established. We previously reported that a bioactive subfraction of Strobilanthes crispus leaves (SCS) exhibit potent cytotoxic activity against human breast cancer cell lines. The current study investigates the effect of this subfraction on cell death activities induced by the antiestrogen drug, tamoxifen, in estrogen receptor-responsive and nonresponsive breast cancer cells. Methods Cytotoxic activity of SCS and tamoxifen in MCF-7 and MDA-MB-231 human breast cancer cells was determined using lactate dehydrogenase release assay and synergism was evaluated using the CalcuSyn software. Apoptosis was quantified by flow cytometry following Annexin V and propidium iodide staining. Cells were also stained with JC-1 dye to determine changes in the mitochondrial membrane potential. Fluorescence imaging using FAM-FLICA assay detects caspase-8 and caspase-9 activities. DNA damage in the non-malignant breast epithelial cell line, MCF-10A, was evaluated using Comet assay. Results The combined SCS and tamoxifen treatment displayed strong synergistic inhibition of MCF-7 and MDA-MB-231 cell growth at low doses of the antiestrogen. SCS further promoted the tamoxifen-induced apoptosis that was associated with modulation of mitochondrial membrane potential and activation of caspase-8 and caspase-9, suggesting the involvement of intrinsic and extrinsic signaling pathways. Interestingly, the non-malignant MCF-10A cells displayed no cytotoxicity or DNA damage when treated with either SCS or SCS-tamoxifen combination. Conclusions The combined use of SCS and lower tamoxifen dose could potentially reduce the side effects/toxicity of the drug. However, further studies are needed to determine the effectiveness and safety of the combination treatment in vivo. PMID:25034326

2014-01-01

103

Effect of hypoxia and Beraprost sodium on human pulmonary arterial smooth muscle cell proliferation: the role of p27kip1  

PubMed Central

Background Hypoxia induces the proliferation of pulmonary arterial smooth muscle cell (PASMC) in vivo and in vitro, and prostacyclin analogues are thought to inhibit the growth of PASMC. Previous studies suggest that p27kip1, a kind of cyclin-dependent kinase inhibitor, play an important role in the smooth muscle cell proliferation. However, the mechanism of hypoxia and the subcellular interactions between p27kip1 and prostacyclin analogues in human pulmonary arterial smooth muscle cell (HPASMC) are not fully understood. Methods We investigated the role of p27kip1 in the ability of Beraprost sodium (BPS; a stable prostacyclin analogue) to inhibit the proliferation of HPASMC during hypoxia. To clarify the biological effects of hypoxic air exposure and BPS on HPASMC, the cells were cultured in a hypoxic chamber under various oxygen concentrations (0.1–21%). Thereafter, DNA synthesis was measured as bromodeoxyuridine (BrdU) incorporation, the cell cycle was analyzed by flow cytometry with propidium iodide staining. The p27kip1 mRNA and protein expression and it's stability was measured by real-time RT-PCR and Western blotting. Further, we assessed the role of p27kip1 in HPASMC proliferation using p27kip1 gene knockdown using small interfering RNA (siRNA) transfection. Results Although severe hypoxia (0.1% oxygen) suppressed the proliferation of serum-stimulated HPASMC, moderate hypoxia (2% oxygen) enhanced proliferation in accordance with enhanced p27kip1 protein degradation, whereas BPS suppressed HPASMC proliferation under both hypoxic and normoxic conditions by suppressing p27kip1 degradation with intracellular cAMP-elevation. The 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), a cAMP analogue, had similar action as BPS in the regulation of p27kip1. Moderate hypoxia did not affect the stability of p27kip1 protein expression, but PDGF, known as major hypoxia-induced growth factors, significantly decreased p27kip1 protein stability. We also demonstrated that BPS and 8-Br-cAMP suppressed HPASMC proliferation under both hypoxic and normoxic conditions by blocking p27kip1 mRNA degradation. Furthermore, p27kip1 gene silencing partially attenuated the effects of BPS and partially restored hypoxia-induced proliferation. Conclusion Our study suggests that moderate hypoxia induces HPASMC proliferation, which is partially dependent of p27kip1 down-regulation probably via the induction of growth factors such as PDGF, and BPS inhibits both the cell proliferation and p27kip1 mRNA degradation through cAMP pathway. PMID:17974037

Kadowaki, Maiko; Mizuno, Shiro; Demura, Yoshiki; Ameshima, Shingo; Miyamori, Isamu; Ishizaki, Takeshi

2007-01-01

104

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

PubMed Central

Background A greater reduction in cancer risk associated with mushroom diet rich in fungus polysaccharides is generally accepted. Meanwhile, edible Pleurotus abalonus as a member of Abalone mushroom family is a popular nutritional supplement that purportedly prevents cancer occurrence. However, these anecdotal claims are supported by limited studies describing tumor-inhibitory responses to the promising polysaccharides, and the molecular mechanisms underlying these properties have not yet been elucidated. Methodology/Principal Findings We here fractionated the crude polysaccharide preparation from the fruiting bodies of P. abalonus into three fractions, namely PAP-1, PAP-2 and PAP-3, and tested these fractions for antiproliferative activity in human breast cancer MCF-7 cells. The largest PAP-3, an acidic polysaccharide fraction with a molecular mass of 3.68×105 Da, was the most active in inhibiting MCF-7 cancer cells with an IC50 of 193 µg/mL. The changes in cell normal morphology were observed by DAPI staining and the PAP-3-induced apoptosis was confirmed by annexin V/propidium iodide staining. The apoptosis was involved in mitochondria-mediated pathway including the loss of mitochondrial membrane potential (??m), the increase of Bax/Bcl-2 ratio, caspase-9/3 activation, and poly(ADP-ribose) polymerase (PARP) degradation, as well as intracellular ROS production. PAP-3 also induced up-regulation of p53, and cell cycle arrest at the S phase. The incubation of MCF-7 cells with antioxidant superoxide dismutase (SOD) and N-acetylcysteine (NAC) significantly attenuated the ROS generation and apoptosis caused by PAP-3, indicating that intracellular ROS plays a pivotal role in cell death. Conclusions/Significance These findings suggest that the polysaccharides, especially acidic PAP-3, are very important nutritional ingredients responsible for, at least in part, the anticancer health benefits of P. abalonus via ROS-mediated mitochondrial apoptotic pathway. It is a major breakthrough bringing new insight of the potential use of the polysaccharides as health-care food or medicine to provide significant natural defense against human cancer. PMID:23691187

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

2013-01-01

105

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.

106

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

107

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

PubMed

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

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

2015-06-01

108

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

109

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

PubMed Central

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

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

2014-01-01

110

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

PubMed

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

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

2014-07-01

111

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

PubMed Central

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

112

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

PubMed

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

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

2015-03-01

113

Application of propidium monoazide quantitative PCR for selective detection of live Escherichia coli O157:H7 in vegetables after inactivation by essential oils.  

PubMed

The use of propidium monoazide (PMA) is enjoying increased popularity among researchers in different fields of microbiology. Its use in combination with real-time PCR (qPCR) represents one of the most successful approaches to detect viable cells. PMA-qPCR has successfully been used to evaluate the efficacy of various disinfection technologies in different microorganisms. Initially, in this study the effect of four essential oils (EOs), cumin, clove, oregano and cinnamon, was evaluated on suspensions of the enterohemorrhagic Escherichia coli O157:H7 by PMA-qPCR, LIVE/DEAD BacLight flow cytometry analysis (LIVE/DEAD-FCM), and plate count. E. coli O157:H7 cells treated with EOs at killing concentrations were permeable to PMA which was confirmed by LIVE/DEAD-FCM. However, the PMA-qPCR assay allows specific quantification among the autochthonous microbiota of food products. Therefore, the PMA-qPCR assay was used to evaluate its applicability in artificially contaminated iceberg lettuce and soya sprouts. Amplification signal was negative for the spiking tests performed with any of the EO-killed E. coli cells. It demonstrates that the PMA-qPCR assay is a suitable technique for monitoring E. coli O157:H7 inactivation by essential oils in fresh-cut vegetables. PMID:23072696

Elizaquível, Patricia; Sánchez, Gloria; Aznar, Rosa

2012-10-01

114

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

PubMed Central

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

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

2014-01-01

115

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

116

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

PubMed

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

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

2015-03-01

117

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

118

[Evaluation of pathogen disinfection efficacy by chlorine and monochloramine disinfection based on quantitative PCR combined with propidium monoazide (PMA-qPCR)].  

PubMed

A novel detection method of quantitative PCR combined with a DNA intercalating dye propidium monoazide (PMA-qPCR) was developed and then applied to analyze inactivation efficacy of chlorine and monochloramine on E. coli as a representative organism. The results shows that PMA removed 99.94% and 99.99% DNA from non-viable E. coli and Salmonella cells respectively and PMA-qPCR could effectively differentiate viable bacteria from non-viable bacteria; According to the first-order kinetic model, the inactivation coefficients on E. coli obtained by PMA-qPCR were 2.24 L x (mg x min)-1 and 0.0175 L x (mg x min)-1 for chlorine and monochloramine respectively, both of which were lower than those obtained by traditional plating counting method. In order to inactivate 99% of E. coli, the ct values by PMA-qPCR were 0.9 mg L(-1) min and more than 100 mg x L(-1) x min for chlorine and monochloramine while those by plating counting method were only 0.6 mg x L(-1) x min and 20 mg x L(-1) min, respectively; E. coli concentration detected by conventional qPCR kept almost the same when ct value increased, indicating that conventional qPCR was unable to evaluate inactivation efficacy of both chlorine and monochloramine disinfection. In summary, PMA-qPCR shows to be a promising method for evaluating disinfection efficacy by chlorine and monochloramine more accurately. PMID:21717757

Tong, Tie-Zheng; Wu, Shu-Xu; Li, Dan; He, Miao; Yang, Tian; Shi, Han-Chang

2011-04-01

119

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

120

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

PubMed Central

Human enteric viruses can be present in untreated and inadequately treated drinking water. Molecular methods, such as the reverse transcriptase PCR (RT-PCR), can detect viral genomes in a few hours, but they cannot distinguish between infectious and noninfectious viruses. Since only infectious viruses are a public health concern, methods that not only are rapid but also provide information on the infectivity of viruses are of interest. The intercalating dye propidium monoazide (PMA) has been used for distinguishing between viable and nonviable bacteria with DNA genomes, but it has not been used to distinguish between infectious and noninfectious enteric viruses with RNA genomes. In this study, PMA in conjunction with RT-PCR (PMA-RT-PCR) was used to determine the infectivity of enteric RNA viruses in water. Coxsackievirus, poliovirus, echovirus, and Norwalk virus were rendered noninfectious or inactivated by treatment with heat (72°C, 37°C, and 19°C) or hypochlorite. Infectious or native and noninfectious or inactivated viruses were treated with PMA. This was followed by RNA extraction and RT-PCR or quantitative RT-PCR (qRT-PCR) analysis. The PMA-RT-PCR results indicated that PMA treatment did not interfere with detection of infectious or native viruses but prevented detection of noninfectious or inactivated viruses that were rendered noninfectious or inactivated by treatment at 72°C and 37°C and by hypochlorite treatment. However, PMA-RT-PCR was unable to prevent detection of enteroviruses that were rendered noninfectious by treatment at 19°C. After PMA treatment poliovirus that was rendered noninfectious by treatment at 37°C was undetectable by qRT-PCR, but PMA treatment did not affect detection of Norwalk virus. PMA-RT-PCR was also shown to be effective for detecting infectious poliovirus in the presence of noninfectious virus and in an environmental matrix. We concluded that PMA can be used to differentiate between potentially infectious and noninfectious viruses under the conditions defined above. PMID:20472736

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

2010-01-01

121

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

Microsoft Academic Search

The objective was to examine the effects of cryoprotectants on oocytes of hard corals (Echinopora spp.) to obtain basic knowledge for cryopreservation procedures. Oocytes were exposed to various concentrations of cryoprotectants (0.25 to 5.0M) for 20min at room temperature (25°C). Two tests were used to assess ovarian follicle viability: fluorescein diacetate (FDA)+propidium iodide (PI) staining, and adenosine triphosphate (ATP) assay.

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

2010-01-01

122

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

PubMed Central

AIM: To determine the effects of BN52021 on platelet-activating factor receptor (PAFR) signaling molecules under lipopolysaccharide (LPS)-induced inflammatory conditions in MS1 cells. METHODS: MS1 cells (a mouse pancreatic islet endothelial cell line) were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 2 mmol/L glutamine and 100 ?g/mL penicillin/streptomycin in 5% CO2 at 37?°C. After growth to confluency in media, the cells were processed for subsequent studies. The MS1 cells received 0, 0.1, 1 and 10 ?g/mL LPS in this experiment. The viability/proliferation of the cells induced by LPS was observed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. Apoptosis and necrosis of the cells under the inflammatory condition described previously were observed using Hoechst 33342-propidium iodide staining. Adenylate cyclase (AC), phospholipase A2 (PLA2), phospholipase C? (PLC?), protein tyrosine kinase (PTK), G protein-coupled receptor kinases (GRK) and p38-mitogen-activated protein kinase (p38 MAPK) mRNA in the PAFR signaling pathway were measured by real-time polymerase chain reaction. The protein expression level of phosphorylated AC (p-AC), phosphorylated PLA2 (p-PLA2), phosphorylated PTK (p-PTK), phosphorylated p38 MAPK (p-p38 MAPK), PLC? and GRK was measured using Western blotting analysis. RESULTS: The activity of MS1 cells incubated with different concentrations of LPS for 6 h decreased significantly in the 1 ?g/mL LPS group (0.49 ± 0.10 vs 0.67 ± 0.13, P < 0.05) and 10 ?g/mL LPS group (0.44 ± 0.10 vs 0.67 ± 0.13, P < 0.001), but not in 0.1 ?g/mL group. When the incubation time was extended to 12 h (0.33 ± 0.05, 0.32 ± 0.03 and 0.25 ± 0.03 vs 0.69 ± 0.01) and 24 h (0.31 ± 0.01, 0.29 ± 0.03 and 0.25 ± 0.01 vs 0.63 ± 0.01), MS1 cell activity decreased in all LPS concentration groups compared with the blank control (P < 0.001). BN52021 significantly improved the cell activity when its concentration reached 50 ?mol/L compared with the group that received LPS treatment alone, which was consistent with the results obtained from fluorescence staining. The mRNAs levels of AC (4.02 ± 0.14 vs 1.00 ± 0.13), GRK (2.63 ± 0.03 vs 1.00 ± 0.12), p38 MAPK (3.87 ± 0.07 vs 1.00 ± 0.17), PLA2 (3.31 ± 0.12 vs 1.00 ± 0.12), PLC? (2.09 ± 0.08 vs 1.00 ± 0.06) and PTK (1.85 ± 0.07 vs 1.00 ± 0.11) were up-regulated after LPS stimulation as compared with the blank control (P < 0.05). The up-regulated mRNAs including AC (2.35 ± 0.13 vs 3.87 ± 0.08), GRK (1.17 ± 0.14 vs 2.65 ± 0.12), p38 MAPK (1.48 ± 0.18 vs 4.30 ± 0.07), PLC? (1.69 ± 0.10 vs 2.41 ± 0.13) and PLA2 (1.87 ± 0.11 vs 2.96 ± 0.08) were significantly suppressed by BN52021 except for that of PTK. The level of p-AC (1.11 ± 0.12 vs 0.65 ± 0.08), GRK (0.83 ± 0.07 vs 0.50 ± 0.03), PLC? (0.83 ± 0.16 vs 0.50 ± 0.10) and p-p38 MAPK (0.74 ± 0.10 vs 0.38 ± 0.05) was up-regulated after LPS stimulation as compared with the blank control (P < 0.05). The up-regulated proteins, including p-AC (0.65 ± 0.15 vs 1.06 ± 0.14), GRK (0.47 ± 0.10 vs 0.80 ± 0.06), PLC? (0.47 ± 0.04 vs 0.80 ± 0.19) and p-p38 MAPK (0.30 ± 0.10 vs 0.97 ± 0.05), was significantly suppressed by BN52021, but p-PLA2 and p-PTK protein level were not suppressed. CONCLUSION: BN52021 could effectively inhibit LPS-induced inflammation by down-regulating the mRNA and protein levels of AC, GRK, p38 MAPK, PLA2 and PLC? in the PAFR signaling pathway. PMID:23840141

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

2013-01-01

123

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 ...

124

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)-...

125

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...

126

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

PubMed

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

Kralik, Petr; Babak, Vladimir; Dziedzinska, Radka

2014-09-01

127

Apoptosis of tumor cells in lectin-dependent lymphokine-activated killer cell-mediated cytotoxicity  

Microsoft Academic Search

By using DNA electrophoresis and propidium iodide (PI) staining flow cytometry (FACS) analysis, we studied the mechanisms\\u000a of lymphokine-activated killer (LAK) cell-mediated cytotoxicity. In the presence of pokeweed mitogen (PWM), human LAK cells\\u000a induced DNA fragmentation of two leukemic cell lines (U937 cells and Raji cells) and two solid tumor cell lines (SW1116 cells\\u000a and Hep-2 cells), a hallmark of

Dong Haidong; Xing Rong; Guo Lianying; Yang Cuihong; Qian Zhenchao

1996-01-01

128

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

PubMed

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

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

2010-03-15

129

Cell Proliferation and Death Derek Davies, Cancer Research UK  

E-print Network

Cell Proliferation and Death Derek Davies, Cancer Research UK http Cells must be permeable - can use detergent or fixation (ethanol is best) Basic protocol - fix, wash cancer cell line 0 200 400 600 800 1000 Propidium iodide 100 10 1 10 2 103 10 4 BrdUFITC 0 200 400 600

130

Kinetics of Transmembrane Transport of Small Molecules into Electropermeabilized Cells  

E-print Network

The transport of propidium iodide into electropermeabilized Chinese hamster ovary cells was monitored- ieved in the treatment of cutaneous and subcutaneous tumors, where chemotherapeutic drugs are delivered to tumor cells in combination with electric pulses (electrochemotherapy) (23­27), and another application

Ljubljana, University of

131

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

132

Morphological analysis of rat ureteric terminal arterioles in situ.  

PubMed

Confocal imaging of Fluo-4, Propidium iodide, and di-8-Anepps loaded ureter were used to study the morphology of terminal arterioles with an inner diameter <50 ?m in intact rat ureter. Optical sectioning showed that the muscle coat of the terminal arterioles consisted of a monolayer of highly curved smooth muscle cells which run circumferentially around the endothelium. This technique allowed not only to measure the inner diameter of the terminal arterioles but also to define the orientation and number of revolutions an individual smooth muscle cell made around the endothelium. We measured thickness, width, length, and morphological profile of the myocytes and endothelial cells. Propidium iodide staining showed nuclei of individual cells by continuous imaging at high resolution in serial optical sections. Conventional haematoxylin-eosin, Masson's tri-chrome staining, and transmission electron microscopy were also used in this study to compare the measurements obtained from live confocal imaging with histological standard methods. Parameters obtained from live imaging were significantly different. This technique of live staining allowed measuring the cellular and nuclear dimensions of the terminal arterioles in their natural environment which are important in studying the effects of vascular disease or aging on vascular structure. PMID:23450700

Mumtaz, Sadaf

2013-06-01

133

Comparison of results of the manual and automated scoring of micronucleus frequencies in (60)Co-irradiated peripheral blood lymphocytes for triage dosimetry.  

PubMed

Scoring micronuclei in the peripheral blood lymphocytes of individuals exposed to ionizing radiation is a rapid biodosimetry assay. Peripheral blood lymphocytes from five individuals were exposed in vitro to 0-5Gy of (60)Co ?-radiation at a dose rate of 0.76Gy/min. The blood cultures were initiated with RPMI-1640 (80%) supplemented with FBS (20%), stimulated with mitogen and incubated at 37°C for 44h. At the 44th hour, cytochalasin-B (6µg/mL) was added, and the cultures were incubated for 28h more. The cells were harvested with a pre-chilled hypotonic solution (0.075M) and fixed with a Carnoy's solution (methanol/acetic acid 5:1). Giemsa- and propidium-iodide-stained cells affixed to slides for microscopy were scored manually and automatically with the micronucleus scoring software from MetaSystems. The micronucleus frequencies determined in the Giemsa-stained cells by manual and automated scoring were 23.6% different (P<0.0001) with an efficiency of 24.9%. Slides stained with propidium iodide are a better choice for automated scoring than Giemsa-stained ones. PMID:25544665

Tamizh Selvan, G; Chaudhury, N K; Venkatachalam, P

2015-03-01

134

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

135

The effect of sodium hypochlorite and chlorhexidine on cultured human periodontal ligament cells  

Microsoft Academic Search

Objective: The objective of this study was to examine the effects of sodium hypochlorite (NaOCl) and chlorhexidine (CHx) on cultured human periodontal ligament (PDL) cells in vitro. Study Design: The effects of irrigation solutions on human PDL cells were evaluated by propidium iodide fluorescence cytotoxicity assay, protein synthesis assay, and mitochondrial activity. Results: Both NaOCl and CHx were cytotoxic to

Yu-Chao Chang; Fu-Mei Huang; Kuo-Wei Tai; Ming-Yung Chou

2001-01-01

136

Flow cytometric quantitation of phagocytosis in heparinized complete blood with latex particles and Candida albicans.  

PubMed

We report a rapid method for the flow cytometric quantitation of phagocytosis in heparinized complete peripheral blood (HCPB), using commercially available phycoerythrin-conjugated latex particles of 1 micron diameter. The method is faster and shows greater reproducibility than Bjerknes' (1984) standard technique using propidium iodide-stained Candida albicans, conventionally applied to the leukocytic layer of peripheral blood but here modified for HCPB. We also report a modification of Bjerknes' Intracellular Killing Test to allow its application to HCPB. PMID:9428182

Egido, J M; Viñuelas, J

1997-01-01

137

Genetic diversity of viable, injured, and dead fecal bacteria assessed by fluorescence-activated cell sorting and 16S rRNA gene analysis  

Microsoft Academic Search

A novel approach combining a flow cytometric in situ viability assay with 16S rRNA gene analysis was used to study the relationship between diversity and activity of the fecal microbiota. Simultaneous staining with propidium iodide (PI) and SYTO BC provided clear discrimination between intact cells (49%), injured or damaged cells (19%), and dead cells (32%). The three subpopulations were sorted

Kaouther Ben-Amor; G. H. J. Heilig; Hauke Smidt; Elaine E. Vaughan; Tjakko Abee; Vos de W. M

2005-01-01

138

Relationship between Membrane Permeability and Specificity of Human Secretory Phospholipase A2 Isoforms during Cell Death  

PubMed Central

Summary During apoptosis, a number of physical changes occur in the cell membrane including a gradual increase in permeability to vital stains such as propidium iodide. This study explored the possibility that one consequence of membrane changes concurrent with early modest permeability is vulnerability to degradation by secretory phospholipase A2 (sPLA2). The activity of this hydrolytic enzyme toward mammalian cells depends on the health of the cell; healthy cells are resistant, but they become susceptible early during programmed death. Populations of S49 lymphoma cells during programmed death were classified by flow cytometry based on permeability to propidium iodide and susceptibility to sPLA2. The apoptotic inducers thapsigargin and dexamethasone caused modest permeability to propidium iodide and increased staining by merocyanine 540, a dye sensitive to membrane perturbations. Various sPLA2 isozymes (human groups IIa, V, X, and snake venom) preferentially hydrolyzed the membranes of cells that displayed enhanced permeability. In contrast, cells exposed briefly to a calcium ionophore showed the increase in cell staining intensity by merocyanine 540 without accompanying uptake of propidium iodide. Under that condition, only the snake venom and human group × enzymes hydrolyzed cells that were dying. These results suggested that cells showing modest permeability to propidium iodide during the early phase of apoptosis are substrates for sPLA2 and that specificity among isoforms of the enzyme depends on the degree to which the membrane has been perturbed during the death process. This susceptibility to hydrolysis may be important as part of the signal to attract macrophages toward apoptotic cells. PMID:21510917

Nelson, Jennifer; Gibbons, Elizabeth; Pickett, Katalyn R.; Streeter, Michael; Warcup, Ashley O.; Yeung, Celestine H.-Y.; Judd, Allan M.; Bell, John D.

2011-01-01

139

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

PubMed

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

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

2011-11-01

140

IL-29 BINDS TO MELANOMA CELLS INDUCING JAK-STAT SIGNAL TRANSDUCTION AND APOPTOSIS  

PubMed Central

Purpose Interlukin-29 (IL-29) is a member of the type III interferon (IFN) family that has been shown to have antiviral activity and inhibit cell growth. Melanoma cell lines were tested for the expression of the IL-29R and their response to IL-29. Methods Expression of IL-28R1 and IL-10R2, components of the IL-29R, was evaluated using RT-PCR. A combination of immunoblot analysis and flow cytometry was used to evaluate IL-29-induced signal transduction. U133 Plus 2.0 Arrays and real-time PCR were used to evaluate gene expression. Apoptosis was measured using Annexin V/Propridium Iodide staining. In situ PCR for the IL-29R was performed on paraffin-embedded melanoma tumors. Results Both IL-28R1 and IL-10R2 were expressed on the A375, 1106 MEL, Hs294T, 18105 MEL, MEL 39, SK MEL 5 and F01 cell lines. Incubation of melanoma cell lines with IL-29 (10–1000 ng/mL) led to phosphorylation of STAT1 and STAT2. Microarray analysis and qRT-PCR showed a marked increase in transcripts of IFN-regulated genes after treatment with IL-29. In the F01 cell line, bortezomib-induced and temozolomide-induced apoptosis was synergistically enhanced following the addition of IL-29. In situ PCR revealed that IL-10R2 and IL-28R1 were present in six of eight primary human melanoma tumors but were not present in benign nevi specimens. Conclusion IL-29 receptors are expressed on the surface of human melanoma cell lines and patient samples and treatment of these cell lines with IL-29 leads to signaling via the Jak-STAT pathway, the transcription of a unique set of genes, and apoptosis. PMID:20103601

Guenterberg, Kristan D.; Grignol, Valerie P.; Raig, Ene T.; Zimmerer, Jason M.; Chan, Anthony N.; Blaskovits, Farriss M.; Young, Gregory S.; Nuovo, Gerard J.; Mundy, Bethany L.; Lesinski, Gregory B.; Carson, William E.

2010-01-01

141

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

PubMed Central

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

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

2015-01-01

142

Induction of death receptor ligand -mediated apoptosis in epithelial ovarian carcinoma: the search for sensitizing agents  

PubMed Central

Objective To assess the abilities of cisplatin, paclitaxel and flexible heteroarotinoid (Flex-Het) compound (SHetA2) to sensitize ovarian cancer cells to induction of the extrinsic apoptosis pathway by death receptor ligands, tumor necrosis factor ? (TNF?) and TNF-related apoptosis-inducing ligand (TRAIL). Study Design The effects of various combinations of TNF?, TRAIL, cisplatin, paclitaxel and SHetA2 on viability and apoptosis in two established ovarian cancer cell lines, A2780 and SKOV3, and normal human primary endometrial cultures were measured with a cytotoxicity assay, flow cytometric analysis of annexin-V and propidium iodide staining and Western blot analysis of caspase 8 and 3 activation. Results Ovarian cancer and normal cells were resistant to TNF? and TRAIL. Cisplatin and paclitaxel did not increase sensitivity to these agents in either cell type. In contrast, combination of SHetA2 with TNF? or TRAIL induced a synergistic induction of apoptosis in cancer cells that involved activation of the extrinsic pathway caspase 8 and executioner caspase 3. The TRAIL combination was more potent than the TNF? combination. SHetA2 did not harm the viability of normal cells as a single agent or in combination with the death receptor ligands. Conclusions SHetA2, but not cisplatin or paclitaxel, can overcome resistance of ovarian cancer cells to TNF? and TRAIL without increasing sensitivity of normal cells to these death receptor ligands. PMID:19804900

Moxley, KM; Chengedza, S; Benbrook, DM

2009-01-01

143

Determination of yeast viability during a stress-model alcoholic fermentation using reagent-free microscopy image analysis.  

PubMed

A dedicated microscopy imaging system including automated positioning, focusing, image acquisition, and image analysis was developed to characterize a yeast population with regard to cell morphology. This method was used to monitor a stress-model alcoholic fermentation with Saccharomyces cerevisiae. Combination of dark field and epifluorescence microscopy after propidium iodide staining for membrane integrity showed that cell death went along with important changes in cell morphology, with a cell shrinking, the onset of inhomogeneities in the cytoplasm, and a detachment of the plasma membrane from the cell wall. These modifications were significant enough to enable a trained human operator to make the difference between dead and viable cells. Accordingly, a multivariate data analysis using an artificial neural network was achieved to build a predictive model to infer viability at single-cell level automatically from microscopy images without any staining. Applying this method to in situ microscope images could help to detect abnormal situations during a fermentation course and to prevent cell death by applying adapted corrective actions. PMID:21290616

Tibayrenc, Pierre; Ghommidh, Charles; Preziosi-Belloy, Laurence

2011-01-01

144

Synthesis and pharmacological evaluation of novel bisindole derivatives bearing oximes moiety: Identification of novel proapoptotic agents.  

PubMed

In an effort to develop potent anti-cancer chemopreventive agents, a novel series of bisindole derivatives bearing oxime moiety were synthesized. Structures of all compounds were characterized by NMR and HRMS. Anti-proliferative activities for all of these compounds were investigated by the method of MTT assay on 7 human cancer lines and the normal cell lines (HUVEC). Most of them showed a noteworthy anti-cancer activity in vitro, the half maximal inhibitory concentration (IC50) value is 4.31 ?M of 4e against T24. The results from Hoechst 33258 and acridine orange/propidium iodide staining as well as annexinV-FITC assays provided evidence for an apoptotic cell death. The further mechanisms of compound 4e-induced apoptosis in T24 cells demonstrated that compound 4e induced the productions of ROS, and altered anti- and pro-apoptotic proteins, leading to mitochondrial dysfunction and activations of caspase-9 and caspase-3 for causing cell apoptosis. Moreover, the cell cycle analysis and western-blot analysis indicated that compound 4e effectively arrested T24 cells in G1 stage and possibly has an effect on cell cycle regulatory proteins particularly cyclin D1. PMID:25841196

Qu, Hong-En; Huang, Ri-Zhen; Yao, Gui-Yang; Li, Jiu-Ling; Ye, Man-Yi; Wang, Heng-Shan; Liu, Liangxian

2015-05-01

145

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

146

Fluorescein isothiocyanate staining intensity as a probe of hyperthermia-induced changes in chromatin conformation.  

PubMed

In a previous report we presented evidence for large increases in fluorescein isothiocyanate (FITC) fluorescent intensity caused by hyperthermia which were not associated with synthesis of heat-shock proteins. We have now refined and considerably extended the measurements of increases in FITC fluorescent intensity caused by hyperthermia within the range 41.0 degrees C to 50.0 degrees C, and associated these with the extent of cell death caused by the hyperthermia. It appears that cell death ensues when the FITC fluorescent intensity has not returned to its baseline value within the time of one cell cycle. If thermotolerance is induced, there is a concomitant reduction in the increase in FITC staining intensity and the extent of cell death. When hyperthermia is followed by acid extraction, an additional increase in FITC staining intensity (above that due to hyperthermia alone) is observed, indicating separate sites of action on basic nuclear proteins. Hyperthermia and acid extraction have related effects on the relationship between FITC and propidium iodide staining. Hyperthermia-induced increases in FITC staining intensity are almost completely reversed by 6.7 mM formaldehyde with a marginal effect on the control FITC staining at this formaldehyde concentration. We suggest that hyperthermia causes extensive dissociation of basic protein-protein binding within nuclear chromatin, and that this may be a contributory cause of hyperthermia-induced cell death. PMID:2469557

Dyson, J E; Britten, R A; Battersby, I; Surrey, C R

1989-03-01

147

Multicellular spheroid formation and evolutionary conserved behaviors of apple snail hemocytes in culture.  

PubMed

A hemocyte primary culture system for Pomacea canaliculata in a medium mimicking hemolymphatic plasma composition was developed. Hemocytes adhered and spread onto culture dish in the first few hours after seeding but later began forming aggregates. Time-lapse video microscopy showed the dynamics of the early aggregation, with cells both entering and leaving the aggregates. During this period phagocytosis occurs and was quantified. Later (>4 h), hemocytes formed large spheroidal aggregates that increased in size and also merged with adjacent spheroids (24-96 h). Large single spheroids and spheroid aggregates detach from the bottom surface and float freely in the medium. Correlative confocal, transmission electron and phase contrast microscopy showed a peculiar organization of the spheroids, with a compact core, an intermediate zone with large extracellular lacunae and an outer zone of flattened cells; also, numerous round cells emitting cytoplasmic extensions were seen attaching to the spheroids' smooth surface. Dual DAPI/propidium iodide staining revealed the coexistence of viable and non-viable cells within aggregates, in varying proportions. DNA concentration increased during the first 24 h of culture and stabilized afterward. BrdU incorporation also indicated proliferation. Spontaneous spheroid formation in culture bears interesting parallels with spheroidal hemocyte aggregates found in vivo in P. canaliculata, and also with spheroids formed by tumoral or non-tumoral mammalian cells in vitro. PMID:23246811

Cueto, Juan A; Vega, Israel A; Castro-Vazquez, Alfredo

2013-02-01

148

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

NASA Astrophysics Data System (ADS)

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

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

2010-02-01

149

Pro-oxidants ameliorate radiation-induced apoptosis through activation of the calcium-ERK1/2-Nrf2 pathway.  

PubMed

There are no reports describing the ability of pro-oxidants to protect against radiation-induced apoptosis. Activation of the redox-sensitive transcription factor Nrf2 by low levels of ROS is known to protect against oxidative stress-induced cell death. In this study, hydrogen peroxide, diethylmaleate, and 1,4-naphthoquinone (NQ) exhibited complete protection against radiation-induced cell death in lymphocytes as estimated by propidium iodide staining. Radioprotection by NQ was demonstrated by inhibition of caspase activation, decrease in cell size, DNA fragmentation, nuclear blebbing, and clonogenic assay. Interestingly, NQ offered protection to lymphocytes even when added to cells postirradiation. NQ increased intracellular ROS levels and decreased GSH levels. NQ activated Nrf2 and increased the expression of the cytoprotective gene heme oxygenase-1 in lymphocytes. NQ increased ERK phosphorylation, which is upstream of Nrf2, and this ERK activation was through increased intracellular calcium levels. Administration of NQ to mice offered protection against whole-body irradiation (WBI)-induced apoptosis in splenic lymphocytes and loss of viability of spleen and bone marrow cells. It restored WBI-mediated changes in hematological parameters and functional responses of lymphocytes. Importantly, NQ rescued mice against WBI-induced mortality. These results demonstrated that a pro-oxidant such as NQ can protect against radiation-induced apoptosis by activation of multiple prosurvival mechanisms including activation of the calcium-ERK1/2-Nrf2 pathway. PMID:21530647

Khan, Nazir M; Sandur, Santosh K; Checker, Rahul; Sharma, Deepak; Poduval, T B; Sainis, Krishna B

2011-07-01

150

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

151

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

PubMed

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

Liang, Zhanbei; Keeley, Ann

2012-11-15

152

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

153

NF-?B inhibition compromises cardiac fibroblast viability under hypoxia  

PubMed Central

Cardiac fibroblasts are reported to be relatively resistant to stress stimuli compared to cardiac myocytes and fibroblasts of non-cardiac origin. However, the mechanisms that facilitate their survival under conditions of stress remain unclear. We explored the possibility that NF-?B protects cardiac fibroblasts from hypoxia-induced cell death. Further, we examined the expression of the anti- apoptotic cIAP-2 and Bcl-2 in hypoxic cardiac fibroblasts, and their possible regulation by NF-?B. Phase contrast microscopy and propidium iodide staining revealed that cardiac fibroblasts are more resistant than pulmonary fibroblasts to hypoxia. Electrophoretic Mobility Shift Assay showed that hypoxia activates NF-?B in cardiac fibroblasts. Supershift assayindicated that the active NF-?B complex is a p65/p50 heterodimer. An I-?B-super-repressor was constructed that prevented NF-?B activation and compromised cell viability under hypoxic but not normoxic conditions. Similar results were obtained with Bay 11-7085, an inhibitor of NF-?B. Western blot analysis showed constitutive levels of Bcl-2 and hypoxic induction of cIAP-2 in these cells. NF-?B inhibition reduced cIAP-2 but not Bcl-2 levels in hypoxic cardiac fibroblasts. The results show for the first time that NF-?B is an important effector of survival in cardiac fibroblasts under hypoxic stress and that regulation of cIAP-2 expression may contribute to its pro-survival role. PMID:21211536

Sangeetha, M; Pillai, Malini S; Philip, Linda; Lakatta, Edward G; Shivakumar, K

2011-01-01

154

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

155

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

PubMed

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

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

2014-09-15

156

A cyclic peptide accelerates the loading of peptide antigens in major histocompatibility complex class II molecules.  

PubMed

Major histocompatibility complex (MHC)-loading enhancers (MLE) have recently attracted attention because of their ability to enhance the efficacy of peptide immunotherapeutics. As small molecular weight compounds, they influence the loading of peptides in MHC molecules by converting them from a non-receptive to a receptive state. Herein, we report a 14-mer cyclic peptide 1 (CP-1) as a new class of MLE-peptide. This peptide was used to investigate its loading on human leukocyte antigen (HLA)-DR molecules. It was found that CP-1 strongly accelerates peptide-loading on both soluble and cell surface HLA-DR molecules in a dose-dependent manner. The effect was evident for all subsets of HLA-DR tested, including HLA-DRB1*1501, indicating that it acts independently of P1-pocket size, which is the canonical MLE-binding site. Importantly, increased peptide-loading by CP-1 was correlated with improved CD4(+) T cell responses in vitro, while propidium iodide staining indicated low peptide-induced cytotoxicity. Thus, this study revealed a new class of peptide-based enhancers that catalyze peptide-loading by allosteric interactions with MHC molecules. Because of its low cellular cytotoxicity and high MLE activity, it may be useful in stimulating antigen-specific T cell responses for therapeutic purposes. PMID:25522880

Afridi, Saifullah; Shaheen, Farzana; Roetzschke, Olaf; Shah, Zafar Ali; Abbas, Syed Comail; Siraj, Rizwana; Makhmoor, Talat

2015-01-16

157

VEGF depletion enhances bcr-abl-specific sensitivity of arsenic trioxide in chronic myelogenous leukemia.  

PubMed

The development of resistance to imatinib mesylate may partly depend on high bcr-abl expression levels or point mutation(s). Arsenic trioxide (ATO) has bcr-abl suppressing activity in vitro, without cross-resistance to imatinib. Meanwhile, bcr-abl also induces expression of vascular endothelial growth factor (VEGF), which is associated with tumor-related angiogenesis and is involved in chronic myelogenous leukemia (CML) pathogenesis. Here, we investigated ways to improve ATO activity in CML by modulating cellular VEGF levels. K562 and primary CML cells were transfected with a VEGF antisense sequence. Cell viability and survival were assessed using 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide and trypan blue exclusion assays. Apoptotic cells were detected by flow cytometry following annexin V and propidium iodide staining. The results showed that VEGF depletion effectively promotes enhanced ATO antileukemic activity by repressing bcr-abl protein levels. These data provide a rationale for the clinical development of optimized ATO-based regimens that incorporate VEGF modulator for CML treatment. PMID:24129092

Luo, Xiaochuang; Feng, Maoxiao; Zhu, Xuejiao; Li, Yumin; Fei, Jia; Zhang, Yuan

2013-11-01

158

Biofilm formation by Escherichia coli in hypertonic sucrose media.  

PubMed

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

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

2009-06-01

159

Inhibition of S-phase kinase-associated protein 2-mediated p27 degradation suppresses tumorigenesis and the progression of hepatocellular carcinoma.  

PubMed

In order to determine the protein expression of S?phase kinase?associated protein 2 (Skp2) and p27kip1, and to evaluate their possible prognostic values in malignant liver cancer, tissue samples from 50 patients and 40 controls were assessed and analyzed by immunohistochemistry and western blot analysis. Positive expression of Skp2 was observed in 35 (70.0%) of the hepatocellular carcinoma samples; however, the positive expression of p27kip1 was observed in 6 (15.0%) of the hepatocellular carcinoma samples. The expression of Skp2 was significantly negatively correlated with the expression of p27 (P<0.01). The results from Annexin V?propidium iodide staining and MTT assays indicated that interference of Skp2 significantly induced apoptosis and inhibited the proliferation of SSMC?7721 cells. In addition, the levels of endogenous p27 increased in the HepG2 and SSMC?7721 cells following transfection with siRNA specific to Skp2, suggesting that the Skp2?mediated degradation of p27kip1 was important in the proliferation of tumor cells. The present study, therefore, provided a molecular reference for the treatment of liver cancer. PMID:25572801

Qi, Ming; Liu, Dongmei; Zhang, Shuhong; Hu, Peixin; Sang, Tan

2015-05-01

160

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

NASA Astrophysics Data System (ADS)

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

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

2015-01-01

161

Proteinase 3 induces oxidative stress-mediated neuronal death in rat primary cortical neuron.  

PubMed

The recruitment of neutrophils into the cerebral microcirculation occurs, especially, in acute brain diseases like a focal cerebral ischemia and plays important role in pathological processes. Proteinase 3 is one of the three major proteinases expressed in neutrophils but no reports are available whether proteinase 3 can modulate neuronal survival. In this study, treatment of cultured rat primary cortical neuron with proteinase 3 induced overt reactive oxygen species production and decreased total glutathione contents as well as disruption of mitochondrial transmembrane potential. Proteinase 3 induced neuronal cell death as evidenced by MTT analysis as well as propidium iodide staining, which was prevented by pretreatment with an antioxidant, N-acetyl cysteine. Proteinase 3 increased activation of procaspase-3 and altered expression level of apoptotic regulator proteins, such as Bcl-2, Bax, and Bcl-xL. Similar to in vitro data, a direct microinjection of proteinase 3 into striatum of rat brain induced neuronal death, which was mediated by reactive oxygen species. These results suggest that proteinase 3 is new essential regulator of neuronal cell death pathway in a condition of excess neutrophil encounter in neuroinflammatory conditions. PMID:23748041

Kwon, Kyoung Ja; Cho, Kyu Suk; Kim, Jung Nam; Kim, Min Kyeong; Lee, Eun Joo; Kim, Soo Young; Jeon, Se Jin; Kim, Ki Chan; Han, Jeong Eun; Kang, Young Sun; Kim, Soohyun; Kim, Hahn Young; Han, Seol-Heui; Bahn, Geonho; Choi, Ji woong; Shin, Chan Young

2013-08-26

162

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

PubMed

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

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

2012-04-01

163

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

NASA Astrophysics Data System (ADS)

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

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

2011-02-01

164

Comparison of assessment of pigeon sperm viability by contrast-phase microscope (eosin-nigrosin staining) and flow cytometry (SYBR-14/propidium iodide (PI) staining) [evaluation of pigeon sperm viability].  

PubMed

The aim of these experiments was to compare the conventional, microscopic method of evaluating pigeon sperm viability to sperm assessed by flow cytometry. Semen was collected twice a week from two groups of pigeons. In every group were 20 males (Group I: meat-type breed; Group II: fancy pigeon breed). Semen was collected using the lumbosacral and cloacal region massage method. Ejaculates collected from each group were pooled and diluted to 10 × 10(6) sperm/ml in BPSE solution. Samples were divided into three equal parts and estimated after collection as well as after in vitro storage for 3, 6 and 24 h. The first part was using for semen motility evaluation. The proportion of motile spermatozoa (MOT) and progressive movement (PMOT) of fresh and stored semen were evaluated using the CASA-system. The second part was examined subjectively by microscope (eosin-nigrosin (EN), eosin-nigrosin staining), the third one was assessed using dual fluorescence SYBR-14/propidium iodide (PI) and flow cytometry (FC). There were not any significant differences in sperm viability and motility between the groups at 0, 3, 6, and 24 h post collection. The percentage of viable spermatozoa in fresh semen determined by EN and FC was not different in Groups I and II (I - 88.71 ± 5.42 and 84.01 ± 3.19, respectively; II-90.87 ± 6.01 and 87.38 ± 5.57, respectively). Significantly lower percentages of viable spermatozoa were detected by FC compared to the EN method in both groups after 6 h (P ? 0.05) as well as 24 h (P ? 0.01) of storage. Moreover, the dual fluorescent SYBR-14/PI staining allowed for the identification a third population of double stained, moribund spermatozoa. High positive correlations in percentage of live spermatozoa were noted between EN and FC methods in both groups of birds. Evaluation of sperm viability by FC is a rapid, accurate, sensitive, and objective method for the assessment of pigeon sperm viability in fresh as well as stored semen. PMID:22056017

Klimowicz-Bodys, M D; Batkowski, F; Ochrem, A S; Savi?, M A

2012-02-01

165

The mechanism of UVB irradiation induced-apoptosis in cataract.  

PubMed

Cataract is the most common eye disease that causes blindness in patients. Ultraviolet B (UVB) irradiation is considered an important factor leading to cataract by inducing apoptosis in human lens epithelial cells (HLECs), but the mechanism is currently unclear. In this study, we investigated HLECs under different intensities of UVB irradiation and different exposure time. The annexin V-FITC/propidium iodide staining results showed that UVB irradiation could efficiently lead to HLECs apoptosis in time- and dose-dependent manner. The expression of pro-apoptotic Bax gene was promoted by UVB irradiation, while anti-apoptotic Bcl-2 gene expression was inhibited at both transcript and protein levels. Notably, the ratio of Bax/Bcl-2 displayed a high and positive correlation to the proportion of apoptotic HLECs. Mitochondrial dysfunction was also observed with rapid loss of potential (?? m), as well as changes of the levels of reactive oxygen species, malondialdehyde, total antioxidative capabilities, and superoxide dismutase. In caspase pathway, the level of caspase-3 protein increased after UVB irradiation. All these discovered changes may play important roles in UVB-induced HLECs apoptosis, and would be helpful in understanding the mechanism of UVB-induced cataract and providing potential prevention and treatment strategies. PMID:25445170

Ji, Yinghong; Cai, Lei; Zheng, Tianyu; Ye, Hongfei; Rong, Xianfang; Rao, Jun; Lu, Yi

2015-03-01

166

Accumulation of Amphotericin B in Human Macrophages Enhances Activity against Aspergillus fumigatus Conidia: Quantification of Conidial Kill at the Single-Cell Level  

Microsoft Academic Search

A cytofluorometric assay that allowed assessment of damage to phagocytosed Aspergillus fumigatus conidia at the single-cell level was developed. After ingestion by monocyte-derived macrophages (MDMs), conidia were reisolated by treatment of the cells with streptolysin O, a pore-forming toxin with lytic properties on mam- malian cells but not on fungi. The counts obtained by staining of damaged conidia with propidium

BERNHARD JAHN; ALBERT RAMPP; CHRISTIAN DICK; ANDREAS JAHN; MICHAEL PALMER; SUCHARIT BHAKDI

1998-01-01

167

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

168

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

169

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

170

Heterogeneity in Pseudomonas aeruginosa Biofilms Includes Expression of Ribosome Hibernation Factors in the Antibiotic-Tolerant Subpopulation and Hypoxia-Induced Stress Response in the Metabolically Active Population  

PubMed Central

Bacteria growing in biofilms are physiologically heterogeneous, due in part to their adaptation to local environmental conditions. Here, we characterized the local transcriptome responses of Pseudomonas aeruginosa growing in biofilms by using a microarray analysis of isolated biofilm subpopulations. The results demonstrated that cells at the top of the biofilms had high mRNA abundances for genes involved in general metabolic functions, while mRNA levels for these housekeeping genes were low in cells at the bottom of the biofilms. Selective green fluorescent protein (GFP) labeling showed that cells at the top of the biofilm were actively dividing. However, the dividing cells had high mRNA levels for genes regulated by the hypoxia-induced regulator Anr. Slow-growing cells deep in the biofilms had little expression of Anr-regulated genes and may have experienced long-term anoxia. Transcripts for ribosomal proteins were associated primarily with the metabolically active cell fraction, while ribosomal RNAs were abundant throughout the biofilms, indicating that ribosomes are stably maintained even in slowly growing cells. Consistent with these results was the identification of mRNAs for ribosome hibernation factors (the rmf and PA4463 genes) at the bottom of the biofilms. The dormant biofilm cells of a P. aeruginosa ?rmf strain had decreased membrane integrity, as shown by propidium iodide staining. Using selective GFP labeling and cell sorting, we show that the dividing cells are more susceptible to killing by tobramycin and ciprofloxacin. The results demonstrate that in thick P. aeruginosa biofilms, cells are physiologically distinct spatially, with cells deep in the biofilm in a viable but antibiotic-tolerant slow-growth state. PMID:22343293

Williamson, Kerry S.; Richards, Lee A.; Perez-Osorio, Ailyn C.; Pitts, Betsey; McInnerney, Kathleen; Stewart, Philip S.

2012-01-01

171

Concurrent use of flow cytometry and fluorescence in-situ hybridization techniques for detecting faulty meiosis in a human sperm sample.  

PubMed

Routine semen analysis in an infertile patient revealed severe teratospermia associated with malformation of head and tail in 100% of the sperm cells. Flow cytometry and fluorescence in-situ hybridization (FISH) were shown to supplement routine semen analysis by providing information on the sperm chromatin. Using flow cytometry, propidium iodide-stained spermatozoa from the same sperm sample were compared with a normal reference pool, and with human lymphocytes. The results point to a population of diploid sperm cells rather than to mature haploid spermatozoa. Numerical chromosomal abnormalities of the spermatozoa were subsequently evaluated using FISH. A total of 1000 sperm cells were scored for X and Y chromosomes, and an additional 1128 sperm cells for chromosome 18. Aneuploidy of chromosomes X and Y was revealed in 96.9% of the cells and of chromosome 18 in 90.3% of the cells. Non-disjunction of chromosome X and Y in meiosis I and II occurred in 54.8 and 2.7% of the sperm cells respectively. Non-disjunction in both meiosis I and II occurred in 39.4% of the sperm cells. A normal haploid pattern for chromosomes X and Y was observed in only 3.1%, and for chromosome 18 in 9.7%, of the cells. Using three colour FISH for the sex chromosomes and for chromosome 18, diploidy was demonstrated in 19.4% of 500 sperm cells and aneuploidy in virtually all sperm cells (99.2%). The use of flow cytometry and FISH in cases where genetic and developmental chromatin abnormalities are suspected is a valuable adjunct to other available techniques, and can guide the clinicians to decide which samples are unsuitable for intracytoplasmic injection. PMID:9510012

Weissenberg, R; Aviram, A; Golan, R; Lewin, L M; Levron, J; Madgar, I; Dor, J; Barkai, G; Goldman, B

1998-01-01

172

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

173

Acute plasmalemma permeability and protracted clearance of injured cells after controlled cortical impact in mice  

PubMed Central

Cell death after traumatic brain injury (TBI) evolves over days to weeks. Despite advances in understanding biochemical mechanisms that contribute to posttraumatic brain cell death, the time course of cell injury, death, and removal remains incompletely characterized in experimental TBI models. In a mouse controlled cortical impact (CCI) model, plasmalemma permeability to propidium iodide (PI) was an early and persistent feature of posttraumatic cellular injury in cortex and hippocampus. In cortical and hippocampal brain regions known to be vulnerable to traumatic cell death, the number of PI + cells peaked early after CCI, and increased with increasing injury severity in hippocampus but not cortex (P < 0.05). Propidium iodide labeling correlated strongly with hematoxylin and eosin staining in injured cells (r = 0.99, P < 0.001), suggesting that plasmalemma damage portends fatal cellular injury. Using PI pulse labeling to identify and follow the fate of a cohort of injured cells, we found that many PI + cells recovered plasmalemma integrity by 24 h and were terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling negative, but nonetheless disappeared from injured brain by 7 days. Propidium iodide-positive cells in dentate gyrus showed significant ultrastructural damage, including plasmalemma and nuclear membrane damage or overt membrane loss, in all cells when examined by laser capture microdissection and transmission electron microscopy 1 to 24 h after CCI. The data suggest that plasmalemma damage is a fundamental marker of cellular injury after CCI; some injured cells might have an extended window for potential rescue by neuroprotective strategies. PMID:17713463

Whalen, Michael J; Dalkara, Turgay; You, Zerong; Qiu, Jianhua; Bermpohl, Daniela; Mehta, Niyati; Suter, Bernhard; Bhide, Pradeep G; Lo, Eng H; Ericsson, Maria; Moskowitz, Michael A

2009-01-01

174

Biodegradable CaMgZn bulk metallic glass for potential skeletal application.  

PubMed

A low density and high strength alloy, Ca65Mg15Zn20 bulk metallic glass (CaMgZn BMG), was evaluated by both in vitro tests on ion release and cytotoxicity and in vivo implantation, aimed at exploring the feasibility of this new biodegradable metallic material for potential skeletal applications. MTT assay results showed that the experimental CaMgZn BMG extracts had no detectable cytotoxic effects on L929, VSMC and ECV304 cells over a wide range of concentrations (0-50%), whereas for MG63 cells concentrations in the range ~5-20% promoted cell viability. Meanwhile, alkaline phosphatase (ALP) activity results showed that CaMgZn BMG extracts increased alkaline phosphatase (ALP) production by MG63 cells. However, Annexin V-fluorescein isothiocyanate and propidium iodide staining indicated that higher concentrations (50%) might induce cell apoptosis. The fluorescence observation of F-actin and nuclei in MG63 cells showed that cells incubated with lower concentrations (0-50%) displayed no significant change in morphology compared with a negative control. Tumor necrosis factor-? expression by Raw264.7 cells in the presence of CaMgZn BMG extract was significantly lower than that of the positive and negative controls. Animal tests proved that there was no obvious inflammation reaction at the implantation site and CaMgZn BMG implants did not result in animal death. The cortical thickness around the CaMgZn BMG implant increased gradually from 1 to 4 weeks, as measured by in vivo micro-computer tomography. PMID:21571105

Wang, Y B; Xie, X H; Li, H F; Wang, X L; Zhao, M Z; Zhang, E W; Bai, Y J; Zheng, Y F; Qin, L

2011-08-01

175

Kinetics of plasma membrane and mitochondrial alterations in cells undergoing apoptosis  

SciTech Connect

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

Lizard, G.; Fournel, S.; Genestier, L.; Dhedin, N. [Hospital Edouard Herriot, Lyon (France)] [and others

1995-11-01

176

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

177

ATP mediates neuroprotective and neuroproliferative effects in mouse olfactory epithelium following exposure to satratoxin G in vitro and in vivo.  

PubMed

Intranasal aspiration of satratoxin G (SG), a mycotoxin produced by the black mold Stachybotrys chartarum, selectively induces apoptosis in olfactory sensory neurons (OSNs) in mouse olfactory epithelium (OE) through unknown mechanisms. Here, we show a dose-dependent induction of apoptosis 24 h post-SG exposure in vitro as measured by increased activated caspases in the OP6 olfactory placodal cell line and increased propidium iodide staining in primary OE cell cultures. Intranasal aspiration of SG increased TUNEL (Terminal dUTP Nick End Labeling) staining in the neuronal layer of the OE and significantly increased the latency to find a buried food pellet, confirming that SG selectively induces neuronal apoptosis and demonstrating that SG impairs the sense of smell. Next, we investigated whether ATP can prevent SG-induced OE toxicity. ATP did not decrease apoptosis under physiological conditions but significantly reduced SG-induced OSN apoptosis in vivo and in vitro. Furthermore, purinergic receptor inhibition significantly increased apoptosis in OE primary cell culture and in vivo. These data indicate that ATP is neuroprotective against SG-induced OE toxicity. The number of cells that incorporated 5'-bromodeoxyuridine, a measure of proliferation, was significantly increased 3 and 6 days post-SG aspiration. Treatment with purinergic receptor antagonists significantly reduced SG-induced cell proliferation, whereas post-treatment with ATP significantly potentiated SG-induced cell proliferation. These data indicate that ATP is released and promotes cell proliferation via activation of purinergic receptors in SG-induced OE injury. Thus, the purinergic system is a therapeutic target to alleviate or restore the loss of OSNs. PMID:21865290

Jia, Cuihong; Sangsiri, Sutheera; Belock, Bethany; Iqbal, Tania R; Pestka, James J; Hegg, Colleen C

2011-11-01

178

ATP Mediates Neuroprotective and Neuroproliferative Effects in Mouse Olfactory Epithelium following Exposure to Satratoxin G In Vitro and In Vivo  

PubMed Central

Intranasal aspiration of satratoxin G (SG), a mycotoxin produced by the black mold Stachybotrys chartarum, selectively induces apoptosis in olfactory sensory neurons (OSNs) in mouse olfactory epithelium (OE) through unknown mechanisms. Here, we show a dose-dependent induction of apoptosis 24 h post-SG exposure in vitro as measured by increased activated caspases in the OP6 olfactory placodal cell line and increased propidium iodide staining in primary OE cell cultures. Intranasal aspiration of SG increased TUNEL (Terminal dUTP Nick End Labeling) staining in the neuronal layer of the OE and significantly increased the latency to find a buried food pellet, confirming that SG selectively induces neuronal apoptosis and demonstrating that SG impairs the sense of smell. Next, we investigated whether ATP can prevent SG-induced OE toxicity. ATP did not decrease apoptosis under physiological conditions but significantly reduced SG-induced OSN apoptosis in vivo and in vitro. Furthermore, purinergic receptor inhibition significantly increased apoptosis in OE primary cell culture and in vivo. These data indicate that ATP is neuroprotective against SG-induced OE toxicity. The number of cells that incorporated 5?-bromodeoxyuridine, a measure of proliferation, was significantly increased 3 and 6 days post-SG aspiration. Treatment with purinergic receptor antagonists significantly reduced SG-induced cell proliferation, whereas post-treatment with ATP significantly potentiated SG-induced cell proliferation. These data indicate that ATP is released and promotes cell proliferation via activation of purinergic receptors in SG-induced OE injury. Thus, the purinergic system is a therapeutic target to alleviate or restore the loss of OSNs. PMID:21865290

Jia, Cuihong; Sangsiri, Sutheera; Belock, Bethany; Iqbal, Tania R.; Pestka, James J.; Hegg, Colleen C.

2011-01-01

179

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 ...

Mrs. McNees

2010-09-28

180

Microparticles stimulate angiogenesis by inducing ELR+ CXC-chemokines in synovial fibroblasts  

PubMed Central

Abstract Microparticles (MPs) are small membrane-vesicles that accumulate in the synovial fluids of patients with rheumatoid arthritis (RA). In the arthritic joints, MPs induce a pro-inflammatory and invasive phenotype in synovial fibroblasts (SFs). The present study investigated whether activation of SFs by MPs stimulates angiogenesis in the inflamed joints of patients with RA. MPs were isolated from Jurkat cells and U937 cells by differential centrifugation. SFs were co-cultured with increasing numbers of MPs. The effects of supernatants from co-cultures on endothelial cells were studied in vitro and in vivo using MTT assays, annexin V and propidium iodide staining, trans-well migration assays and modified matrigel pouch assays. MPs strongly induced the expression of the pro-angiogenic ELR+ chemokines CXCL1, CXCL2, CXCL3, CXCL5 and CXCL6 in RASFs. Other vascular growth factors were not induced. Supernatants from co-cultures enhanced the migration of endothelial cells, which could be blocked by neutralizing antibodies against ELR+ chemokines. Consistent with the specific induction of ELR+ chemokines, proliferation and viability of endothelial cells were not affected by the supernatants. In the in vivo bio-chamber assay, supernatants from RASFs co-cultured with MPs stimulated angiogenesis with a significant increase of vessels infiltrating into the matrigel chamber. We demonstrated that MPs activate RASFs to release pro-angiogenic ELR+ chemokines. These pro-angiogenic mediators enhance migration of endothelial cells and stimulate the formation of new vessels. Our data suggest that MPs may contribute to the hypervascularization of inflamed joints in patients with rheumatoid arthritis. PMID:20219013

Reich, Nicole; Beyer, Christian; Gelse, Kolja; Akhmetshina, Alfiya; Dees, Clara; Zwerina, Jochen; Schett, Georg; Distler, Oliver; Distler, Jörg HW

2011-01-01

181

Immunomodulatory properties and anti-apoptotic effects of zinc and melatonin in an experimental model of chronic Chagas disease.  

PubMed

The immunomodulatory effects of melatonin and zinc during chronic experimental Chagas' disease were studied. Early and late apoptosis by Annexin V-propidium iodide staining were evaluated. The expression of CD28, CD80, CD86, CD45RA and CD4(+)T and CD8(+)T cells were also evaluated by flow cytometry analysis. The combination of zinc and melatonin notably reduced the apoptotic ratios of splenic cells in the infected and treated animals when compared to untreated rats, during early and late stages of apoptosis. The percentages of CD8(+)T cells in Zn, Mel or Zn and Mel treated rats were reduced when compared to infected and untreated animals. Higher percentages of CD28 expression in CD4(+) and CD8(+) T cell populations were observed in control and infected Zn-treated group as compared to untreated ones. Zn, Mel or the combination of both did not induce any statistically significant differences for B cells when comparing to treated control and infected groups. Zinc or Mel-treated animals presented a lower expression of CD86 when compared to untreated counterparts. According to our data, this work strongly suggest that the modulation of the immune system operated by zinc and melatonin administration affected the balance among T cell immune response, apoptosis and expression of co-stimulatory molecules during chronic Trypanosoma cruzi infection, inducing important changes in the host's immune response against the parasite. Future experiments in this field should be focused in improving our understanding of the key mechanisms underlying the involvement of melatonin and zinc in the immune response during chronic Chagas' disease. PMID:25604665

Brazão, Vânia; Filipin, Marina Del Vecchio; Santello, Fabricia Helena; Azevedo, Angela Palamin; Toldo, Míriam Paula Alonso; de Morais, Fabiana Rossetto; do Prado, José Clóvis

2015-05-01

182

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

183

TFDP3 was expressed in coordination with E2F1 to inhibit E2F1-mediated apoptosis in prostate cancer.  

PubMed

TFDP3 has been previously identified as an inhibitor of E2F molecules. It has been shown to suppress E2F1-induced apoptosis dependent P53 and to play a potential role in carcinogenesis. However, whether it indeed helps cancer cells tolerate apoptosis stress in cancer tissues remains unknown. TFDP3 expression was assessed by RT-PCR, in situ hybridization and immunohistochemistry in normal human tissues, cancer tissues and prostate cancer tissues. The association between TFDP3 and E2F1 in prostate cancer development was analyzed in various stages. Apoptosis was evaluated with annexin-V and propidium iodide staining and flow-cytometry. The results show that, in 96 samples of normal human tissues, TFDP3 could be detected in the cerebrum, esophagus, stomach, small intestine, bronchus, breast, ovary, uterus, and skin, but seldom in the lung, muscles, prostate, and liver. In addition, TFDP3 was highly expressed in numerous cancer tissues, such as brain-keratinous, lung squamous cell carcinoma, testicular seminoma, cervical carcinoma, skin squamous cell carcinoma, gastric adenocarcinoma, liver cancer, and prostate cancer. Moreover, TFDP3 was positive in 23 (62.2%) of 37 prostate cancer samples regardless of stage. Furthermore, immunohistochemistry results show that TFDP3 was always expressed in coordination with E2F1 at equivalent expression levels in prostate cancer tissues, and was highly expressed particularly in samples of high stage. When E2F1 was extrogenously expressed in LNCap cells, TFDP3 could be induced, and the apoptosis induced by E2F1 was significantly decreased. It was demonstrated that TFDP3 was a broadly expressed protein corresponding to E2F1 in human tissues, and suggested that TFDP3 is involved in prostate cancer cell survival by suppressing apoptosis induced by E2F1. PMID:24406621

Ma, Yueyun; Xin, Yijuan; Li, Rui; Wang, Zhe; Yue, Qiaohong; Xiao, Fengjing; Hao, Xiaoke

2014-03-10

184

Preclinical Evaluation of Novel Triphenylphosphonium Salts with Broad-Spectrum Activity  

PubMed Central

Background Recently, there has been a surge of interest in developing compounds selectively targeting mitochondria for the treatment of neoplasms. The critical role of mitochondria in cellular metabolism and respiration supports this therapeutic rationale. Dysfunction in the processes of energy production and metabolism contributes to attenuation of response to pro-apoptotic stimuli and increased ROS production both of which are implicated in the initiation and progression of most human cancers. Methodology/Principal Findings A high-throughput MTT-based screen of over 10,000 drug-like small molecules for anti-proliferative activity identified the phosphonium salts TP187, 197 and 421 as having IC50 concentrations in the submicromolar range. TP treatment induced cell cycle arrest independent of p53 status, as determined by analysis of DNA content in propidium iodide stained cells. In a mouse model of human breast cancer, TP-treated mice showed significantly decreased tumor growth compared to vehicle or paclitaxel treated mice. No toxicities or organ damage were observed following TP treatment. Immunohistochemical staining of tissue sections from TP187-treated tumors demonstrated a decrease in cellular proliferation and increased caspase-3 cleavage. The fluorescent properties of analog TP421 were exploited to assess subcellular uptake of TP compounds, demonstrating mitochondrial localization. Following mitochondrial uptake cells exhibited decreased oxygen consumption and concomittant increase in mitochondrial superoxide production. Proteomics analysis of results from a 600 target antibody microarray demonstrated that TP compounds significantly affected signaling pathways relevant to growth and proliferation. Conclusions/Significance Through our continued interest in designing compounds targeting cancer-cell metabolism, the Warburg effect, and mitochondria we recently discovered a series of novel, small-molecule compounds containing a triphenylphosphine moiety that show remarkable activity in a panel of cancer cell lines as well as in a mouse model of human breast cancer. The mechanism of action includes mitochondrial localization causing decreased oxygen consumption, increased superoxide production and attenuated growth factor signaling. PMID:20957228

Millard, Melissa; Pathania, Divya; Shabaik, Yumna; Taheri, Laleh; Deng, Jinxia; Neamati, Nouri

2010-01-01

185

Total alkaloids of Rubus alceifolius Poir shows anti-angiogenic activity in vivo and in vitro.  

PubMed

Total alkaloids is an active ingredient of the natural plant Rubus alceifolius Poir, commonly used for the treatment of various cancers. Antitumor effects may be mediated through anti-angiogenic mechanisms. As such, the goal of the present study was to investigate and evaluate the effect of total alkaloids in Rubus alceifolius Poir (TARAP) on tumor angiogenesis and investigate the underlying molecular mechanisms of TARAP action in vivo and in vitro. A chick embryo chorioallantoic membrane (CAM) assay was used to assess angiogenesis in vivo. An MTT assay was performed to determine the viability of human umbilical vein endothelial cells (HUVECs) with and without treatment. Cell cycle progression of HUVECs was examined by FACS analysis with propidium iodide staining. HUVEC migration was determined using a scratch wound method. Tube formation of HUVECs was assessed with an ECMatrix gel system, and mRNA and protein expression of VEGF-A in both HUVECs and HepG2 human hepatocellular carcinoma cells were examined by RT-PCR and ELISA, respectively. Our results showed that TARAP inhibited angiogenesis in the CAM model in vivo and inhibited HUVEC proliferation via blocking cell cycle G1 to S progression in a dose- and time-dependent manners in vitro. Moreover, TARAP inhibited HUVEC migration and tube formation and downregulated mRNA and protein expression of VEGF-A in both HepG2 cells and HUVECs. Our findings suggest that the anti-angiogenic activity of TARAP may partly contribute to its antitumor properties and may be valuable for the treatment of diseases involving pathologic angiogenesis such as cancer. PMID:25148840

Zhao, Jinyan; Lin, Wei; Zhuang, Qunchuan; Zhong, Xiaoyong; Cao, Zhiyun; Hong, Zhenfeng; Peng, Jun

2014-11-01

186

Sonoporation of endothelial cells by vibrating targeted microbubbles.  

PubMed

Molecular imaging using ultrasound makes use of targeted microbubbles. In this study we investigated whether these microbubbles could also be used to induce sonoporation in endothelial cells. Lipid-coated microbubbles were targeted to CD31 and insonified at 1 MHz at low peak negative acoustic pressures at six sequences of 10 cycle sine-wave bursts. Vibration of the targeted microbubbles was recorded with the Brandaris-128 high-speed camera (~13 million frames per second). In total, 31 cells were studied that all had one microbubble (1.2-4.2 micron in diameter) attached per cell. After insonification at 80 kPa, 30% of the cells (n=6) had taken up propidium iodide, while this was 20% (n=1) at 120 kPa and 83% (n=5) at 200 kPa. Irrespective of the peak negative acoustic pressure, uptake of propidium iodide was observed when the relative vibration amplitude of targeted microbubbles was greater than 0.5. No relationship was found between the position of the microbubble on the cell and induction of sonoporation. This study shows that targeted microbubbles can also be used to induce sonoporation, thus making it possible to combine molecular imaging and drug delivery. PMID:21514333

Kooiman, Klazina; Foppen-Harteveld, Miranda; van der Steen, Antonius F W; de Jong, Nico

2011-08-25

187

The ER Stress-Mediated Mitochondrial Apoptotic Pathway and MAPKs Modulate Tachypacing-Induced Apoptosis in HL-1 Atrial Myocytes  

PubMed Central

Background and Object Cell apoptosis is a contributing factor in the initiation, progression and relapse of atrial fibrillation (AF), a life-threatening illness accompanied with stroke and heart failure. However, the regulatory cascade of apoptosis is intricate and remains unidentified, especially in the setting of AF. The aim of this study was to explore the roles of endoplasmic reticulum (ER) stress, mitochondrial apoptotic pathway (MAP), mitogen-activated protein kinases (MAPKs), and their cross-talking in tachypacing-induced apoptosis. Methods and Results HL-1 cells were cultured in the presence of tachypacing for 24 h to simulate atrial tachycardia remodeling. Results showed that tachypacing reduced cell viability measured by the cell counting kit-8, dissipated mitochondrial membrane potential detected by JC-1 staining and resulted in approximately 50% apoptosis examined by Hoechst staining and annexin V/propidium iodide staining. In addition, the proteins involved in ER stress, MAP and MAPKs were universally up-regulated or activated via phosphorylation, as confirmed by western blotting; and reversely silencing of ER stress, caspase-3 (the ultimate executor of MAP) and MAPKs with specific inhibitors prior to pacing partially alleviated apoptosis. An inhibitor of ER stress was applied to further investigate the responses of mitochondria and MAPKs to ER stress, and results indicated that suppression of ER stress comprehensively but incompletely attenuated the activation of MAP and MAPKs aroused by tachypacing, with the exception of ERK1/2, one branch of MAPKs. Conclusions Our study suggested tachypacing-induced apoptosis is regulated by ER stress-mediated MAP and MAPKs. Thus, the above three components are all promising anti-apoptotic targets in AF patients and ER stress appears to play a dominant role due to its comprehensive effects. PMID:25689866

Shi, Jiaojiao; Jiang, Qi; Ding, Xiangwei; Xu, Wenhua; Wang, Dao W.; Chen, Minglong

2015-01-01

188

Effect of a glutathione S-transferase inhibitor on oxidative stress and ischemia-reperfusion-induced apoptotic signalling of cultured cardiomyocytes  

PubMed Central

Oxidative stress and ischemia-reperfusion (I/R) injury are crucial in the pathogenesis of cardiovascular diseases. The antioxidant glutathione S-transferase (GST) is responsible for the high-capacity metabolic inactivation of electrophilic compounds and toxic substrates. The main objective of the present study was to examine the effect of GST inhibition (with the administration of ethacrynic acid [EA]) on the viability and apoptosis of cardiomyocytes when these cells are exposed to various stress components of I/R and mitogen-activated protein kinase (c-Jun N-terminal kinase, p38 and extracellular signal-regulated kinase [ERK]) inhibitors. The primary culture of neonatal rat cardiomyocytes was divided into six experimental groups: control group of cells (group 1), cells exposed to H2O2 (group 2), I/R (group 3), I/R and EA (group 4), H2O2 coupled with EA (group 5), and EA alone (group 6). The viability of cardiomyocytes was determined using a colorimetric MTT assay. The apoptosis ratio was evaluated via fluorescein isothiocyanate-labelled annexin V and propidium iodide staining. c-Jun N-terminal kinase, p38, Akt/protein kinase B and ERK/p42-p44 transcription factors were monitored with flow cytometry. c-Jun N-terminal kinase activation increased due to GST inhibition during I/R. EA administration led to a significant increase in p38 activation following both H2O2 treatment and I/R. ERK phosphorylation increased when GST was exposed to I/R. A pronounced decrease in Akt phosphorylation was observed when cells were cotreated with EA and H2O2. GST plays an important role as a regulator of mitogen-activated protein kinase pathways in I/R injury. PMID:22065940

Röth, E; Marczin, N; Balatonyi, B; Ghosh, S; Kovács, V; Alotti, N; Borsiczky, B; Gasz, B

2011-01-01

189

The effects of oncolytic reovirus in canine lymphoma cell lines.  

PubMed

Reovirus is a potent oncolytic virus in many human neoplasms that has reached phase II and III clinical trials. Our laboratory has previously reported the oncolytic effects of reovirus in canine mast cell tumour (MCT). In order to further explore the potential of reovirus in veterinary oncology, we tested the susceptibility of reovirus in 10 canine lymphoma cell lines. Reovirus-induced cell death, virus replication and infectivity were confirmed in four cell lines with variable levels of susceptibility. The level of Ras activation varied among the cell lines with no correlation with reovirus susceptibility. Reovirus-susceptible cell lines underwent apoptosis as proven by propidium iodide (PI) staining, Annexin V-FITC/PI assay, cleavage of PARP and inhibition of cell death by caspase inhibitor. A single intratumoral injection of reovirus suppressed the growth of canine lymphoma subcutaneous tumour in NOD/SCID mice. Unlike canine MCT, canine lymphoma is less susceptible to reovirus. PMID:25319493

Hwang, C C; Umeki, S; Igase, M; Coffey, M; Noguchi, S; Okuda, M; Mizuno, T

2014-10-15

190

Cell death induced by application of time-varying magnetic fields on nanoparticle-loaded dendritic cells  

E-print Network

Aim: To assess the capability of monocyte-derived dendritic cells (DCs) to take Fe3O4 magnetic nanoparticles (MNPs), keeping their viability. To provoke cell death on these MNPs-loaded DCs using an external alternating magnetic field (AMF). Material & methods: Peripheral blood mononuclear cells were isolated from normal blood and platelets removed by centrifugation. Immunoselected CD14+ cells were cultured for 5 days, and the resulting cell phenotype was determined against several markers using flow cytometry. Co-cultures of DCs and MNPs were done overnight. The amount of Fe3O4 nanoparticles incorporated by DCs was quantified by magnetization measurements. MNPs-loaded DCs were exposed to AMF for 30 min and then cell viability was measured using trypan blue and FACS (annexin-propidium iodide) protocols. Morphological changes were investigated using scanning electron microscopy. Results: No significant decrease in cell viability of MNP/loaded DCs was observed up to five days, as compared against control sam...

Marcos-Campos, I; Torres, T E; Marquina, C; Tres, A; Ibarra, M R; Goya, G F

2010-01-01

191

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

192

Flow cytometric method for measuring chromatin fragmentation in fixed sperm from yellow perch (Perca flavescens).  

PubMed

Declining harvests of yellow perch, Perca flavescens, in urbanized watersheds of Chesapeake Bay have prompted investigations of their reproductive fitness. The purpose of this study was to establish a flow cytometric technique for DNA analysis of fixed samples sent from the field to provide reliable gamete quality measurements. Similar to the sperm chromatin structure assay, measures were made on the susceptibility of nuclear DNA to acid-induced denaturation, but used fixed rather than live or thawed cells. Nuclei were best exposed to the acid treatment for 1 minute at 37 °C followed by the addition of cold (4 °C) propidium iodide staining solution before flow cytometry. The rationale for protocol development is presented graphically through cytograms. Field results collected in 2008 and 2009 revealed DNA fragmentation up to 14.5%. In 2008, DNA fragmentation from the more urbanized watersheds was significantly greater than from reference sites (P = 0.026) and in 2009, higher percentages of haploid testicular cells were noted from the less urbanized watersheds (P = 0.032) indicating better reproductive condition at sites with less urbanization. For both years, total and progressive live sperm motilities by computer-assisted sperm motion analysis ranged from 19.1% to 76.5%, being significantly higher at the less urbanized sites (P < 0.05). This flow cytometric method takes advantage of the propensity of fragmented DNA to be denatured under standard conditions, or 1 minute at 37 °C with 10% buffered formalin-fixed cells. The study of fixed sperm makes possible the restrospective investigation of germplasm fragmentation, spermatogenic ploidy patterns, and chromatin compaction levels from samples translocated over distance and time. The protocol provides an approach that can be modified for other species across taxa. PMID:25559842

Jenkins, J A; Draugelis-Dale, R O; Pinkney, A E; Iwanowicz, L R; Blazer, V S

2015-03-15

193

Flow cytometric method for measuring chromatin fragmentation in fixed sperm from yellow perch (Perca flavescens)  

USGS Publications Warehouse

Declining harvests of yellow perch, Perca flavescens, in urbanized watersheds of Chesapeake Bay have prompted investigations of their reproductive fitness. The purpose of this study was to establish a flow cytometric technique for DNA analysis of fixed samples sent from the field to provide reliable gamete quality measurements. Similar to the sperm chromatin structure assay, measures were made on the susceptibility of nuclear DNA to acid-induced denaturation, but used fixed rather than live or thawed cells. Nuclei were best exposed to the acid treatment for 1 minute at 37 °C followed by the addition of cold (4 °C) propidium iodide staining solution before flow cytometry. The rationale for protocol development is presented graphically through cytograms. Field results collected in 2008 and 2009 revealed DNA fragmentation up to 14.5%. In 2008, DNA fragmentation from the more urbanized watersheds was significantly greater than from reference sites (P = 0.026) and in 2009, higher percentages of haploid testicular cells were noted from the less urbanized watersheds (P = 0.032) indicating better reproductive condition at sites with less urbanization. For both years, total and progressive live sperm motilities by computer-assisted sperm motion analysis ranged from 19.1% to 76.5%, being significantly higher at the less urbanized sites (P < 0.05). This flow cytometric method takes advantage of the propensity of fragmented DNA to be denatured under standard conditions, or 1 minute at 37 °C with 10% buffered formalin–fixed cells. The study of fixed sperm makes possible the restrospective investigation of germplasm fragmentation, spermatogenic ploidy patterns, and chromatin compaction levels from samples translocated over distance and time. The protocol provides an approach that can be modified for other species across taxa.

Jenkins, Jill A.; Draugelis-Dale, Rassa O.; Pinkney, Alfred E.; Iwanowicz, Luke R.; Blazer, Vicki S.

2015-01-01

194

Ceiling culture of mature human adipocytes: use in studies of adipocyte functions.  

PubMed

Adipocytes contain large lipid droplets in their cytoplasm. When cultured, they float on top of the medium, clump together, and do not gain equal and sufficient access to the medium. Morphological changes cannot be observed and the majority of adipocytes undergo cell lysis within 72 h of isolation. We have used a ceiling culture method for human mature adipocytes which uses their buoyant property to allow them to adhere to a floating glass surface, where they remain viable for several weeks. Using confocal immunofluorescence microscopy we showed the cellular expression and subcellular localization of leptin in ceiling-cultured adipocytes. The secretion of leptin was increased from ceiling cultures following tumour necrosis factor-alpha treatment. Proliferation of mature human adipocytes in serum-containing medium was demonstrated by incorporation of bromodeoxyuridine, 2% of adipocytes showing positive incorporation after 4 h labelling. Proliferation was also evident from the budding of daughter cells. Apoptosis in the ceiling cultures was increased by 48 h serum deprivation (30-35 vs 10-15% in the control) and was assayed by propidium iodide staining and terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick-end labelling. Lipolysis, analysed by liquid scintillation counting, was increased by forskolin (10 microM for 90 min) and lipogenesis, shown by autoradiography, was stimulated by insulin (10 and 100 nM for 4 h). These findings indicate that ceiling-cultured adipocytes maintain adipocyte-specific functions and that ceiling culture, which overcomes the shortcomings of adipocyte suspension culture, can be used to study adipocyte cell biology. PMID:10657847

Zhang, H H; Kumar, S; Barnett, A H; Eggo, M C

2000-02-01

195

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

196

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

197

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

198

Induction of Eosinophil Apoptosis by the Cyclin-Dependent Kinase Inhibitor AT7519 Promotes the Resolution of Eosinophil-Dominant Allergic Inflammation  

PubMed Central

Background Eosinophils not only defend the body against parasitic infection but are also involved in pathological inflammatory allergic diseases such as asthma, allergic rhinitis and contact dermatitis. Clearance of apoptotic eosinophils by macrophages is a key process responsible for driving the resolution of eosinophilic inflammation and can be defective in allergic diseases. However, enhanced resolution of eosinophilic inflammation by deliberate induction of eosinophil apoptosis using pharmacological agents has not been previously demonstrated. Here we investigated the effect of a novel cyclin-dependent kinase inhibitor drug, AT7519, on human and mouse eosinophil apoptosis and examined whether it could enhance the resolution of a murine model of eosinophil-dominant inflammation in vivo. Methodology/Principal Findings Eosinophils from blood of healthy donors were treated with AT7519 and apoptosis assessed morphologically and by flow-cytometric detection of annexin-V/propidium iodide staining. AT7519 induced eosinophil apoptosis in a concentration dependent manner. Therapeutic administration of AT7519 in eosinophil-dominant allergic inflammation was investigated using an established ovalbumin-sensitised mouse model of allergic pleurisy. Following ovalbumin challenge AT7519 was administered systemically at the peak of pleural inflammation and inflammatory cell infiltrate, apoptosis and evidence of macrophage phagocytosis of apoptotic eosinophils assessed at appropriate time points. Administration of AT7519 dramatically enhanced the resolution of allergic pleurisy via direct induction of eosinophil apoptosis without detriment to macrophage clearance of these cells. This enhanced resolution of inflammation was shown to be caspase-dependent as the effects of AT7519 were reduced by treatment with a broad spectrum caspase inhibitor (z-vad-fmk). Conclusions Our data show that AT7519 induces human eosinophil apoptosis and enhances the resolution of a murine model of allergic pleurisy by inducing caspase-dependent eosinophil apoptosis and enhancing macrophage ingestion of apoptotic eosinophils. These findings demonstrate the utility of cyclin-dependent kinase inhibitors such as AT7519 as potential therapeutic agents for the treatment of eosinophil dominant allergic disorders. PMID:21984938

Alessandri, Ana L.; Duffin, Rodger; Leitch, Andrew E.; Lucas, Christopher D.; Sheldrake, Tara A.; Dorward, David A.; Hirani, Nik; Pinho, Vanessa; de Sousa, Lirlândia Pires; Teixeira, Mauro M.; Lyons, John F.; Haslett, Christopher; Rossi, Adriano G.

2011-01-01

199

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

Veselý, Pavel; Bureš, Petr; Šmarda, Petr

2013-01-01

200

Celastrol Potentiates Radiotherapy by Impairment of DNA Damage Processing in Human Prostate Cancer  

SciTech Connect

Purpose: Celastrol is an active ingredient of traditional herbal medicine and has recently been identified as a potent natural proteasome inhibitor. In the present study, we evaluated the radiosensitizing potential of celastrol in the human prostate cancer PC-3 model. Methods and Materials: Clonogenic assays were performed to determine the radiosensitizing effect of celastrol. Apoptosis was examined by flow cytometry using Annexin V and propidium iodide staining and by a caspase-3 activation assay. DNA damage processing was examined by immunofluorescent staining and Western blot for phosphorylated H2AX ({gamma}H2AX). The PC-3 xenograft model in the athymic nude mouse was used for the determination of the in vivo efficacy of celastrol combined with radiotherapy. The tumor samples were also analyzed for apoptosis and angiogenesis. Results: Celastrol sensitized PC-3 cells to ionizing radiation (IR) in a dose- and schedule-dependent manner, in which pretreatment with celastrol for 1 h followed by IR achieved maximal radiosensitization. Celastrol significantly prolonged the presence of IR-induced {gamma}H2AX and increased IR-induced apoptosis. Celastrol, combined with fractionated radiation, significantly inhibited PC-3 tumor growth in vivo without obvious systemic toxicity. The combination treatment increased {gamma}H2AX levels and apoptosis, induced cleavage of poly(adenosine diphosphate-ribose)polymerase and Mcl-1, and reduced angiogenesis in vivo compared with either treatment alone. Conclusion: Celastrol sensitized PC-3 cells to radiation both in vitro and in vivo by impairing DNA damage processing and augmenting apoptosis. Celastrol might represent a promising new adjuvant regimen for the treatment of hormone-refractory prostate cancer.

Dai Yao; DeSano, Jeffrey T.; Meng Yang; J, Qing; Ljungman, Mats; Lawrence, Theodore S. [Department of Radiation Oncology, Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI (United States); Xu Liang [Department of Radiation Oncology, Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI (United States)], E-mail: liangxu@umich.edu

2009-07-15

201

Silymarin component 2,3-dehydrosilybin attenuates cardiomyocyte damage following hypoxia/reoxygenation by limiting oxidative stress.  

PubMed

Ischemic postconditioning and remote conditioning are potentially useful tools for protecting ischemic myocardium. This study tested the hypothesis that 2,3-dehydrosilybin (DHS), a flavonolignan component of Silybum marianum, could attenuate cardiomyocyte damage following hypoxia/reoxygenation by decreasing the generation of reactive oxygen species (ROS). After 5-6 days of cell culture in normoxic conditions the rat neonatal cardiomyocytes were divided into four groups. Control group (9 h at normoxic conditions), hypoxia/reoxygenation group (3 h at 1 % O(2), 94 % N(2) and 5 % CO(2) followed by 10 min of 10 micromol·l(-1) DHS and 6 h of reoxygenation in normoxia) and postconditioning group (3 h of hypoxia, three cycles of 5 min reoxygenation and 5 min hypoxia followed by 6 h of normoxia). Cell viability assessed by propidium iodide staining was decreased after DHS treatment consistent with increased levels of lactatedehydrogenase (LDH) after reoxygenation. LDH leakage was significantly reduced when cardiomyocytes in the H/Re group were exposed to DHS. DHS treatment reduced H(2)O(2) production and also decreased the generation of ROS in the H/Re group as evidenced by a fluorescence indicator. DHS treatment reduces reoxygenation-induced injury in cardiomyocytes by attenuation of ROS generation, H(2)O(2) and protein carbonyls levels. In addition, we found that both the postconditioning protocol and the DHS treatment are associated with restored ratio of phosphorylated/total protein kinase C epsilon, relative to the H/Re group. In conclusion, our data support the protective role of DHS in hypoxia/reperfusion injury and indicate that DHS may act as a postconditioning mimic. PMID:25194130

Gabrielová, E; K?en, V; Jab?rek, M; Modrianský, M

2015-03-01

202

Micromanipulation and physiological monitoring of cells using two-photon excited fluorescence in cw laser tweezers  

NASA Astrophysics Data System (ADS)

We report the observation of two-photon fluorescence excitation and cell confinement, simultaneously, in a continuous-wave (cw) single-beam gradient force optical trap, and demonstrate its use as an in-situ probe to study the physiological state of an optically confined cell sample. At the wavelength of 1064 nm, a single focused gaussian laser beam is used to simultaneously confine, and excite visible fluorescence from, a human sperm cell that has been tagged with propidium iodide, a exogenous fluorescent dye that functions as a viability assay of cellular physiological state. The intensity at the dye peak emission wavelength of 620 nm exhibits a near-square-law dependence on incident trapping beam photon laser power, a behavior consistent with a two-photon absorption process. In addition, for a sperm cell held stationary in the optical tweezers for a period of several minutes at a constant trapping power, red fluorescence emission was observed to increase the time, indicating that the cell has gradually transitioned between a live and dead state. Two-photon excited fluorescence was also observed in chinese hamster ovary cells that were confined by cw laser tweezers and stained with either propidium iodide or Snarf, a pH-sensitive dye probe. These results suggest that, for samples suitably tagged with fluorescent probes and vital stains, optical tweezers can be used to generate their own in-situ diagnostic optical probes of cellular viability or induced photodamage, via two-photon processes.

Sonek, Gregory J.; Liu, Yagang; Berns, Michael W.; Tromberg, Bruce J.

1996-05-01

203

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

204

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

205

Interplay between Ca2+ cycling and mitochondrial permeability transition pores promotes reperfusion-induced injury of cardiac myocytes  

PubMed Central

Abstract Uncontrolled release of Ca2+ from the sarcoplasmic reticulum (SR) contributes to the reperfusion-induced cardiomyocyte injury, e.g. hypercontracture and necrosis. To find out the underlying cellular mechanisms of this phenomenon, we investigated whether the opening of mitochondrial permeability transition pores (MPTP), resulting in ATP depletion and reactive oxygen species (ROS) formation, may be involved. For this purpose, isolated cardiac myocytes from adult rats were subjected to simulated ischemia and reperfusion. MPTP opening was detected by calcein release and by monitoring the ??m. Fura-2 was used to monitor cytosolic [Ca2+]i or mitochondrial calcium [Ca2+]m, after quenching the cytosolic compartment with MnCl2. Mitochondrial ROS [ROS]m production was detected with MitoSOX Red and mag-fura-2 was used to monitor Mg2+ concentration, which reflects changes in cellular ATP. Necrosis was determined by propidium iodide staining. Reperfusion led to a calcein release from mitochondria, ??m collapse and disturbance of ATP recovery. Simultaneously, Ca2+ oscillations occurred, [Ca2+]m and [ROS]m increased, cells developed hypercontracture and underwent necrosis. Inhibition of the SR-driven Ca2+ cycling with thapsigargine or ryanodine prevented mitochondrial dysfunction, ROS formation and MPTP opening. Suppression of the mitochondrial Ca2+ uptake (Ru360) or MPTP (cyclosporine A) significantly attenuated Ca2+ cycling, hypercontracture and necrosis. ROS scavengers (2-mercaptopropionyl glycine or N-acetylcysteine) had no effect on these parameters, but reduced [ROS]m. In conclusion, MPTP opening occurs early during reperfusion and is due to the Ca2+ oscillations originating primarily from the SR and supported by MPTP. The interplay between Ca2+ cycling and MPTP promotes the reperfusion-induced cardiomyocyte hypercontracture and necrosis. Mitochondrial ROS formation is a result rather than a cause of MPTP opening. PMID:21199327

Abdallah, Yaser; Kasseckert, Sascha A; Iraqi, Wisam; Said, Maher; Shahzad, Tayyab; Erdogan, Ali; Neuhof, Christiane; Gündüz, Dürsün; Schlüter, Klaus-Dieter; Tillmanns, Harald; Piper, H Michael; Reusch, H Peter; Ladilov, Yury

2011-01-01

206

Increased Susceptibility to Oxidative Death of Lymphocytes from Alzheimer Patients Correlates with Dementia Severity  

PubMed Central

We previously reported on enhanced susceptibility to death of lymphocytes from Alzheimer’s disease (AD) patients when exposed to hydrogen peroxide (H2O2)-induced oxidative stress and an increased resistance to death in those of patients with a history of skin cancer. This is consistent with our hypothesis proposing that the cellular machinery controlling cell death is deregulated in opposite directions in Alzheimer’s disease (AD) and cancer, to explain the inverse association observed in epidemiological studies. Here we investigated whether the observed increased susceptibility correlates with the degree of dementia severity. Peripheral lymphocytes from 23 AD patients, classified using the Clinical Dementia Rating (CDR) into severe dementia (CDR 3, n=10) and mild-to-moderate dementia (CDR 1–2, n=13), and 15 healthy controls (HC) (CDR 0), were exposed to H2O2 for 20 hours. Lymphocyte death was determined by flow cytometry and propidium iodide staining. The greatest susceptibility to H2O2-induced death was observed for lymphocytes from severe dementia patients, whereas those with mild-to-moderate dementia exhibited intermediate values, compared to healthy controls. A significant increase in the apoptosis/necrosis ratio was found in AD patients. Poly (ADP-ribosyl) polymerase-1 (PARP-1) inhibition significantly protected from H2O2-induced death of lymphocytes, whereby a lower degree of protection was observed in severe AD patients. Moreover, inhibition of PARP-1 abolished the differences in apoptosis/necrosis ratios observed between the three groups of patients. These results support the notion that AD is a systemic disorder, whereby enhanced susceptibility to H2O2-induced death in peripheral lymphocytes correlates with dementia severity and enhanced death in AD patients is attributable to a PARP-dependent increase in the apoptosis/necrosis ratio. PMID:25274115

Ponce, Daniela P.; Salech, Felipe; SanMartin, Carol D.; Silva, Monica; Xiong, Chengjie; Roe, Catherine M.; Henriquez, Mauricio; Quest, Andrew F.; Behrens, Maria I.

2015-01-01

207

Damage to F-actin and cell death induced by chromium VI and nickel in primary monolayer cultures of rat hepatocytes.  

PubMed

The toxicity of hexavalent chromium and nickel was investigated using primary cultures of hepatocytes as an in vitro system. Cr VI and Ni are widely used in the steel and orthopaedic implant industry. Although their toxicity has been extensively investigated, the mechanism(s) of action is/are not fully understood. Monolayer cultures of hepatocytes (10(5) cells/cm2) were exposed to various concentrations of Cr VI and Ni for 24 h. Cells were stained with phalloidin-FITC for the detection of the cytoskeletal component, F-actin, and Annexin V-FITC and propidium iodide for the detection of the mode of cell death. Exposure of cells to Cr VI (1, 5, 10 and 50 microM) resulted in the loss of the cell cytoskeleton, and this was accompanied by membrane blebbing and shrinking of the cell. Ni, on the other hand, induced detectable damage to the cytoskeleton only at 500 microM. Staining of the cells with Annexin V and propidium iodide showed that Cr VI induces apoptosis at low concentrations (5 microM), and necrosis at higher concentrations (25 and 50 microM). Ni almost exclusively induced necrosis at 500 microM with very few cells undergoing apoptosis. Below this concentration it had no discernable effect on hepatocytes. Damage to the cell cytoskeleton caused by Cr VI may be an early indication of apoptosis in hepatocytes. PMID:15046770

Gunaratnam, Mekala; Grant, Mary Helen

2004-06-01

208

Prediction of clinical toxicity in locally advanced head and neck cancer patients by radio-induced apoptosis in peripheral blood lymphocytes (PBLs)  

PubMed Central

Head and neck cancer is treated mainly by surgery and radiotherapy. Normal tissue toxicity due to x-ray exposure is a limiting factor for treatment success. Many efforts have been employed to develop predictive tests applied to clinical practice. Determination of lymphocyte radio-sensitivity by radio-induced apoptosis arises as a possible method to predict tissue toxicity due to radiotherapy. The aim of the present study was to analyze radio-induced apoptosis of peripheral blood lymphocytes in head and neck cancer patients and to explore their role in predicting radiation induced toxicity. Seventy nine consecutive patients suffering from head and neck cancer, diagnosed and treated in our institution, were included in the study. Toxicity was evaluated using the Radiation Therapy Oncology Group scale. Peripheral blood lymphocytes were isolated and irradiated at 0, 1, 2 and 8 Gy during 24 hours. Apoptosis was measured by flow cytometry using annexin V/propidium iodide. Lymphocytes were marked with CD45 APC-conjugated monoclonal antibody. Radiation-induced apoptosis increased in order to radiation dose and fitted to a semi logarithmic model defined by two constants: ? and ?. ?, as the origin of the curve in the Y axis determining the percentage of spontaneous cell death, and ?, as the slope of the curve determining the percentage of cell death induced at a determined radiation dose, were obtained. ? value was statistically associated to normal tissue toxicity in terms of severe xerostomia, as higher levels of apoptosis were observed in patients with low toxicity (p = 0.035; Exp(B) 0.224, I.C.95% (0.060-0.904)). These data agree with our previous results and suggest that it is possible to estimate the radiosensitivity of peripheral blood lymphocytes from patients determining the radiation induced apoptosis with annexin V/propidium iodide staining. ? values observed define an individual radiosensitivity profile that could predict late toxicity due to radiotherapy in locally advanced head and neck cancer patients. Anyhow, prospective studies with different cancer types and higher number of patients are needed to validate these results. PMID:20109191

2010-01-01

209

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,

210

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

211

VvpM Induces Human Cell Death via Multifarious Modes Including Necroptosis and Autophagy.  

PubMed

VvpM, one of the extracellular metalloproteases produced by Vibrio vulnificus, induces apoptotic cell death via a pathway consisting of ERK activation, cytochrome c release, and activation of caspases-9 and -3. VvpM-treated cells also showed necrotic cell death as stained by propidium iodide (PI). The percentage of PI-stained cells was decreased by pretreatment with Necrostatin-1, indicating that VvpM-mediated cell death occurs through necroptosis. The appearance of autophagic vesicles and lipidated form of light-chain-3B in rVvpM-treated cells suggests an involvement of autophagy in this process. Therefore, the multifarious action of VvpM might be one of the factors responsible for V. vulnificus pathogenesis. PMID:25649984

Lee, Mi-Ae; Kim, Jeong-A; Shin, Mee-Young; Lee, Jeong K; Park, Soon-Jung; Lee, Kyu-Ho

2015-02-28

212

Parallel single-cell light-induced electroporation and dielectrophoretic manipulation  

PubMed Central

Electroporation is a common technique for the introduction of exogenous molecules across the, otherwise, impermeant cell membrane. Conventional techniques are limited by either low throughput or limited selectivity. Here we present a novel technique whereby we use patterned light to create virtual electrodes which can induce the parallel electroporation of single cells. This technique seamlessly integrates with optoelectronic tweezers to provide a single cell manipulation platform as well. We present evidence of parallel, single cell electroporation using this method through use of fluorescent dyes and dielectrophoretic responses. Additionally, through the use of integrated microfluidic channels, we show that cells remain viable following treatment in the device. Finally, we determine the optimal field dosage to inject propidium iodide into a HeLa cell and maintain cellular viability. PMID:19495455

Valley, Justin K.; Neale, Steven; Hsu, Hsan-Yin; Ohta, Aaron T.; Jamshidi, Arash; Wu, Ming C.

2009-01-01

213

Local electroporation of a single cell using a scanning ion conductance microscope  

NASA Astrophysics Data System (ADS)

We developed a novel electroporation technique for molecular delivery into a single cell. A nanopipette, a thermally pulled glass capillary, is prepared as to act as a pair of tiny electrodes for single-cell electroporation. An Ag/AgCl wire is inserted into the nanopipette, and the outside edge of the nanopipette is coated by Ag sputtering. Electric pulses are applied between the outside and inside electrodes to form a local electric field at the edge of the nanopipette. To position the pipette edge in the vicinity of the cell membrane, we control the probe-surface distance using a scanning ion conductance microscope (SICM). The SICM technique achieves non-contact approach of the nanopipette edge on the cell membrane, which allows low-invasive electroporation of a single cell. As a demonstration of this technique, a fluorescent molecule of propidium iodide was successfully delivered into a single HeLa cell.

Iwata, Futoshi; Yamazaki, Koji; Ishizaki, Kimihiro; Ushiki, Tatuo

2014-03-01

214

Xanthoangelol, a major chalcone constituent of Angelica keiskei, induces apoptosis in neuroblastoma and leukemia cells.  

PubMed

Xanthoangelol, a major chalcone constituent of the stem exudates of Angelica keiskei, was evaluated for cell toxicity and apoptosis-inducing activity in human neuroblastoma (IMR-32) and leukemia (Jurkat) cells. Xanthoangelol concentration-dependently reduced the survival rates of both cell lines as revealed by the trypan blue exclusion test. Early apoptosis induced by 4 h incubation with xanthoangelol was detected using flow cytometry after double-staining with annexin V and propidium iodide (PI). Western blot analysis showed that xanthoangelol markedly reduced the level of precursor caspase-3 and increased the level of cleaved caspase-3, but Bax and Bcl-2 proteins were not affected. These results suggest that xanthoangelol induces apoptotic cell death by activatation of caspase-3 in neuroblastoma and leukemia cells through a mechanism that does not involve Bax/Bcl-2 signal transduction. Therefore, xanthoangelol may be applicable as an effective drug for treatment of neuroblastoma and leukemia. PMID:16079483

Tabata, Keiichi; Motani, Kou; Takayanagi, Noriya; Nishimura, Reiko; Asami, Satoru; Kimura, Yumiko; Ukiya, Motohiko; Hasegawa, Daisuke; Akihisa, Toshihiro; Suzuki, Takashi

2005-08-01

215

Characterization of embryonic cortical neuron death in prolonged cell suspension.  

PubMed

Cell transplantation may be an effective therapeutic strategy for many neurodegenerative diseases. However, difficulty in obtaining a sufficient amount of donor cells and low graft survival are two major limiting factors. Dissociation of cells from tissues or culture is an inevitable step for cell transplantation, and cell viability in suspension may influence the outcome of the cell therapy. To this end, we asked whether the suspension time of freshly dissociated neurons in vitro affects their viability. Following 4-24h cell suspension, primary cortical neurons underwent cell death. Interestingly, the neurons exhibited only marginal caspase-3 immunoreactivity with very few sub-G1 apoptotic cell proportions in flow cytometry. In addition, the suppression of caspase-3 or Bax action failed to prevent cell death of primary cortical neurons, indicating minimal apoptotic cell death. On the other hand, there was a marked increase in the TdT-mediated dUTP nick end labeling-positive and propidium iodide-labeled necrotic cells (?50%) with enhanced poly [ADP-ribose] polymerase-1 activity. Therefore, prevention against necrosis rather than apoptosis may be required for optimal benefits in cell transplantation. PMID:23127849

Moon, Younghye; Kim, Hyun; Sun, Woong

2012-12-01

216

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

PubMed

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

Uemura, Makoto; Ishiguro, Hiroshi

2015-04-01

217

PACAP protects against TNF?-induced cell death in olfactory epithelium and olfactory placodal cell lines.  

PubMed

In mouse olfactory epithelium (OE), pituitary adenylate cyclase-activating peptide (PACAP) protects against axotomy-induced apoptosis. We used mouse OE to determine whether PACAP protects neurons during exposure to the inflammatory cytokine TNF?. Live slices of neonatal mouse OE were treated with 40 ng/ml TNF? ± 40nM PACAP for 6h and dying cells were live-labeled with 0.5% propidium iodide. TNF? significantly increased the percentage of dying cells while co-incubation with PACAP prevented cell death. PACAP also prevented TNF?-mediated cell death in the olfactory placodal (OP) cell lines, OP6 and OP27. Although OP cell lines express all three PACAP receptors (PAC1, VPAC1,VPAC2), PACAP's protection of these cells from TNF? was mimicked by the specific PAC1 receptor agonist maxadilan and abolished by the PAC1 antagonist PACAP6-38. Treatment of OP cell lines with blockers or activators of the PLC and AC/MAPKK pathways revealed that PACAP-mediated protection from TNF? involved both pathways. PACAP may therefore function through PAC1 receptors to protect neurons from cell death during inflammatory cytokine release in vivo as would occur upon viral infection or allergic rhinitis-associated injury. PMID:20654718

Kanekar, Shami; Gandham, Mahendra; Lucero, Mary T

2010-12-01

218

K562 cells display different vulnerability to H?O? induced oxidative stress in differing cell cycle phases.  

PubMed

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

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

2015-02-01

219

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

220

Deoxynivalenol induces apoptosis in mouse thymic epithelial cells through mitochondria-mediated pathway.  

PubMed

Deoxynivalenol (DON) is a mycotoxin produced as a secondary metabolite by fungal species. In this report, we investigated the apoptotic effect of DON in mouse thymic epithelial cell line 1 (MTEC1). MTEC1 cell apoptosis induced by DON was confirmed by nuclei morphology change, TUNEL positive staining, annexin V/propidium iodide positive staining and increased protein levels of caspase-3, caspase-8, caspase-9 and poly(ADP-ribose) polymerase (PARP). The effects of DON on reactive oxygen species (ROS) levels and mitochondrial membrane potential were investigated via fluorescence microscope and flow cytometry, respectively. In addition, DON could significantly increase the protein levels of p53 and Bax/Bcl-2 ratio in MTEC1 cells. Taken together, our results suggest that DON causes the activation of p53, increased levels of ROS and the induction of mitochondrial dysfunction, which may contribute to DON-induced apoptosis in MTEC1 cells. PMID:24952344

Li, Daotong; Ma, Haoran; Ye, Yaqiong; Ji, Changyun; Tang, Xiaohong; Ouyang, Dan; Chen, Jian; Li, Yugu; Ma, Yongjiang

2014-07-01

221

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

PubMed

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

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

2013-02-01

222

Morphological changes of post-isolation of caprine pancreatic islet.  

PubMed

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

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

2015-02-01

223

Oxygen\\/glucose deprivation increases the integration of recombinant P2X 7 receptors into the plasma membrane of HEK293 cells  

Microsoft Academic Search

Recombinant human P2X7 receptors, C-terminally labelled with enhanced green fluorescent protein (P2X7–EGFP), were transiently expressed in HEK293 cells. Activation of these receptors by their preferential agonist 2?,3?-O-(4-benzoylbenzoyl)-ATP (BzATP) induced inward currents and propidium ion uptake indicating the opening of cationic channels and of large pores permeable for dye molecules, respectively. Two mutants of P2X7 receptors (P2X7–EGFP–I568N, –E496A) representing polymorphisms in

Doreen Milius; Helke Gröger-Arndt; Doychin Stanchev; Christine Lange-Dohna; Steffen Rossner; Beata Sperlagh; Kerstin Wirkner; Peter Illes

2007-01-01

224

Effects of oxymatrine on the apoptosis and proliferation of gallbladder cancer cells.  

PubMed

Gallbladder carcinoma is the most common malignancy of the biliary tract and is associated with a very poor outcome. The aim of the present study was to investigate the effects of oxymatrine (OM) on gallbladder cancer cells and the possible mechanism of its effects. The effects of OM on the proliferation of gallbladder cancer cells (GBC-SD and SGC-996) were investigated using cell counting kit-8 and colony formation assays. Annexin V/propidium iodide double staining was performed to investigate whether OM could induce apoptosis in gallbladder cancer cells. The mitochondrial membrane potential (??m) and expression of apoptosis-associated proteins were evaluated to identify a mechanism for the effects of OM. In addition, the RNA expression of relevant genes was measured by qRT-PCR using the SYBR Green method. Finally, a subcutaneous implantation model was used to verify the effects of OM on tumor growth in vivo. We found that OM inhibited the proliferation of gallbladder cancer cells. In addition, Annexin V/propidium iodide double staining showed that OM induced apoptosis after 48?h and the ??m decreased in a dose-dependent manner after OM treatment. Moreover, the activation of caspase-3 and Bax and downregulation of Bcl-2 and nuclear factor ?B were observed in OM-treated cells. Finally, OM potently inhibited in-vivo tumor growth following subcutaneous inoculation of SGC-996 cells in nude mice. In conclusion, OM treatment reduced proliferation and induced apoptosis in gallbladder cancer cells, which suggests that this drug may serve as a novel candidate for adjuvant treatment in patients with gallbladder cancer. PMID:24869760

Wu, Xiang-Song; Yang, Tian; Gu, Jun; Li, Mao-Lan; Wu, Wen-Guang; Weng, Hao; Ding, Qian; Mu, Jia-Sheng; Bao, Run-Fa; Shu, Yi-Jun; Cao, Yang; Wang, Xu-an; Ding, Qi-Chen; Dong, Ping; Xie, Shun-Feng; Liu, Ying-Bin

2014-10-01

225

Intramitochondrial pyruvate attenuates hydrogen peroxide-induced apoptosis in bovine pulmonary artery endothelium.  

PubMed

In the hydrogen peroxide (H2O2) apoptosis model of the murine thymocyte, redox reactant and antioxidant pyruvate prevents programmed cell death. We tested the hypothesis that such protection was mediated, at least in part, via pyruvate handling by mitochondrial metabolism. Cultured bovine pulmonary artery endothelial cells were incubated for 30 min with 0.5 mM H2O2 in the absence and presence of 0.5 mM alpha-cyano-3-hydroxycinnamate, as a selective inhibitor of the mitochondrial pyruvate transporter. In controls H2O2 decreased cell viability by 30% within 24 h; this was associated with apoptosis-like bodies, nuclear condensation, and biochemical DNA damage consistent with programmed cell death. Pyruvate (0.1-20 mM) enhanced cell viability in a dose-dependent manner, with > or = 85% viable cells at > or = 3 mM and no DNA laddering, no positive nick-end labeling (TUNEL), and no detectable Annexin V or propidium iodide staining. In contrast, using > or = 5 mM L-lactate as a cytosolic reductant or acetate as a redox-neutral substrate, cell death increased to approximately 40%, which was associated with intense DNA laddering, positive TUNEL and Hoechst 33258 assays. Alpha-cyano-3-hydroxycinnamate alone did not significantly decrease endothelial viability but reduced viability from 85+/-3 to 71+/-4% (p = 0.023) in presence of 3 mM pyruvate plus H2O2; pathological cell morphology and DNA laddering under the same conditions suggested loss of pyruvate protection against apoptosis. Since alpha-cyano-3-hydroxycinnamate re-distributed medium pyruvate and L-lactate consistent with selective blockade of pyruvate uptake into the mitochondria, the findings support the hypothesis that pyruvate protection against H2O2 apoptosis is mediated in part via the mitochondrial matrix compartment. Possible mediators include anti-apoptotic bcl-2 and/or products of mitochondrial pyruvate metabolism such as citrate that affect metabolic regulation and anti-oxidant status in the cytoplasm. PMID:11216862

Kang, Y H; Chung, S J; Kang, I J; Park, J H; Bünger, R

2001-01-01

226

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

227

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

228

Pseudolaric Acid B Induced Cell Cycle Arrest, Autophagy and Senescence in Murine Fibrosarcoma L929 Cell  

PubMed Central

Objective: PAB induced various cancer cell apoptosis, cell cycle arrest and senescence. But in cell line murine fibrosarcoma L929, PAB did not induce apoptosis, but autophagy, therefore it was thought by us as a good model to research the relationship of cell cycle arrest, autophagy and senescence bypass apoptosis. Methods: Inhibitory ratio was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. Phase contrast microscopy visualized cell morphology. Hoechst 33258 staining for nuclear change, propidium iodode (PI) staining for cell cycle, monodansylcadaverine (MDC) staining for autophagy, and rodanmine 123 staining for mitochondrial membrane potential (MMP) were measured by fluorescence microscopy or flowcytometry. Apoptosis was determined by DNA ladder test. Protein kinase C (PKC) activity was detected by PKC assay kit. SA-?-galactosidase assay was used to detect senescence. Protein expression was examined by western blot. Results: PAB inhibited L929 cell growth in time-and dose-dependent manner. At 12 h, 80 ?mol/L PAB induced obvious mitotic arrest; at 24 h, PAB began to induce autophagy; at 36 h, cell-treated with PAB slip into G1 cell cycle; and 3 d PAB induced senescence. In time sequence PAB induced firstly cell cycle arrest, then autophagy, then slippage into G1 phase, lastly senescence. Senescent cells had high level of autophagy, inhibiting autophagy led to apoptosis, and no senescence. PAB activated PKC activity to induce cell cycle arrest, autophagy and senescence, inhibiting PKC activity suppressed cell cycle arrest, autophagy and senescence. Conclusion: PAB induced cell cycle arrest, autophagy and senescence in murine fibrosarcoma L929 cell through PKC. PMID:23630435

hua Yu, Jing; yu Liu, Chun; bin Zheng, Gui; Zhang, Li Ying; hui Yan, Ming; yan Zhang, Wen; ying Meng, Xian; fang Yu, Xiao

2013-01-01

229

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

SciTech Connect

Bacteroides fragilis is an anaerobic bacteria component of human intestinal microbiota and agent of infections. In the host B. fragilis interacts with macrophages, which produces toxic radicals like NO. The interaction of activated mice peritoneal macrophages with four strains of B. fragilis was evaluated on this study. Previously was shown that such strains could cause metabolic and morphologic alterations related to macrophage death. In this work propidium iodide staining showed the strains inducing macrophage necrosis in that the labeling was evident. Besides nitroblue tetrazolium test showed that B. fragilis stimulates macrophage to produce oxygen radicals. In vivo assays performed in BalbC mice have results similar to those for in vitro tests as well as scanning electron microscopy, which showed the same surface pore-like structures observed in vitro before. The results revealed that B. fragilis strains studied lead to macrophage death by a process similar to necrosis.

Vieira, J.M.B.D., E-mail: jmanya@terra.com.br [Laboratorio de Tecnologia em Cultura de Celulas, UEZO, Rio de Janeiro (Brazil) [Laboratorio de Tecnologia em Cultura de Celulas, UEZO, Rio de Janeiro (Brazil); Laboratorio de Biologia de Anaerobios, IMPPG, UFRJ, Rio de Janeiro (Brazil); Seabra, S.H. [Laboratorio de Tecnologia em Cultura de Celulas, UEZO, Rio de Janeiro (Brazil)] [Laboratorio de Tecnologia em Cultura de Celulas, UEZO, Rio de Janeiro (Brazil); Vallim, D.C. [Instituto Oswaldo Cruz, Rio de Janeiro (Brazil)] [Instituto Oswaldo Cruz, Rio de Janeiro (Brazil); Americo, M.A.; Fracallanza, S.E.L. [Laboratorio de Bacteriologia Medica, IMPPG, UFRJ, Rio de Janeiro (Brazil)] [Laboratorio de Bacteriologia Medica, IMPPG, UFRJ, Rio de Janeiro (Brazil); Vommaro, R.C. [Laboratorio de Ultra-estrutura Celular Hertha Meyer, IBCCF, UFRJ (Brazil)] [Laboratorio de Ultra-estrutura Celular Hertha Meyer, IBCCF, UFRJ (Brazil); Domingues, R.M.C.P. [Laboratorio de Biologia de Anaerobios, IMPPG, UFRJ, Rio de Janeiro (Brazil)] [Laboratorio de Biologia de Anaerobios, IMPPG, UFRJ, Rio de Janeiro (Brazil)

2009-10-02

230

Clostridium perfringens Beta-Toxin Induces Necrostatin-Inhibitable, Calpain-Dependent Necrosis in Primary Porcine Endothelial Cells  

PubMed Central

Clostridium perfringens ?-toxin (CPB) is a ?-barrel pore-forming toxin and an essential virulence factor of C. perfringens type C strains, which cause fatal hemorrhagic enteritis in animals and humans. We have previously shown that CPB is bound to endothelial cells within the intestine of affected pigs and humans, and that CPB is highly toxic to primary porcine endothelial cells (pEC) in vitro. The objective of the present study was to investigate the type of cell death induced by CPB in these cells, and to study potential host cell mechanisms involved in this process. CPB rapidly induced lactate dehydrogenase (LDH) release, propidium iodide uptake, ATP depletion, potassium efflux, a marked rise in intracellular calcium [Ca2+]i, release of high-mobility group protein B1 (HMGB1), and caused ultrastructural changes characteristic of necrotic cell death. Despite a certain level of caspase-3 activation, no appreciable DNA fragmentation was detected. CPB-induced LDH release and propidium iodide uptake were inhibited by necrostatin-1 and the two dissimilar calpain inhibitors PD150606 and calpeptin. Likewise, inhibition of potassium efflux, chelation of intracellular calcium and treatment of pEC with cyclosporin A also significantly inhibited CPB-induced LDH release. Our results demonstrate that rCPB primarily induces necrotic cell death in pEC, and that necrotic cell death is not merely a passive event caused by toxin-induced membrane disruption, but is propagated by host cell-dependent biochemical pathways activated by the rise in intracellular calcium and inhibitable by necrostatin-1, consistent with the emerging concept of programmed necrosis (“necroptosis”). PMID:23734212

Popoff, Michel; D’Herde, Katharina; Christen, Stephan; Posthaus, Horst

2013-01-01

231

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

232

Optical injection of mammalian cells using a microfluidic platform  

PubMed Central

The use of a focused laser beam to create a sub-micron hole in the plasma membrane of a cell (photoporation), for the selective introduction of membrane impermeable substances (optical injection) including nucleic acids (optical transfection), is a powerful technique most commonly applied to treat single cells. However, particularly for femtosecond photoporation, these studies have been limited to low throughput, small-scale studies, because they require sequential dosing of individual cells. Herein, we describe a microfluidic photoporation system for increased throughput and automated optical injection of cells. Hydrodynamic focusing is employed to direct a flow of single-file cells through a focused femtosecond laser beam for photoporation. Upon traversing the beam, a number of transient pores potentially open across the extracellular membrane, which allows the uptake of the surrounding fluid media into the cytoplasm, also containing the chosen injection agent. The process is entirely automated and a rate of 1 cell/sec could readily be obtained, enabling several thousand cells to be injected per hour using this system. The efficiency of optically injecting propidium iodide into HEK293 mammalian cells was found to be 42 ± 8%, or 28 ± 4% taking into account the requirement of post-injection viability, as tested using Calcein AM. This work now opens the way for combining photoporation with microfluidic analyses, sorting, purification or on-chip cell culture studies. PMID:21258487

Marchington, Robert F.; Arita, Yoshihiko; Tsampoula, Xanthi; Gunn-Moore, Frank J.; Dholakia, Kishan

2010-01-01

233

Mefloquine induces cell death in prostate cancer cells and provides a potential novel treatment strategy in vivo  

PubMed Central

Mefloquine (MQ) is currently in clinical use as a prophylactic treatment for malaria. Previous studies have shown that MQ induces oxidative stress in vitro. The present study investigated the anticancer effects of MQ treatment in PC3 cells. The cell viability was evaluated using sulphorhodamine-B (SRB) staining, while annexin V and propidium iodide (PI) were used as an assay for cell death. Reactive oxygen species (ROS) formation was detected with 2?,7?-dichlorofluorescein-diacetate (DCFH-DA), a sensitive intracellular probe, and the alteration of cellular status was defined by trypan blue staining. The results of the present study indicated that MQ has a high cytotoxicity that causes cell death in PC3 cells. MQ markedly inhibited the PC3 cells through non-apoptotic cell death. MQ also induced significant ROS production. The MQ treatment mediated G1 cell cycle arrest and cyclin D1 accumulation through p21 upregulation in the PC3 cells. Moreover, the use of MQ improved the survival of the treatment group compared with the control group in the experimental mice. The present study indicates that MQ possesses potential therapeutic efficacy for the treatment of prostate cancer (PCa) in vivo. These findings provide insights that may aid the further optimization and application of new and existing therapeutic options. PMID:23759954

YAN, KUN-HUANG; LIN, YUNG-WEI; HSIAO, CHI-HAO; WEN, YU-CHING; LIN, KE-HSUN; LIU, CHUNG-CHI; HSIEH, MAO-CHIH; YAO, CHIH-JUNG; YAN, MING-DE; LAI, GI-MING; CHUANG, SHUANG-EN; LEE, LIANG-MING

2013-01-01

234

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

235

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

236

Cyclic GMP protects human macrophages against peroxynitrite-induced apoptosis  

PubMed Central

Background Nitric oxide (NO) can be both pro- and anti-apoptotic in various cell types, including macrophages. This apparent paradox may result from the actions of NO-related species generated in the microenvironment of the cell, for example the formation of peroxynitrite (ONOO-). In this study we have examined the ability of NO and ONOO- to evoke apoptosis in human monocyte-derived macrophages (MDM?), and investigated whether preconditioning by cyclic guanosine monophosphate (cGMP) is able to limit apoptosis in this cell type. Methods Characterisation of the NO-related species generated by (Z)-1- [2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO) and 1,2,3,4-oxatriazolium, 5-amino-3-(3,4-dichlorophenyl)-, chloride (GEA-3162) was performed by electrochemistry using an isolated NO electrode and electron paramagnetic resonance (EPR) spectrometry. Mononuclear cells were isolated from peripheral blood of healthy volunteers and cultured to allow differentiation into MDM?. Resultant MDM? were treated for 24 h with DETA/NO (100 – 1000 ?M) or GEA-3162 (10 – 300 ?M) in the presence or absence of BAY 41–2272 (1 ?M), isobutylmethylxanthine (IBMX; 1 ?M), 1H- [1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 20 ?M) or 8-bromo-cGMP (1 mM). Apoptosis in MDM? was assessed by flow cytometric analysis of annexin V binding in combination with propidium iodide staining. Results Electrochemistry and EPR revealed that DETA/NO liberated free NO radical, whilst GEA-3162 concomitantly released NO and O2-, and is therefore a ONOO- generator. NO (DETA/NO) had no effect on cell viability, but ONOO- (GEA-3162) caused a concentration-dependent induction of apoptosis in MDM?. Preconditioning of MDM? with NO in combination with the phosphodiesterase inhibitor, 3-Isobutyl-1-methylxanthine (IBMX), or the NO-independent stimulator of soluble guanylate cyclase, BAY 41–2272, significantly attenuated ONOO--induced apoptosis in a cGMP-dependent manner. Conclusion These results demonstrate disparities between the ability of NO and ONOO- to induce apoptosis in human MDM?. Furthermore, this study provides evidence for a novel cGMP-dependent pre-conditioning mechanism to limit ONOO--induced apoptosis in human MDM?. PMID:19422695

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

2009-01-01

237

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

238

Curcumin Inhibits Glyoxalase 1—A Possible Link to Its Anti-Inflammatory and Anti-Tumor Activity  

PubMed Central

Background Glyoxalases (Glo1 and Glo2) are involved in the glycolytic pathway by detoxifying the reactive methylglyoxal (MGO) into D-lactate in a two-step reaction using glutathione (GSH) as cofactor. Inhibitors of glyoxalases are considered as anti-inflammatory and anti-carcinogenic agents. The recent finding that various polyphenols modulate Glo1 activity has prompted us to assess curcumin's potency as an Glo1 inhibitor. Methodology/Principal Findings Cultures of whole blood cells and tumor cell lines (PC-3, JIM-1, MDA-MD 231 and 1321N1) were set up to investigate the effect of selected polyphenols, including curcumin, on the LPS-induced cytokine production (cytometric bead-based array), cell proliferation (WST-1 assay), cytosolic Glo1 and Glo2 enzymatic activity, apoptosis/necrosis (annexin V-FITC/propidium iodide staining; flow cytometric analysis) as well as GSH and ATP content. Results of enzyme kinetics revealed that curcumin, compared to the polyphenols quercetin, myricetin, kaempferol, luteolin and rutin, elicited a stronger competitive inhibitory effect on Glo1 (Ki?=?5.1±1.4 µM). Applying a whole blood assay, IC50 values of pro-inflammatory cytokine release (TNF-?, IL-6, IL-8, IL-1?) were found to be positively correlated with the Ki-values of the aforementioned polyphenols. Moreover, whereas curcumin was found to hamper the growth of breast cancer (JIMT-1, MDA-MB-231), prostate cancer PC-3 and brain astrocytoma 1321N1 cells, no effect on growth or vitality of human primary hepatocytes was elucidated. Curcumin decreased D-lactate release by tumor cells, another clue for inhibition of intracellular Glo1. Conclusions/Significance The results described herein provide new insights into curcumin's biological activities as they indicate that inhibition of Glo1 by curcumin may result in non-tolerable levels of MGO and GSH, which, in turn, modulate various metabolic cellular pathways including depletion of cellular ATP and GSH content. This may account for curcumin's potency as an anti-inflammatory and anti-tumor agent. The findings support the use of curcumin as a potential therapeutic agent. PMID:18946510

Santel, Thore; Pflug, Gabi; Hemdan, Nasr Y. A.; Schäfer, Angelika; Hollenbach, Marcus; Buchold, Martin; Hintersdorf, Anja; Lindner, Inge; Otto, Andreas; Bigl, Marina; Oerlecke, Ilka; Hutschenreuter, Antje; Sack, Ulrich; Huse, Klaus; Groth, Marco; Birkemeyer, Claudia; Schellenberger, Wolfgang; Gebhardt, Rolf; Platzer, Mathias; Weiss, Thomas; Vijayalakshmi, Mookambeswaran A.; Krüger, Monika; Birkenmeier, Gerd

2008-01-01

239

Oridonin inhibits BxPC-3 cell growth through cell apoptosis.  

PubMed

Oridonin, an ent-kaurene diterpenoid extracted from the traditional Chinese herb Rabdosia rubescens, has multiple biological and pharmaceutical functions and has been used clinically for many years. While the antitumor function of oridonin has been corroborated by numerous lines of evidence, its anticancer mechanism has not been well documented. In this study, the pancreatic cancer cell line BxPC-3 was used as a model to investigate a possible anticancer mechanism of oridonin through examining its effects on cell viability. The results showed that oridonin affected cell viability in a time- and dose-dependent manner. After exposure to different oridonin concentrations, growth rates and cell cycle arrest of BxPC-3 cells were significantly reduced compared with untreated cells, suggesting its effects on proliferation inhibition. Detailed signaling pathway analysis by western blot analysis revealed that low-dose oridonin treatment inhibited BxPC-3 cell proliferation by up-regulating p53 and down-regulating cyclin-dependent kinase 1 (CDK1), which led to cell cycle arrest in the G2/M phase. A high-dose oridonin not only arrested BxPC-3 cells in the G2/M phase but also induced cell accumulation in the S phase, presumably through ?H2AX up-regulation and DNA damage. In addition, our results showed that a cell subpopulation was stained with propidium iodide after oridonin treatment. Protein quantification showed that cleaved poly(ADP-ribose) polymerase (PARP) expression was increased after a high-dose oridonin treatment, especially after long-term exposure. Accompanied by the increased level of deactivated PARP in BxPC-3 cells, the apoptosis initiators caspase-3 and caspase-7 expressions were also significantly increased, suggesting that caspase-mediated apoptosis contributed to cell death. PMID:25651847

Xu, Bin; Shen, Wen; Liu, Xing; Zhang, Ting; Ren, Jun; Fan, Yongjun; Xu, Jian

2015-03-01

240

Mechanisms of programmed cell death during oogenesis in Drosophila virilis.  

PubMed

We describe the features of programmed cell death occurring in the egg chambers of Drosophila virilis during mid-oogenesis and late oogenesis. During mid-oogenesis, the spontaneously degenerating egg chambers exhibit typical characteristics of apoptotic cell death. As revealed by propidium iodide, rhodamine-conjugated phalloidin staining, and the TUNEL assay, respectively, the nurse cells contain condensed chromatin, altered actin cytoskeleton, and fragmented DNA. In vitro caspase activity assays and immunostaining procedures demonstrate that the atretic egg chambers possess high levels of caspase activity. Features of autophagic cell death are also observed during D. virilis mid-oogenesis, as shown by monodansylcadaverine staining, together with an ultrastructural examination by transmission electron microscopy. During the late stages of oogenesis in D. virilis, once again, the two mechanisms, viz., nurse cell cluster apoptosis and autophagy, operate together, manifesting features of cell death similar to those detailed above. Moreover, an altered form of cytochrome c seems to be released from the mitochondria in the nurse cells proximal to the oocyte. We propose that apoptosis and autophagy function synergistically during oogenesis in D. virilis in order to achieve a more efficient elimination of the degenerated nurse cells and abnormal egg chambers. PMID:17004067

Velentzas, Athanassios D; Nezis, Ioannis P; Stravopodis, Dimitrios J; Papassideri, Issidora S; Margaritis, Lukas H

2007-02-01

241

Adaptation to alkalosis induces cell cycle delay and apoptosis in cortical collecting duct cells: role of Aquaporin-2.  

PubMed

Collecting ducts (CD) not only constitute the final site for regulating urine concentration by increasing apical membrane Aquaporin-2 (AQP2) expression, but are also essential for the control of acid-base status. The aim of this work was to examine, in renal cells, the effects of chronic alkalosis on cell growth/death as well as to define whether AQP2 expression plays any role during this adaptation. Two CD cell lines were used: WT- (not expressing AQPs) and AQP2-RCCD(1) (expressing apical AQP2). Our results showed that AQP2 expression per se accelerates cell proliferation by an increase in cell cycle progression. Chronic alkalosis induced, in both cells lines, a time-dependent reduction in cell growth. Even more, cell cycle movement, assessed by 5-bromodeoxyuridine pulse-chase and propidium iodide analyses, revealed a G2/M phase cell accumulation associated with longer S- and G2/M-transit times. This G2/M arrest is paralleled with changes consistent with apoptosis. All these effects appeared 24 h before and were always more pronounced in cells expressing AQP2. Moreover, in AQP2-expressing cells, part of the observed alkalosis cell growth decrease is explained by AQP2 protein down-regulation. We conclude that in CD cells alkalosis causes a reduction in cell growth by cell cycle delay that triggers apoptosis as an adaptive reaction to this environment stress. Since cell volume changes are prerequisite for the initiation of cell proliferation or apoptosis, we propose that AQP2 expression facilitates cell swelling or shrinkage leading to the activation of channels necessary to the control of these processes. PMID:20432437

Rivarola, Valeria; Flamenco, Pilar; Melamud, Luciana; Galizia, Luciano; Ford, Paula; Capurro, Claudia

2010-08-01

242

Spica Prunellae extract inhibits the proliferation of human colon carcinoma cells via the regulation of the cell cycle.  

PubMed

Spica Prunellae has long been used as a significant component in numerous traditional Chinese medicine (TCM) formulas to clinically treat cancers. Previously, Spica Prunellae was shown to promote cancer cell apoptosis and inhibit angiogenesis in vivo and in vitro. To further elucidate the precise mechanism of its tumoricidal activity, the effect of the ethanol extract of Spica Prunellae (EESP) on the proliferation of human colon carcinoma HT-29 cells was elucidated and the underlying molecular mechanisms were investigated. The proliferation of HT-29 cells was evaluated using 3-(4, 5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation analyses. The cell cycle was determined using fluorescence-activated cell sorting (FACS) with propidium iodide (PI) staining. The mRNA and protein expression of cyclin-dependent kinase 4 (CDK4) and cyclin D1 was examined using RT-PCR and western blotting, respectively. EESP was observed to inhibit HT-29 viability and survival in a dose- and time-dependent manner. Furthermore, EESP treatment blocked G1/S cell cycle progression and reduced the expression of pro-proliferative cyclin D1 and CDK4 at the transcriptional and translational levels. Altogether, these data suggest that the inhibition of cell proliferation via G1/S cell cycle arrest may be one of the mechanisms through which Spica Prunellae treats cancer. PMID:24137475

Lin, Wei; Zheng, Liangpu; Zhuang, Qunchuan; Shen, Aling; Liu, Liya; Chen, Youqin; Sferra, Thomas J; Peng, Jun

2013-10-01

243

Sublethal heat shock induces premature senescence rather than apoptosis in human mesenchymal stem cells.  

PubMed

Stem cells in adult organism are responsible for cell turnover and tissue regeneration. The study of stem cell stress response contributes to our knowledge on the mechanisms of damaged tissue repair. Previously, we demonstrated that sublethal heat shock (HS) induced apoptosis in human embryonic stem cells. This study aimed to investigate HS response of human adult stem cells. Human mesenchymal stem cells (MSCs) cultivated in vitro were challenged with sublethal HS. It was found that sublethal HS did not affect the cell viability assessed by annexin V/propidium staining. However, MSCs subjected to severe HS exhibited features of stress-induced premature senescence (SIPS): irreversible cell cycle arrest, altered morphology, increased expression of senescence-associated ?-galactosidase (SA-?-gal) activity, and induction of cyclin-dependent kinase inhibitor p21 protein. High level of Hsp70 accumulation induced by sublethal HS did not return to the basal level, at least, after 72 h of the cell recovery when most cells exhibited SIPS hallmarks. MSCs survived sublethal HS, and resumed proliferation sustained the properties of parental MSCs: diploid karyotype, replicative senescence, expression of the cell surface markers, and capacity for multilineage differentiation. Our results showed for the first time that in human MSCs, sublethal HS induced premature senescence rather than apoptosis or necrosis. MSC progeny that survived sublethal HS manifested stem cell properties of the parental cells: limited replicative life span and multilineage capacity. PMID:24078383

Alekseenko, Larisa L; Zemelko, Victoria I; Domnina, Alisa P; Lyublinskaya, Olga G; Zenin, Valery V; Pugovkina, Nataly A; Kozhukharova, Irina V; Borodkina, Alexandra V; Grinchuk, Tatiana M; Fridlyanskaya, Irina I; Nikolsky, Nikolay N

2014-05-01

244

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

PubMed

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

Siemieniuch, Marta; Dubiel, Andrzej

2007-05-01

245

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

246

Schwann-like cell differentiation from rat bone marrow stem cells  

PubMed Central

Introduction The main purpose of this study was differentiation of bone marrow stem cells (BMSCs) into Schwann-like cells and to determine the intensity of apoptosis in BMSCs during the differentiation process. Material and methods Bone marrow stem cells were isolated from the femur of adult rats and the identity of the undifferentiated BMSCs was confirmed by the detection of specific cell surface markers. The BMSCs were differentiated by sequential administration of ?-mercaptoethanol and all-trans-retinoic acid as pre-inducer factors and a mixture of forskolin, basic fibroblast growth factor, platelet-derived growth factor-AA and heregulin-b1 as inducer factors. The immunocytochemical properties of differentiated Schwann-like cells were examined at a specified time point. Reverse transcription-polymerase chain reaction (RT-PCR) was used to investigate the gene expression of the undifferentiated and differentiated BMSCs. Cell apoptosis and viability were assessed with annexin V and propidium iodide double staining and dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide (MTT) assay. Results Immunocytochemistry staining and RT-PCR analysis revealed that the induced BMSCs exhibited Schwann cell-specific markers such as S-100, P75 and glial fibrillary acidic protein (GFAP) at the 14th day of differentiation. MTT assay and flow cytometry revealed that of the total BMSCs in the differentiation medium, 40% to 50% of the cells died by apoptosis, but the remaining cell population remained strongly attached to the substrate and differentiated. Conclusions These findings indicated that BMSCs could differentiate into Schwann-like cells. As a side effect of differentiation an increased cell death rate was noted and our findings indicate that the principle mode of cell death is by apoptosis. PMID:22291732

Kashani, Iraj Ragerdi; Golipoor, Zolikha; Akbari, Mohammad; Mahmoudi, Reza; Azari, Shahram; Shirazi, Reza; Bayat, Mohammad; Ghasemi, Soudabeh

2011-01-01

247

Effects of curine in HL-60 leukemic cells: cell cycle arrest and apoptosis induction.  

PubMed

Curine is a natural alkaloid isolated from Chondrodendron platyphyllum and it has been reported that this alkaloid has vasodilatory and anti-inflammatory effects. The aim of this study is to analyze the cytotoxic effects of curine in cancer cell lines HL-60, K562, and HT-29, and in primary cultures of peripheral blood mononuclear cells (PBMC). Cells were treated with curine (from 3 to 15 µM) for 24 and 48 h. Cell viability was analyzed by the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and flow cytometry with propidium iodide (PI) assay. To assess the type of cell death induced in HL-60, the cell cycle, morphological, and biochemical alterations were analyzed, which were determined by differential staining with acridine orange/ethidium bromide, and annexin V/PI double-labeling and change in mitochondrial membrane potential assays. Curine demonstrated a potent cytotoxic effect on leukemic cell lines (HL-60 and K562). Its cytotoxic effects in HL-60 cells was related to plasma membrane damage and cell cycle arrest at the G1 phase from 43.4 ± 1.0 to 56.7 ± 1.4 % (p < 0.05). Curine (15 ?M) also increased the apoptotic cells number by around 60 % in HL-60 cells and caused phosphatidylserine externalization, inducing about 57 % of apoptosis. Moreover, this alkaloid provoked 20 % of mitochondrial membrane depolarization. We conclude that curine presented a cytotoxic effect and induced apoptosis in HL-60 cells. Thus, it can be considered a promising pharmacological drug. PMID:25616501

Dantas, Bruna Braga; Faheina-Martins, Gláucia Veríssimo; Coulidiati, Tangbadioa Hervé; Bomfim, Caio César Barbosa; da Silva Dias, Celidarque; Barbosa-Filho, José Maria; Araújo, Demetrius Antônio Machado

2015-04-01

248

Induction of apoptosis in NK cells by monocyte-derived reactive oxygen metabolites.  

PubMed

Human NK cells (with CD3-/56+ phenotype) acquired features characteristic of apoptosis after incubation with autologous monocytes, as revealed by apoptotic nuclear morphology, degradation of DNA into oligonucleosomal fragments, and reduced nuclear interchalation of propidium iodide. In contrast, T cells (CD3+/56-) remained non-apoptotic. The monocyte-induced apoptosis in NK cells was prevented by catalase, a scavenger of hydrogen peroxide; whereas superoxide dismutase (a scavenger of superoxide anion), hydroxyl radical scavengers such as mannitol and deferoxamine, or the hypochlorus acid scavenger taurine did not prevent apoptosis. Sodium azide, a myeloperoxidase inhibitor, substantially reduced the monocyte-induced apoptosis in NK cells. Exogenous hydrogen peroxide, at concentrations exceeding 1 microns, induced apoptosis in both NK and T cells. Apoptosis induced by hydrogen peroxide occurred independently of synthesis of protein or mRNA and was blocked by the endonuclease inhibitor aurin tricarboxylic acid. Furthermore, oxidatively induced apoptosis in NK cells was inhibited by herbimycin A, indicating that apoptosis was dependent on protein kinases. Two to five times more hydrogen peroxide was required to induce apoptosis in T cells compared with NK cells. Similarly, NK cells were considerably more susceptible to apoptosis induced by the topoisomerase II inhibitor etoposide or by gamma-irradiation than were T cells. We conclude that monocyte-derived reactive oxygen metabolites kill NK cells by apoptosis and that NK cells are unusually sensitive to oxidatively as well as non-oxidatively induced apoptosis. PMID:8598491

Hansson, M; Asea, A; Ersson, U; Hermodsson, S; Hellstrand, K

1996-01-01

249

Effects of microwave exposure and Gemcitabine treatment on apoptotic activity in Burkitt's lymphoma (Raji) cells.  

PubMed

Abstract We investigated the effects of 1.8?MHz Global System for Mobile Communications (GSM)-modulated microwave (MW) radiation on apoptotic level and cell viability of Burkitt's lymphoma (Raji) cells with or without Gemcitabine, which exhibits cell phase specificity, primarily killing cells undergoing DNA synthesis (S-phase). Raji cells were exposed to 1.8?GHz GSM-modulated MW radiation at a specific absorption rate (SAR) of 0.350?W/kg in a CO2 incubator. The duration of the exposure was 24?h. The amount of apoptotic cells was analyzed using Annexin V-FITC and propidium iodide (PI) staining with flow cytometer. The apoptotic activity of MW exposed Raji cells was increased significantly. In addition, cell viability of exposed samples was significantly decreased. Combined exposure of MW and Gemcitabine increased the amount of apoptotic cells than MW radiation alone. Moreover, viability of MW?+?Gemcitabine exposed cells was lower than that of cells exposed only to MW. These results demonstrated that MW radiation exposure and Gemcitabine treatment have a synergistic effect on apoptotic activity of Raji cells. PMID:24901461

Canseven, Ay?e G; Esmekaya, Meric Arda; Kayhan, Handan; Tuysuz, Mehmet Zahid; Seyhan, Nesrin

2014-06-01

250

Pro-apoptotic Bax is the major and Bak an auxiliary effector in cytokine deprivation-induced mast cell apoptosis  

PubMed Central

The process of apoptosis in immune cells like mast cells is essential to regain homeostasis after an inflammatory response. The intrinsic pathway of apoptosis is ultimately controlled by the pro-apoptotic Bcl-2 family members Bax and Bak, which upon activation oligomerize to cause increased permeabilization of the mitochondria outer membrane leading to cell death. We examined the role of Bax and Bak in cytokine deprivation-induced apoptosis in mast cells using connective tissue-like mast cells and mucosal-like mast cells derived from bax?/?, bak?/? and bax?/?bak?/? mice. Although both Bax and Bak were expressed at readily detectable protein levels, we found a major role for Bax in mediating mast cell apoptosis induced by cytokine deprivation. We analyzed cell viability by propidium iodide exclusion and flow cytometry after deprivation of vital cytokines for each mast cell population. Upon cytokine withdrawal, bak?/? mast cells died at a similar rate as wild type, whereas bax?/? and bax?/?bak?/? mast cells were partially or completely resistant to apoptosis, respectively. The total resistance seen in bax?/?bak?/? mast cells is comparable with mast cells deficient of both pro-apoptotic Bim and Puma or mast cells overexpressing anti-apoptotic Bcl-2. These results show that Bax has a predominant and Bak a minor role in cytokine deprivation-induced apoptosis in both connective tissue-like and mucosal-like mast cells. PMID:21364649

Karlberg, M; Ekoff, M; Labi, V; Strasser, A; Huang, D; Nilsson, G

2010-01-01

251

Induction of apoptosis and cell cycle arrest in human HCC MHCC97H cells with Chrysanthemum indicum extract  

PubMed Central

AIM: To investigate the effects of Chrysanthemum indicum extract (CIE) on inhibition of proliferation and on apoptosis, and the underlying mechanisms, in a human hepatocellular carcinoma (HCC) MHCC97H cell line. METHODS: Viable rat hepatocytes and human endothelial ECV304 cells were examined by trypan blue exclusion and MTT assay, respectively, as normal controls. The proliferation of MHCC97H cells was determined by MTT assay. The cellular morphology of MHCC97H cells was observed by phase contrast microscopy. Flow cytometry was performed to analyze cell apoptosis with annexin V/propidium iodide (PI), mitochondrial membrane potential with rhodamine 123 and cell cycle with PI in MHCC97H cells. Apoptotic proteins such as cytochrome C, caspase-9, caspase-3 and cell cycle proteins, including P21 and CDK4, were measured by Western blotting. RESULTS: CIE inhibited proliferation of MHCC97H cells in a time- and dose-dependent manner without cytotoxicity in rat hepatocytes and human endothelial cells. CIE induced apoptosis of MHCC97H cells in a concentration-dependent manner, as determined by flow cytometry. The apoptosis was accompanied by a decrease in mitochondrial membrane potential, release of cytochrome C and activation of caspase-9 and caspase-3. CIE arrested the cell cycle in the S phase by increasing P21 and decreasing CDK4 protein expression. CONCLUSION: CIE exerted a significant apoptotic effect through a mitochondrial pathway and arrested the cell cycle by regulation of cell cycle-related proteins in MHCC97H cells without an effect on normal cells. The cancer-specific selectivity shown in this study suggests that the plant extract could be a promising novel treatment for human cancer. PMID:19777612

Li, Zong-Fang; Wang, Zhi-Dong; Ji, Yuan-Yuan; Zhang, Shu; Huang, Chen; Li, Jun; Xia, Xian-Ming

2009-01-01

252

5-Episinuleptolide acetate, a norcembranoidal diterpene from the formosan soft coral Sinularia sp., induces leukemia cell apoptosis through Hsp90 inhibition.  

PubMed

5-Episinuleptolide acetate (5EPA), a cytotoxic norcembranoidal diterpene recently identified from the Formosan soft coral Sinularia sp., exhibited potent activity against the K562, Molt 4 and HL 60 cancer cell lines. The antiproliferative assay, as well as the annexin V-FITC/propidium iodide (PI) apoptotic assay, indicated that the HL 60 cell line is the most sensitive one towards 5EPA. This diterpenoid led to caspases -3, -8, and -9 activation as well as PARP cleavage. It also induced ROS generation, calcium accumulation and disruption of mitochondrial membrane potential. Additionally, the expression levels of Hsp90 protein and several client proteins were downregulated in response to 5EPA treatment. These results suggest that 5EPA's cytotoxic effect on HL 60 cells may be attributed to the inhibition of Hsp90 as well as the induction of mitochondrial stress which finally results in apoptotic cell death. PMID:23459302

Huang, Kao-Jean; Chen, Yu-Cheng; El-Shazly, Mohamed; Du, Ying-Chi; Su, Jui-Hsin; Tsao, Chia-Wei; Yen, Wei-Hsuan; Chang, Wen-Been; Su, Yin-Di; Yeh, Yao-Tsung; Lu, Mei-Chin

2013-01-01

253

Time courses of mammalian cell electropermeabilization observed by millisecond imaging of membrane property changes during the pulse.  

PubMed Central

Time courses of electropermeabilization were analyzed during the electric field application using a rapid fluorescent imaging system. Exchanges of calcium ions through electropermeabilized membrane of Chinese hamster ovary cells were found to be asymmetrical. Entry of calcium ions during a millisecond pulse occurred on the anode-facing cell hemisphere. Entry through the region facing the cathode was observed only after the pulse. Leakage of intracellular calcium ions from electropermeabilized cell in low-calcium content medium was observed only from the anode-facing side. The exchanges during the pulse were mostly due to diffusion-driven processes, i.e., governed by the concentration gradient. Interaction of propidium iodide, a dye sensitive to the structural alteration of membrane, with cell membrane was asymmetrical during electropermeabilization. Localized enhancement of the dye fluorescence was observed during and after the pulsation on the cell surface. Specific staining of a limited anode-facing part of the membrane was observed as soon as the pulse was applied. The membrane fluorescence level increased during and immediately after the pulse whereas the geometry of the staining was unchanged. The membrane regions stained by propidium iodide were the same as those where calcium exchanges occurred. The fraction of the membrane on which structural alterations occurred was defined by the field strength. The density of defects was governed by the pulse duration. Electropermeabilization is a localized but asymmetrical process. The membrane defects are created unequally on the two cell sides during the pulse, implying a vectorial effect of the electric field on the membrane. PMID:10096909

Gabriel, B; Teissié, J

1999-01-01

254

Overexpression of interleukin-18 protein reduces viability and induces apoptosis of tongue squamous cell carcinoma cells by activation of glycogen synthase kinase-3? signaling  

PubMed Central

The aim of this study was to investigate the effects of interleukin-18 (IL-18) expression on regulating the viability and apoptosis of tongue squamous cell carcinoma (TSCC) cells in vitro and examine the underlying molecular events. Human IL-18 cDNA was cloned into the vector pcDNA3.1 (+) and transfected into CRL-1623™ cells. Quantitative reverse transcription-PCR (RT-qPCR), western blot analysis, immunofluorescence, cell viability MTT assay, flow cytometric Annexin V/propidium iodide (PI), Giemsa staining, and caspase-3 activity assay were performed. The data showed that overexpression of IL-18 protein reduced TSCC cell viability by inducing apoptosis. Compared with cells transfected with the control vector, IL-18 expression activated caspase-3, -7, and -9 by inducing their cleavage and increased the expression of interferon (IFN)-? and cytochrome c mRNA, but reduced cyclin D1 and A1 expression in TSCC cells. IL-18 expression upregulated the expression and phosphorylation of glycogen synthase kinase (GSK)-3? protein in CRL1623 cells, whereas the selective GSK-3? inhibitor kenpaullone antagonized the effects of IL-18 protein on TSCC cells in vitro. The results indicated that IL-18 played an important role in the inhibition of TSCC cell growth and may be further investigated as a novel therapeutic target against TSCC. PMID:25591548

LIU, WEIWEI; HU, MIN; WANG, YUMEI; SUN, BAOZHEN; GUO, YU; XU, ZHIMIN; LI, JIA; HAN, BING

2015-01-01

255

Overexpression of interleukin-18 protein reduces viability and induces apoptosis of tongue squamous cell carcinoma cells by activation of glycogen synthase kinase-3? signaling.  

PubMed

The aim of this study was to investigate the effects of interleukin-18 (IL-18) expression on regulating the viability and apoptosis of tongue squamous cell carcinoma (TSCC) cells in vitro and examine the underlying molecular events. Human IL-18 cDNA was cloned into the vector pcDNA3.1 (+) and transfected into CRL-1623™ cells. Quantitative reverse transcription-PCR (RT-qPCR), western blot analysis, immunofluorescence, cell viability MTT assay, flow cytometric Annexin V/propidium iodide (PI), Giemsa staining, and caspase-3 activity assay were performed. The data showed that overexpression of IL-18 protein reduced TSCC cell viability by inducing apoptosis. Compared with cells transfected with the control vector, IL-18 expression activated caspase-3, -7, and -9 by inducing their cleavage and increased the expression of interferon (IFN)-? and cytochrome c mRNA, but reduced cyclin D1 and A1 expression in TSCC cells. IL-18 expression upregulated the expression and phosphorylation of glycogen synthase kinase (GSK)-3? protein in CRL1623 cells, whereas the selective GSK-3? inhibitor kenpaullone antagonized the effects of IL-18 protein on TSCC cells in vitro. The results indicated that IL-18 played an important role in the inhibition of TSCC cell growth and may be further investigated as a novel therapeutic target against TSCC. PMID:25591548

Liu, Weiwei; Hu, Min; Wang, Yumei; Sun, Baozhen; Guo, Yu; Xu, Zhimin; Li, Jia; Han, Bing

2015-03-01

256

Inhibitory Effects of Megakaryocytic Cells in Prostate Cancer Skeletal Metastasis  

PubMed Central

Prostate cancer cells commonly spread through the circulation, but few successfully generate metastatic foci in bone. Osteoclastic cellular activity has been proposed as an initiating event for skeletal metastasis. Megakaryocytes (MKs) inhibit osteoclastogenesis, which could have an impact on tumor establishment in bone. Given the location of mature MKs at vascular sinusoids, they may be the first cells to physically encounter cancer cells as they enter the bone marrow. Identification of the interaction between MKs and prostate cancer cells was the focus of this study. K562 (human MK precursors) and primary MKs derived from mouse bone marrow hematopoietic precursor cells potently suppressed prostate carcinoma PC-3 cells in coculture. The inhibitory effects were specific to prostate carcinoma cells and were enhanced by direct cell-cell contact. Flow cytometry for propidium iodide (PI) and annexin V supported a proapoptotic role for K562 cells in limiting PC-3 cells. Gene expression analysis revealed reduced mRNA levels for cyclin D1, whereas mRNA levels of apoptosis-associated specklike protein containing a CARD (ASC) and death-associated protein kinase 1 (DAPK1) were increased in PC-3 cells after coculture with K562 cells. Recombinant thrombopoietin (TPO) was used to expand MKs in the marrow and resulted in decreased skeletal lesion development after intracardiac tumor inoculation. These novel findings suggest a potent inhibitory role of MKs in prostate carcinoma cell growth in vitro and in vivo. This new finding, of an interaction of metastatic tumors and hematopoietic cells during tumor colonization in bone, ultimately will lead to improved therapeutic interventions for prostate cancer patients. © 2011 American Society for Bone and Mineral Research. PMID:20684002

Li, Xin; Koh, Amy J; Wang, Zhengyan; Soki, Fabiana N; Park, Serk In; Pienta, Kenneth J; McCauley, Laurie K

2011-01-01

257

Disruption of the actin cortex contributes to susceptibility of mammalian cells to nanosecond pulsed electric fields.  

PubMed

Nanosecond pulsed electric fields (nsPEFs) perturb membranes of cultured mammalian cells in a dose-dependent manner with different types of cells exhibiting characteristic survivability. Adherent cells appear more robust than non-adherent cells during whole-cell exposure. We hypothesize that cellular elasticity based upon the actin cytoskeleton is a contributing parameter, and the alteration of a cell's actin cortex will significantly affect viability upon nsPEF exposure. Chinese hamster ovary (CHO) cells that are (a) untreated, (b) treated with latrunculin A to inhibit actin polymerization, or (c) exposed to nsPEFs have been probed using atomic force microscopy (AFM) force-indentations. Exposure to 50 or 100 pulses of 10?ns duration and 150?kV/cm in a single dosage approximately lowers average CHO cell elastic modulus by half, whereas latrunculin lowers it more than 75%. Latrunculin pre-treatment disrupts the actin cortex enough that it negates cumulative damage by equally fractionated (i.e., two rounds of 50 pulses each, separated by 10?min) dosages of nsPEFs as seen in untreated and dimethyl sulfoxide (DMSO)-treated cells with propidium uptake, phosphatidylserine externalization, and 24?h viability according to MTT and CellTiter Glo assays. These results suggest a correlation among cell stiffness, cytoskeletal integrity, and susceptibility to recurrent exposures to nsPEFs, which emphasizes a mechanobiological underpinning of nsPEF bioeffects. PMID:24619788

Thompson, Gary L; Roth, Caleb; Tolstykh, Gleb; Kuipers, Marjorie; Ibey, Bennett L

2014-05-01

258

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

PubMed Central

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

Erman, Andreja; Zupan?i?, Daša; Jezernik, Kristijan

2009-01-01

259

Antiproliferative and cell apoptosis-inducing activities of compounds from Buddleja davidii in Mgc-803 cells  

PubMed Central

Background Buddleja davidii is widely distributed in the southwestern region of China. We have undertaken a systematic analysis of B. davidii as a Chinese traditional medicine with anticancer activity by isolating natural products for their activity against the human gastric cancer cell line Mgc-803 and the human breast cancer cell line Bcap-37. Results Ten compounds were extracted and isolated from B. davidii, among which colchicine was identified in B. davidii for the first time. The inhibitory activities of these compounds were investigated in Mgc-803, Bcap-37 cells in vitro by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, and the results showed that luteolin and colchicine had potent inhibitory activities against the growth of Mgc-803 cells. Subsequent fluorescence staining and flow cytometry analysis indicated that these two compounds could induce apoptosis in Mgc-803 cells. The results also showed that the percentages of early apoptotic cells (Annexin V+/PI-, where PI is propidium iodide) and late apoptotic cells (Annexin V+/PI+) increased in a dose- and time-dependent manner. After 36 h of incubation with luteolin at 20 ?M, the percentages of cells were approximately 15.4% in early apoptosis and 43.7% in late apoptosis; after 36 h of incubation with colchicine at 20 ?M, the corresponding values were 7.7% and 35.2%, respectively. Conclusions Colchicine and luteolin from B. davidii have potential applications as adjuvant therapies for treating human carcinoma cells. These compounds could also induce apoptosis in tumor cells. PMID:22938042

2012-01-01

260

Paris chinensis dioscin induces G2/M cell cycle arrest and apoptosis in human gastric cancer SGC-7901 cells  

PubMed Central

AIM: To investigate the anti-tumor effects of Paris chinensis dioscin (PCD) and mechanisms regarding cell cycle regulation and apoptosis in human gastric cancer SGC-7901 cells. METHODS: Cell viability was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Cell apoptosis was evaluated by flow cytometry and laser scanning confocal microscope (LSCM) using Annexin-V/propidium iodide (PI) staining, and the cell cycle was evaluated using PI staining with flow cytometry. Intracellular calcium ions were detected under fluorescence microscope. The expression of cell cycle and apoptosis-related proteins cyclin B1, CDK1, cytochrome C and caspase-3 was measured by immunohistochemical staining. RESULTS: PCD had an anti-proliferation effect on human gastric cancer SGC-7901 cells in a dose- and time-dependent manner. After treatment of SGC-7901 cells with PCD, apoptosis appeared in SGC-7901 cells. Morphological changes typical of apoptosis were also observed with LSCM by Annexin V/PI staining, and the cell number of the G0/G1 phase was decreased, while the number of cells in the G2/M phase was increased. Cell cycle-related proteins, such as cyclin B1 and CDK1, were all down-regulated, but caspase-3 and cytochrome C were up-regulated. Moreover, intracellular calcium accumulation occurred in PCD-treated cells. CONCLUSION: G2/M phase arrest and apoptosis induced by PCD are associated with the inhibition of CDK-activating kinase activity and the activation of Ca2+-related mitochondrion pathway in SGC-7901 cells. PMID:22110264

Gao, Lin-Lin; Li, Fu-Rong; Jiao, Peng; Yang, Ming-Feng; Zhou, Xiao-Jun; Si, Yan-Hong; Jiang, Wen-Jian; Zheng, Ting-Ting

2011-01-01

261

Brazilian marine sponge Polymastia janeirensis induces apoptotic cell death in human U138MG glioma cell line, but not in a normal cell culture.  

PubMed

Marine sponges have been prominently featured in the area of cancer research. Here, we examined the anti-proliferative effects of crude extracts (aqueous and organic) of the Brazilian marine sponge Polymastia janeirensis in the U138MG human glioma cell line. Moreover, we examined the effects of extracts on selective cytotoxicity in the glioma cells in comparison with a normal cell culture. Exposure of glioma cells to treatments (24 h) resulted in cell number decrease at all doses tested, with both aqueous and organic extracts (IC(50) <20 and <30 microg/ml, respectively). Parallel to this result, sponge extracts reduced glioma cell viability (IC(50) <15 microg/ml for both extracts). However, higher doses (50 and 100 microg/ml) induced a stronger cytotoxic effect when compared to the lower dose tested (10 microg/ml), inhibiting more than 80% of cellular growth and viability. Propidium iodide uptake and flow cytometry analysis further showed that sponge extracts caused necrosis in the glioma cell line at higher doses, while a high percentage of apoptotic glioma cells were observed at 10 microg/ml. Moreover, apoptosis was prevented by the pan-caspase inhibitor Z-VAD, suggesting that marine sponge extracts, at lower doses, induce caspase-dependent apoptosis in U138MG glioma cells. Surprisingly the extracts herein tested were more effective than temozolomide, a potent inductor of apoptosis used for the treatment of malignant gliomas. Furthermore, our results suggested a selectivity cytotoxic effect on glioma cell line in comparison with a normal cell culture, since the effect on viability found in glioma cells was not observed in astrocyte cultures with the lower dose (10 microg/ml). Thus, this marine sponge may be considered a good candidate for development of new cancer medicines with antitumor activity against gliomas. PMID:18454276

da Frota, Mario Luiz Conte; Braganhol, Elizandra; Canedo, Andrés Delgado; Klamt, Fabio; Apel, Miriam Anders; Mothes, Beatriz; Lerner, Cléa; Battastini, Ana Maria Oliveira; Henriques, Amélia Teresinha; Moreira, José Cláudio Fonseca

2009-02-01

262

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

263

A new flow cytometric method for discrimination of apoptotic cells and detection of their cell cycle specificity through staining of F-actin and DNA.  

PubMed

Drug-initiated apoptosis of human leukemia HL-60, THP-1, and U-937 cells was studied via multiparameter flow cytometry and cell sorting. A new flow cytometric method that allows both identification and quantitation of apoptotic cells and estimation of their cell cycle specificity is presented. The method is based on paraformaldehyde fixation followed by staining of F-actin and DNA with fluorescein isothiocyanate (FITC)-phalloidin and propidium iodide (PI), respectively. Bivariate green fluorescence (F-actin) vs. side scatterplots of HL-60 cells treated with 10 microM etoposide for 4 h showed two cell populations, one with high green fluorescence and low side scatter and one with low green fluorescence and high side scatter. Sorting revealed cells with intact nuclei in the high green fluorescence/low side scatter population and cells with fragmented nuclei in the low green fluorescence/high side scatter population, demonstrating that the cells in the latter population were apoptotic. Exposure of HL-60 cells to 10 microM etoposide for 4 h resulted in S-phase selective apoptosis, whereas 5 micrograms/ml cycloheximide initiated apoptosis mainly in G0/G1-phase and S-phase cells. The apoptotic response of HL-60 cells to 20 GY gamma-irradiation was selective for S-phase and G2 + M-phase cells. The present method offers the opportunity to estimate the cell cycle distributions of both the apoptotic and the nonapoptotic cell populations, which is especially valuable when apoptosis occurs in association with cell cycle perturbations. A similar shift from one to two cell populations in green fluorescence vs. side scatter-plots, similar to that observed for HL-60 cells, was observed in the THP-1 and U-937 cell lines secondary to etoposide treatment. PMID:7545098

Endresen, P C; Prytz, P S; Aarbakke, J

1995-06-01

264

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

265

Heme Oxygenase-1 Induction Improves Cardiac Function following Myocardial Ischemia by Reducing Oxidative Stress  

PubMed Central

Background Oxidative stress plays a key role in exacerbating diabetes and cardiovascular disease. Heme oxygenase-1 (HO-1), a stress response protein, is cytoprotective, but its role in post myocardial infarction (MI) and diabetes is not fully characterized. We aimed to investigate the protection and the mechanisms of HO-1 induction in cardiomyocytes subjected to hypoxia and in diabetic mice subjected to LAD ligation. Methods In vitro: cultured cardiomyocytes were treated with cobalt-protoporphyrin (CoPP) and tin protoporphyrin (SnPP) prior to hypoxic stress. In vivo: CoPP treated streptozotocin-induced diabetic mice were subjected to LAD ligation for 2/24 h. Cardiac function, histology, biochemical damage markers and signaling pathways were measured. Results HO-1 induction lowered release of lactate dehydrogenase (LDH) and creatine phospho kinase (CK), decreased propidium iodide staining, improved cell morphology and preserved mitochondrial membrane potential in cardiomyocytes. In diabetic mice, Fractional Shortening (FS) was lower than non-diabetic mice (35±1%vs.41±2, respectively p<0.05). CoPP-treated diabetic animals improved cardiac function (43±2% p<0.01), reduced CK, Troponin T levels and infarct size compared to non-treated diabetic mice (P<0.01, P<0.001, P<0.01 respectively). CoPP-enhanced HO-1 protein levels and reduced oxidative stress in diabetic animals, as indicated by the decrease in superoxide levels in cardiac tissues and plasma TNF? levels (p<0.05). The increased levels of HO-1 by CoPP treatment after LAD ligation led to a shift of the Bcl-2/bax ratio towards the antiapoptotic process (p<0.05). CoPP significantly increased the expression levels of pAKT and pGSK3? (p<0.05) in cardiomyocytes and in diabetic mice with MI. SnPP abolished CoPP's cardioprotective effects. Conclusions HO-1 induction plays a role in cardioprotection against hypoxic damage in cardiomyocytes and in reducing post ischemic cardiac damage in the diabetic heart as proved by the increased levels of pAKT with a concomitant inhibition of pGSK3? leading to preserved mitochondrial membrane potential. PMID:24658657

Issan, Yossi; Kornowski, Ran; Aravot, Dan; Shainberg, Asher; Laniado-Schwartzman, Michal; Sodhi, Komal; Abraham, Nader G.; Hochhauser, Edith

2014-01-01

266

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

2014-01-01

267

Potentiation of Anticancer Drugs: Effects of Pentoxifylline on Neoplastic Cells  

PubMed Central

The drug efflux activity of P-glycoprotein (P-gp, a product of the mdr1 gene, ABCB1 member of ABC transporter family) represents a mechanism by which tumor cells escape death induced by chemotherapeutics. In this study, we investigated the mechanisms involved in the effects of pentoxifylline (PTX) on P-gp-mediated multidrug resistance (MDR) in mouse leukemia L1210/VCR cells. Parental sensitive mouse leukemia cells L1210, and multidrug-resistant cells, L1210/VCR, which are characterized by the overexpression of P-gp, were used as experimental models. The cells were exposed to 100 ?mol/L PTX in the presence or absence of 1.2 ?mol/L vincristine (VCR). Western blot analysis indicated a downregulation of P-gp protein expression when multidrug-resistant L1210/VCR cells were exposed to PTX. The effects of PTX on the sensitization of L1210/VCR cells to VCR correlate with the stimulation of apoptosis detected by Annexin V/propidium iodide apoptosis necrosis kit and proteolytic activation of both caspase-3 and caspase-9 monitored by Western blot analysis. Higher release of matrix metalloproteinases (MMPs), especially MMP-2, which could be attenuated by PTX, was found in L1210/VCR than in L1210 cells by gelatin zymography in electrophoretic gel. Exposure of resistant cells to PTX increased the content of phosphorylated Akt kinase. In contrast, the presence of VCR eliminated the effects of PTX on Akt kinase phosphorylation. Taken together, we conclude that PTX induces the sensitization of multidrug-resistant cells to VCR via downregulation of P-gp, stimulation of apoptosis and reduction of MMPs released from drug-resistant L1210/VCR cells. These facts bring new insights into the mechanisms of PTX action on cancer cells. PMID:22312258

Barancik, Miroslav; Bohacova, Viera; Gibalova, Lenka; Sedlak, Jan; Sulova, Zdena; Breier, Albert

2012-01-01

268

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. PMID:25435975

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

2015-01-01

269

Cytotoxicity of Cyclodipeptides from Pseudomonas aeruginosa PAO1 Leads to Apoptosis in Human Cancer Cell Lines  

PubMed Central

Pseudomonas aeruginosa is an opportunistic pathogen of plants and animals, which produces virulence factors in order to infect or colonize its eukaryotic hosts. Cyclodipeptides (CDPs) produced by P. aeruginosa exhibit cytotoxic properties toward human tumor cells. In this study, we evaluated the effect of a CDP mix, comprised of cyclo(L-Pro-L-Tyr), cyclo(L-Pro-L-Val), and cyclo(L-Pro-L-Phe) that were isolated from P. aeruginosa, on two human cancer cell lines. Our results demonstrated that the CDP mix promoted cell death in cultures of the HeLa cervical adenocarcinoma and Caco-2 colorectal adenocarcinoma cell lines in a dose-dependent manner, with a 50% inhibitory concentration (IC50) of 0.53 and 0.66?mg/mL, for HeLa and Caco-2 cells, respectively. Flow cytometric analysis, using annexin V and propidium iodide as apoptosis and necrosis indicators, respectively, clearly showed that HeLa and Caco-2 cells exhibited apoptotic characteristics when treated with the CDP mix at a concentration <0.001?mg/mL. IC50 values for apoptotic cells in HeLa and Caco-2 cells were 6.5?×?10?5 and 1.8?×?10?4?mg/mL, respectively. Our results indicate that an apoptotic pathway is involved in the inhibition of cell proliferation caused by the P. aeruginosa CDP mix.

Vázquez-Rivera, Dolores; González, Omar; Guzmán-Rodríguez, Jaquelina; Díaz-Pérez, Alma L.; Ochoa-Zarzosa, Alejandra; López-Bucio, José; Meza-Carmen, Víctor; Campos-García, Jesús

2015-01-01

270

The effect of centrifugation condition on mature adipocytes and adipose stem cell viability.  

PubMed

Different researchers have recommended different lipoaspirate centrifugation speeds and times, probably due to the limits in fat cell viability assays. We assessed fat cell viability using a fluorescein diacetate and propidium iodide (FDA-PI) stain and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay after harvesting syringe liposuction and spun with different centrifugation speeds to determine the optimal conditions. Lipoaspirates, harvested from 13 donors, were transferred into a centrifuge tube and spun at 1000, 3000, and 4000 rpm for 3 minutes. Mature adipocytes and adipose stem cells were isolated and tested with a direct counting of FDA-PI-stained cells under fluorescence microscope and XTT assay. We incubated adipocytes and adipose stem cells for 1 day and 3 days, and we compared both of them with fresh samples to evaluate the influence of culturing condition on fat cell viability. Centrifugation speeds from 1000 rpm to 4000 rpm for 3 minutes showed no change in the percentage of adipocytes and adipose stem cell viability not only in the fresh samples but also in the cultured samples (1 day and 3 days). Centrifugation speeds under 4000 rpm do not change the percentage of fat cell viability. To differentiate viable cells from dying or dead mature adipocytes and oil accurately, combinations of viability tests are essential. PMID:23636113

Son, Daegu; Choi, Taehyun; Yeo, Hyeonjung; Kim, Junhyung; Han, Kihwan

2014-05-01

271

IGF-1/IGFBP-1 increases blastocyst formation and total blastocyst cell number in mouse embryo culture and facilitates the establishment of a stem-cell line  

PubMed Central

Background Apoptosis occurs frequently for blastocysts cultured in vitro, where conditions are suboptimal to those found in the natural environment. Insulin-like growth factor-1 (IGF-1) plays an important role in preventing apoptosis in the early development of the embryo, as well as in the progressive regulation of organ development. We hypothesize that IGF-1 and its dephosphorylated binding protein (IGFBP-1) may be able to improve embryo culture with an associated reduced cell death, and that the resultant increase in the total cell number of the embryo could increase the chances of establishing an embryonic stem-cell line. Results In vivo fertilized zygotes were cultured in medium containing supplementary IGF-1, or IGFBP-1/IGF-1. The stages of the resultant embryos were evaluated at noon on day five post-hCG injection. The extent of apoptosis and necrosis was evaluated using Annexin V and propidium iodine staining under fluorescent microscopy. The establishment of embryonic stem-cell lines was performed using the hatching blastocysts that were cultured in the presence of IGF-1 or IGFBP-1/IGF-1. The results show that the rate of blastocyst formation in a tissue-culture system in the presence of IGF-1 was 88.7% and IGFBP-1/IGF-1 it was 94.6%, respectively, and that it was significantly greater than the figure for the control group (81.9%). IGFBP-1/IGF-1 also resulted in a higher hatching rate than was the case for the control group (68.8% vs. 48.6% respectively). IGF-1 also increased the number of Annexin V-free and propidium iodine-free blastocysts in culture (86.8% vs. 75.9% respectively). Total cell number of blastocyst in culture was increased by 18.9% for those examples cultured with dephosphorylated IGFBP-1/IGF-1. For subsequent stem-cell culture, the chances of the successful establishment of a stem-cell line was increased for the IGF-1 and IGFBP-1/IGF-1 groups (IGF-1 vs. IGFBP-1/IGF-1 vs. control: 45.8% vs. 59.6% vs. 27.3% respectively). Conclusion IGF-1 or dephosphorylated IGFBP-1/IGF-1 supplement does result in an anti-apoptotic effect for early embryo development in culture, with a subsequent increased total cell number resulting from cell culture. The effect is beneficial for the later establishment of a stem-cell line. PMID:14499003

Lin, Ta-Chin; Yen, Jui-Mei; Gong, Kun-Bing; Hsu, Teng-Tsao; Chen, Lih-Ren

2003-01-01

272

Screening of Compounds Toxicity against Human Monocytic cell line-THP-1 by Flow Cytometry  

PubMed Central

The worldwide rapid increase in bacterial resistance to numerous antibiotics requires on-going development of new drugs to enter the market. As the development of new antibiotics is lengthy and costly, early monitoring of compound's toxicity is essential in the development of novel agents. Our interest is in a rapid, simple, high throughput screening method to assess cytotoxicity induced by potential agents. Some intracellular pathogens, such as Mycobacterium tuberculosis primary site of infection is human alveolar macrophages. Thus, evaluation of candidate drugs for macrophage toxicity is crucial. Protocols for high throughput drug toxicity screening of macrophages using flow cytometry are lacking in the literature. For this application we modified a preexisting technique, propidium iodide (PI) exclusion staining and utilized it for rapid toxicity tests. Samples were prepared in 96 well plates and analyzed by flow cytometry, which allowed for rapid, inexpensive and precise assessment of compound's toxicity associated with cell death. PMID:15472722

Pick, Neora; Cameron, Scott; Arad, Dorit

2004-01-01

273

Formation of 5-aminolevulinic-acid (ALA) induced protoporphyrin IX (PPIX) and photodynamic effectiveness in human urothelial cell lines  

NASA Astrophysics Data System (ADS)

Background: To optimize differential effectiveness of aminulevulinic acid (ALA)-induced protoporphyrin IX (PPIX)- mediated photodynamic therapy (PDT) on normal and tumor urothelial cells, aspects of PPIX formation and parameters responsible for treatment efficiency were examined in vitro. Material and Methods: Plateau phase cells of a bladder cancer and a normal urothelial cell line were incubated using various incubation conditions and analyzed with respect to their PPIX content and cellular sensitizer distribution. PDT was performed using incoherent light from a Xenon coldlight projector. Photo toxicity was investigated using flow cytometric analysis of propidium iodide exclusion and analysis of cell size and number. Results: Following 3h incubation intervals, both cell lines showed similar PPIX localization with an amount of sensitizer three times higher in RT4 tumor cells. 1h incubation times resulted in the same ratio of PPIX amount but lead to different cellular PPIX distribution. After 3h incubation, PDT resulted in complete tumor cell kill accomplished by a marked fraction of damaged normal urothelial cells. TR4 cell kill with significantly reduced damage of UROtsa cells could be achieved using 1h incubation times. Discussion: Besides sensitizer amount, cellular localization is crucial for PDT effectiveness. Differential effectiveness of tumor and normal cells can be enhanced utilizing the finding of different PPIX distribution after short incubation times.

Seidl, Juergen; Krieg, Rene C.; Rauch, Joachim; Waidelich, Raphaela M.; Stepp, Herbert G.; Knuechel, Ruth

2001-01-01

274

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

275

Human RPE cell apoptosis induced by activated monocytes is mediated by caspase-3 activation.  

PubMed Central

PURPOSE: To determine the effects of activated monocytes on the induction of human retinal pigment epithelial (HRPE) cell reactive oxygen metabolite (ROM) production and apoptosis. METHODS: HRPE cells were co-cultured with interferon-gamma (IFN-gamma)-stimulated human monocytes. HRPE apoptosis was detected by propidium iodide, proliferating cell nuclear antigen (PCNA) and TdT-mediated dUTP nick end labeling (TUNEL) staining, caspase-3 activation, and Western blot analysis. HRPE cell ROMs were imaged using the fluorescent marker dihydrotetramethylrosamine (H2TMRos). RESULTS: IFN-gamma-activated monocytes in direct contact with HRPE cells elicited significant increases in TUNEL-positive (P < .0001) and decreases in PCNA-positive (P < .0001) HRPE cells. The activated monocytes also induced HRPE cell caspase-3 activation, which was inhibited by inhibitor Z-DEVD-fmk. Co-incubations, in which monocytes were either prevented from direct contact with HRPE cells or separated from HRPE cells after 30 minutes of direct contact, did not induce significant HRPE cell apoptosis. Anti-CD18 and anti-ICAM-1 antibodies significantly reduced activated monocyte-induced TUNEL-positive HRPE cells, by 48% (P = .0051) and 38% (P = .046), respectively, and caspase-3 activity by 56% (P < .0001) and 45% (P < .0001), respectively. Overlay of monocytes induced HRPE cell ROM that was inhibited by anti-CD18 and anti-ICAM-1 antibodies, but not by superoxide dismutase (SOD) or nitric oxide (NO) inhibitors. Accordingly, neither SOD nor NO inhibitors had significant effects on HRPE cell apoptosis or caspase-3 activation. CONCLUSIONS: We demonstrated that IFN-gamma-activated monocytes may induce ROM in HRPE cells through cell-to-cell contact, in part via CD18 and ICAM-1, and promote HRPE cell apoptosis via caspase-3 activation. These mechanisms may compromise HRPE cell function and survival in retinal diseases in which mononuclear phagocyte infiltration at the HRPE interface is observed. PMID:14971566

Eliner, Susan G; Yoshida, Ayako; Bian, Zong-Mei; Kindezelskii, Andrei L; Petty, Howard R; Elner, Victor M

2003-01-01

276

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

2010-12-01

277

Delta-aminolevulinic acid as a photosensitizer precursor for the treatment of hepatoma cells in vitro  

NASA Astrophysics Data System (ADS)

Delta-aminolevulinic acid ((delta) -ALA) has been recently proposed as a tumor photosensitizer precursor with increased selectivity and decreased toxicity for the treatment of neoplasms. We investigated the conversion and cytotoxicity of (delta) -ALA in a human hepatoma cell line to determine its clinical potential. SK-HEP-1 (ATCC) cells were plated on 35 mm coverslips in media for use in a digital fluorescence microscopic imaging system. (delta) -ALA was added to achieve final concentrations between 0-5 mM. Cells were excited with 450-490 nm light while a 610 nm long pass filter was used to assess fluorescence from conversion to protoporphyrin IX, the putative photosensitizer. After maximal fluorescence was obtained at each initial concentration of (delta) -ALA, cells were radiated with 10 J/cm2 of light from a xenon lamp fitted with a 515 nm band pass filter. After photoradiation, cell death was assessed by flow cytometry using propidium iodide labeling. Protoporphyrin IX accumulation was constant at Ksequals0.001 until a plateau was achieved 2 hours after the addition of (delta -ALA. Photoradiation with 10 J/cm2 at a concentration of 1 mM (delta ALA resulted in a linear increase in cell death over time with 5% cell death at 2 hours and 12% at 5 hours compared to controls. Interestingly, controls with (delta) -ALA alone demonstrated a cytoprotective effect with a logarithmic relationship between increasing cell survival and increasing dose of drug.

Laukka, Mark A.; Wang, Kenneth K.

1994-07-01

278

Surface plasmon coupled emission in micrometer-scale cells: a leap from interface to bulk targets.  

PubMed

Surface plasmon coupled emission (SPCE) technique has attracted increasing attention in biomolecular interaction analysis and cell imaging because of its high sensitivity, low detection volume and low fluorescence background. Typically, the working range of SPCE is limited at nanometers to an interface. For micrometer-scale samples, new SPCE properties are expected because of complex coupling modes. In this work, cells with different subregions labeled were studied using a SPCE spectroscopy system. Angular and p-polarized emission was observed for cell membrane, cytoplasm, and nucleus labeled with DiI, Nile Red, and propidium iodide, respectively. The SPCE signals were always partially p-polarized, and the maximum emission angle did not shift, regardless of variations in emission wavelength, fluorophore distribution and stained layer thickness. Additionally, increased polarization and a broader angle distribution were also observed with an increase in sample thickness. We also investigated the impact of metallic substrates on the SPCE properties of cells. Compared with Au and Ni substrates, Al substrates presented better polarization and angle distribution. Moreover, the real-time detection of the cell labeling process was achieved by monitoring SPCE intensity. These findings expand SPCE from a surface technique to a 3D method for investigating bulk targets beyond the nanoscale interfaces, providing a basis to apply this technique to study cell membrane fluidity and biomolecule interactions inside the cell and to distinguish between cell subregions. PMID:25581118

Liu, Qian; Cao, Shuo-Hui; Cai, Wei-Peng; Liu, Xiao-Qing; Weng, Yu-Hua; Xie, Kai-Xin; Huo, Si-Xin; Li, Yao-Qun

2015-02-19

279

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

280

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

281

Clostridium septicum alpha-toxin forms pores and induces rapid cell necrosis.  

PubMed

Alpha-toxin is the unique lethal virulent factor produced by Clostridium septicum, which causes traumatic or non-traumatic gas gangrene and necrotizing enterocolitis in humans. Here, we analyzed channel formation of the recombinant septicum alpha-toxin and characterized its activity on living cells. Recombinant septicum alpha-toxin induces the formation of ion-permeable channels with a single-channel conductance of about 175pS in 0.1M KCl in lipid bilayer membranes, which is typical for a large diffusion pore. Septicum alpha-toxin channels remained mostly in the open configuration, displayed no lipid specificity, and exhibited slight anion selectivity. Septicum alpha-toxin caused a rapid decrease in the transepithelial electrical resistance of MDCK cell monolayers grown on filters, and induced a rapid cell necrosis in a variety of cell lines, characterized by cell permeabilization to propidium iodide without DNA fragmentation and activation of caspase-3. Septicum alpha-toxin also induced a rapid K(+) efflux and ATP depletion. Incubation of the cells in K(+)-enriched medium delayed cell death caused by septicum alpha-toxin or epsilon-toxin, another potent pore-forming toxin, suggesting that the rapid loss of intracellular K(+) represents an early signal of pore-forming toxins-mediated cell necrosis. PMID:19632260

Knapp, Oliver; Maier, Elke; Mkaddem, Sanae Ben; Benz, Roland; Bens, Marcelle; Chenal, Alexandre; Geny, Blandine; Vandewalle, Alain; Popoff, Michel R

2010-01-01

282

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

283

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

284

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

PubMed Central

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

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

2011-01-01

285

Mycobacterial Di-O-Acyl-Trehalose Inhibits Mitogen- and Antigen-Induced Proliferation of Murine T Cells In Vitro  

PubMed Central

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

Saavedra, Rafael; Segura, Erika; Leyva, Rosario; Esparza, Luis A.; López-Marín, Luz M.

2001-01-01

286

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

NASA Astrophysics Data System (ADS)

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

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

2013-03-01

287

The GANT61, a GLI inhibitor, induces caspase-independent apoptosis of SK-N-LO cells.  

PubMed

GANT61 is a small-molecule inhibitor of glioma-associated oncogene 1 (GLI1)- and GLI2-mediated transcription at the nuclear level that exerts its effect by preventing DNA binding. It has been demonstrated to induce cell death against Ewing's sarcoma family tumor (ESFT) cell lines in a dose-dependent manner. The most sensitive cell line was SK-N-LO, which expresses the EWS-FLI1 fusion gene. SK-N-LO cells treated with GANT61 showed cellular and nuclear morphological changes, including cell shrinkage, chromatin condensation and nuclear fragmentation, in a concentration-dependent manner, as visualized by Hoechst 33342 staining. Furthermore, annexin V-propidium iodide (PI) double-staining revealed a significant increase in the number of late apoptotic cells. GANT61 induced a significant decrease in the proportion of cells in the S phase. Significant decrease of the protein levels of GLI2, survivin, cyclin A and claspin, and significant increase of p21 expression was also observed in the cells treated with GANT61. Moreover, poly (ADP-ribose) polymerase (PARP) cleavage was observed, but no cleavage of caspase-3 or -7, or any change in the expressions of Bcl-2 or p53 were observed. These findings suggest that GANT61 induces cell death of SK-N-LO cells in a caspase-independent manner, by inhibiting DNA replication in the S phase. PMID:24694609

Matsumoto, Takahiro; Tabata, Keiichi; Suzuki, Takashi

2014-01-01

288

Silica Nanoparticles Sensitize Human Multiple Myeloma Cells to Snake (Walterinnesia aegyptia) Venom-Induced Apoptosis and Growth Arrest  

PubMed Central

Background. Multiple myeloma (MM), an almost incurable disease, is the second most common blood cancer. Initial chemotherapeutic treatment could be successful; however, resistance development urges the use of higher toxic doses accompanied by hematopoietic stem cell transplantation. The establishment of more effective treatments that can overcome or circumvent chemoresistance has become a priority. We recently demonstrated that venom extracted from Walterinnesia aegyptia (WEV) either alone or in combination with silica nanoparticles (WEV+NPs) mediated the growth arrest and apoptosis of prostate cancer cells. In the present study, we evaluated the impact of WEV alone and WEV+NP on proliferation and apoptosis of MM cells. Methods. The impacts of WEV alone and WEV+NP were monitored in MM cells from 70 diagnosed patients. The influences of WEV and WEV+NP were assessed with flow cytometry analysis. Results. WEV alone and WEV+NP decreased the viability of MM cells. Using a CFSE proliferation assay, we found that WEV+NP strongly inhibited MM cell proliferation. Furthermore, analysis of the cell cycle using the propidium iodide (PI) staining method indicated that WEV+NP strongly altered the cell cycle of MM cells and enhanced the induction of apoptosis. Conclusions. Our data reveal the biological effects of WEV and WEV+NP on MM cells that enable these compounds to function as effective treatments for MM. PMID:23304253

Sayed, Douaa; Al-Sadoon, Mohamed K.

2012-01-01

289

Dual AO/EB Staining to Detect Apoptosis in Osteosarcoma Cells Compared with Flow Cytometry  

PubMed Central

Background The aim of this study was to evaluate the ability of dual acridine orange/ethidium bromide (AO/EB) staining to detect tumor cell apoptosis. According to apoptosis-associated changes of cell membranes during the process of apoptosis, a clear distinction is made between normal cells, early and late apoptotic cells, and necrotic cells. Material/Method We cultured human osteosarcoma cells with 30, 60, and 120 ?g/ml kappa-selenocarrageenan. To assess the rates of cell proliferation and apoptosis, cells were fluorescently stained with acridine orange/ethidium bromide (AO/EB) or stained with propidium iodide (PI) and analyzed by flow cytometry. All experiments were repeated at least 3 times. Result Normal tumor cells, early and late apoptotic cells, and necrotic cells were examined using fluorescent microscopy. Early-stage apoptotic cells were marked by crescent-shaped or granular yellow-green acridine orange nuclear staining. Late-stage apoptotic cells were marked with concentrated and asymmetrically localized orange nuclear ethidium bromide staining. Necrotic cells increased in volume and showed uneven orange-red fluorescence at their periphery. Cells appeared to be in the process of disintegrating. The percentage of apoptotic osteosarcoma cells detected by dual acridine orange/ethidium bromide (AO/EB) staining was not significantly different from that detected using flow cytometry (P>0.05). Conclusions Our results suggest that dual acridine orange/ethidium bromide staining is an economic and convenient method to detect apoptosis in tumor cells and to test tumor chemosensitivity compared with flow cytometry. PMID:25664686

Liu, Kuan; Liu, Peng-cheng; Liu, Run; Wu, Xing

2015-01-01

290

Wogonoside induces cell cycle arrest and mitochondrial mediated apoptosis by modulation of Bcl-2 and Bax in osteosarcoma cancer cells  

PubMed Central

Osteosarcoma (OS) is the most common bone cancer with a great tendency for local invasion and distant metastasis. Restricted by the severe toxicity of conventional drugs, the therapeutic challenge of osteosarcoma still remains unconquered. The objective of the present research work was to investigate the antiproliferative activity of wogonoside against human osteosarcoma (SaOS-2) cell line. Cell viability after wogonoside treatment was evaluated by MTT assay. Phase contrast microscopy was used to evaluate the change in cell morphology following drug treatment. The effect of wogonoside on cell cycle phase distribution and mitochondrial membrane potential was investigated by flow cytometry using propidium iodide (PI) and rhodamine-123 DNA-binding fluorescent dyes respectively. Western blotting was used to evaluate the effect of wogonoside on cell cycle-related proteins as well as on the expression levels of Bcl-2, Bax, cytosolic and mitochondrial cytochrome c and apoptotic protease activating factor-1 (Apaf-1). Wogonoside induced a dose-dependent as well as time-dependent growth inhibitory effects on cell proliferation of SaOS-2 cancer cells. Wogonoside induced G2/M cell cycle arrest as well as loss in mitochondrial membrane potential in these cells. Within 48 h of incubation, approximately 4.36%, 6.72%, 11.54%, 21.88% and 15.54% of the cells underwent early apoptosis after treatment with 0, 5, 10, 25 and 75 ?M of wogonoside respectively. Wogonoside led to reduced Bcl-2 expression and increased Bax expression, while as it led to s decrease in the levels of mitochondrial cytochrome c and an increase in cytosolic fraction and expressions of cytosolic apoptotic protease activating factor-1 (Apaf-1). PMID:25755693

Wang, Yang; Yin, Ruo-Feng; Teng, Jia-Song

2015-01-01

291

Wogonoside induces cell cycle arrest and mitochondrial mediated apoptosis by modulation of Bcl-2 and Bax in osteosarcoma cancer cells.  

PubMed

Osteosarcoma (OS) is the most common bone cancer with a great tendency for local invasion and distant metastasis. Restricted by the severe toxicity of conventional drugs, the therapeutic challenge of osteosarcoma still remains unconquered. The objective of the present research work was to investigate the antiproliferative activity of wogonoside against human osteosarcoma (SaOS-2) cell line. Cell viability after wogonoside treatment was evaluated by MTT assay. Phase contrast microscopy was used to evaluate the change in cell morphology following drug treatment. The effect of wogonoside on cell cycle phase distribution and mitochondrial membrane potential was investigated by flow cytometry using propidium iodide (PI) and rhodamine-123 DNA-binding fluorescent dyes respectively. Western blotting was used to evaluate the effect of wogonoside on cell cycle-related proteins as well as on the expression levels of Bcl-2, Bax, cytosolic and mitochondrial cytochrome c and apoptotic protease activating factor-1 (Apaf-1). Wogonoside induced a dose-dependent as well as time-dependent growth inhibitory effects on cell proliferation of SaOS-2 cancer cells. Wogonoside induced G2/M cell cycle arrest as well as loss in mitochondrial membrane potential in these cells. Within 48 h of incubation, approximately 4.36%, 6.72%, 11.54%, 21.88% and 15.54% of the cells underwent early apoptosis after treatment with 0, 5, 10, 25 and 75 ?M of wogonoside respectively. Wogonoside led to reduced Bcl-2 expression and increased Bax expression, while as it led to s decrease in the levels of mitochondrial cytochrome c and an increase in cytosolic fraction and expressions of cytosolic apoptotic protease activating factor-1 (Apaf-1). PMID:25755693

Wang, Yang; Yin, Ruo-Feng; Teng, Jia-Song

2015-01-01

292

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

SciTech Connect

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

Souslova, Tatiana [Departement de Chimie et de Biochimie (TOXEN), Universite du Quebec a Montreal, Succursale Centre Ville, Montreal, Quebec (Canada); Averill-Bates, Diana A. [Departement de Chimie et de Biochimie (TOXEN), Universite du Quebec a Montreal, Succursale Centre Ville, Montreal, Quebec (Canada)]. E-mail: averill.diana@uqam.ca

2004-12-01

293

Growth Inhibition and Apoptosis Induction of Salvia chloroleuca on MCF-7 Breast Cancer Cell Line  

PubMed Central

Fragrant species of the genus Salvia have been attributed many medicinal properties, which include anticancer activity. In the present study, cytotoxic properties of total methanol extract of Salvia chloroleuca Rech. f. & Aellen and its fractions were investigated on MCF- 7, a breast carcinoma cell line. Malignant and non-malignant cells were cultured in RPMI medium and incubated with different concentrations of plant extracts. Cell viability was quantitated by 3-(4,5-dimethylthiazol-2-yl) -5-(3-carboxymethoxyphenyl) -2-(4-sulphophenyl) -2H-tetrazolium (MTS) assay. Apoptotic cells were determined using propidium iodide (PI) staining of DNA fragmentation by flow cytometry (sub-G1 peak). S. chloroleuca inhibited the growth of malignant cells in a dose-dependent manner. Among solvent fractions of S. chloroleuca, the n-hexane and methylene chloride fractions were found to be more toxic compared to other fractions. S. chloroleuca-induced a sub-G1 peak in flow cytometry histogram of treated cells compared to control and DNA fragmentation suggested the induction of apoptosis. Administration of N-acetyl cysteine and vitamin C two ROS scavengers also resulted in significant inhibition of cytotoxicity induced by S. chloroleuca. These results support a mechanism whereby S. chloroleuca induces apoptosis of MCF-7 human breast cells through a ROS-mediated pathway. PMID:24523759

Tayarani-Najaran, Zahra; Asili, Javad; Aioubi, Ehsan; Emami, Seyed Ahmad

2013-01-01

294

Subcellular location and photodynamic therapeutic effect of chlorin e6 in the human tongue squamous cell cancer Tca8113 cell line  

PubMed Central

The present study aimed to investigate the distribution and photodynamic therapeutic effect of chlorin e6 (Ce6) in the human tongue squamous cell carcinoma Tca8113 cell line in vitro. The distribution of Ce6 in the Tca8113 cells was observed in situ combined with mitochondrial and lysosomal fluorescent probes. Next, 630-nm semiconductor laser irradiation was performed. The MTS colorimetric method was used to determine cell survival. Annexin V fluorescein isothiocyanate/propidium iodide (PI) double staining was used to detect early apoptosis following photodynamic therapy (PDT). The flow cytometer was used to analyze the DNA content subsequent to PI-staining. It was observed that Ce6 could combine with the cellular membrane following 30 min of incubation with the Tca8113 cells. As the length of incubation increased, Ce6 gradually entered the cells in a particular distribution and reached saturation by 3 h. Co-localization analysis demonstrated that Ce6 was more likely to be present in the mitochondria than in the lysosomes. The cells incubated with 5 ?g/ml Ce6 for 24 h exhibited a low toxicity of 5%, however, following light irradiation, Ce6-PDT was able to kill the Tca8113 cells in vitro. The cell toxicity was positively correlated with Ce6 concentration and light dose, therefore, the effect of Ce6 was concentration/dose-dependent (P<0.01). The lower Ce6 concentrations and light doses could significantly induce apoptosis in the Tca8113 cells, while higher doses increased necrosis/percentage of dead cells. In summary, Ce6 saturated the Tca8113 cells following 3 h of incubation. Furthermore, Ce6-PDT effectively killed the cultured Tca8113 cells in vitro at a safe concentration. At a low concentration and light dose, Ce6 is more likely to induce cell apoptosis via the mitochondria than the lysosomes. PMID:25621023

LUO, WEI; LIU, RONG-SEN; ZHU, JIAN-GUO; LI, YING-CHAO; LIU, HONG-CHEN

2015-01-01

295

Calcium-mediated pore expansion and cell death following nanoelectroporation.  

PubMed

Opening of long-lived pores in the cell membrane is the principal primary effect of intense, nanosecond pulsed electric field (nsPEF). Here we demonstrate that the evolution of pores, cell survival, the time and the mode of cell death (necrotic or apoptotic) are determined by the level of external Ca(2+) after nsPEF. We also introduce a novel, minimally disruptive technique for nsEP exposure of adherent cells on indium tin oxide (ITO)-coated glass coverslips, which does not require cell detachment and enables fast exchanges of bath media. Increasing the Ca(2+) level from the nominal 2-5?M to 2mM for the first 60-90min after permeabilization by 300-nsPEF increased the early (necrotic) death in U937, CHO, and BPAE cells. With nominal Ca(2+), the inhibition of osmotic swelling rescued cells from the early necrosis and increased caspase 3/7 activation later on. However, the inhibition of swelling had a modest or no protective effect with 2mM Ca(2+) in the medium. With the nominal Ca(2+), most cells displayed gradual increase in YO-PRO-1 and propidium (Pr) uptake. With 2mM Ca(2+), the initially lower Pr uptake was eventually replaced by a massive and abrupt Pr entry (necrotic death). It was accompanied by a transient acceleration of the growth of membrane blebs due to the increase of the intracellular osmotic pressure. We conclude that the high-Ca(2+)-dependent necrotic death in nsPEF-treated cells is effected by a delayed, sudden, and osmotically-independent pore expansion (or de novo formation of larger pores), but not by the membrane rupture. PMID:24978108

Pakhomova, Olga N; Gregory, Betsy; Semenov, Iurii; Pakhomov, Andrei G

2014-10-01

296

Selection of tumour cell subpopulations occurs during cultivation of human tumours in soft agar. A DNA flow cytometric study.  

PubMed Central

To examine whether selection of tumour cell subpopulations occurs during cultivation in soft agar, we compared in 23 human tumours of different histological types the DNA content of cells from colonies formed in soft agar (method of Courtenay and Mills, 1978) with that of the original tumour cells. The ploidy as well as the fraction of cells in S phase were determined from DNA histograms after staining of the nuclei with a propidium-iodide procedure and flow cytometric recordings. In 8 of 17 aneuploid tumours analysed, specific aneuploid subpopulations disappeared during cultivation or new aneuploid populations, not demonstrable in the original cell suspensions, appeared in the colonies. In 9 cases identical aneuploid populations were found in the colonies and the tumours. In one of 6 diploid tumours examined, aneuploid cell populations not revealed in the original cell suspension, were found in addition to diploid cells, whereas 5 tumours gave rise to colonies containing a purely diploid population. The results show that in a variety of human malignant tumours cultivation in soft agar may select specific aneuploid tumour cell populations. PMID:4063145

Tveit, K. M.; Pettersen, E. O.; Fosså, S. D.; Pihl, A.

1985-01-01

297

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

298

Cisplatin in combination with programmed cell death protein 5 increases antitumor activity in prostate cancer cells by promoting apoptosis.  

PubMed

Prostate cancer is the most common type of cancer affecting males. The aim of the present study was to investigate the antitumor effect of cisplatin in combination with programmed cell death protein 5 (Pdcd5) on Du145 prostate cancer cells and to elucidate the underlying mechanisms of action. An MTT cell viability assay was performed in order to determine the proliferation rate of Du145 cells. The results demonstrated that Du145 cells treated with cisplatin for 48 h had an IC50 value >200 µM; however, following transfection of Pdcd5 in combination with treatment with various concentrations of cisplatin, the proliferation rates of Du145 and PC3 prostate cancer cells were significantly decreased in a dose?dependent manner, with IC50 values of 114.1 and 50.6 µM, respectively. Annexin V?fluorescein isothiocyanate/propidium iodide dual labeling analyses demonstrated a significant increase in the apoptotic rate of Du145 cells following transfection of Pcdc5 in combination with cisplatin treatment. Furthermore, western blot analysis revealed a marked increase in activated caspase?3 expression in Du145 cells as well as a decreased ratio of Bcl?2/Bax. In conclusion, the results of the present study demonstrated that Pdcd5 increased the chemosensitivity of prostate cancer cells and decreased the toxicity of cisplatin via activation of the receptor?associated apoptotic pathway; this may therefore indicate the combined use of cisplatin and Pdcd5 as a novel therapeutic strategy for the treatment of prostate cancer. PMID:25625867

Zhu, Wenbin; Li, Yang; Gao, Lei

2015-06-01

299

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

300

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

301

Microenvironment mesenchymal cells protect ovarian cancer cell lines from apoptosis by inhibiting XIAP inactivation  

PubMed Central

Epithelial ovarian carcinoma is characterized by high frequency of recurrence (70% of patients) and carboplatin resistance acquisition. Carcinoma-associated mesenchymal stem cells (CA-MSC) have been shown to induce ovarian cancer chemoresistance through trogocytosis. Here we examined CA-MSC properties to protect ovarian cancer cells from carboplatin-induced apoptosis. Apoptosis was determined by Propidium Iodide and Annexin-V-FITC labelling and poly-ADP-ribose polymerase cleavage analysis. We showed a significant increase of inhibitory concentration 50 and a 30% decrease of carboplatin-induced apoptosis in ovarian cancer cells incubated in the presence of CA-MSC-conditioned medium (CM). A molecular analysis of apoptosis signalling pathway in response to carboplatin revealed that the presence of CA-MSC CM induced a 30% decrease of effector caspases-3 and -7 activation and proteolysis activity. CA-MSC secretions promoted Akt and X-linked inhibitor of apoptosis protein (XIAP; caspase inhibitor from inhibitor of apoptosis protein (IAP) family) phosphorylation. XIAP depletion by siRNA strategy permitted to restore apoptosis in ovarian cancer cells stimulated by CA-MSC CM. The factors secreted by CA-MSC are able to confer chemoresistance to carboplatin in ovarian cancer cells through the inhibition of effector caspases activation and apoptosis blockade. Activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway and the phosphorylation of its downstream target XIAP underlined the implication of this signalling pathway in ovarian cancer chemoresistance. This study reveals the potentialities of targeting XIAP in ovarian cancer therapy. PMID:24176845

Castells, M; Milhas, D; Gandy, C; Thibault, B; Rafii, A; Delord, J-P; Couderc, B

2013-01-01

302

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

PubMed Central

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 (15 minutes) 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; Pakhomova, Olga N.; Roth, Caleb C.; Semenov, Iurri; Beier, Hope T.; Tarango, Melissa; Xiao, Shu; Schoenbach, Karl; Pakhomov, Andrei G.

2014-01-01

303

Inhibitory effects of sea buckthorn procyanidins on fatty acid synthase and MDA-MB-231 cells.  

PubMed

Fatty acid synthase (FAS) is overexpressed in many human cancers including breast cancer and is considered to be a promising target for therapy. Sea buckthorn has long been used to treat a variety of maladies. Here, we investigated the inhibitory effect of sea buckthorn procyanidins (SBPs) isolated from the seeds of sea buckthorn on FAS and FAS overexpressed human breast cancer MDA-MB-231 cells. The FAS activity and FAS inhibition were measured by a spectrophotometer at 340 nm of nicotinamide adenine dinucleotide phosphate (NADPH) absorption. We found that SBP potently inhibited the activity of FAS with a half-inhibitory concentration (IC50) value of 0.087 ?g/ml. 3-4,5-Dimethylthiazol-2-yl-2,3-diphenyl tetrazolium bromide (MTT) assay was used to test the cell viability. SBP reduced MDA-MB-231 cell viability with an IC50 value of 37.5 ?g/ml. Hoechst 33258/propidium iodide dual staining and flow cytometric analysis showed that SBP induced MDA-MB-231 cell apoptosis. SBP inhibited intracellular FAS activity with a dose-dependent manner. In addition, sodium palmitate could rescue the cell apoptosis induced by SBP. These results showed that SBP was a promising FAS inhibitor which could induce the apoptosis of MDA-MB-231 cells via inhibiting FAS. These findings suggested that SBP might be useful for preventing or treating breast cancer. PMID:24957042

Wang, Yi; Nie, Fangyuan; Ouyang, Jian; Wang, Xiaoyan; Ma, Xiaofeng

2014-10-01

304

Apoptosis induced by sonodynamic therapy in human osteosarcoma cells in vitro.  

PubMed

The aim of the present study was to investigate the potential effect of hematoporphyrin monomethyl ether-sonodynamic therapy (HMME?SDT) on MG?63 osteosarcoma cells. The HMME concentration was kept constant at 20 µg/ml and the MG?63 osteosarcoma cell line was exposed to ultrasound with an intensity of 1.0 W/cm2 for 30 sec. Cell cytotoxicity was quantified using an MTT assay 6 h after HMME?SDT. The intracellular localization of HMME was imaged using inverted confocal laser scanning microscopy. Apoptosis was investigated using flow cytometry with Annexin V?fluorescein isothiocyanate and propidium iodine staining. The cytotoxicity of HMME?mediated sonodynamic action on MG?63 cells was significantly higher than that of other treatments, including ultrasound alone, HMME alone and sham treatment. Flow cytometry demonstrated that HMME?SDT action markedly enhanced the apoptotic rate of MG?63 cells. The mechanisms of apoptosis were analyzed by measuring the protein expression of poly ADP?ribose polymerase (PARP), cleaved PARP, procaspase?3, cleaved caspase?3 and cleaved caspase?9. The data demonstrated that HMME?SDT action markedly induced the apoptosis of MG?63 cells. PMID:25778820

Liu, Xing; Li, Wei; Geng, Shuo; Meng, Qing-Gang; Bi, Zheng-Gang

2015-07-01

305

Nordamnacanthal potentiates the cytotoxic effects of tamoxifen in human breast cancer cells  

PubMed Central

Tamoxifen (TAM) is the mainline drug treatment for breast cancer, despite its side effects and the development of resistance. As an alternative approach, in the present study a novel combination therapy was established through combining TAM with nordamnacanthal (NDAM) in order to investigate the additive effect of these drugs in MCF-7 human breast cancer cells. A significant dose-dependent reduction in cell viability and an increase in apoptosis were observed in the MCF-7 cells cotreated with TAM and NDAM compared with the untreated control cells or the cells treated with TAM and NDAM alone (P<0.05). The cytotoxic influence of the combination of TAM and NDAM was found to be two-fold that of the individual agents. Annexin V/propidium iodide double-staining revealed the typical nuclear features of apoptosis. Furthermore, an increase in the proportion of apoptotic, Annexin V-positive cells was observed with the combination therapy. Moreover, this apoptotic induction was associated with a collapse of the mitochondrial membrane potential and the generation of reactive oxygen species. To the best of our knowledge, the findings of the present study are the first to suggest that combining TAM with NDAM may be a potential combination therapy for the treatment of breast cancer and may have the potential to minimize or eliminate the side effects associated with high doses of TAM. PMID:25435988

SUBRAMANI, TAMILSELVAN; YEAP, SWEE KEONG; HO, WAN YANG; HO, CHAI LING; OSMAN, CHE PUTEH; ISMAIL, NOR HADIANI; RAHMAN, NIK MOHD AFIZAN NIK ABDUL; ALITHEEN, NOORJAHAN BANU

2015-01-01

306

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

307

Nordamnacanthal potentiates the cytotoxic effects of tamoxifen in human breast cancer cells.  

PubMed

Tamoxifen (TAM) is the mainline drug treatment for breast cancer, despite its side effects and the development of resistance. As an alternative approach, in the present study a novel combination therapy was established through combining TAM with nordamnacanthal (NDAM) in order to investigate the additive effect of these drugs in MCF-7 human breast cancer cells. A significant dose-dependent reduction in cell viability and an increase in apoptosis were observed in the MCF-7 cells cotreated with TAM and NDAM compared with the untreated control cells or the cells treated with TAM and NDAM alone (P<0.05). The cytotoxic influence of the combination of TAM and NDAM was found to be two-fold that of the individual agents. Annexin V/propidium iodide double-staining revealed the typical nuclear features of apoptosis. Furthermore, an increase in the proportion of apoptotic, Annexin V-positive cells was observed with the combination therapy. Moreover, this apoptotic induction was associated with a collapse of the mitochondrial membrane potential and the generation of reactive oxygen species. To the best of our knowledge, the findings of the present study are the first to suggest that combining TAM with NDAM may be a potential combination therapy for the treatment of breast cancer and may have the potential to minimize or eliminate the side effects associated with high doses of TAM. PMID:25435988

Subramani, Tamilselvan; Yeap, Swee Keong; Ho, Wan Yang; Ho, Chai Ling; Osman, Che Puteh; Ismail, Nor Hadiani; Rahman, Nik Mohd Afizan Nik Abdul; Alitheen, Noorjahan Banu

2015-01-01

308

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

309

Apoptosis and necrosis of human breast cancer cells by an aqueous extract of garden cress (Lepidium sativum) seeds.  

PubMed

Conventional treatments for breast cancer are costly and have serious side effects. Non-conventional natural treatments have gained wide acceptance due to their promise of a cure with minimal or no side effects, but little scientific evidence exists. One such common remedy is the seed of the Lepidium sativum plant. Presented here is the first reported use of the aqueous extract of Lepidium sativum seeds on breast cancer cells. The ability of the extract to induce apoptosis and necrosis in the human breast cancer cell line MCF-7, compared to normal human skin fibroblasts (HFS), was determined by morphological changes in the cells using light microscopy, DNA fragmentation assay, and florescent stains (Annexin V and propidium iodide) using flow cytometry and fluorescent microscopy. Apoptosis was induced in both cells, and more in MCF-7, when they were treated with 25% and 50% extract, while necrosis was observed mainly after exposure to elevated extract concentrations (75%). DNA fragmentation resulted for both cells, in a time and dose-dependent manner. Both cells, at all extract concentrations, showed no significant differences in the number of living, dead, apoptotic, and necrotic cells. Finally, the results may indicate that apoptotic changes in MCF-7 may be independent of caspase-3, which is involved in apoptosis and is lacking in MCF-7 cells. PMID:23961228

Mahassni, Sawsan Hassan; Al-Reemi, Roaa Mahdi

2013-04-01

310

Apoptosis and necrosis of human breast cancer cells by an aqueous extract of garden cress (Lepidium sativum) seeds  

PubMed Central

Conventional treatments for breast cancer are costly and have serious side effects. Non-conventional natural treatments have gained wide acceptance due to their promise of a cure with minimal or no side effects, but little scientific evidence exists. One such common remedy is the seed of the Lepidium sativum plant. Presented here is the first reported use of the aqueous extract of Lepidium sativum seeds on breast cancer cells. The ability of the extract to induce apoptosis and necrosis in the human breast cancer cell line MCF-7, compared to normal human skin fibroblasts (HFS), was determined by morphological changes in the cells using light microscopy, DNA fragmentation assay, and florescent stains (Annexin V and propidium iodide) using flow cytometry and fluorescent microscopy. Apoptosis was induced in both cells, and more in MCF-7, when they were treated with 25% and 50% extract, while necrosis was observed mainly after exposure to elevated extract concentrations (75%). DNA fragmentation resulted for both cells, in a time and dose-dependent manner. Both cells, at all extract concentrations, showed no significant differences in the number of living, dead, apoptotic, and necrotic cells. Finally, the results may indicate that apoptotic changes in MCF-7 may be independent of caspase-3, which is involved in apoptosis and is lacking in MCF-7 cells. PMID:23961228

Mahassni, Sawsan Hassan; Al-Reemi, Roaa Mahdi

2013-01-01

311

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

312

Protective effect of carboxymethylated chitosan on hydrogen peroxide-induced apoptosis in nucleus pulposus cells  

PubMed Central

Although the etiology of intervertebral disc degeneration is poorly understood, one approach to prevent this process may be to inhibit apoptosis. In the current study, the anti-apoptotic effects of carboxymethylated chitosan (CMCS) in nucleus pulposus (NP) cells were investigated with the aim to enhance disc cell survival. Rat NP cells were isolated and cultured in vitro, and hydrogen peroxide (H2O2) was used to build the NP cell apoptosis model. Cell viability was assessed with a cell counting kit-8 assay. The ratio of apoptotic cells was surveyed by annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) double staining analysis, and the morphology was observed by Hoechst 33342 staining. The mitochondrial membrane potential of NP cells was evaluated by rhodamine 123 fluorescence staining. Reverse transcription (RT)-quantitative polymerase chain reaction (qPCR) was performed to measure mRNA levels of inducible nitric oxide synthase (iNOS), caspase-3, B-cell lymphoma (Bcl)-2, type II collagen and aggrecan. Western blot analysis was performed to detect protein levels of iNOS and Bcl-2. The annexin V-FITC/PI and Hoechst 33342 staining results indicated that CMCS was able to prevent NP cells from apoptosis in a dose-dependent manner. Rhodamine 123 staining clarified that CMCS reduced the impairment of the mitochondrial membrane potential in H2O2-treated NP cells. Reduced caspase-3 and increased Bcl-2 activity were detected in CMCS-treated NP cells by RT-qPCR and western blot analysis. CMCS also promoted the proliferation and secretion of type II collagen and aggrecan in H2O2-treated NP cells. CMCS was indicated to be effective in preventing apoptotic cell death in vitro, demonstrating the potential advantages of this therapeutic approach in regulating disc degeneration. PMID:25394560

HE, BIN; TAO, HAIYING; LIU, SHIQING; WEI, AILIN

2015-01-01

313

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

SciTech Connect

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.

Asare, Nana [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo (Norway)], E-mail: nana.asare@fhi.no; Landvik, Nina E. [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo (Norway); Lagadic-Gossmann, Dominique; Rissel, Mary; Tekpli, Xavier [Inserm U620, Group Toxicity of Environmental Contaminants labellisee Ligue contre le Cancer, 2 av Pr. Leon Bernard, 35043 Rennes Cedex (France); Universite Rennes 1, IFR 140, 2 av Pr. Leon Bernard, 35043 Rennes Cedex (France); Ask, Kjetil [Department of Pathology and Molecular Medicine, Centre for Gene Therapeutics, McMaster University, Hamilton, Ontario (Canada); Lag, Marit; Holme, Jorn A. [Division of Environmental Medicine, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo (Norway)

2008-07-15

314

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; Låg, Marit; Holme, Jørn A

2008-07-15

315

Cytotoxic Effect of Ethanol Extract of Microalga, Chaetoceros calcitrans, and Its Mechanisms in Inducing Apoptosis in Human Breast Cancer Cell Line  

PubMed Central

Marine microalgae have been prominently featured in cancer research. Here, we examined cytotoxic effect and apoptosis mechanism of crude ethanol extracts of an indigenous microalga, Chaetoceros calcitrans (UPMAAHU10) on human breast cell lines. MCF-7 was more sensitive than MCF-10A with IC50 value of 3.00 ± 0.65, whilst the IC50 value of Tamoxifen against MCF-7 was 12.00 ± 0.52??g/mL after 24 hour incubation. Based on Annexin V/Propidium iodide and cell cycle flow cytometry analysis, it was found that inhibition of cell growth by EEC on MCF-7 cells was through the induction of apoptosis without cell cycle arrest. The apoptotic cells at subG0/G1 phase in treated MCF-7 cells at 48 and 72 hours showed 34 and 16 folds increased compared to extract treated MCF-10A cells which showed only 6 and 7 folds increased at the same time points, respectively. Based on GeXP study, EEC induced apoptosis on MCF-7 cells via modulation of CDK2, MDM2, p21Cip1, Cyclin A2, Bax and Bcl-2. The EEC treated MCF-7 cells also showed an increase in Bax/Bcl-2 ratio that in turn activated the caspase-dependent pathways by activating caspase 7. Thus, marine microalga, Chaetoceros calcitrans may be considered a good candidate to be developed as a new anti-breast cancer drug. PMID:23509778

Ebrahimi Nigjeh, Siyamak; Yusoff, Fatimah Md; Mohamed Alitheen, Noorjahan Banu; Rasoli, Mehdi; Keong, Yeap Swee; Omar, Abdul Rahman bin

2013-01-01

316

Enhanced detection of fluorescence quenching in labeled cells  

DOEpatents

A method is provided for quantifying BrdU labeled DNA in cells. The BrdU is incorporated into 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 that 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 substracted 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.

Crissman, Harry A. (Los Alamos, NM); Steinkamp, John A. (Los Alamos, NM)

1992-01-01

317

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

318

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

PubMed

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

2013-03-01

319

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

320

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

321

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

322

Cell cycle-dependent cytotoxicity and induction of apoptosis by liposomal N4-hexadecyl-1-beta-D-arabinofuranosylcytosine.  

PubMed Central

The clonogenic growth inhibition, the cell cycle dependence of N4-hexadecyl-1-beta-D-arabinofuranosylcytosine (NHAC) cytotoxicity and the capability to induce apoptosis in ara-C-sensitive and -resistant HL-60 cells were investigated and compared with arabinofuranosylcytosine (ara-C). In the clonogenic assay with sensitive HL-60 cells, ara-C was slightly more effective than a liposomal preparation of NHAC, whereas in the resistant cells, NHAC revealed its potency to overcome ara-C resistance, resulting in a 23-fold lower 50% inhibitory concentration compared with ara-C. Cell cycle dependent cytotoxicity and induction of apoptosis were studied by flow cytometry, using the bromodeoxyuridine-propidium iodide and terminal transferase method respectively. In contrast to ara-C, NHAC exerted no phase-specific toxicity at low concentrations (< 40 microM). At higher concentrations the S-phase-specific toxicity increased, probably resulting from ara-C formed from NHAC. NHAC induced apoptosis at higher drug concentrations than ara-C, however apoptosis appeared not to be limited to the S-phase cells. Apoptosis occurred in both cell lines within 2-4 h after drug exposure. These results give further evidence that NHAC exerts its cytotoxicity by different mechanisms of action than ara-C and might therefore be active in ara-C-resistant tumours. Images Figure 4 PMID:7577448

Horber, D. H.; von Ballmoos, P.; Schott, H.; Schwendener, R. A.

1995-01-01

323

Induction of intrinsic and extrinsic apoptosis pathways in the human leukemic MOLT-4 cell line by terpinen-4-ol.  

PubMed

Terpinen-4-ol is a terpene found in the rhizome of Plai (Zingiber montanum (Koenig) Link ex Dietr.). In this study apoptogenic activity and mechanisms of cell death induced by terpinen-4-ol were investigated in the human leukemic MOLT-4 cell line. Terpinen-4-ol exhibited cytotoxicity in MOLT-4 cells, with characteristic morphological features of apoptosis by Wright's staining. The mode of cell death was confirmed to be apoptosis by flow cytometric analysis after staining with annexin V-FITC and propidium iodide. A sub-G1 peak in DNA histograms of cell cycle assays was observed. Terpinen-4-ol induced-MOLT-4 cell apoptosis mediated through an intrinsic pathway involving the loss of mitochondrial transmembrane potential (MTP) and release of cytochrome c into the cytosol. In addition, terpinen-4-ol also induced apoptosis via an extrinsic pathway by caspase-8 activation resulting in the cleavage of cytosolic Bid. Truncated-Bid (tBid) translocated to mitochondria and activated the mitochondrial pathway in conjunction with down-regulation of Bcl-2 protein expression. Caspase-3 activity also increased. In conclusion, terpinen-4-ol can induce human leukemic MOLT-4 cell apoptosis via both intrinsic and extrinsic pathways. PMID:22994712

Khaw-on, Patompong; Banjerdpongchai, Ratana

2012-01-01

324

Effect of Celastrol on Growth Inhibition of Prostate Cancer Cells through the Regulation of hERG Channel In Vitro  

PubMed Central

Objective. To explore the antiprostate cancer effects of Celastrol on prostate cancer cells' proliferation, apoptosis, and cell cycle distribution, as well as the correlation to the regulation of hERG. Methods. DU145 cells were treated with various concentrations of Celastrol (0.25–16.0??mol/L) for 0–72 hours. MTT assay was used to evaluate the inhibition effect of Celastrol on the growth of DU145 cells. Cell apoptosis was detected through both Annexin-V FITC/PI double-labeled cytometry and Hoechst 33258. Cell cycle regulation was examined by a propidium iodide method. Western blot and RT-PCR technologies were applied to assess the expression level of hERG in DU145 cells. Results. Celastrol presented striking growth inhibition and apoptosis induction potency on DU145 cells in vitro in a time- and dose-dependent manner. The IC50 value of Celastrol for 24 hours was 2.349 ± 0.213??mol/L. Moreover, Celastrol induced DU145 cell apoptosis in a cell cycle-dependent manner, which means Celastrol could arrest DU145 cells in G0/G1 phase; accordingly, cells in S phase decreased gradually and no obvious changes were found in G2/M phase cells. Through transmission electron microscope, apoptotic bodies containing nuclear fragments were found in Celastrol-treated DU145 cells. Overexpression of hERG channel was found in DU145 cells, while Celastrol could downregulate it at both protein and mRNA level in a dose-dependent manner (P < 0.01). Conclusions. Celastrol exhibits its antiprostate cancer effects partially through the downregulation of the expression level of hERG channel in DU145 cells, suggesting that Celastrol may be a potential agent against prostate cancer with a mechanism of blocking the hERG channel. PMID:25866772

Ji, Nan; Li, Jinjun; Wei, Zexiong; Kong, Fanhu; Jin, Hongyan; Chen, Xiaoya; Li, Yan; Deng, Youping

2015-01-01

325

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

326

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

2014-10-01

327

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

328

Analysis of the antitumor activity of gemcitabine and carboplatin against ovarian clear-cell carcinoma using the DNA damage marker ?H2AX  

PubMed Central

Background Differences in the incidence and type of DNA damage induced by antitumor agents for ovarian clear-cell carcinoma (CCC) were determined in two CCC cell lines, using ?H2AX. Materials and methods The antitumor activity of gemcitabine (GEM) and carboplatin (CBDCA) were examined using cultured cell lines of CCC (OVISE and RMG-I). Each cell line was treated with GEM and CBDCA, the cells were collected, fixed, and then reacted with anti-?H2AX antibody. ?H2AX and nuclear DNA were then simultaneously detected by flow cytometry using fluorescein isothiocyanate and propidium iodide, respectively, to determine the amounts of ?H2AX formed in each cell-cycle phase. Results After administration of GEM, both cell lines showed DNA damage and cell-cycle arrest in the S and G2/M phases, and increased apoptosis. Similarly, with CBDCA, OVISE showed S- and G2/M-phase arrest, while RMG-I showed G2/M-phase arrest. Conclusion The mechanism of action of GEM and CBDCA in CCC cell lines was elucidated using ?H2AX as a DNA damage marker. Our findings suggested that concomitant use of GEM plus CBDCA may be effective in the treatment of CCC. PMID:23898228

Takatori, Eriko; Shoji, Tadahiro; Sawai, Takashi; Kurose, Akira; Sugiyama, Toru

2013-01-01

329

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

330

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

331

Photodynamic damage of glial cells in crayfish ventral nerve cord  

NASA Astrophysics Data System (ADS)

Photodynamic therapy (PDT) is a promising method for treatment of brain tumors, the most of which are of glial origin. In the present work we studied PDT-mediated injury of glial cells in nerve tissue, specifically, in abdominal connectives in the crayfish ventral nerve cord. The preparation was photosensitized with alumophthalocyanine Photosens and irradiated 30 min with the diode laser (670 nm, 0.1 or 0.15 W/cm2). After following incubation in the darkness during 1- 10 hours it was fluorochromed with Hoechst 33342 and propidium iodide to reveal nuclei of living, necrotic and apoptotic cells. The chain-like location of the glial nuclei allowed visualization of those enveloping giant axons and blood vessels. The level of glial necrosis in control preparations was about 2-5 %. Apoptosis was not observed in control preparations. PDT significantly increased necrosis of glial cells to 52 or 67 % just after irradiation with 0.1 or 0.15 W/cm2, respectively. Apoptosis of glial cells was observed only at 10 hours after light exposure. Upper layers of the glial envelope of the connectives were injured stronger comparing to deep ones: the level of glial necrosis decreased from 100 to 30 % upon moving from the connective surface to the plane of the giant axon inside the connective. Survival of glial cells was also high in the vicinity of blood vessels. One can suggest that giant axons and blood vessels protect neighboring glial cells from photodynamic damage. The mechanism of such protective action remains to be elucidated.

Kolosov, M. S.; Duz, E.; Uzdensky, A. B.

2011-03-01

332

Photodynamic damage of glial cells in crayfish ventral nerve cord  

NASA Astrophysics Data System (ADS)

Photodynamic therapy (PDT) is a promising method for treatment of brain tumors, the most of which are of glial origin. In the present work we studied PDT-mediated injury of glial cells in nerve tissue, specifically, in abdominal connectives in the crayfish ventral nerve cord. The preparation was photosensitized with alumophthalocyanine Photosens and irradiated 30 min with the diode laser (670 nm, 0.1 or 0.15 W/cm2). After following incubation in the darkness during 1- 10 hours it was fluorochromed with Hoechst 33342 and propidium iodide to reveal nuclei of living, necrotic and apoptotic cells. The chain-like location of the glial nuclei allowed visualization of those enveloping giant axons and blood vessels. The level of glial necrosis in control preparations was about 2-5 %. Apoptosis was not observed in control preparations. PDT significantly increased necrosis of glial cells to 52 or 67 % just after irradiation with 0.1 or 0.15 W/cm2, respectively. Apoptosis of glial cells was observed only at 10 hours after light exposure. Upper layers of the glial envelope of the connectives were injured stronger comparing to deep ones: the level of glial necrosis decreased from 100 to 30 % upon moving from the connective surface to the plane of the giant axon inside the connective. Survival of glial cells was also high in the vicinity of blood vessels. One can suggest that giant axons and blood vessels protect neighboring glial cells from photodynamic damage. The mechanism of such protective action remains to be elucidated.

Kolosov, M. S.; Duz, E.; Uzdensky, A. B.

2010-10-01