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1

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

EPA Science Inventory

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

2

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

Quantification of viable Legionella pneumophila cells using propidium monoazide combined with quantitative PCR  

Microsoft Academic Search

One of the greatest challenges of implementing fast molecular detection methods as part of Legionella surveillance systems is to limit detection to live cells. In this work, a protocol for sample treatment with propidium monoazide (PMA) in combination with quantitative PCR (qPCR) has been optimized and validated for L. pneumophila as an alternative of the currently used time-consuming culture method.

M. Adela Yáñez; Andreas Nocker; Elena Soria-Soria; Raquel Múrtula; Lorena Martínez; Vicente Catalán

2011-01-01

4

Comparison of ethidium monoazide and propidium monoazide for the selective detection of viable Legionella cells.  

PubMed

Ethidium monoazide (EMA) and propidium monoazide (PMA) have been utilized for selective PCR amplification of DNA from viable bacterial cells. In this study, we compared the abilities of EMA and PMA, together with real-time PCR, to specifically distinguish dead Legionella cells from viable cells. Several experiments showed that PMA or EMA treatment could specifically prevent the PCR amplification of DNA from dead Legionella cells in water samples. However, a 4-fold higher concentration of PMA than EMA was required to achieve this effect. EMA may therefore be more useful for practical environmental investigations of Legionella. PMID:20332575

Chang, Bin; Taguri, Toshitsugu; Sugiyama, Kanji; Amemura-Maekawa, Junko; Kura, Fumiaki; Watanabe, Haruo

2010-03-01

5

Quantification of viable Legionella pneumophila cells using propidium monoazide combined with quantitative PCR.  

PubMed

One of the greatest challenges of implementing fast molecular detection methods as part of Legionella surveillance systems is to limit detection to live cells. In this work, a protocol for sample treatment with propidium monoazide (PMA) in combination with quantitative PCR (qPCR) has been optimized and validated for L. pneumophila as an alternative of the currently used time-consuming culture method. Results from PMA-qPCR were compared with culture isolation and traditional qPCR. Under the conditions used, sample treatment with 50 ?M PMA followed by 5 min of light exposure were assumed optimal resulting in an average reduction of 4.45 log units of the qPCR signal from heat-killed cells. When applied to environmental samples (including water from cooling water towers, hospitals, spas, hot water systems in hotels, and tap water), different degrees of correlations between the three methods were obtained which might be explained by different matrix properties, but also varying degrees of non-culturable cells. It was furthermore shown that PMA displayed substantially lower cytotoxicity with Legionella than the alternative dye ethidium monoazide (EMA) when exposing live cells to the dye followed by plate counting. This result confirmed the findings with other species that PMA is less membrane-permeant and more selective for the intact cells. In conclusion, PMA-qPCR is a promising technique for limiting detection to intact cells and makes Legionella surveillance data substantially more relevant in comparison with qPCR alone. For future research it would be desirable to increase the method's capacity to exclude signals from dead cells in difficult matrices or samples containing high numbers of dead cells. PMID:21329735

Yáñez, M Adela; Nocker, Andreas; Soria-Soria, Elena; Múrtula, Raquel; Martínez, Lorena; Catalán, Vicente

2011-05-01

6

Flow cytometric phase-resolved discrimination of damaged/dead cells by propidium iodide uptake in macrophages having phagocytized fluorescent microspheres  

NASA Astrophysics Data System (ADS)

Instilled particle burdens of uniform green-yellow fluorescent microspheres phagocytized by rat alveolar (lung) macrophages and cell viability, as indexed by propidium iodide uptake (red fluorescence), were assessed using flow cytometry. Since the spectral emission from phagocytized microspheres partially overlapped the propidium iodide fluorescence and interfered with the conventional flow cytometric measurement of damaged/dead cells without subtractive compensation, this caused errors when estimating the percentage of non-viable, propidium iodide positive, phagocytic macrophages. The interference was eliminated by employing phase-sensitive detection in the red fluorescence measurement channel based on differences in lifetimes between the fluorescent microspheres and propidium iodide. In addition, intrinsic cellular autofluorescence, whose fluorescence lifetime is approximately the same as the phagocytized microspheres, also was eliminated in the measurement process. Since there was no detectable spectral interference of propidium iodide in the green fluorescence (particle phagocytosis) measurement channel, conventional fluorescence detection was employed.

Steinkamp, John A.; Valdez, Yolanda E.; Lehnert, Bruce E.

2001-05-01

7

Multiplex polymerase chain reaction using ethidium monoazide and propidium monoazide for distinguishing viable and dead cells of arcobacters in biofilm.  

PubMed

This paper concerns the formation of biofilm in bacteria of the genus Arcobacter. A multiplex polymerase chain reaction (PCR) method was introduced and optimized for detecting biofilm while using the intercalating dyes ethidium monoazide (EMA) and propidium monoazide (PMA), first for analysis of strains of the genus Arcobacter from a collection, and then applied to samples of prepared biofilms. The results of the study indicate considerable variability among species of bacteria within the genus Arcobacter. The EMA-PMA PCR method can distinguish viable cells from dead cells and is therefore suitable for determining the viability of cells. PMID:24313452

Hrušková, Lucie; Mot'ková, Petra; Vyt?asová, Jarmila

2013-12-01

8

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

9

Kinetic analysis of nanoparticulate polyelectrolyte complex interactions with endothelial cells.  

PubMed

A non-toxic, nanoparticulate polyelectrolyte complex (PEC) drug delivery system was formulated to maintain suitable physicochemical properties at physiological pH. Toxicity, binding, and internalization were evaluated in relevant microvascular endothelial cells. PEC were non-toxic, as indicated by cell proliferation studies and propidium iodide staining. Inhibitor studies revealed that PEC were bound, in part, via heparan sulfate proteoglycans and internalized through macropinocytosis. A novel, flow cytometric, Scatchard protocol was established and showed that PEC, in the absence of surface modification, bind cells non-specifically with positive cooperativity, as seen by graphical transformations. PMID:17560645

Hartig, Sean M; Greene, Rachel R; Carlesso, Gianluca; Higginbotham, James N; Khan, Wasif N; Prokop, Ales; Davidson, Jeffrey M

2007-09-01

10

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

Microsoft Academic Search

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

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

2010-01-01

11

The detection of viable vegetative cells of Bacillus sporothermodurans using propidium monoazide with semi-nested PCR.  

PubMed

Bacillus sporothermodurans produces highly heat-resistant spores that can survive ultra-high temperature (UHT) treatment in milk. Therefore, we developed a rapid, specific and sensitive semi-nested touchdown PCR assay combined with propidium monoazide (PMA) treatment for the detection of viable B. sporothermodurans vegetative cells. The semi-nested touchdown PCR alone proved to be specific for B. sporothermodurans, and the achieved detection limit was 4 CFU/mL from bacterial culture and artificially contaminated UHT milk. This method combined with PMA treatment was shown to amplify DNA specifically from viable cells and presented a detection limit of 10(2) CFU/mL in UHT milk. The developed PMA-PCR assay shows applicability for the specific detection of viable cells of B. sporothermodurans from UHT milk. This method is of special significance for applications in the food industry by reducing the time required for the analysis of milk and dairy products for the presence of this microorganism. PMID:23498198

Cattani, F; Ferreira, C A S; Oliveira, S D

2013-05-01

12

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

PubMed

The lack of differentiation between viable and nonviable bacterial cells limits the implementation of PCR-based methods for routine diagnostic approaches. Recently, the combination of a quantitative real-time PCR (qPCR) and ethidium monoazide (EMA) or propidium monoazide (PMA) pretreatment has been described to circumvent this disadvantage. In regard to the suitability of this approach for Campylobacter spp., conflicting results have been reported. Thus, we compared the suitabilities of EMA and PMA in various concentrations for a Campylobacter viability qPCR method. The presence of either intercalating dye, EMA or PMA, leads to concentration-dependent shifts toward higher threshold cycle (CT) values, especially after EMA treatment. However, regression analysis resulted in high correlation coefficient (R(2)) values of 0.99 (EMA) and 0.98 (PMA) between Campylobacter counts determined by qPCR and culture-based enumeration. EMA (10 ?g/ml) and PMA (51.10 ?g/ml) removed DNA selectively from nonviable cells in mixed samples at viable/nonviable ratios of up to 1:1,000. The optimized EMA protocol was successfully applied to 16 Campylobacter jejuni and Campylobacter coli field isolates from poultry and indicated the applicability for field isolates as well. EMA-qPCR and culture-based enumeration of Campylobacter spiked chicken leg quarters resulted in comparable bacterial cell counts. The correlation coefficient between the two analytical methods was 0.95. Nevertheless, larger amounts of nonviable cells (>10(4)) resulted in an incomplete qPCR signal reduction, representing a serious methodological limitation, but double staining with EMA considerably improved the signal inhibition. Hence, the proposed Campylobacter viability EMA-qPCR provides a promising rapid method for diagnostic applications, but further research is needed to circumvent the limitation. PMID:24487529

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

2014-04-01

13

Quantitative study of viable Vibrio parahaemolyticus cells in raw seafood using propidium monoazide in combination with quantitative PCR.  

PubMed

In this study we developed a specific and sensitive quantitative PCR (qPCR) method combined with a propidium monoazide (PMA) sample treatment to quantify tdh-positive viable cells of V. parahaemolyticus in raw seafood (PMA-qPCR). The high selectivity of primers and probes were demonstrated by using purified DNA from 57 strains belonging to 18 species. Using these primers and probes for qPCR and in artificial contamination samples, a good correlation was obtained between Ct values and log CFU/reaction in the range of 12-1.2×10(6)CFU/reaction both from qPCR and PMA-qPCR with R(2) values of 0.9973 and 0.9919, respectively. The optimization of PMA concentration showed that 8 ?g/mL was considered optimal to achieve a compromise between minimal impact on intact cells and maximal signal reduction in compromised cells. However, turbidity and cell concentration experiments showed that PMA treatment was not effective in samples where turbidities were ?10 NTU and OD(600 nm) values were ?0.8. PMA-qPCR was compared with culture isolation and traditional qPCR in environmental samples (including oyster, scallop, shrimp, and crab). The PMA-qPCR resulted in lower numbers of log CFUg(-1) than qPCR, with values having better agreement with numbers determined by culture isolation. In conclusion, this method is an effective tool for producing reliable quantitative data on viable V. parahaemolyticus in raw seafood. PMID:22677606

Zhu, Ru-Gang; Li, Tuo-Ping; Jia, You-Feng; Song, Li-Feng

2012-09-01

14

Pharmacological concentrations of ascorbate radiosensitize glioblastoma multiforme primary cells by increasing oxidative DNA damage and inhibiting G2/M arrest.  

PubMed

Glioblastoma multiforme (GBM) has a very poor prognosis because of its chemo- and radiation therapy resistance. Here we investigated the ability of pharmacological concentrations of ascorbate to radiosensitize primary cells isolated from six GBM patients, mouse astrocytoma cells, and mouse astrocytes. We measured cell viability by trypan blue exclusion, generation of double-stranded DNA breaks by H2AX phosphorylation using fluorescently labeled antibodies and FACS analysis, apoptosis by annexin V/propidium iodide staining, inhibition of autophagy by 3-methyladenine, and cell cycle progression by propidium iodide staining of permeabilized cells. We showed that 5 mM ascorbate in combination with 6 Gy radiation killed more GBM primary cells by generating significantly more double-stranded breaks than either treatment alone (p<0.05). Combined treatment affected viability and double-stranded break generation in normal astrocytes to a much smaller extent. Radiation, but not 5 mM ascorbate, caused G2/M arrest in GBM cells and ascorbate prevented radiation-induced G2/M arrest in combined treatment. Cell death in response to 5 mM ascorbate or combination treatment was not mediated by apoptosis or autophagy. In conclusion, pharmacological concentrations of ascorbate radiosensitize GBM primary cells to a much greater extent than astrocytes; this large therapeutic ratio may be of clinical significance in radiation-resistant cancers. PMID:22342518

Herst, Patries M; Broadley, Kate W R; Harper, Jacquie L; McConnell, Melanie J

2012-04-15

15

Resveratrol inhibits proliferation in human colorectal carcinoma cells by inducing G1/S-phase cell cycle arrest and apoptosis through caspase/cyclin-CDK pathways  

PubMed Central

The present study compared the effect of resveratrol on HCT116 and Caco-2 human colon cancer cells. Annexin V/propidium iodide staining, MTT assay and western blot analysis revealed that resveratrol induced cycle arrest in the two cell lines, which was evidenced by cell cycle analysis and changes in the expression of the cell cycle proteins cyclin-dependent kinase (CDK) 2, CDK4, cyclin D1, proliferating cell nuclear antigen and P21. Furthermore, resveratrol was found to have a strong apoptosis-inducing effect, which was evidenced through the high percentage of annexin V positive cells and high protein expression of cleaved-caspase-7, cleaved-caspase-9 and cleaved-poly(ADP-ribose) polymerase in the resveratrol-treated cancer cells. In conclusion, these results demonstrated that resveratrol had greater growth inhibitory and cell cycle arrest effects on Caco-2 cells than HCT116 cells, through caspase-dependent and cyclin-CDK pathways. PMID:25050564

LIU, BIN; ZHOU, ZHONGYOU; ZHOU, WEI; LIU, JIE; ZHANG, QINGYU; XIA, JUAN; LIU, JUNTAO; CHEN, NIANPING; LI, MINGYI; ZHU, RUNZHI

2014-01-01

16

Resveratrol inhibits proliferation in human colorectal carcinoma cells by inducing G1/S?phase cell cycle arrest and apoptosis through caspase/cyclin?CDK pathways.  

PubMed

The present study compared the effect of resveratrol on HCT116 and Caco-2 human colon cancer cells. Annexin V/propidium iodide staining, MTT assay and western blot analysis revealed that resveratrol induced cycle arrest in the two cell lines, which was evidenced by cell cycle analysis and changes in the expression of the cell cycle proteins cyclin-dependent kinase (CDK) 2, CDK4, cyclin D1, proliferating cell nuclear antigen and P21. Furthermore, resveratrol was found to have a strong apoptosis-inducing effect, which was evidenced through the high percentage of annexin V positive cells and high protein expression of cleaved?caspase?7, cleaved?caspase?9 and cleaved?poly(ADP-ribose) polymerase in the resveratrol-treated cancer cells. In conclusion, these results demonstrated that resveratrol had greater growth inhibitory and cell cycle arrest effects on Caco-2 cells than HCT116 cells, through caspase-dependent and cyclin-CDK pathways. PMID:25050564

Liu, Bin; Zhou, Zhongyou; Zhou, Wei; Liu, Jie; Zhang, Qingyu; Xia, Juan; Liu, Juntao; Chen, Nianping; Li, Mingyi; Zhu, Runzhi

2014-10-01

17

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

18

The Arctic Alzheimer mutation enhances sensitivity to toxic stress in human neuroblastoma cells.  

PubMed

The E693G (Arctic) mutation of the amyloid precursor protein was recently found to lead to early-onset Alzheimer's disease in a Swedish family. In the present study, we report that the Arctic mutation decreases cell viability in human neuroblastoma cells. The cell viability, as measured by the MTT assay and propidium iodide staining, was further compromised following exposure to calcium ionophore A23187, microtubule-binding colchicine or oxidative stress inducer hydrogen peroxide. The manner of cell death was found to be apoptotic. During apoptosis, cells with the Arctic mutation also decreased their secretion of beta-secretase cleaved amyloid precursor protein. The enhanced sensitivity to toxic stress in cells with the Arctic mutation most likely contributes to the pathogenic pathway leading to Alzheimer's disease. PMID:12052536

Sennvik, Kristina; Nilsberth, Camilla; Stenh, Charlotte; Lannfelt, Lars; Benedikz, Eirikur

2002-06-21

19

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

PubMed Central

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

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

2014-01-01

20

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

21

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

PubMed Central

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

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

2006-01-01

22

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

PubMed Central

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

2013-01-01

23

Endoplasmic reticulum stress-mediated aldosterone-induced apoptosis in vascular endothelial cells.  

PubMed

The aim of this study was to examine the effects of endoplasmic reticulum (ER) stress on aldosterone (Aldo)-induced apoptosis of endothelial cells. Glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP, a hallmark of ER-associated apoptosis) were used to evaluate ER stress. Western blotting and real-time PCR were used to analyze indicators of ER molecule. Apoptosis was detected by annexin V/propidium iodide staining and flow cytometry. Human umbilical vein endothelial cells (HUVECs) were stimulated with different concentrations of Aldo for different durations. Aldo promoted apoptosis of HUVECs and induced ER stress, as evidenced by increased expression of GRP78 and CHOP. siRNA knockdown of CHOP attenuated Aldo-mediated apoptosis. These results indicate that ER stress may be involved in Aldo-induced apoptosis of HUVECs. PMID:25480576

Lu, Jin-Ping; Li, Xia; Jin, Ya-Lei; Chen, Mei-Xiang

2014-12-01

24

Lysosomal membrane permeabilization induces cell death in human mast cells.  

PubMed

Mast cells (MC) have pathogenic roles in numerous disorders, and strategies that stabilize MC or induce MC apoptosis are therefore emerging as possible therapeutic regimens. A typical feature of MC is their high content of secretory lysosomes (granules), containing numerous components such as biogenic amines, cytokines, serglycin proteoglycan and proteases. Damage to the secretory lysosomes will thus lead to leakage of these compounds, including the proteases, into the cytosol, and this could potentially trigger apoptosis. Here, we evaluated whether MC are sensitive to cell death induced by secretory lysosome destabilization, induced by the lysosomotropic agent Leu-Leu-OMe (LLME). Human MC were sensitive to LLME-induced cell death. In contrast, fibroblasts and HEK-293 cells were largely resistant. As judged by Annexin V/propidium iodide staining, LLME caused apoptotic cell death, and this was supported by induction of caspase-3-like activity, detection of activated caspase-3 by immunoblot analysis and reduced cell death in the presence of a caspase inhibitor. In support of a role for serglycin in regulating LLME-induced cell death, the survival rate of various cell types correlated negatively with the level of serglycin expression. In summary, this study introduces the concept of using lysosomotropic agents to induce cell death of human MC. PMID:21645032

Melo, F R; Lundequist, A; Calounova, G; Wernersson, S; Pejler, G

2011-10-01

25

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

26

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

27

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

28

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

PubMed

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

Wang, Bing; Li, Hongbo; Yang, Rui; Zhou, Shunchang; Zou, Shengquan

2014-11-01

29

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

PubMed Central

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

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

2014-01-01

30

Cytotoxicity of absorption enhancers in Caco-2 cell monolayers.  

PubMed

This study was performed to evaluate the utility of absorption enhancers with reference to mucosal cell cytotoxicity. Overall assessment of the damage to plasma, lysosomal and nuclear membranes by three absorption enhancers, sodium deoxycholate, sodium caprate and dipotassium glycyrrhizinate, was performed on Caco-2 cell monolayers. The cytotoxicities of sodium deoxycholate (0.02-0.1% w/v), sodium caprate (0.1-0.5% w/v) and dipotassium glycyrrhizinate (0.5-2% w/v) were evaluated by the trypan blue-exclusion test, the protein-release test, the neutral-red assay, the DNA--propidium iodide staining test and the test for recovery of transepithelial electrical resistance (TEER) up to 24 h after treatment with each enhancer. Sodium dodecyl sulphate (SDS; 0.1% w/v), a potent surfactant, was used as positive control. SDS at this level was significantly cytotoxic whereas dipotassium glycyrrhizinate was not cytotoxic in any tests. Results from the trypan blue-exclusion and protein-release tests showed that high concentrations of sodium caprate (0.5% w/v) and sodium deoxycholate (0.1% w/v) were significantly cytotoxic to the plasma membrane. The neutral-red assay, an indicator of damage to lysosomal membranes, revealed that 0.5% (w/v) sodium caprate had no effect whereas the uptake of neutral red was slightly increased by treatment with 0.1% (w/v) sodium deoxycholate, implying that the compound had cell-growth-enhancing activity. Nuclear-membrane damage, as evaluated by the DNA--propidium iodide staining test, was severe in cell monolayers treated with 0.5% (w/v) sodium caprate compared with that induced by 0.1% (w/v) sodium deoxycholate. In the TEER recovery test, TEER failed to recover 24 h after treatment with 0.5% (w/v) sodium caprate and 0.1% (w/v) SDS, but recovered after treatment with 0.1% (w/v) sodium deoxycholate. The recovery of TEER might be related to nuclear membrane damage and cell-growth-enhancing activity. These results indicate that of the three classes of enhancer, dipotassium glycyrrhizinate was not cytotoxic and that high concentrations of sodium caprate and sodium deoxycholate could damage plasma and nuclear membranes. PMID:9821655

Sakai, M; Imai, T; Ohtake, H; Otagiri, M

1998-10-01

31

Inhibitory effects of evodiamine on human osteosarcoma cell proliferation and apoptosis  

PubMed Central

Osteosarcoma is a primary malignancy of bone, which is characterized by the proliferation of malignant mesenchymal cells, particularly in children and adolescents. Evodiamine is extracted from a variety of traditional Chinese medicines, which has been reported to induce apoptosis in certain tumors, including cervical, prostate and breast cancer, however, its effect on oestosarcoma cells remains unclear. The aim of the present study was to investigate the effect of evodiamine on osteosarcoma cell proliferation and apoptosis, and explore the associated underlying molecular mechanism. A Cell Counting Kit 8 assay was performed to detect the effects of evodiamine on the proliferation of human osteosarcoma U2OS cells. Annexin V-fluorescein isothiocyanate/propidium iodide staining was performed to analyze the apoptotic rate of the cells. The effect of evodiamine on the protein expression levels of B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), caspase-3 and survivin were detected by performing western blot analysis. Evodiamine inhibited the growth of human osteosarcoma U2OS cells by inhibiting cell proliferation and inducing cell apoptosis. Western blotting demonstrated that evodiamine downregulated the expression of Bcl-2, caspase-3 and survivin, and upregulated the expression of Bax in human osteosarcoma cells. Evodiamine effectively inhibited proliferation and induced apoptosis of osteosarcoma cells in a dose-dependent manner via downregulation of Bcl-2, caspase-3 and survivin protein expression levels and upregulation of Bax protein expression levels. PMID:25621054

BAI, XIAODONG; MENG, HAI; MA, LIFENG; GUO, AI

2015-01-01

32

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

33

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

34

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

35

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

PubMed Central

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

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

2007-01-01

36

Structural basis for autoantibody recognition of phosphatidylserine-?2 glycoprotein I and apoptotic cells  

PubMed Central

Apoptotic cells contain nuclear autoantigens that may initiate a systemic autoimmune response. To explore the mechanism of antibody binding to apoptotic cells, 3H9, a murine autoantibody with dual specificity for phospholipids and DNA, was used. H chain mutants of 3H9 were constructed, expressed as single-chain Fv (scFv) in Escherichia coli, and assessed for binding to phosphatidylserine, an antigen expressed on apoptotic cells. Both 3H9 and its germline revertant bound to dioleoyl phosphatidylserine in ELISA, and binding was enhanced by ?2 glycoprotein I (?2GPI), a plasma protein that selectively binds to apoptotic cells. Higher relative affinity for DOPS-?2GPI was achieved by the introduction of Arg residues into the 3H9 H chain variable region at positions previously shown to mediate DNA binding. Specificity of the two structurally most diverse scFv for apoptotic cells was shown by flow cytometry, and two populations of scFv-bound cells were identified by differences in propidium iodide staining. The results suggest that, in autoimmunity, B cells with Ig receptors for apoptotic cells and DNA are positively selected, and that the antibodies they produce have the potential to affect the clearance and processing of apoptotic cells. PMID:11717440

Cocca, Brian A.; Seal, Samarendra N.; D'Agnillo, Paolo; Mueller, Yvonne M.; Katsikis, Peter D.; Rauch, Joyce; Weigert, Martin; Radic, Marko Z.

2001-01-01

37

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

38

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

39

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

PubMed

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

Kathiravan, Govindarajan; Sureban, Sripathi M

2009-12-01

40

Comparative studies of cytotoxic and apoptotic properties of different extracts and the essential oil of Lavandula angustifolia on malignant and normal cells.  

PubMed

Lavender (Lavandula angustifolia Mill.) is a bush-like shrub from Lamiaceae. The herb has been used in alternative medicine for several centuries. In this study, the cytotoxicity and the mechanisms of cell death induced by 3 different extracts of aerial parts and the essential oil of L. angustifolia were compared in normal and cancerous human cells. Malignant (HeLa and MCF-7 cell lines) and nonmalignant (human fibroblasts) cells were incubated with different concentrations of the plant extracts. Cell viability was quantified by MTS assay. Apoptotic cells were determined using propidium iodide staining of DNA fragmentation by flow cytometry (sub-G1 peak). The molecules as apoptotic signal translation, including Bax and cleaved PARP, were identified by Western blot. Ethanol and n-hexane extracts and essential oil exhibited significant cytotoxicity to malignant cells but marginal cytotoxicity to human fibroblasts in vitro and induced a sub-G1 peak in flow cytometry histogram of treated cells compared to the control. Western blot analysis demonstrated that EtOH and n-hexane extracts upregulated Bax expression, also it induced cleavage of PARP in HeLa cells compared to the control. In conclusion, L. angustifolia has cytotoxic and apoptotic effects in HeLa and MCF-7 cell lines, and apoptosis is proposed as the possible mechanism of action. PMID:24571090

Tayarani-Najaran, Zahra; Amiri, Atefeh; Karimi, Gholamreza; Emami, Seyed Ahmad; Asili, Javad; Mousavi, Seyed Hadi

2014-01-01

41

Apoptotic effects of 7,8-dihydroxyflavone in human oral squamous cancer cells through suppression of Sp1.  

PubMed

7,8-Dihydroxyflavone (7,8-DHF) is a member of the flavonoid family and has recently been identified as a brain-derived neurotrophic factor mimetic that selectively activates tropomyosin-receptor kinase B with high affinity. The antioxidant and anticancer effects of 7,8-DHF have been reported. However, the pharmacological mechanisms of 7,8-DHF in oral cancer are unclear. Thus, we investigated the mechanisms of the antiproliferative action of 7,8-DHF on HN22 and HSC4 oral squamous cell carcinoma cell lines. We demonstrated that 7,8-DHF decreased cell growth and induced apoptosis in the HN22 and HSC4 cells through regulation of specificity protein 1 (Sp1) using the MTS assay, DAPI staining, Annexin V, propidium iodide staining, reverse transcription-polymerase chain reaction, immunocytochemistry, pull-down assay and western blot analysis. The results showed that the Sp1 protein bound with 7,8-DHF in the HN22 and HSC4 cells. Taken together, the results suggest that 7,8-DHF could modulate Sp1 transactivation and induce apoptotic cell death by regulating the cell cycle and suppressing antiapoptotic proteins. Furthermore, 7,8-DHF may be valuable for cancer prevention and better clinical outcomes. PMID:25434704

Lee, Ra Ham; Shin, Jae-Cheon; Kim, Ka-Hwi; Choi, Yung Hyun; Chae, Jung-Il; Shim, Jung-Hyun

2015-02-01

42

GFP-transfected tumor cells are useful in examining early metastasis in vivo, but immune reaction precludes long-term tumor development studies in immunocompetent mice.  

PubMed

To develop effective therapeutic strategies aimed at treating tumor metastasis, critical steps in this process must be better understood. For this purpose we have established a new model to visualize and quantify early metastasis. Murine CT-26 colon adenocarcinoma cells were stably transfected with green fluorescent protein (GFP). Tumor cells were intraportally delivered to the liver of Balb/c mice and subsequently tracked by intravital fluorescence microscopy. Coinjection of fluorescent beads and in vivo propidium iodide staining allowed examination of initial tumor cell arrest, extravasation, viability and proliferation. Results showed that GFP-transfection compared to conventional labeling procedures (Calcein, cytoplasmic microspheres) did not alter early metastatic properties. However, the long-term development of liver metastases expressing GFP was markedly reduced compared to wild type CT-26 tumor cells. An increase in the size and the number of liver metastases in T- and B-cell-deficient SCID mice suggested an immune response to the GFP transfected cells responsible for the reduced metastatic growth in wild-type mice. Based on our findings, this model can be used to examine the early steps of metastasis in vivo. However, in immunocompetent mice, the use of GFP-labeled tumor cells should be limited to tracking cell arrest and extravasation, whereas evaluations of long-term metastatic growth should be performed in immunodeficient mice. PMID:12705634

Steinbauer, Markus; Guba, Markus; Cernaianu, Grigore; Köhl, Gudrun; Cetto, Michaela; Kunz-Schughart, Leoni A; Geissler, Edward K; Falk, Werner; Jauch, Karl-Walter

2003-01-01

43

Growth inhibition and apoptosis induction by Scutellaria pinnatifida A. Ham. on HL-60 and K562 leukemic cell lines.  

PubMed

A rich flavonoid plant extensively used in traditional medicine is Scutellaria (Lamiaceae). In this study the cytotoxic effects of different extracts of Scutellaria pinnatifida were explored on two different cancer cell lines including K562 and HL-60 and a normal cell line. Also, the role of apoptosis on this toxicity was explored. The cell viability was quantitated by alamarBlue(®) assay. S. pinnatifida could effectively decrease the viability of malignant cells and the CH2Cl2 extract of S. pinnatifida had showed the most cytotoxic effects among other extracts. Apoptosis was confirmed after propidium iodide staining of DNA fragments and detection of the sub-G1 peak in the related flow cytometry histogram of cells. The results of western blot assay showed that CH2Cl2 extract has been able to increase the amount of Bax and cleavage of PARP protein after a 48h contact with cells. Neobaicalein (skullcap?avone II) and wogonin were identified in the extract of S. pinnatifida as the active components. The result of the present study confirmed the putative role of neobaicalein (skullcap?avone II) and wogonin as cytotoxic components in the CH2Cl2 extract of S. pinnatifida. PMID:25546119

Boozari, Motahareh; Mohammadi, Ameneh; Asili, Javad; Emami, Seyed Ahmad; Tayarani-Najaran, Zahra

2015-01-01

44

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 concentrationdependent 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

45

Rituximab synergizes with hydroxyurea or vincristine in the killing of Ramos Burkitt's lymphoma B cell line.  

PubMed

Rituximab is an effective immunotherapy for CD20-positive B-cell non-Hodgkin's lymphoma. However, some patients show resistance, particularly those suffering from more aggressive lymphoma types, such as Burkitt's lymphoma. Hence, Rituximab is commonly combined with several chemotherapeutic drugs. With a view to reduce the number of such drugs, we examined the effect of combining Rituximab individually with hydroxyurea, vincristine, or etoposide on the killing of Ramos Burkitt lymphoma cell line type I. Cell death was examined by using Annexin-V/propidium iodide staining. Combining Rituximab with hydroxyurea or vincristine resulted in a synergistic effect, whereas combining it with etoposide resulted in a subadditive effect. In single treatments, the percentage of cell death ranged from 23% (Rituximab) to 36% (hydroxyurea). Combining Rituximab with hydroxyurea or vincristine resulted in a synergistic effect (83% and 74% killing, respectively). In contrast, only a subadditive effect was noticed with etoposide (36%). We conclude that the synergistic effect of Rituximab with hydroxyurea or vincristine is worthy of further study, and that further in vitro screening of chemotherapeutics might identify chemo-immunotherapeutic combinations that are effective in vivo but less toxic than currently used regimens. PMID:24256491

Deyab, Mohamed; Elbanani, Abdulrhman; Tabal, Salah; Geriani, Hajer; Lamami, Yosra; Bredan, Amin; Abulayha, Abdulmunem

2014-03-01

46

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

47

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

48

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

49

Ca(2+) movement and apoptosis induced by deltamethrin in Madin-Darby canine kidney canine renal tubular cells.  

PubMed

This study explored the effect of deltamethrin, a pesticide, on free Ca(2+) concentration [Ca(2+)]i, viability, and apoptosis in Madin-Darby canine kidney (MDCK) canine renal tubular cells. Deltamethrin at concentrations between 10?M and 40?M evoked [Ca(2+)]i rises in a concentration-dependent manner. The Ca(2+) entry was inhibited by nifedipine, econazole, phorbol 12-myristate 13-acetate, and SKF96365. Treatment with the endoplasmic reticulum Ca(2+) pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) in a Ca(2+)-free medium abolished deltamethrin-induced [Ca(2+)]i rise. Treatment with deltamethrin also abolished BHQ-induced [Ca(2+)]i rise. Inhibition of phospholipase C (PLC) activity with U73122 abolished deltamethrin-evoked [Ca(2+)]i rise. Deltamethrin killed cells at 30-60?M in a concentration-dependent manner. The cytotoxic effect of deltamethrin was not reversed by prechelating cytosolic Ca(2+) with the acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Annexin V/propidium iodide staining data suggest that 30-50?M deltamethrin induced apoptosis. Together, in MDCK renal tubular cells, deltamethrin induced [Ca(2+)]i rises that involved Ca(2+) entry through protein kinase C-mediated store-operated Ca(2+) channels, and PLC-dependent Ca(2+) release from the endoplasmic reticulum. Deltamethrin also induced Ca(2+)-independent cell death that might involve apoptosis. PMID:25600914

Liu, Fang-Jin; Chou, Chiang-Ting; Cheng, Jin Shiung; Chang, Hong-Tai; Liang, Wei-Zhe; Kuo, Chun-Chi; Kuo, Soong-Yu; Kuo, Daih-Huang; Shieh, Pochuen; Chang, Fang-Rong; Jan, Chung-Ren

2015-01-01

50

Toxicity of surface-modified PLGA nanoparticles toward lung alveolar epithelial cells.  

PubMed

In vitro cytotoxicity and inflammatory response following exposure to nanoparticles (NPs) made of poly(lactide-co-glycolide) (PLGA) have been investigated on A549 human lung epithelial cells. Three different PLGA NPs (230 nm) were obtained using different stabilizers (polyvinyl alcohol, chitosan, or Pluronic(®) F68) to form respectively neutral, positively or negatively charged NPs. Polystyrene NPs were used as polymeric but non-biodegradable NPs, and titanium dioxide (anatase and rutile) as inorganic NPs, for comparison. Cytotoxicity was evaluated through mitochondrial activity as well as membrane integrity (lactate dehydrogenase release, trypan blue exclusion, propidium iodide staining). The cytotoxicity of PLGA-based and polystyrene NPs was lower or equivalent to the one observed after exposure to titanium dioxide NPs. The inflammatory response, evaluated through the release of the IL-6, IL-8, MCP-1, TNF-? cytokines, was low for all NPs. However, some differences were observed, especially for negative PLGA NPs that led to a higher inflammatory response, which can be correlated to a higher uptake of these NPs. Taken together, these results show that both coating of PLGA NPs and the nature of the core play a key role in cell response. PMID:23747506

Grabowski, Nadège; Hillaireau, Hervé; Vergnaud, Juliette; Santiago, Letícia Aragão; Kerdine-Romer, Saadia; Pallardy, Marc; Tsapis, Nicolas; Fattal, Elias

2013-10-01

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

Human Tumor Cell Proliferation Evaluated Using Manganese-Enhanced MRI  

PubMed Central

Background Tumor cell proliferation can depend on calcium entry across the cell membrane. As a first step toward the development of a non-invasive test of the extent of tumor cell proliferation in vivo, we tested the hypothesis that tumor cell uptake of a calcium surrogate, Mn2+ [measured with manganese-enhanced MRI (MEMRI)], is linked to proliferation rate in vitro. Methodology/Principal Findings Proliferation rates were determined in vitro in three different human tumor cell lines: C918 and OCM-1 human uveal melanomas and PC-3 prostate carcinoma. Cells growing at different average proliferation rates were exposed to 1 mM MnCl2 for one hour and then thoroughly washed. MEMRI R1 values (longitudinal relaxation rates), which have a positive linear relationship with Mn2+ concentration, were then determined from cell pellets. Cell cycle distributions were determined using propidium iodide staining and flow cytometry. All three lines showed Mn2+-induced increases in R1 compared to cells not exposed to Mn2+. C918 and PC-3 cells each showed a significant, positive correlation between MEMRI R1 values and proliferation rate (p?0.005), while OCM-1 cells showed no significant correlation. Preliminary, general modeling of these positive relationships suggested that pellet R1 for the PC-3 cells, but not for the C918 cells, could be adequately described by simply accounting for changes in the distribution of the cell cycle-dependent subpopulations in the pellet. Conclusions/Significance These data clearly demonstrate the tumor-cell dependent nature of the relationship between proliferation and calcium influx, and underscore the usefulness of MEMRI as a non-invasive method for investigating this link. MEMRI is applicable to study tumors in vivo, and the present results raise the possibility of evaluating proliferation parameters of some tumor types in vivo using MEMRI. PMID:22363447

Braun, Rod D.; Bissig, David; North, Robert; Vistisen, Kerry S.; Berkowitz, Bruce A.

2012-01-01

54

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.

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

2015-01-01

55

Anti-tumor activity of safranal against neuroblastoma cells  

PubMed Central

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

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

2014-01-01

56

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

57

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

58

rILYd4, a Human CD59 Inhibitor Enhances Complement Dependent Cytotoxicity of Ofatumumab against Rituximab-resistant B-cell Lymphoma Cells and Chronic Lymphocytic Leukemia  

PubMed Central

Purpose Ofatumumab (OFA) is an anti-CD20 antibody recently approved for treatment of fludarabine and alemtuzumab refractory chronic lymphocytic lymphomia (CLL); it mediates much stronger complement-dependent cytotoxicity (CDC) than Rituximab (RTX). Human CD59, a key membrane complement regulator that inhibits CDC, is highly expressed in B-cell malignancies and its up-regulation is an important determinant of the sensitivity of B-cell malignancies to RTX treatment. Previously, we have demonstrated that the potent CD59 inhibitor rILYd4 sensitizes RTX-resistant lymphoma cells to RTX-mediated CDC. Here, we further investigated whether rILYd4 can sensitize B-cell malignancies to OFA-mediated CDC and whether either OFA-mediated CDC or rILYd4-enhanced OFA-mediated CDC correlates with CD20 or CD59 expression, known biomarkers involved in RTX activity. Experimental Design RTX-resistant cell lines and primary CLL cells were used to investigate the antitumor efficacy of the combination of rILYd4 with OFA or RTX. Propidium iodide staining or Alamar blue assay were employed to evaluate the CDC effect. The levels of CD20 and CD59 on the cell membrane were analyzed by flow cytometry. Results rILYd4 enhanced CDC effects mediated by OFA or RTX on RTX-resistant lymphoma cells and primary CLL cells in vitro. The sensitivity to CDC effects mediated by OFA positively correlated with the ratio of CD20/CD59 and negatively correlated with CD59 levels on CLL cells. The degree to which rILYd4 enhanced CDC correlated positively with the CD59 levels on CLL cells. Conclusions These data suggest that rILYd4 may enhance the anti-cancer activity of OFA and RTX in B-cell malignancies that have relapsed after prior antibody-based therapies. PMID:21918174

Ge, Xiaowen; Wu, Lin; Hu, Weiguo; Fernandes, Stacey; Wang, Chun; Li, Xu; Brown, Jennifer R; Qin, Xuebin

2011-01-01

59

Isophilippinolide A arrests cell cycle progression and induces apoptosis for anticancer inhibitory agents in human melanoma cells.  

PubMed

Three new butanolides, isophilippinolide A, philippinolide A, and philippinolide B, and an amide, cinnaretamine, were isolated from the roots of Cinnamomum philippinense to be identified by spectroscopic analysis. Four isolated compounds were screened to examine their radical-scavenging ability, metal-chelating power, and ferric-reducing antioxidant power assay (FRAP). Cinnaretamine showed powerful antioxidative properties in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay and a reducing activity; all compounds presented minor inhibition of metal-chelating capacities. The effects of anti-tyrosinase of C. philippinense constituents were determined by the level of the suppression of hydroxylation that turned from L-tyrosine to L-dopa through an in vitro mushroom tyrosinase assay, and all testing samples illustrated slight mushroom tyrosinase inhibitory properties. Isophilippinolide A exhibited inhibitory effectivenesses against the A375.S2 melanoma cell line in a cell viability assay at concentrations ranging from 0 to 200 ?M for 24 h. Propidium iodide staining and flow cytometry analyses were applied to assess cell cycle accumulative distribution. It was discovered that isophilippinolide A caused sub-G1 phase accumulation in positive correlation for apoptosis to inhibit cell growth. Further investigation revealed that isophilippinolide A induced A375.S2 cells with an increase of caspase-dependent apoptotic proteins to trigger correlated pathway mechanisms according to Western blotting results. Finally, isophilippinolide A displayed only low cytotoxicities to human normal epidermal cells (melanocytes) and dermal cells (fibroblasts). Altogether, the results implied C. philippinense compounds could be considered functional ingredients in cosmetics, foods, and pharmaceutical products, particularly for their anticancer ability on human skin melanoma cells. PMID:24359513

Wang, Hui-Min David; Chen, Chung-Yi; Wu, Pei-Fang

2014-02-01

60

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

PubMed

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

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

2007-01-01

61

High throughput characterization of adult stem cells engineered for delivery of therapeutic factors for neuroprotective strategies.  

PubMed

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system. Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair. PMID:25590859

Sharma, Anup D; Brodskiy, Pavel A; Petersen, Emma M; Dagdeviren, Melih; Ye, Eun-Ah; Mallapragada, Surya K; Sakaguchi, Donald

2015-01-01

62

Crocetin induces cytotoxicity and enhances vincristine-induced cancer cell death via p53-dependent and -independent mechanisms  

PubMed Central

Aim: To investigate the anticancer effect of crocetin, a major ingredient in saffron, and its underlying mechanisms. Methods: Cervical cancer cell line HeLa, non-small cell lung cancer cell line A549 and ovarian cancer cell line SKOV3 were treated with crocetin alone or in combination with vincristine. Cell proliferation was examined using MTT assay. Cell cycle distribution and sub-G1 fraction were analyzed using flow cytometric analysis after propidium iodide staining. Apoptosis was detected using the Annexin V-FITC Apoptosis Detection Kit with flow cytometry. Cell death was measured based on the release of lactate dehydrogenase (LDH). The expression levels of p53 and p21WAF1/Cip1 as well as caspase activation were examined using Western blot analysis. Results: Treatment of the 3 types of cancer cells with crocetin (60-240 ?mol/L) for 48 h significantly inhibited their proliferation in a concentration-dependent manner. Crocetin (240 ?mol/L) significantly induced cell cycle arrest through p53-dependent and -independent mechanisms accompanied with p21WAF1/Cip1 induction. Crocetin (120-240 ?mol/L) caused cytotoxicity in the 3 types of cancer cells by enhancing apoptosis in a time-dependent manner. In the 3 types of cancer cells, crocetin (60 ?mol/L) significantly enhanced the cytotoxicity induced by vincristine (1 ?mol/L). Furthermore, this synergistic effect was also detected in the vincristine-resistant breast cancer cell line MCF-7/VCR. Conclusion: Ccrocetin is a potential anticancer agent, which may be used as a chemotherapeutic drug or as a chemosensitizer for vincristine. PMID:21986580

Zhong, Ying-jia; Shi, Fang; Zheng, Xue-lian; Wang, Qiong; Yang, Lan; Sun, Hong; He, Fan; Zhang, Lin; Lin, Yong; Qin, Yong; Liao, Lin-chuan; Wang, Xia

2011-01-01

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

Dose-dependent and sequence-dependent cytotoxicity of erlotinib and docetaxel in head and neck squamous cell carcinoma.  

PubMed

The purpose of this study was to determine whether the efficacy of taxoid treatment combined with epidermal growth factor receptor (EGFR) inhibition is dose and sequence dependent in head and neck squamous cell carcinoma. Three head and neck squamous cell carcinoma cell lines, chosen on the basis of their diverse EGFR expression levels, were treated with docetaxel, erlotinib, or both. The combination index was calculated using the Chou-Talalay equation. Propidium iodide staining with fluorescence-activated cell sorting analysis was used to evaluate the effects of drugs on cell cycle changes. Western blot analysis was used to determine the effects of agents on cell signaling pathways. Administration of low-dose docetaxel (0.1-3 nmol/l) concurrently or before erlotinib had additive cytotoxic effects in two cell lines but was antagonistic in one line, whereas low-dose docetaxel after erlotinib was synergistic in all cell lines. In contrast, high-dose docetaxel (40 nmol/l) resulted in more apoptosis when given before, rather than after or concurrently with, erlotinib. Low-dose docetaxel induced an accumulation of cells in the sub-G0 phase of the cell cycle with no mitotic arrest or apoptosis, whereas high-dose docetaxel induced mitotic arrest and apoptosis. The low and high doses of docetaxel had opposite effects on EGFR expression: a decrease and an increase, respectively. The dose of docetaxel affects sequence-dependent cytotoxicity when docetaxel is combined with an EGFR inhibitor. The mechanism for this difference is a combination of the dose-dependent effects of docetaxel on the mode of cell death and on EGFR expression. PMID:18418213

Saigal, Babita; Glisson, Bonnie S; Johnson, Faye M

2008-06-01

65

Insulin-like growth factor binding protein 7 exhibits tumor suppressive and vessel stabilization properties in U87MG and T98G glioblastoma cell lines.  

PubMed

Insulin-like growth factor binding protein 7 (IGFBP7) is downregulated in several solid cancers. IGFBP7 has been proposed to act as a tumor suppressor gene through mechanisms involving senescence and apoptotic pathways. The tumor suppressor effect of IGFBP7 in glioblastoma multiforme (GBM) was examined in this study using two human GBM cell lines, U87MG and T98G. Exogenously applied IGFBP7 (20 and 100 nM) significantly reduced U87MG (~70 and ~75%, respectively) and T98G (~37 and ~50%, respectively) cell growth in soft agar. IGFBP7 stimulated senescence-associated ?-galactosidase in both U87MG and T98G cells without stimulating apoptosis (annexin V and propidium iodide staining, expression of SMARCB1 or BNIP3L and caspase cleavage) or affecting phosphorylation of p44/42 MAPK. The inhibitory effect of IGFBP7 on U87MG cell growth was further assessed in vivo using U87MG cells grafted on the chick chorioallantoic membrane. In this model, U87MG cells formed solid and highly vascularized tumors that were reduced in size (~40%) when treated with 500 nM IGFBP7 compared with control tumors. Vessels in IGFBP7-treated tumors were clustered, unevenly distributed and associated with higher number of ?-SMA positive cells compared with those in untreated tumors. IGFBP7 induced both aortic smooth muscle cell (AoSMC) chemoattraction and endothelial cell (EC) transdifferentiation into a SM-like cell phenotype. U87MG conditioned media-induced IGFBP7 expression in ECs was significantly inhibited by the cross-talk/interaction with SMCs. This study indicates that IGFBP7 suppresses U87MG tumor cell growth, induces cell senescence and participates in tumor vessel stabilization by promoting SMC/pericyte recruitment and differentiation. PMID:21795858

Pen, Ally; Durocher, Yves; Slinn, Jacqueline; Rukhlova, Marina; Charlebois, Claudie; Stanimirovic, Danica B; Moreno, Maria J

2011-10-01

66

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

PubMed Central

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

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

2013-01-01

67

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

PubMed

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

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

2014-12-01

68

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

PubMed

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

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

2007-05-01

69

Bradykinin Preconditioning Improves Therapeutic Potential of Human Endothelial Progenitor Cells in Infarcted Myocardium  

PubMed Central

Objectives Stem cell preconditioning (PC) is a powerful approach in reducing cell death after transplantation. We hypothesized that PC human endothelial progenitor cells (hEPCs) with bradykinin (BK) enhance cell survival, inhibit apoptosis and repair the infarcted myocardium. Methods The hEPCs were preconditioned with or without BK. The hEPCs apoptosis induced by hypoxia along with serum deprivation was determined by annexin V-fluorescein isothiocyanate/ propidium iodide staining. Cleaved caspase-3, Akt and eNOS expressions were determined by Western blots. Caspase-3 activity and vascular endothelial growth factor (VEGF) levels were assessed in hEPCs. For in vivo studies, the survival and cardiomyocytes apoptosis of transplanted hEPCs were assessed using 1,1?-dioctadecyl-3,3,3?,3?-tetramethylindodi- carbocyanine,4-chlorobenzenesul-fonate salt labeled hEPCs and TUNEL staining. Infarct size and cardiac function were measured at 10 days after transplantation, and the survival of transplanted hEPCs were visualized using near-infrared optical imaging. Results In vitro data showed a marked suppression in cell apoptosis following BK PC. The PC reduced caspase-3 activation, increased the Akt, eNOS phosphorylation and VEGF levels. In vivo data in preconditioned group showed a robust cell anti-apoptosis, reduction in infarct size, and significant improvement in cardiac function. The effects of BK PC were abrogated by the B2 receptor antagonist HOE140, the Akt and eNOS antagonists LY294002 and L-NAME, respectively. Conclusions The activation of B2 receptor-dependent PI3K/Akt/eNOS pathway by BK PC promotes VEGF secretion, hEPC survival and inhibits apoptosis, thereby improving cardiac function in vivo. The BK PC hEPC transplantation for stem cell-based therapies is a novel approach that has potential for clinical used. PMID:24312554

Li, Yefei; Yan, Fengdi; Huang, Jie; Ma, Genshan

2013-01-01

70

Age-associated decline in cdk1 activity delays cell cycle progression of human T lymphocytes.  

PubMed

Despite the repeatedly observed impaired proliferative response of T lymphocytes from aged donors, the precise molecular basis underlying such a defect is still poorly understood. The aim of this study was to determine whether cyclin-dependent kinase 1 (cdk1), a serine-threonine kinase required for entry into mitosis, is implicated in this age-associated dysregulation of the cell cycle. T lymphocytes derived from young and elderly donors were blocked in S phase by hydroxyurea after a 48-h activation by anti-CD3 Abs. Under these experimental conditions, only the cells that were already located beyond the S phase were able to complete the cell cycle, decreasing their DNA content from 4n to 2n chromosomes. Using this procedure, a delay in the accomplishment of mitosis could be observed in cells from elderly individuals, as evidenced by propidium iodide staining. In this age group, only a minimal cdk1 activity could be immunoprecipitated from cells sorted in G2/M after nocodazole block. The decrease in cdk1 activity observed in T lymphocytes from aged donors could be accounted for by at least three mechanisms: 1) a failure of these cells to express a sufficient amount of cdk1, 2) a reduced level of the associated cyclin B1, and 3) an incomplete dephosphorylation of the kinase on tyrosine. This low cdk1 activity is likely to postpone the progression through the G2/M transition and participates in the dysfunction of the cell cycle during the process of aging. PMID:9820491

Quadri, R A; Arbogast, A; Phelouzat, M A; Boutet, S; Plastre, O; Proust, J J

1998-11-15

71

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

72

Acetyl-11-keto-beta-boswellic acid induces apoptosis in HL-60 and CCRF-CEM cells and inhibits topoisomerase I.  

PubMed

Antiproliferative action of different pentacyclic triterpenes has repeatedly been reported, and some lipoxygenase inhibitors have been shown to induce cell death in various cell systems. Acetyl-11-keto-beta-boswellic acid (AKBA) is a pentacyclic triterpene that inhibits 5-lipoxygenase in a selective, enzymedirected, nonredox, and noncompetitive manner. To investigate a possible effect of AKBA on leukemic cell growth, proliferation of HL-60 and CCRF-CEM cells was assayed in the presence of AKBA and a structural analog without effect on 5-lipoxygenase, amyrin. Cell counts and [3H]thymidine incorporation were significantly reduced in a dose-dependent manner in the presence of AKBA (IC50 = 30 microM) but not amyrin. An additive effect of AKBA with the crosslinking of the CD95 receptor was also observed. Flow cytometric analysis of propidium iodide-stained cells indicated that the cells underwent apoptosis. This was confirmed by flow cytometric detection of sub-G1 peaks in AKBA-treated cells and by DNA laddering. However, because HL-60 and CCRF-CEM do not express 5-lipoxygenase mRNA constitutively, a mechanism distinct from inhibition of 5-lipoxygenase must account for the effect of AKBA. In a DNA relaxation assay with phiX174RF DNA, AKBA inhibited topoisomerase I from calf thymus at concentrations of >/=10 microM. A semiquantitative cDNA polymerase chain reaction approach was used to estimate the relative level of expression of topoisomerases in both cell lines. The data suggest that induction of apoptosis in HL-60 and CCRF-CEM by AKBA may be due to inhibition of topoisomerase I in these cells. PMID:9918566

Hoernlein, R F; Orlikowsky, T; Zehrer, C; Niethammer, D; Sailer, E R; Simmet, T; Dannecker, G E; Ammon, H P

1999-02-01

73

Anticancer Activities of Pterostilbene-Isothiocyanate Conjugate in Breast Cancer Cells: Involvement of PPAR?  

PubMed Central

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

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

2014-01-01

74

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

75

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

PubMed

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

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

2014-12-01

76

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

PubMed

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

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

2012-11-01

77

Effects of Thymol on Ca²? Homeostasis and Apoptosis in MDCK Renal Tubular Cells.  

PubMed

Thymol is a natural essential oil present in many plants and has many different effects in various cell types. However, the effect of thymol on the physiology of MDCK renal tubular cells is unknown. The action of the phytochemical thymol on cytosolic Ca²? concentrations ([Ca²?]i) and apoptosis in Madin-Darby canine kidney (MDCK) renal tubular cells was explored. Fura-2, a Ca²?-sensitive fluorescent dye, was used to assess [Ca²?]i. Thymol at concentrations of 200-500 ?M caused a [Ca²?]i rise in a concentration-dependent manner. Removal of extracellular Ca²? partially reduced the effects of thymol. Thymol-induced Ca²? entry was inhibited by nifedipine, econazole, SK&F96365 and protein kinase C modulators. In a Ca²?-free medium, treatment with the endoplasmic reticulum Ca²? pump inhibitor thapsigargin inhibited thymol-induced [Ca²?]i increases. Treatment with thymol also inhibited thapsigargin-induced [Ca²?]i rise. Thymol killed cells at concentrations of 300-500 ?M in a concentrationdependent fashion. Chelating cytosolic Ca²? with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM (BAPTA/AM) did not prevent thymol cytotoxicity. Thymol (400 and 500 ?M) induced apoptosis detected by using Annexin V/propidium iodide staining. At 400 or 500 ?M, thymol increased levels of reactive oxygen species. Together, in MDCK cells, thymol induced a [Ca²?]i rise by inducing Ca²? release from the endoplasmic reticulum and Ca²? entry via protein kinase C-sensitive store-operated Ca²? channels. Our data suggest that thymol-induced apoptosis might involve reactive oxygen species (ROS) production. PMID:24694198

Chang, Hong-Tai; Chou, Chiang-Ting; Liang, Wei-Zhe; Lu, Ti; Kuo, Daih-Huang; Shieh, Pochuen; Ho, Chin-Man; Jan, Chung-Ren

2014-04-30

78

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

79

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

PubMed Central

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

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

2011-01-01

80

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

81

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

PubMed Central

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

Berrington, Danielle; Lall, Namrita

2012-01-01

82

Complement factor H-derived short consensus repeat 18–20 enhanced complement-dependent cytotoxicity of ofatumumab on chronic lymphocytic leukemia cells  

PubMed Central

The antitumor activity of monoclonal antibodies in the treatment of chronic lymphocytic leukemia is mediated mainly by antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. Unfortunately, the efficacy of complement-dependent cytotoxicity is strongly restricted due to the expression and acquisition of regulators of complement activation by lymphocytic leukemia cells. Whereas the role of membrane regulators of complement activation, such as CD55 and CD59, has been investigated in detail in chronic lymphocytic leukemia, the involvement of soluble regulators of complement activation, such as complement factor H, has not yet been reported. Propidium iodide staining was performed to investigate the efficacy of ofatumumab and factor H-derived short-consensus-repeat 18–20 in the induction of complement-dependent cytotoxicity on primary chronic lymphocytic leukemia cells from 20 patients. Deposition of complement C3 fragments was monitored by western blot analysis. Expression of CD20, CD55 or CD59 was determined by FACS analysis. Replacement of factor H with short consensus repeat 18–20 significantly increased the susceptibility of primary chronic lymphocytic leukemia cells to ofatumumab-induced complement-dependent cytotoxicity. More importantly, addition of short-consensus-repeat 18–20 was able to overcome complement- resistance occurring during treatment with ofatumumab alone. Use of short consensus repeat 18–20 is likely to prolong the turnover time of active C3b fragments generated on the target cells following ofatumumab-induced complement activation, thereby improving specific killing of chronic lymphocytic leukemia cells by complement-dependent cytotoxicity. The relative contribution of factor H to the protection of chronic lymphocytic leukemia cells against complement-dependent cytotoxicity was comparable to that of CD55. Our data suggest that, by abrogating factor H function, short consensus repeat 18–20 may provide a novel approach that improves the complement-dependent efficacy of therapeutic monoclonal antibodies. PMID:23850806

Hörl, Susanne; Banki, Zoltan; Huber, Georg; Ejaz, Asim; Müllauer, Brigitte; Willenbacher, Ella; Steurer, Michael; Stoiber, Heribert

2013-01-01

83

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

PubMed

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

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

2013-01-01

84

(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

85

Paris saponin I induces apoptosis via increasing the Bax/Bcl-2 ratio and caspase-3 expression in gefitinib-resistant non-small cell lung cancer in vitro and in vivo.  

PubMed

Polyphyllins, a major component of Rhizoma paridis, have been extensively used in non-small cell lung cancer (NSCLC). The aim of the present study was to evaluate the effects of Paris saponin I (PSI) on a panel of gefitinib-resistant NSCLC cell lines and its inhibition of tumor growth in a nude mouse model. The MTT assay was used to assess growth inhibition. The cell cycle was analyzed using flow cytometry and apoptosis was assessed using Annexin V/propidium iodide staining. The morphology of the apoptotic cells was determined by transmission electron microscopy. The protein expression levels of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) and caspase-3 were detected using western blot analysis. In addition, the glucose metabolism in tumor-bearing mice was evaluated using 18F-fludeoxyglucose (FDG) micro-positron emission tomography imaging. The PSI-induced growth inhibition rate was observed to significantly increase in a time- and dose-dependent manner. Furthermore, PSI induced significant G2/M-phase arrest and apoptosis. The expression levels of Bcl-2 decreased, while those of Bax and caspase-3 increased following PSI treatment. 18F-FDG-uptake in the PSI treatment groups was significantly decreased compared with that in the control group in vivo. In conclusion, PSI is a potent antitumor agent that acts by inhibiting the proliferation of gefitinib-resistant cells, and has potential as a candidate for a natural drug for gefitinib-resistant therapy. PSI-induced apoptosis, which occurred via multiple pathways, including G2/M-phase arrest and upregulation of the Bax/Bcl-2 ratio and caspase-3 expression, ultimately led to cell death and tumor inhibition. PMID:24718383

Jiang, Hao; Zhao, Peng-Jun; Su, Dan; Feng, Jianguo; Ma, Sheng-Lin

2014-06-01

86

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

87

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

PubMed

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

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

2014-06-01

88

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

PubMed

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

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

2013-01-01

89

Cell kinetics in leukaemia and solid tumours studied with in vivo bromodeoxyuridine and flow cytometry.  

PubMed Central

During a 15-month period, we used in vivo bromodeoxyuridine (BUDR) infusion to study cell kinetics in 112 consecutive patients with various types of malignant tumours: acute leukaemia (50 patients), gastric cancer (42) and brain gliomas (20). The in vivo BUDR method requires that a single tumour sample be taken 4-6 h after infusion and that bivariate flow cytometry (FCM) be employed to measure simultaneously the percentage of BUDR-labelled cells (which are identified with a green fluorescent anti-BUDR monoclonal antibody) and their mean DNA content (following propidium iodide staining). This technique rapidly furnishes the labelling index (LI) and the DNA synthesis time (TS), from which the tumour potential doubling time (Tpot) and production rate (fractional turnover rate, FTR) are calculated. The procedure took 6-9 h to complete and there was no immediate toxicity from BUDR administration. Successful LI and TS determinations were obtained in 89 (80%) and 80 (72%) of the 112 patients, respectively. Correlations were sought between kinetic parameters and a number of pathological and clinical ones. In 34 patients with acute non-lymphoblastic leukaemias who were uniformly treated for remission (CR) induction and maintenance, proliferative activity, as measured by Tpot and FTR, was greater in responsive than in non-responsive patients, and in those who experienced CR for over 8 months than in those who had a shorter CR. Proliferative activity was also greater in patients with advanced gastric cancers than in those with more limited disease. No correlations between kinetic and clinical and pathological parameters were found in gliomas. These data indicate the in vivo BUDR infusion coupled with FCM measurements can be performed in clinical settings to obtain kinetic data rapidly in quite large patient series. This will probably allow the inclusion of kinetic data in clinical trials aimed at evaluating the prognostic relevance of these data. PMID:2736227

Riccardi, A.; Danova, M.; Dionigi, P.; Gaetani, P.; Cebrelli, T.; Butti, G.; Mazzini, G.; Wilson, G.

1989-01-01

90

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

91

Selective quantification of viable Escherichia coli bacteria in biosolids by quantitative PCR with propidium monoazide modification.  

PubMed

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

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

2011-07-01

92

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

PubMed Central

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

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

2011-01-01

93

Transfection of the c-myc oncogene into normal Epstein-Barr virus- harboring B cells results in new phenotypic and functional features resembling those of Burkitt lymphoma cells and normal centroblasts  

PubMed Central

Activated c-myc gene was introduced into the cells of three normal Epstein-Barr virus (EBV)-positive lymphoblastoid B cell lines (LCL). The cells were monitored for the appearance of new phenotypic and functional features compared with the control LCL cells transfected with plasmid that did not contain the c-myc gene. The LCL-expressing c- myc constitutively did not arrest growth in low serum concentration. However, the cell number in the cultures failed to increase because of substantial cell death. Death was due to apoptosis as demonstrated by flow cytometric analysis of propidium iodide-stained cells, by typical DNA laddering in gel electrophoresis, and by the inspection of Giemsa- stained cell smears. Apoptosis was also induced by exposing the transfected cells to antibodies directed to the immunoglobulin mu chain (a-mu-ab) irrespective of the serum concentration in the culture. Exposure of the cells to CD40 ligand (CD40L) or CD40 monoclonal antibody prevented cell apoptosis. Upon transfection with c-myc, the LCL cells acquired a vacuolated morphology that was never observed in control cells. Moreover, the expression of CD10 and CD38 was upregulated, while that of CD39 and especially CD23 was downregulated. Unlike that observed in certain Burkitt lymphoma (BL) cell lines that share the same surface phenotype (CD10+CD38+CD23-CD39-), the c-myc- transfected cells expressed lymphocyte function-associated (LFA) 1, LFA- 3, and intercellular adhesion molecule 1 and grew in large clumps rather than single-cell layers. Expression of CD10 and CD38 was particularly evident on the cells undergoing apoptosis, thus suggesting a correlation between the presence of these markers and the apoptotic process. Cells placed in conditions favoring in vitro apoptosis displayed downregulation of Bcl-2 protein. Bcl-2 expression was, however, upregulated when the cells were exposed to CD40L. These data indicate that the B cells expressing c-myc constitutively acquire some of the features of normal centroblasts and of BL cells, including the expression of CD10 and CD38, and the propensity to undergo apoptosis, which can be prevented by exposure to CD40L. Therefore, these cells can serve as a model system to study both BL lymphomagenesis as well as the process of B cell selection occurring in the germinal centers. PMID:7836923

1995-01-01

94

Selenium suppresses oxidative-stress-enhanced vascular smooth muscle cell calcification by inhibiting the activation of the PI3K/AKT and ERK signaling pathways and endoplasmic reticulum stress.  

PubMed

Vascular calcification is a prominent feature of many diseases, including atherosclerosis, and it has emerged as a powerful predictor of cardiovascular morbidity and mortality. A number of studies have examined the association between selenium and risk of cardiovascular diseases, but little is known about the role of selenium in vascular calcification. To determine the role of selenium in regulating vascular calcification, we assessed the effect of sodium selenite on oxidative-stress-enhanced vascular smooth muscle cell (VSMC) calcification and the underlying mechanism. Oxidative stress induced by xanthine/xanthine oxidase increased apoptosis, as determined by Hoechst 33342 staining and annexin V/propidium iodide staining, and it enhanced osteoblastic differentiation and calcification of VSMCs, on the basis of alkaline phosphatase activity, the expression of Runx2 and type I collagen, and calcium deposition. These effects of oxidative stress were significantly inhibited by selenite. The following processes may explain the inhibitory effects of selenite: (1) selenite significantly suppressed oxidative stress, as evidenced by the decrease of the oxidative status of the cell and lipid peroxidation levels, as well as by the increase of the total protein thiol content and the activity of the antioxidant selenoenzyme glutathione peroxidase; (2) selenite significantly attenuated oxidative-stress-induced activation of the phosphatidylinositol 3-kinase/AKT and extracellular-signal-regulated kinase signaling pathways, resulting in decreased osteoblastic differentiation of VSMCs; (3) selenite significantly inhibited oxidative-stress-activated endoplasmic reticulum stress, thereby leading to decreased apoptosis. Our results suggest a potential role of selenium in the prevention of vascular calcification, which may provide more mechanistic insights into the relationship between selenium and cardiovascular diseases. PMID:24390545

Liu, Hongmei; Li, Xiaoming; Qin, Fei; Huang, Kaixun

2014-03-01

95

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

PubMed Central

One of the prerequisites of making ecological conclusions derived from genetic fingerprints is that bacterial community profiles reflect the live portion of the sample of interest. Propidium monoazide is a membrane-impermeant dye that selectively penetrates cells with compromised membranes, which can be considered dead. Once inside the cells, PMA intercalates into the DNA and can be covalently cross-linked to it, which strongly inhibits PCR amplification. By using PCR after PMA treatment, the analysis of bacterial communities can theoretically be limited to cells with intact cell membranes. Four experiments were performed to study the usefulness of PMA treatment of mixed bacterial communities comprising both intact and compromised cells in combination with end-point PCR by generating community profiles from the following samples: (i) defined mixtures of live and isopropanol-killed cells from pure cultures of random environmental isolates, (ii) wastewater treatment plant influent spiked with defined ratios of live and dead cells, (iii) selected environmental communities, and (iv) a water sediment sample exposed to increasing heat stress. Regions of 16S rRNA genes were PCR amplified from extracted genomic DNA, and PCR products were analyzed by using denaturing gradient gel electrophoresis (DGGE). Results from the first two experiments show that PMA treatment can be of value with end-point PCR by suppressing amplification of DNA from killed cells. The last two experiments suggest that PMA treatment can affect banding patterns in DGGE community profiles and their intensities, although the intrinsic limitations of end-point PCR have to be taken into consideration. PMID:17586667

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

2007-01-01

96

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

97

1,25-Dihydroxyvitamin D? and cisplatin synergistically induce apoptosis and cell cycle arrest in gastric cancer cells.  

PubMed

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] plays an anticancer role in multiple types of cancer and potentiates the cytotoxic effects of several common chemotherapeutic agents. The hypercalcemia caused by 1,25(OH)2D3 alone or resistance to cisplatin weaken the anticancer effects of vitamin D. Thus, in this study, we aimed to investigate the synergistic effects of 1,25(OH)2D3 and cisplatin on the apoptosis and cell cycle progression of gastric cancer cells. BGC-823 human gastric cancer cells were treated with 1,25(OH)2D3 or cisplatin alone, or a combination of both agents. Cell apoptosis was assessed by TUNEL assay and flow cytometry. The expression of the apoptosis-related proteins, poly(ADP-ribose) polymerase (PARP), Bax, Bcl-2, caspase-3 and caspase-8, was examined using immunoblot analysis. ERK and AKT phosphorylation were examined by immunoblot analysis. The cell cycle distribution was determined by propidium iodide staining and flow cytometric analysis. p21 and p27 protein expression was also examined using immunoblot analysis. Our results revealed that co-treatment with 1,25(OH)2D3 enhanced cisplatin-induced apoptosis and upregulated the expression of Bax, and promoted the cleavage of PARP and caspase-3. The phosphorylation levels of ERK and AKT were reduced following combined treatment with 1,25(OH)2D3 and cisplatin. The percentage of cells in the G0/G1 phase was greater in the cells treated with the combined treatment than in those treated with either 1,25(OH)2D3 or cisplatin alone. p21 and p27 expression was upregulated following co-treatment with both agents. The results of this study suggest that 1,25(OH)2D3 potentiates cisplatin-mediated cell growth inhibition and cell apoptosis, which involves the upregulation of Bax, a decrease in ERK and AKT phosphorylation levels, and increased p21 and p27 levels. PMID:24573222

Bao, Anyu; Li, Yan; Tong, Yongqing; Zheng, Hongyun; Wu, Wei; Wei, Chuandong

2014-05-01

98

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

PubMed

Current studies were aimed to elucidate influence of pulsed electromagnetic field stimulation on cell viability and apoptosis induction pathways. For the experimental model we have chosen monocytic cell line MonoMac6 and several apoptosis inducers with different mechanism of death induction like puromycin, colchicine, cyclophosphamide, minocycline and hydrogen peroxide. MonoMac6 cell line was grown at density 1x10(5) cells/well in 96-well culture plates. To induce cell death cell cultures were treated with different apoptosis inducers like puromycin, colchicine, cyclophosphamide, minocycline, hydrogen peroxide and at the same time with pulsed electromagnetic field 50 Hz, 45±5 mT (PEMF) for 4 hour per each stimulation, three times, in 24 hours intervals. Afterwards, cells were harvested for flow cytometry analysis of cell viability measured by annexin V-APC labeled and propidium iodide staining. Expression of apoptosis related genes was evaluated by semi quantitative reverse transcription (RT)-PCR assay. NuPAGE Novex Western blot analysis was carried out for apoptosis inducing factor (AIF) abundance in cytosolic and nuclear extracts of MonoMac6 cells. Puromycin, colchicine and minocycline activated cells and simultaneously treated with PEMF have shown out diminished percentage of annexinV positive (AnV+) cells comparing to controls without PEMF stimulation. MonaMac6 cells puromycin/colchicyne and PEMF treated were to a higher extent double stained (AnV+,PI+), which means increased late apoptotic as well as necrotic (PI+) cells, than non-stimulated controls. On the other hand, minocycline activated cells prior to PEMF treatment showed diminished amount of apoptotic and necrotic (annexin V, annexin V and propidium iodide, propidium iodide positive staining) cells. The opposite effect of PEMF on the percentage of annexin V positively stained cells has been achieved after treatment of MonoMac6 culture with cyclophoshamide and hydrogen peroxide. PEMF enhanced early phase of apoptosis induced by both apoptosis inducing agents. The analysis of expression of the apoptosis related genes in MonoMac6 cultures treated with puromycin and exposed to PEMF performed in reverse transcription of polymerase chain reaction (PCR) assay has shown changes in mRNA of genes engaged in intrinsic apoptotic pathway and pathway with AIF abundance. The most influenced was expression of gene belonging to pro-apoptotic family of Bcl-2 and AIF agent. Examination of immunoblots developed with anti-AIF antibody showed that cytosol content of AIF protein was diminished after puromycin and PEMF treatment of MonoMac6 cells. The obtained results indicate that PEMF affects induction of apoptosis in MonoMac6 cells stimulated to death with inducing agents to a different extent. Main finding of the current results is that, PEMF stimulation of MonoMac6 cells simultaneously treated with puromycin caused changes in the Bcl-family genes expression as well as in caspase independent pathway of apoptosis inducing factor (AIF). PMID:23211308

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

2012-10-01

99

Survivin gene silencing sensitizes prostate cancer cells to selenium growth inhibition  

PubMed Central

Background Prostate cancer is a leading cause of cancer-related death in men worldwide. Survivin is a member of the inhibitor of apoptosis (IAP) protein family that is expressed in the majority of human tumors including prostate cancer, but is barely detectable in terminally differentiated normal cells. Downregulation of survivin could sensitize prostate cancer cells to chemotherapeutic agents in vitro and in vivo. Selenium is an essential trace element. Several studies have shown that selenium compounds inhibit the growth of prostate cancer cells. The objective of this study is to investigate whether survivin gene silencing in conjunction with selenium treatment could enhance the therapeutic efficacy for prostate cancer and to elucidate the underlying mechanisms. Methods Expression of survivin was analyzed in a collection of normal and malignant prostatic tissues by immunohistochemical staining. In vitro studies were conducted in PC-3M, C4-2B, and 22Rv1 prostate cancer cells. The effect of selenium on survivin expression was analyzed by Western blotting and semi-quantitative RT-PCR. Survivin gene knockdown was carried out by transfecting cells with a short hairpin RNA (shRNA) designed against survivin. Cell proliferation was quantitated by the 3-(4,5-Dimethylthiazol-2-yl)- 2,5-Diphenyltetrazolium Bromide (MTT) assay and apoptosis by propidium iodide staining followed by flow cytometry analysis. Finally, in vivo tumor growth assay was performed by establishing PC-3M xenograft in nude mice and monitoring tumor growth following transfection and treatment. Results We found that survivin was undetectable in normal prostatic tissues but was highly expressed in prostate cancers. Survivin knockdown or selenium treatment inhibited the growth of prostate cancer cells, but the selenium effect was modest. In contrast to what have been observed in other cell lines, selenium treatment had little or no effect on survivin expression in several androgen-independent prostate cancer cell lines. Survivin knockdown sensitized these cells to selenium growth inhibition and apoptosis induction. In nude mice bearing PC-3M xenografts, survivin knockdown synergizes with selenium in inhibiting tumor growth. Conclusions Selenium could inhibit the growth of hormone-refractory prostate cancer cells both in vitro and in vivo, but the effects were modest. The growth inhibition was not mediated by downregulating survivin expression. Survivin silencing greatly enhanced the growth inhibitory effects of selenium. PMID:20698994

2010-01-01

100

Involvement of Mitogen-Activated Protein Kinase Pathway in T-2 Toxin-Induced Cell Cycle Alteration and Apoptosis in Human Neuroblastoma Cells.  

PubMed

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

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

2014-08-01

101

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

PubMed Central

Introduction Rapamycin, an inhibitor of the serine/threonine kinase target of rapamycin, induces G1 arrest and/or apoptosis. Although rapamycin and its analogues are attractive candidates for cancer therapy, their sensitivities with respect to growth inhibition differ markedly among various cancer cells. Using human breast carcinoma cell line MCF-7 as an experimental model system, we examined the growth-inhibitory effects of combinations of various agents and rapamycin to find the agent that most potently enhances the growth-inhibitory effect of rapamycin. Method We evaluated the growth-inhibitory effect of rapamycin plus various agents, including cotylenin A (a novel inducer of differentiation of myeloid leukaemia cells) to MCF-7 cells, using either MTT assay or trypan blue dye exclusion test. The cell cycle was analyzed using propidium iodide-stained nuclei. Expressions of several genes in MCF-7 cells with rapamycin plus cotylenin A were studied using cDNA microarray analysis and RT-PCR. The in vitro results of MCF-7 cells treated with rapamycin plus cotylenin A were further confirmed in vivo in a mouse xenograft model. Results We found that the sensitivity of rapamycin to MCF-7 cells was markedly affected by cotylenin A. This treatment induced growth arrest of the cells at the G1 phase, rather than apoptosis, and induced senescence-associated ?-galactosidase activity. We examined the gene expression profiles associated with exposure to rapamycin and cotylenin A using cDNA microarrays. We found that expressions of cyclin G2, transforming growth factor-?-induced 68 kDa protein, BCL2-interacting killer, and growth factor receptor-bound 7 were markedly induced in MCF-7 cells treated with rapamycin plus cotylenin A. Furthermore, combined treatment with rapamycin and cotylenin A significantly inhibited the growth of MCF-7 cells as xenografts, without apparent adverse effects. Conclusion Rapamycin and cotylenin A cooperatively induced growth arrest in breast carcinoma MCF-7 cells in vitro, and treatment with rapamycin and cotylenin A combined more strongly inhibited the growth of MCF-7 cells as xenografts in vivo than treatment with rapamycin or cotylenin A alone, suggesting that this combination may have therapeutic value in treating breast cancer. We also identified several genes that were markedly modulated in MCF-7 cells treated with rapamycin plus cotylenin A. PMID:16457690

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

2005-01-01

102

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

PubMed Central

The use of a suitable food carrier such as cheese could significantly enhance probiotic viability during storage. The main goal of this study was to assess viability of commercial probiotic strains during Cheddar cheesemaking and ripening (4–6?months) by comparing the efficiency of microbiological and molecular approaches. Molecular methods such as quantitative PCR (qPCR) allow bacterial quantification, and DNA-blocking molecules such as propidium monoazide (PMA) select only the living cells’ DNA. Cheese samples were manufactured with a lactococci starter and with one of three probiotic strains (Bifidobacterium animalis subsp. lactis BB-12, Lactobacillus rhamnosus RO011, or Lactobacillus helveticus RO052) or a mixed culture containing B. animalis subsp. lactis BB-12 and L. helveticus RO052 (MC1), both lactobacilli strains (MC2), or all three strains (MC3). DNA extractions were then carried out on PMA-treated and non-treated cell pellets in order to assess PMA treatment efficiency, followed by quantification using the 16S rRNA gene, the elongation factor Tu gene (tuf) or the transaldolase gene (tal). Results with intact/dead ratios of bacteria showed that PMA-treated cheese samples had a significantly lower bacterial count than non-treated DNA samples (P?

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

2012-01-01

103

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

104

Action Mechanisms of Lithium Chloride on Cell Infection by Transmissible Gastroenteritis Coronavirus  

PubMed Central

Transmissible gastroenteritis virus (TGEV) is a porcine coronavirus. Lithium chloride (LiCl) has been found to be effective against several DNA viruses, such as Herpes simplex virus and vaccinia virus. Recently, we and others have reported the inhibitory effect of LiCl on avian infectious bronchitis coronavirus (IBV) infection, an RNA virus. In the current study, the action mechanism of LiCl on cell infection by TGEV was investigated. Plaque assays and 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenyl tetrazoliumbromide (MTT) assays showed that the cell infection by TGEV was inhibited in a dose-dependent manner, when LiCl was added to virus-infected cells; the cell infection was not affected when either cells or viruses were pretreated with the drug. The inhibition of TGEV infection in vitro by LiCl was observed at different virus doses and with different cell lines. The inhibitory effect of LiCl against TGEV infection and transcription was confirmed by RT-PCR and real-time PCR targeting viral S and 3CL-protease genes. The time-of-addition effect of the drug on TGEV infection indicated that LiCl acted on the initial and late stage of TGEV infection. The production of virus was not detected at 36 h post-infection due to the drug treatment. Moreover, immunofluorescence (IF) and flow cytometry analyses based on staining of Annexin V and propidium iodide staining of nuclei indicated that early and late cell apoptosis induced by TGEV was inhibited efficiently. The ability of LiCl to inhibit apoptosis was investigated by IF analysis of caspase-3 expression. Our data indicate that LiCl inhibits TGEV infection by exerting an anti-apoptotic effect. The inhibitory effect of LiCl was also observed with porcine epidemic diarrhea coronavirus. Together with other reports concerning the inhibitory effect of lithium salts on IBV in cell culture, our results indicate that LiCl may be a potent agent against porcine and avian coronaviruses. PMID:21573100

Ren, Xiaofeng; Meng, Fandan; Yin, Jiechao; Li, Guangxing; Li, Xunliang; Wang, Chao; Herrler, Georg

2011-01-01

105

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

106

Antitumor effect of a copper (II) complex of a coumarin derivative and phenanthroline on lung adenocarcinoma cells and the mechanism of action.  

PubMed

In order to investigate the effect of a copper (II) complexes of a coumarin derivative and phenanthroline (hereinafter referred to as the coumarin-copper drug) on lung adenocarcinoma cells in vivo and in vitro, along with the mechanism of action, LA795 lung adenocarcinoma cells were treated with different concentrations of coumarin-copper drug. An MTT assay was performed to determine the cell proliferation ratio, cell apoptosis was detected by Annexin V/propidium iodide staining with flow cytometric analysis and western blot analysis was employed to evaluate the expression levels of apoptosis-associated proteins. In addition, an LA795 cell xenograft tumor model was established in nude mice, with mice receiving intraperitoneal injection once a week for three weeks of either 2 or 4 mg/kg in three divided doses coumarin?copper drug, or phosphate?buffered saline. The tumor growth curves were drawn and the tumor growth inhibition rates were calculated. The apoptotic index of subcutaneously transplanted tumor cells was determined by terminal deoxynucleotidyl?transferase?mediated dUTP nick end?labeling assay. The coumarin-copper drug effectively inhibited the proliferation of LA795 cells in a dose? and time?dependent manner, with the half maximal inhibitory concentration equaling 2.0 µmol/l. The coumarin-copper drug also significantly induced LA795 cell apoptosis in a time-dependent manner (P<0.05), which was accompanied by upregulation p35 and B-cell lymphoma-2 (Bcl-2)-associated X protein (Bax), and downregulation of Bcl-2. Furthermore, the coumarin?copper drug significantly inhibited the growth of LA795 tumors in a dose dependent manner (P<0.05), in accordance with the apoptotic index. In conclusion, the coumarin-copper drug may inhibit the proliferation of LA795 cells through the induction of cell apoptosis, which may be associated with the upregulation of p53 and Bax, with concurrent downregulation of Bcl-2. PMID:25176185

Zhu, Taofeng; Chen, Ruhua; Yu, Hao; Feng, Yan; Chen, Jianqiang; Lu, Qin; Xie, Jing; Ding, Weiliang; Ma, Tieliang

2014-11-01

107

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.

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

2015-01-01

108

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

PubMed Central

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

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

2014-01-01

109

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

PubMed

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

Peng, Jing; Chen, Xinlian; Hu, Qian; Yang, Mei; Liu, Hongqian; Wei, Wei; Liu, Shanling; Wang, He

2014-11-01

110

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

111

Rapid detection of viable Escherichia coli O157 by coupling propidium monoazide with loop-mediated isothermal amplification.  

PubMed

Conventional molecular detection methods cannot distinguish between viable and dead Escherichia coli O157 cells. In this study, the loop-mediated isothermal amplification (LAMP) method combined with propidium monoazide (PMA) treatment was developed to selectively detect viable E. coli O157 cells. Four primers, including outer primers and inner primers, were specially designed for the recognition of six distinct sequences on the serogroups (O157) of the specific rfbE gene of the E. coli O157 genome. PMA selectively penetrated through the compromised cell membranes and intercalated into DNA. Amplification of DNA from dead cells was completely inhibited by 3.0 ?g/ml PMA, whereas the DNA derived from viable cells was amplified remarkably within 1 h by PMA-LAMP. Exhibiting high sensitivity and specificity, PMA-LAMP is a suitable method for evaluating the inactivation efficacy of slightly acidic electrolyzed water in broth. PMA-LAMP can selectively detect viable E. coli O157 cells. This study offers a novel molecular detection method to distinguish between viable and dead E. coli O157 cells. PMID:24002453

Zhao, Xihong; Wang, Jun; Forghani, Fereidoun; Park, Joong-Hyun; Park, Myoung-Su; Seo, Kun-Ho; Oh, Deog-Hwan

2013-12-01

112

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

Microsoft Academic Search

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

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

1994-01-01

113

Rapid glycation with D-ribose induces globular amyloid-like aggregations of BSA with high cytotoxicity to SH-SY5Y cells  

PubMed Central

Background D-ribose in cells and human serum participates in glycation of proteins resulting in advanced glycation end products (AGEs) that affect cell metabolism and induce cell death. However, the mechanism by which D-ribose-glycated proteins induce cell death is still unclear. Results Here, we incubated D-ribose with bovine serum albumin (BSA) and observed changes in the intensity of fluorescence at 410 nm and 425 nm to monitor the formation of D-ribose-glycated BSA. Comparing glycation of BSA with xylose (a control for furanose), glucose and fructose (controls for pyranose), the rate of glycation with D-ribose was the most rapid. Protein intrinsic fluorescence (335 nm), Nitroblue tetrazolium (NBT) assays and Western blotting with anti-AGEs showed that glycation of BSA incubated with D-ribose occurred faster than for the other reducing sugars. Protein intrinsic fluorescence showed marked conformational changes when BSA was incubated with D-ribose. Importantly, observations with atomic force microscopy showed that D-ribose-glycated BSA appeared in globular polymers. Furthermore, a fluorescent assay with Thioflavin T (ThT) showed a remarkable increase in fluorescence at 485 nm in the presence of D-ribose-glycated BSA. However, ThT fluorescence did not show the same marked increase in the presence of xylose or glucose. This suggests that glycation with D-ribose induced BSA to aggregate into globular amyloid-like deposits. As observed by Hoechst 33258 staining, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cell counting kit-8 (CCK-8) assay, lactate dehydrogenase (LDH) activity assay, flow cytometry using Annexin V and Propidium Iodide staining and reactive oxygen species (ROS) measurements, the amyloid-like aggregation of glycated BSA induced apoptosis in the neurotypic cell line SH-SY5Y. Conclusion Glycation with D-ribose induces BSA to misfold rapidly and form globular amyloid-like aggregations which play an important role in cytotoxicity to neural cells. PMID:19216769

Wei, Yan; Chen, Lan; Chen, Ji; Ge, Lin; He, Rong Qiao

2009-01-01

114

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

Microsoft Academic Search

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

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

2000-01-01

115

Rise of [Ca²?]i and apoptosis induced by M-3M3FBS in SCM1 human gastric cancer cells.  

PubMed

M-3M3FBS (2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide is a presumed phospholipase C activator which induced Ca²? movement and apoptosis in different cell models. How- ever, the effect of m-3M3FBS on cytosolic free Ca²? concentrations ([Ca²?]i) and apoptosis in SCM1 human gastric cancer cells is unclear. This study explored whether m-3M3FBS elevated basal [Ca²?]i levels in suspended cells by using fura-2 as a Ca²?-sensitive fluorescent dye. M-3M3FBS at concentrations between 5-50 ?M increased [Ca²?]i in a concentration-dependent manner. The Ca²? signal was reduced by half by removing extracellular Ca²?. M-3M3FBS-induced Ca²? influx was inhibited by nifedipine, econazole, SK&F96365, aristolochic acid, and GF109203X. In Ca²?-free medium, 50 ?M m-3M3FBS pretreatment inhibited the [Ca²?]i rise induced by the endoplasmic reticulum Ca²? pump inhibitor thapsigargin. Conversely, pretreatment with thapsigargin partly reduced m-3M3FBS-induced [Ca²?]i rise. Suppression of inositol 1,4,5-trisphosphate production with U73122 did not change m-3M3FBS- induced [Ca²?]i rise. At concentrations between 25 and 50 ?M m-3M3FBS killed cells in a concentration- dependent manner. The cytotoxic effect of m-3M3FBS was not reversed by prechelating cytosolic Ca²? with acetoxy-methyl ester of bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA/AM). Annexin V/propidium iodide staining data suggest that m-3M3FBS induced apoptosis at 25 and 50 ?M. M-3M3FBS also increased levels of superoxide. Together, in human gastric cancer cells, m-3M3FBS induced a [Ca²?]i rise by inducing phospholipase C-independent Ca²? release from the endoplasmic reticulum and Ca²? entry via protein kinase C-sensitive store-operated Ca²? channels. M-3M3FBS induced cell death that might involve apoptosis via reactive oxygen species production. PMID:24621336

Chen, Wei-Chuan; Chou, Chiang-Ting; Liou, Wen-Chin; Liu, Shiuh-Inn; Lin, Ko-Long; Lu, Ti; Lu, Yi-Chau; Hsu, Shu-Shong; Tsai, Jeng-Yu; Liao, Wei-Chuan; Liang, Wei-Zhe; Jan, Chung-Ren

2014-02-28

116

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

PubMed

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

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

2012-02-01

117

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

118

Discrimination of infectious hepatitis A viruses by propidium monoazide real-time RT-PCR.  

PubMed

The discrimination of infectious and inactivated viruses remains a key obstacle when using quantitative RT-PCR (RT-qPCR) to quantify enteric viruses. In this study, propidium monoazide (PMA) and RNase pretreatments were evaluated for the detection and quantification of infectious hepatitis A virus (HAV). For thermally inactivated HAV, PMA treatment was more effective than RNase treatment for differentiating infectious and inactivated viruses, with HAV titers reduced by more than 2.4 log(10) units. Results showed that combining 50 ?M of PMA and RT-qPCR selectively quantify infectious HAV in media suspensions. Therefore, PMA treatment previous to RT-qPCR detection is a promising alternative to assess HAV infectivity. PMID:23412764

Sánchez, Gloria; Elizaquível, Patricia; Aznar, Rosa

2012-03-01

119

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

120

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

EPA Science Inventory

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

121

Selective detection of viable Helicobacter pylori using ethidium monoazide or propidium monoazide in combination with real-time polymerase chain reaction.  

PubMed

Because Helicobacter pylori has a role in the pathogenesis of gastric cancer, chronic gastritis and peptic ulcer disease, detection of its viable form is very important. The objective of this study was to optimize a PCR method using ethidium monoazide (EMA) or propidium monoazide (PMA) for selective detection of viable H. pylori cells in mixed samples of viable and dead bacteria. Before conducting the real-time PCR using SodB primers of H. pylori, EMA or PMA was added to suspensions of viable and/or dead H. pylori cells at concentrations between 1 and 100 ?M. PMA at a concentration of 50 ?M induced the highest DNA loss in dead cells with little loss of genomic DNA in viable cells. In addition, selective detection of viable cells in the mixtures of viable and dead cells at various ratios was possible with the combined use of PMA and real-time PCR. In contrast, EMA penetrated the membranes of both viable and dead cells and induced degradation of their genomic DNA. The findings of this study suggest that PMA, but not EMA, can be used effectively to differentiate viable H. pylori from its dead form. PMID:22004535

Nam, Sehee; Kwon, Soonbok; Kim, Min-jeong; Chae, Jong-Chan; Jae Maeng, Pil; Park, Jong-Geun; Lee, Gyu-Cheol

2011-12-01

122

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

123

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

PubMed Central

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

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

2003-01-01

124

Rapid quantification of viable Campylobacter bacteria on chicken carcasses, using real-time PCR and propidium monoazide treatment, as a tool for quantitative risk assessment.  

PubMed

A number of intervention strategies against Campylobacter-contaminated poultry focus on postslaughter reduction of the number of cells, emphasizing the need for rapid and reliable quantitative detection of only viable Campylobacter bacteria. We present a new and rapid quantitative approach to the enumeration of food-borne Campylobacter bacteria that combines real-time quantitative PCR (Q-PCR) with simple propidium monoazide (PMA) sample treatment. In less than 3 h, this method generates a signal from only viable and viable but nonculturable (VBNC) Campylobacter bacteria with an intact membrane. The method's performance was evaluated by assessing the contributions to variability by individual chicken carcass rinse matrices, species of Campylobacter, and differences in efficiency of DNA extraction with differing cell inputs. The method was compared with culture-based enumeration on 50 naturally infected chickens. The cell contents correlated with cycle threshold (C(T)) values (R(2) = 0.993), with a quantification range of 1 x 10(2) to 1 x 10(7) CFU/ml. The correlation between the Campylobacter counts obtained by PMA-PCR and culture on naturally contaminated chickens was high (R(2) = 0.844). The amplification efficiency of the Q-PCR method was not affected by the chicken rinse matrix or by the species of Campylobacter. No Q-PCR signals were obtained from artificially inoculated chicken rinse when PMA sample treatment was applied. In conclusion, this study presents a rapid tool for producing reliable quantitative data on viable Campylobacter bacteria in chicken carcass rinse. The proposed method does not detect DNA from dead Campylobacter bacteria but recognizes the infectious potential of the VBNC state and is thereby able to assess the effect of control strategies and provide trustworthy data for risk assessment. PMID:20562292

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

2010-08-01

125

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

126

Synthesis, characterization, and biological affinity of a near-infrared-emitting conjugated oligoelectrolyte.  

PubMed

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

Thomas, Alexander W; Henson, Zachary B; Du, Jenny; Vandenberg, Carol A; Bazan, Guillermo C

2014-03-12

127

Fibroblasts from Idiopathic Pulmonary Fibrosis and Normal Lungs Differ in Growth Rate, Apoptosis, and Tissue Inhibitor of Metalloproteinases Expression  

Microsoft Academic Search

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disorder characterized by fibroblast proliferation and extracellular ma- trix accumulation. However, studies on fibroblast growth rate and collagen synthesis have given contradictory results. Here we analyzed fibroblast growth rate by a formazan-based chro- mogenic assay; fibroblast apoptosis by in situ end labeling (ISEL) and propidium iodide staining; percent of a -smooth muscle

Carlos Ramos; Martha Montaño; Jorge García-Alvarez; Víctor Ruiz; Bruce D. Uhal; Moises Selman; Annie Pardo

128

Use of propidium monoazide for the enumeration of viable Brettanomyces bruxellensis in wine and beer by quantitative PCR.  

PubMed

Brettanomyces bruxellensis is a current problem in winemaking all over the world, and the question if B. bruxellensis has a positive or negative impact on wine is one of the most controversial discussions in the world. The presence of live B. bruxellensis cells represents the risk of growth and an increase in cell numbers, which is related to the potential production of volatile phenols. In this work, the optimisation of a PMA-quantitative PCR (qPCR) method to enumerate only viable cells was carried out using the standard strain B. bruxellensis DSMZ 70726. The obtained detection limits were 0.83 log CFU/mL in red wine, 0.63 log CFU/mL in white wine and 0.23 log CFU/mL in beer. Moreover, the quantification was also performed by Reverse Transcription quantitative PCR (RT-qPCR), and the results showed a higher detection limit for all of the trials. PMID:24929737

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

2014-09-01

129

Flow Cytometry for Determination of the Efficacy of Contact Lens Disinfecting Solutions against Acanthamoeba spp.  

PubMed Central

Flow cytometric analyses of cellular staining with fluorescent viability dyes and direct microscopic observations of methylene blue exclusion were compared for evaluation of the effects of a chlorhexidine gluconate-based contact lens disinfectant solution and a polyhexamethylene biguanide solution against cysts and trophozoites of Acanthamoeba castellanii and Acanthamoeba polyphaga. The flow cytometric procedure with propidium iodide (used to stain dead cells) indicated that more than 90% of trophozoites of both species (inocula of 105 to 106/ml) at 22°C lost their viability after 4 h of exposure to chlorhexidine. When propidium iodide was used in combination with fluorescein diacetate (for live cells), the apparent number of propidium iodide-stained cells was reduced, but the relative efficacies of the two biguanide solutions appeared unchanged from those evident with the single dyes; the chlorhexidine solution was more effective than the polyhexamethylene biguanide solution. Similar data were obtained with the more cumbersome methylene blue exclusion procedure. Flow cytometric analyses provided a statistically reproducible and rapid procedure for determining the relative antiamoebal efficacies of the disinfecting solutions. PMID:10698771

Borazjani, Roya N.; May, Lauren L.; Noble, Judith A.; Avery, Simon V.; Ahearn, Donald G.

2000-01-01

130

Application of propidium monoazide-qPCR to evaluate the ultrasonic inactivation of Escherichia coli O157:H7 in fresh-cut vegetable wash water.  

PubMed

The efficacy of sanitizing technologies in produce or in vegetable wash water is generally evaluated by plate count in selective media. This procedure is time consuming and can lead to misinterpretations because environmental conditions and sanitizing processes may affect bacterial growth or culturable capability. Thus, the aim of this study was to determine the applicability of a propidium monoazide real-time PCR (PMA-qPCR) method to monitor the inactivation by ultrasound treatment of foodborne bacteria in fresh-cut vegetable wash water. To this aim, lettuce wash water was artificially inoculated with Escherichia coli O157:H7 (10? CFU/mL) and treated by means of a continuous ultrasonic irradiation with a power density of 0.280 kW/L. Quantification data obtained by PMA-qPCR and plate counts were statistically similar during the viability reduction of 99.996% which corresponds to 4.4 log reductions. Further reductions of E. coli O157:H7 were not detected by the PMA-qPCR method due to the limit of detection of this technique (20 CFU/mL). Inactivation data obtained by both techniques successfully fitted a linear model, giving no significant differences in kinetic parameters. These results indicate that the PMA-qPCR method is a suitable technique for evaluating ultrasonic disinfection of vegetable wash water, being able to distinguish between live and dead bacteria. PMID:22265318

Elizaquível, P; Sánchez, G; Selma, M V; Aznar, R

2012-05-01

131

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

132

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

E-print Network

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

Sheridan, Jennifer

133

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

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

135

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

PubMed Central

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

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

2014-01-01

136

Melanoma differentiation associated gene-7 (mda-7): a novel anti-tumor gene for cancer gene therapy.  

PubMed Central

BACKGROUND: The mda-7 gene (melanoma differentiation associated gene-7) is a novel tumor suppressor gene. The anti-proliferative activity of MDA-7 has been previously reported. In this report, we analyze the anti-tumor efficacy of Ad-mda7 in a broad spectrum of cancer lines. MATERIALS AND METHODS: Ad-mda7-transduced cancer or normal cell lines were assayed for cell proliferation (tritiated thymidine incorporation assay, Alamar blue assay, and trypan-blue exclusion assay), apoptosis (TUNEL, and Annexin V staining visualized by fluorescent microscopy or FACs analysis), and cell cycle regulation (Propidium Iodide staining and FACs analysis). RESULTS: Ad-mda7 treatment of tumor cells resulted in growth inhibition and apoptosis in a temporal and dose-dependent manner. The anti-tumor effects were independent of the genomic status of p53, RB, p16, ras, bax, and caspase 3 in these cells. In addition, normal cell lines did not show inhibition of proliferation or apoptotic response to Ad-mda7. Moreover, Ad-mda7-transduced cancer cells secreted a soluble form of MDA-7 protein. Thus, Ad-mda7 may represent a novel gene-therapeutic agent for the treatment of a variety of cancers. CONCLUSIONS: The potent and selective killing activity of Ad-mda7 in cancer cells but not in normal cells makes this vector a potential candidate for cancer gene therapy. PMID:11471572

Mhashilkar, A. M.; Schrock, R. D.; Hindi, M.; Liao, J.; Sieger, K.; Kourouma, F.; Zou-Yang, X. H.; Onishi, E.; Takh, O.; Vedvick, T. S.; Fanger, G.; Stewart, L.; Watson, G. J.; Snary, D.; Fisher, P. B.; Saeki, T.; Roth, J. A.; Ramesh, R.; Chada, S.

2001-01-01

137

Calibrating the imaging and therapy performance of magneto-fluorescent gold nanoshells for breast cancer  

NASA Astrophysics Data System (ADS)

Gold nanoshells with NIR plasmon resonance can be modified to simultaneously enhance conjugated NIR fluorescence dyes and T2 contrast of embedded iron-oxide nanoparticles, and molecularly targeted to breast and other cancers. We calibrated the theranostic performance of magneto-fluorescent nanoshells, and contrasted the performance of molecularly targeted and untargeted nanoshells for breast cancer therapy, employing MCF-7L and their HER2 overexpressing derivative MCF-7/HER2-18 breast cancer cells as in vitro model systems. Silica core gold nanoshells with plasmon resonance on ~810 nm were doped with NIR dye ICG and ~10 nm iron-oxide nanoparticles in a ~20 nm epilayer of silica. A subset of nanoshells was conjugated to antibodies targeting HER2. Cell viability with varying laser power levels in presence and absence of bare and HER2-targeted nanoshells was assessed by calcein and propidium iodide staining. For MCF-7L cells, increasing power resulted in increased cell death (F=5.63, p=0.0018), and bare nanoshells caused more cell death than HER2-targeted nanoshells or laser treatment alone (F=30.13, p<0.001). For MCF-7/HER2-18 cells, death was greater with HER2-targeted nanoshells and was independent of laser power. This study demonstrates the capability of magneto-fluorescent nanocomplexes for imaging and therapy of breast cancer cells, and the advantages of targeting receptors unique to cancer cells.

Dowell, Adam; Chen, Wenxue; Biswal, Nrusingh; Ayala-Orozco, Ciceron; Giuliano, Mario; Schiff, Rachel; Halas, Naomi J.; Joshi, Amit

2012-03-01

138

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

PubMed

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

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

2014-03-15

139

Method to detect only viable cells in microbial ecology  

Microsoft Academic Search

Propidium monoazide can limit the analysis of microbial communities derived from genetic fingerprints to viable cells with\\u000a intact cell membranes. However, PMA treatment cannot completely suppress polymerase chain reaction (PCR) amplification when\\u000a the targeted gene is too short. PMA treatment in combination with two-step nested PCR was designed to overcome this problem.\\u000a Four experiments were performed to determine the limitation

Jian-Fei Luo; Wei-Tie Lin; Yong Guo

2010-01-01

140

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

141

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

PubMed Central

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

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

2008-01-01

142

Enhanced gene disruption by programmable nucleases delivered by a minicircle vector.  

PubMed

Targeted genetic modification using programmable nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) is of great value in biomedical research, medicine and biotechnology. Minicircle vectors, which lack extraneous bacterial sequences, have several advantages over conventional plasmids for transgene delivery. Here, for the first time, we delivered programmable nucleases into human cells using transient transfection of a minicircle vector and compared the results with those obtained using a conventional plasmid. Surrogate reporter assays and T7 endonuclease analyses revealed that cells in the minicircle vector group displayed significantly higher mutation frequencies at the target sites than those in the conventional plasmid group. Quantitative PCR and reverse transcription-PCR showed higher vector copy number and programmable nuclease transcript levels, respectively, in 293T cells after minicircle versus conventional plasmid vector transfection. In addition, tryphan blue staining and flow cytometry after annexin V and propidium iodide staining showed that cell viability was also significantly higher in the minicircle group than in the conventional plasmid group. Taken together, our results show that gene disruption using minicircle vector-mediated delivery of ZFNs and TALENs is a more efficient, safer and less toxic method than using a conventional plasmid, and indicate that the minicircle vector could serve as an advanced delivery method for programmable nucleases. PMID:25142139

Dad, A-B K; Ramakrishna, S; Song, M; Kim, H

2014-11-01

143

Frequency of M-Cadherin-stained Satellite Cells Declines in Human Muscles During Aging  

Microsoft Academic Search

To answer the question of whether the satellite cell pool in human muscle is reduced during aging, we detected satellite cells in 30-?m-thick transverse sections under the confocal microscope by binding of M-cadherin antibody. The basal lamina was detected with laminin. Nuclei were stained with bisbenzimide or propidium iodide. Satellite cells were counted by applying the disector method and unbiased

Špela Sajko; Lucie Kubínová; Erika Cvetko; Marko Kreft; Anton Wernig; Ida Eržen

2004-01-01

144

Flow cytometric analysis of the DNA content in cultured human brain tumor cells  

Microsoft Academic Search

Summary  Analysis of DNA content in cultured cells derived from 5 benign and 8 malignant human brain tumors was performed by flow cytometry,\\u000a using propidium iodide as fluorochrome. Normal, non-stimulated human lymphocytes were used as controls. Cells harvested from\\u000a the first confluent subculture had DNA distribution histograms similar to those of a replicating, non-synchronous population\\u000a of diploid cells. This observation was

Keiji Kawamoto; Fritz Herz; Robert C. Wolley; Asao Hirano; Leopold G. Koss

1980-01-01

145

Bufalin exerts inhibitory effects on IL-1?-mediated proliferation and induces apoptosis in human rheumatoid arthritis fibroblast-like synoviocytes.  

PubMed

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

Chang, Yue-wen; Zhao, Yong-fang; Cao, Yue-long; Gu, Wei; Pang, Jian; Zhan, Hong-sheng

2014-10-01

146

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

147

Flow cytometric quantification of proliferating coelomocytes non-invasively retrieved from the earthworm, Dendrobaena veneta.  

PubMed

Earthworms irritated naturally (e.g. by predators) or experimentally extrude coelomocyte-containing coelomic fluid through the dorsal pores of the body wall. In the present study, the earthworms, Dendrobaena veneta, experimentally depleted of free-floating coelomocytes by multiple electric shocks (1 min, 4.5 V) remained fully vital and coelomocyte depletion was followed by the extensive cell replenishment, which was more efficient in the case of amoebocytes than autofluorescent eleocytes/chloragocytes, quantified by flow cytometry. Immunohistochemical procedure with antibodies against human Ki-67 proliferation antigens revealed proliferating cells on cytospin preparations of coelomocytes extruded by electric shock. Quantification of proliferating cells in the suspension of extruded coelomocytes was performed by flow cytometry on FL-2 profiles of propidium iodide-stained samples; riboflavin-derived autofluorescence of eleocytes/chloragocytes was lost during detergent treatment. As expected, the percentage of coelomocytes proliferating in coelomic fluid was increased during restoration of coelomocyte number after experimental depletion. The method described here may be very useful for investigations of antigen-driven proliferation of earthworm coelomocytes. PMID:17544121

Homa, Joanna; Bzowska, Malgorzata; Klimek, Malgorzata; Plytycz, Barbara

2008-01-01

148

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

149

Proteasome inhibition reverses hedgehog inhibitor and taxane resistance in ovarian cancer.  

PubMed

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

Steg, Adam D; Burke, Mata R; Amm, Hope M; Katre, Ashwini A; Dobbin, Zachary C; Jeong, Dae Hoon; Landen, Charles N

2014-08-30

150

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

PubMed

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

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

2014-02-28

151

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

152

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

153

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.

2014-02-01

154

The role of reactive oxygen species and hemeoxygenase-1 expression in the cytotoxicity, cell cycle alteration and apoptosis of dental pulp cells induced by BisGMA  

Microsoft Academic Search

Biocompatibility of dentin bonding agents (DBAs) and resin composite is important to preserve the pulp vitality after operative restoration. Bisphenol-glycidyl-methacrylate (BisGMA) is one common monomer adding into DBAs and resin. In this study, we found that exposure of human dental pulp cells to BisGMA (>0.1 mm) led to cytotoxicity, G2\\/M cell cycle arrest and apoptosis as analyzed by propidium iodide (PI)

Mei-Chi Chang; Lin-I. Chen; Chiu-Po Chan; Jang-Jaer Lee; Tong-Mei Wang; Ting-Ting Yang; Po-Shuen Lin; Hsueh-Jen Lin; Hsiao-Hua Chang; Jiiang-Huei Jeng

2010-01-01

155

A Peptide That Binds Specifically to the ?-Amyloid of Alzheimer's Disease: Selection and Assessment of Anti-?-Amyloid Neurotoxic Effects  

PubMed Central

The accumulation of the amyloid-? peptide (A?) into amyloid plaques, an essential event in Alzheimer's disease (AD) pathogenesis, has caused researchers to seek compounds that physiologically bind A? and modulate its aggregation and neurotoxicity. In order to develop new A?-specific peptides for AD, a randomized 12-mer peptide library with A?1-10 as the target was used to identify peptides in the present study. After three rounds of selection, specific phages were screened, and their binding affinities to A?1-10 were found to be highly specific. Finally, a special peptide was synthesized according to the sequences of the selected phages. In addition, the effects of the special peptide on A? aggregation and A?-mediated neurotoxicity in vitro and in vivo were assessed. The results show that the special peptide not only inhibited the aggregation of A? into plaques, but it also alleviated A?-induced PC12 cell viability and apoptosis at appropriate concentrations as assessed by the cell counting kit-8 assay and propidium iodide staining. Moreover, the special peptide exhibited a protective effect against A?-induced learning and memory deficits in rats, as determined by the Morris water maze task. In conclusion, we selected a peptide that specifically binds A?1-10 and can modulate A? aggregation and A?-induced neuronal damage. This opens up possibilities for the development of a novel therapeutic approach for the treatment of AD. PMID:22102917

Wang, Fang; Zhou, Xian-Ling; Yang, Qi-Gang; Xu, Wen-Hua; Wang, Fei; Chen, Yong-Ping; Chen, Gui-Hai

2011-01-01

156

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

157

Lithium chloride promotes host resistance against Pseudomonas aeruginosa keratitis  

PubMed Central

Purpose To explore the role of lithium chloride (LiCl) in Pseudomonas aeruginosa (PA) keratitis. Methods B6 mice were subconjunctivally injected with LiCl in contrast to appropriate control sodium chloride (NaCl), and then routinely infected with PA. Clinical score, slit-lamp photography, hematoxylin and eosin (H&E) staining, and bacterial plate counts were used to determine the role of LiCl in PA keratitis. Messenger ribonucleic acid and protein levels of inflammatory cytokines in PA-challenged mouse corneas and in vitro cultured macrophages and neutrophils were measured with real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. Apoptosis of the infiltrating inflammatory cells in the PA-infected murine corneas was assessed using terminal deoxynucleotidyl transferase-mediated uridine 5?-triphosphate-biotin nick end labeling staining and propidium iodide staining associated with flow cytometry. In cultured murine macrophages and neutrophils, cell apoptosis was determined with annexin V/propidium iodide double staining associated with flow cytometry and western blot analysis for cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase. Results Treatment with LiCl reduced the severity of corneal disease by reducing corneal inflammatory response and bacterial burden. Moreover, LiCl increased anti-inflammatory cytokine interleukin-10 levels, decreased proinflammatory cytokine tumor necrosis factor-? levels, and enhanced apoptosis of infiltrating macrophages and neutrophils in the PA-infected mouse corneas. In vitro studies further confirmed that LiCl elevated anti-inflammatory cytokine expression but reduced proinflammatory cytokine production, as well as promoted cell apoptosis in murine macrophages and neutrophils. Conclusions This study demonstrates a protective role of LiCl in PA keratitis. LiCl promotes host resistance against PA infection by suppressing inflammatory responses, enhancing inflammatory cell apoptosis, and promoting bacterial clearance. PMID:23878501

Chen, Kang; Wu, Yongjian; Zhu, Min; Deng, Qiuchan; Nie, Xinxin; Li, Meiyu; Wu, Minhao

2013-01-01

158

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

PubMed

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

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

2014-02-01

159

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

PubMed

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

Chai, Qiuxia; Li, Benchang; Xu, Ziqin

2014-03-01

160

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

161

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

PubMed Central

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

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

2001-01-01

162

Somatostatin and opioid receptors do not regulate proliferation or apoptosis of the human multiple myeloma U266 cells  

Microsoft Academic Search

BACKGROUND: opioid and somatostatin receptors (SSTRs) that can assemble as heterodimer were individually reported to modulate malignant cell proliferation and to favour apoptosis. Materials and methods: SSTRs and opioid receptors expression were examined by RT-PCR, western-blot and binding assays, cell proliferation was studied by XTT assay and propidium iodide (PI) staining and apoptosis by annexin V-PI labelling. RESULTS: almost all

Céline Kerros; Thibault Cavey; Brigitte Sola; Philippe Jauzac; Stéphane Allouche

2009-01-01

163

Polymerase chain reaction amplification length-dependent ethidium monoazide suppression power for heat-killed cells of Enterobacteriaceae  

Microsoft Academic Search

The polymerase chain reaction (PCR) can confirm the presence of bacteria, but it is unable to differentiate between live and dead bacteria. Although ethidium monoazide (EMA)- and propidium monoazide (PMA)-based PCR have been evaluated, a quantity of ?103cells\\/ml dead cells produces a false-positive reading at 40 to 50cycles (K. Rudi et al., Appl. Environ. Microbiol. 71 (2005) 1018–1024). After confirming

Takashi Soejima; Frank Schlitt-Dittrich; Shin-ichi Yoshida

2011-01-01

164

Flow Cytometry Approach Study of Enterococcus faecalis Vancomycin Resistance by Detection of Vancomycin@FL Binding to the Bacterial Cells  

Microsoft Academic Search

We studied the usefulness of flow cytometry for detection of vancomycin resistance in Enterococcus faecalis by direct binding of commercially available fluorescent vancomycin to cells obtained from culture. The cells were stained\\u000a with Vancomycin@FL, sonicated and additionally stained with propidium iodide (PI). Regarding to inductive mechanism of vanA-mediated vancomycin resistance, resistant reference strain was also pre-incubated with vancomycin. PI staining

Tomasz JarzembowskiAgnieszka Jozwik; Agnieszka Jó?wik; Katarzyna Wi?niewska; Jacek Witkowski

2010-01-01

165

Cytotoxic Effects of Loperamide Hydrochloride on Canine Cancer Cells  

PubMed Central

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

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

2014-01-01

166

Confocal microscopy and 3-D distribution of dead cells in cryopreserved pancreatic islets  

Microsoft Academic Search

Our laboratory is involved in studies of changes in shape and size of biological specimens under osmotic stress at ambient and sub-zero temperatures. This paper describes confocal microscopy, image processing and analysis of 3-D distribution of cells in acridine orange\\/propidium iodide (AO\\/PI) fluorescent stained frozen-thawed islet of Langerhans. Isolated and cultured rat pancreatic islets were frozen and thawed in 2

Fatima A. Merchant; Shanti J. Aggarwal; Kenneth R. Diller; Keith A. Bartels; Alan C. Bovik

1992-01-01

167

Combination of bladder cancer-specific oncolytic adenovirus gene therapy with cisplatin on bladder cancer in vitro.  

PubMed

Bladder cancer-specific oncolytic adenovirus Ad/PSCAE/UPII/E1A, carrying E1A gene regulated by human Uroplakin II (UPII) promoter and prostate stem cell antigen enhancer (PSCAE), could kill bladder tumor cells preferentially. The aim of this study was to examine the effects of Ad/PSCAE/UPII/E1A combined with cisplatin on human bladder cancer cells and to identify the underlying mechanisms. The combined effects of Ad/PSCAE/UPII/E1A and cisplatin on EJ, 5637, and BIU-87 bladder cancer cells were evaluated by MTT cell proliferation assay. Cell apoptosis was detected by flow cytometry with fluorescein isothiocyanate-conjugated annexin V (annexin V-FITC) and propidium iodide staining. The activation of the caspase pathway and the expression of Bcl-2 family proteins were determined by western blot assay. Ad/PSCAE/UPII/E1A adenovirus vector could infect bladder cancer cell lines selectively and induce growth inhibition effectively. Of note, the combination treatment of cisplatin and Ad/PSCAE/UPII/E1A could inhibit the proliferation of bladder cancer cells significantly compared with the "alone" treatment. Furthermore, Ad/PSCAE/UPII/E1A plus cisplatin combined treatment resulted in enhanced apoptosis in bladder cancer cells. The enhanced antitumor effects in vitro elicited by Ad/PSCAE/UPII/E1A plus cisplatin were closely related to the increased Fas expression and cleavage of caspase-8 and Bid and decrease in the ratio of anti- to pro-apoptotic proteins followed by activation of caspase-9 and caspase-3, which may contribute to the activation of extrinsic and intrinsic apoptotic pathways. Our results indicate that the combination of Ad/PSCAE/UPII/E1A with cisplatin exerts a synergistic antitumor effect on human bladder cancer cells and is a potential combined treatment strategy for bladder cancer. PMID:25085582

Wang, Li; Zhang, Yunxin; Zhao, Jinxia; Xiao, Erlong; Lu, Jianzhong; Fu, Shengjun; Wang, Zhiping

2014-11-01

168

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

PubMed Central

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

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

2006-01-01

169

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

170

Enhanced egress of intracellular Eimeria tenella sporozoites by splenic lymphocytes from coccidian-infected chickens.  

PubMed

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

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

2011-08-01

171

Enhanced Egress of Intracellular Eimeria tenella Sporozoites by Splenic Lymphocytes from Coccidian-Infected Chickens?  

PubMed Central

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

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

2011-01-01

172

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

PubMed

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

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

2012-09-01

173

Short Communication: Apoptosis Pathways in HIV-1-Infected Patients Before and After Highly Active Antiretroviral Therapy: Relevance to Immune Recovery.  

PubMed

Investigations into apoptotic pathways, intrinsic and extrinsic, and the effects of highly active antiretroviral therapy (HAART) on T cell death via those pathways may provide insight into the mechanisms of and barriers to immune recovery. HIV-1-infected patients were enrolled into a randomized, controlled study of the immune effects of a lopinavir/ritonavir (LPV/r)-based versus an efavirenz (EFV)-based HAART regimen in antiretroviral-naive subjects with CD4(+) counts <350 cells/mm(3). Patients were randomized to receive TDF/FTC/EFZ or TDF/FTC plus LPV/r. Fourteen patients were enrolled and 10 patients completed 6 months of therapy as per the protocol. CD4(+) counts were measured before and during HAART therapy. We isolated T cell subsets to measure ex vivo apoptosis by propidium iodide staining. We also assessed caspase activation for the intrinsic and extrinsic pathways of apoptosis, as well as effector caspase activation. We also measured mitochondrial membrane potential. Cells were analyzed by flow cytometry. All patients had increased activation of caspase 8 (extrinsic pathway), caspase 9 (intrinsic pathway), effector caspases 3/7, and low mitochondrial membrane potential at baseline compared to controls. By 4 weeks, there was a decrease in activation of all caspases, but little further decrease by week 24. T cell mitochondrial membrane potential did not increase until week 12, but continued to increase until week 24. The only predictor of CD4(+) count increase was the increase in mitochondrial membrane potential of naive cells at 6 months (r=0.66, p=0.038). This suggests that positive selection of naive CD4(+) T cells in the thymus is the major determinant of CD4(+) recovery. PMID:25386736

Pitrak, David L; Novak, Richard M; Estes, Randee; Tschampa, Jean; Abaya, Christina D; Martinson, Jeffrey; Bradley, Kirsten; Tenorio, Allan R; Landay, Alan L

2015-02-01

174

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

175

Cells  

NSDL National Science Digital Library

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

Mcnees, Mrs.

2010-09-28

176

Structure-activity relationship of trifluoromethyl-containing metallocenes: electrochemistry, lipophilicity, cytotoxicity, and ROS production.  

PubMed

We report the synthesis of trifluoromethylated metallocenes (M=Fe, Ru) and related metal-free compounds for comparison of their biological properties with the aim to establish structure-activity relationships toward the anti-proliferative activity of this compound class. All new compounds were comprehensively characterized by NMR spectroscopy ((1) H, (13) C, (19) F), mass spectrometry, IR spectroscopy, and elemental analysis. A single-crystal X-ray structure was obtained on the Ru derivative, 1-(1-hydroxy-1-hexafluoromethylethyl)ruthenocene (3). The cytotoxicity of all compounds was tested on MCF-7, HT-29, and PT-45 cells, and IC50 values as low as 12 ?M were observed. Both the metallocene moiety and the hydroxy function are crucial for cytotoxicity. In addition, the activity decreased sharply even if only one trifluoromethyl group was replaced with a methyl group. Electrochemical investigations by cyclic voltammetry revealed that all CF3 -containing compounds are harder to oxidize than the unsubstituted metallocenes. Moreover, log?P determination by RP-HPLC showed the fluorinated derivatives to have higher lipophilicity, with log?P values up to 4.6. At the same time, the generation of reactive oxygen species (ROS) in Jurkat cells by these compounds was investigated by flow cytometry. Strong ROS production was shown exclusively for the bis-CF3 derivative 1-(1-hydroxy-1-hexafluoromethylethyl)ferrocene (1) after 6 and 24 h. Also on the Jurkat cell line, only compound 1 strongly induces necrosis after 24 and 48 h, as shown by annexin V/propidium iodide staining. No induction of apoptosis was observed. We propose that compound 1 is more efficiently incorporated into cancer cells relative to all other derivatives, causing significant induction of oxidative stress within the cell, which ultimately leads to cell death. PMID:24838930

Maschke, Marcus; Alborzinia, Hamed; Lieb, Max; Wölfl, Stefan; Metzler-Nolte, Nils

2014-06-01

177

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

178

Isolation of a glucosamine binding leguminous lectin with mitogenic activity towards splenocytes and anti-proliferative activity towards tumor cells.  

PubMed

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 IC(50) 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

179

Protective effect of hydrogen-rich saline against radiation-induced immune dysfunction.  

PubMed

Recent studies showed that hydrogen can be used as an effective radioprotective agent through scavenging free radicals. This study was undertaken to evaluate the radioprotective effects of hydrogen on immune system in mice. H(2) was dissolved in physiological saline using an apparatus produced by our department. Spleen index and histological analysis were used to evaluate the splenic structural damage. Spleen superoxide dismutase, GSH, MDA were measured to appraise the antioxidant capacity and a DCF assay for the measurement of radical oxygen species. Cell apoptosis was evaluated by an Annexin V-FITC and propidium iodide staining method as well as the apoptotic proteins such as Bcl-2, Bax, caspase-3 and c-caspase-3. CD4+ and CD8+ T cells subtypes were detected by flow cytometry with FITC-labelled antimouse CD4 and PE antimouse CD8 staining. Real-time PCR was utilized to determine the CD4+ T cell subtypes and related cytokines. Our study demonstrated that pre-treatment with H(2) could increase the spleen index and attenuate the radiation damage on splenic structure. Radical oxygen species level was also reduced by H(2) treatment. H(2) also inhibited radiation-induced apoptosis in splenocytes and down-regulated pro-apoptotic proteins in living mice. Radiation-induced imbalance of T cells was attenuated by H(2). Finally, we found that H(2) could regulate the polarization of CD4+ T cells and the level of related cytokines. This study suggests H(2) as an effective radioprotective agent on immune system by scavenging reactive oxygen species. PMID:24618260

Zhao, Sanhu; Yang, Yanyong; Liu, Wen; Xuan, Zhiqiang; Wu, Shouming; Yu, Shunfei; Mei, Ke; Huang, Yijuan; Zhang, Pei; Cai, Jianming; Ni, Jin; Zhao, Yaoxian

2014-05-01

180

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

181

Green tea (Camellia sinensis) extract inhibits both the metastasis and osteolytic components of mammary cancer 4T1 lesions in mice.  

PubMed

Green tea (Camellia sinensis, CS), a kind of Chinese tea commonly consumed as a healthy beverage, has been demonstrated to have various biological activities, including antioxidation, antiobesity and anticancer. Our study aims to investigate the antitumor, antimetastasis and antiosteolytic effects of CS aqueous extract both in vitro and in vivo using metastasis-specific mouse mammary carcinoma 4T1 cells. Our results showed that treatment of 4T1 cells with CS aqueous extract resulted in significant inhibition of 4T1 cell proliferation. CS extract induced 4T1 apoptosis in a dose-dependent manner as assessed by annexin-V and propidium iodide staining and caspase-3 activity. Western blot analysis showed that CS increased the expression of Bax-to-Bcl-2 ratio and activated caspase-8 and caspase-3 to induce apoptosis. CS also inhibited 4T1 cell migration and invasion at 0.06-0.125 mg/ml. In addition, CS extract (0.6 g/kg, orally fed daily for 4 weeks) was effective in decreasing the tumor weight by 34.8% in female BALB/c mice against water treatment control (100%). Apart from the antitumor effect, CS extract significantly decreased lung and liver metastasis in BALB/c mice bearing 4T1 tumors by 54.5% and 72.6%, respectively. Furthermore, micro-computed tomography and in vitro osteoclast staining analysis suggested that CS extract was effective in bone protection against breast cancer-induced bone destruction. In conclusion, the present study demonstrated that the CS aqueous extract, which closely mimics green tea beverage, has potent antitumor and antimetastasis effects in breast cancer and could protect the bone from breast cancer-induced bone destruction. PMID:24561153

Luo, Ke-Wang; Ko, Chun-Hay; Yue, Grace Gar-Lee; Lee, Julia Kin-Ming; Li, Kai-Kai; Lee, Michelle; Li, Gang; Fung, Kwok-Pui; Leung, Ping-Chung; Lau, Clara Bik-San

2014-04-01

182

Strong anticancer potential of nano-triterpenoid from Phytolacca decandra against A549 adenocarcinoma via a Ca(2+)-dependent mitochondrial apoptotic pathway.  

PubMed

We isolated a triterpenoid from an ethanolic extract of Phytolacca decandra and nanoencapsulated it with biodegradable nontoxic polymers of poly(lactide-co-glycolide) to examine if the nanoform of this hitherto unexplored betulinic-acid derivative (NdBA) could produce a stronger anticancer effect by rendering better drug bioavailability and targeted delivery than the nonencapsulated betulinic-acid derivative (dBA). The nanoparticles were characterized with the help of physicochemical and morphological studies involving dynamic light scattering and atomic force microscopy. A549 cancer cells were exposed to NdBA and dBA at the IC50 doses of 50 ?g/mL and 100 ?g/mL, respectively. Mitochondrial dysfunction-mediated apoptosis was determined by examining the changes in the intracellular calcium content, the reactive oxygen species accumulation, the cytochrome c release, the upregulation of Bcl-2-associated-X protein (Bax) and caspase 3, the downregulation of B cell lymphoma 2, and the mitochondrial membrane potential (??m) depolarization. Apoptosis was also verified by acridine orange staining observed under fluorescence microscopy and annexin V-fluorescein isothiocyanate/propidium iodide staining through flow cytometric studies. The levels of intracellular adenosine triphosphate/adenosine diphosphate ratio decreased, and the ATPase activity increased more strikingly in A549 cells exposed to NdBA than in A549 cells exposed to dBA. Overall results showed that both drugs directly target the mitochondrial oxidative phosphorylation system, with NdBA having a stronger effect, indicating NdBA to be a better candidate for the development of an anticancer drug for use against lung adenocarcinomas. PMID:24929458

Das, Jayeeta; Das, Sreemanti; Paul, Avijit; Samadder, Asmita; Khuda-Bukhsh, Anisur Rahman

2014-06-01

183

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

PubMed Central

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

2013-01-01

184

A pan caspase inhibitor decreases caspase-1, IL-1? and IL-1?, and protects against necrosis of cisplatin-treated freshly isolated proximal tubules.  

PubMed

Abstract Caspase-1, IL-1?, and IL-1? are known to be activated in the NLRP3 inflammasome. The inflammasome is activated mostly in inflammatory cells. The presence of inflammasome proteins in proximal tubules (PTs) and the effect of cisplatin-treatment or caspase inhibition on inflammasome proteins in PTs are not known. The aim of this study was to investigate the effect of cisplatin on inflammasome proteins in freshly isolated PTs and also to determine the effect of caspase inhibition on inflammasome proteins and PT injury. PTs were isolated using collagenase digestion and Percoll centrifugation. After recovery period, freshly isolated PTs were incubated with vehicle, 50?µM cisplatin or 50?µM cisplatin plus 50?µM pan caspase inhibitor, QVD-OPH. PTs treated with 50?µM cisplatin showed Propidium Iodide staining indicative of necrosis. Necrotic cells (%) were 2.2 in Vehicle-treated, 37.7 in Cisplatin-treated (p?cell membrane damage seen in necrosis was 7.1 in Vehicle-treated, 39.7 in Cisplatin-treated (p?

Lee, Dong Won; Faubel, Sarah; Edelstein, Charles L

2015-02-01

185

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

PubMed

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

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

2009-07-01

186

The Calpain Inhibitor MDL28170 Induces the Expression of Apoptotic Markers in Leishmania amazonensis Promastigotes  

PubMed Central

Background Human cutaneous leishmaniasis is caused by distinct species, including Leishmania amazonensis. Treatment of cutaneous leishmaniasis is far from satisfactory due to increases in drug resistance and relapses, and toxicity of compounds to the host. As a consequence for this situation, the development of new leishmanicidal drugs and the search of new targets in the parasite biology are important goals. Methodology/Principal Findings In this study, we investigated the mechanism of death pathway induced by the calpain inhibitor MDL28170 on Leishmania amazonensis promastigote forms. The combined use of different techniques was applied to contemplate this goal. MDL28170 treatment with IC50 (15 µM) and two times the IC50 doses induced loss of parasite viability, as verified by resazurin assay, as well as depolarization of the mitochondrial membrane, which was quantified by JC-1 staining. Scanning and transmission electron microscopic images revealed drastic alterations on the parasite morphology, some of them resembling apoptotic-like death, including cell shrinking, surface membrane blebs and altered chromatin condensation pattern. The lipid rearrangement of the plasma membrane was detected by Annexin-V labeling. The inhibitor also induced a significant increase in the proportion of cells in the sub-G0/G1 phase, as quantified by propidium iodide staining, as well as genomic DNA fragmentation, detected by TUNEL assay. In cells treated with MDL28170 at two times the IC50 dose, it was also possible to observe an oligonucleossomal DNA fragmentation by agarose gel electrophoresis. Conclusions/Significance The data presented in the current study suggest that MDL28170 induces apoptotic marker expression in promastigotes of L. amazonensis. Altogether, the results described in the present work not only provide a rationale for further exploration of the mechanism of action of calpain inhibitors against trypanosomatids, but may also widen the investigation of the potential clinical utility of calpain inhibitors in the chemotherapy of leishmaniases. PMID:24498160

Marinho, Fernanda A.; Gonçalves, Keyla C. S.; Oliveira, Simone S. C.; Gonçalves, Diego S.; Matteoli, Filipe P.; Seabra, Sergio H.; Oliveira, Ana Carolina S.; Bellio, Maria; Oliveira, Selma S.; Souto-Padrón, Thaïs; d'Avila-Levy, Claudia M.; Santos, André L. S.; Branquinha, Marta H.

2014-01-01

187

A flow cytometric method for viability assessment of Staphylococcus aureus and Burkholderia cepacia in mixed culture.  

PubMed

Mixed bacterial communities are commonly encountered in microbial infections of humans. Knowledge on the composition of species and viability of each species in these communities allows for a detailed description of the complexity of interspecies dynamics and contributes to the assessment of the severity of infections. Several assays exist for quantification of specific species in mixed communities, including analysis of quantitative terminal restriction fragment length polymorphisms. While this method allows for species-specific cell enumeration, it cannot provide viability data. In this study, flow cytometry was applied to assess the viability of Staphylococcus aureus and Burkholderia cepacia in mixed culture by membrane integrity analysis using SYBR® Green I and propidium iodide staining. Both bacteria are relevant to pulmonary infections of cystic fibrosis patients. Fluorescence staining was optimized separately for each species in pure culture due to differences between species in cell wall structure and metabolic capabilities. To determine viability of species in mixed culture, a protocol was established as a compromise between optimum conditions determined before for pure cultures. This protocol allowed the detection of viable and dead cells of both species, exhibiting an intact and a permeabilized membrane, respectively. To discriminate between S. aureus and B. cepacia, the protocol was combined with Gram-specific fluorescent staining using wheat germ agglutinin. The established three-color staining method was successfully tested for viability determination of S. aureus and B. cepacia in mixed culture cultivations. In addition, growth of both species was monitored by quantitative terminal restriction fragment length polymorphisms. The obtained data revealed alterations in viability during cultivations for different growth phases and suggest interspecies effects in mixed culture. Overall, this method allows for rapid simultaneous Gram-differentiation and viability assessment of bacterial mixed cultures and is therefore suitable for the analysis of dynamics of mixed communities of medical, environmental, and biotechnological relevance. PMID:23081865

Rüger, Marc; Bensch, Gerald; Tüngler, Ralf; Reichl, Udo

2012-12-01

188

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

189

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

PubMed Central

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

2013-01-01

190

Protection of ginsenoside Rg1 on chondrocyte from IL-1?-induced mitochondria-activated apoptosis through PI3K/Akt signaling.  

PubMed

Chondrocyte apoptosis is closely related to the development and progression of osteoarthritis. Ginsenoside Rg1 protects cells by antagonizing apoptosis. This study aimed to investigate the protective effect of Rg1 on interleukin 1? (IL-1?)-induced chondrocyte apoptosis and the underlying molecular mechanisms. Chondrocytes were harvested from the joints of 1-week-old Sprague-Dawley rats. After treated with 10 ?g/mL Rg1 for 2 h, the chondrocytes were cultured with 10 ng/mL IL-1? to induce cytotoxicity. Cell viability was assessed with MTT assays. Annexin V/propidium iodide staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling were used to detect chondrocyte apoptosis. The contents of total Akt, phosphorylated Akt (p-Akt), Bcl-2, Bax, and cytochrome C (Cyt c) were determined by Western blotting assay. A quantitative colorimetric assay was used to determine caspase-3 activity. Our present findings have shown that pre-treatment of chondrocytes with Rg1 reduces IL-1? induced cytotoxicity/apoptosis. Rg1 pretreatment also decreases the activity of IL-1? that reduces expression of Bcl-2 and level of p-Akt, and increases Bax activity, Cyt c release, and caspase-3 activation. It also reverses the activity of IL-1? that reduces the expression of tissue inhibitor of metalloproteinase-1 expression and increased the synthesis of matrix metalloproteinase-13, with the net effect of inhibiting extracellular matrix degradation. These results indicate that Rg1 may protect chondrocytes from IL-1?-induced apoptosis via the phosphatidylinositol 3-kinase/protein kinase B signaling pathway, through preventing caspase-3 release. PMID:24671491

Huang, Yumin; Wu, Dongying; Fan, Weimin

2014-07-01

191

Overcoming multidrug resistance in 2D and 3D culture models by controlled drug chitosan-graft poly(caprolactone)-based nanoparticles.  

PubMed

The principal limitations of chemotherapy are dose-limiting systemic toxicity and the development of multidrug-resistant phenotypes. The aim of this study was to investigate the efficiency of a new sustained drug delivery system based on chitosan and ?-caprolactone to overcome multidrug resistance in monolayer and drug resistance associated with the three-dimensional (3D) tumor microenvironment in our established 3D models. The 5-fluorouracil (5-FU)-loaded nanoparticles (NPs) were characterized by transmission electron microscope and dynamic light scattering, and its released property was determined at different pH values. 5-FU/NPs exhibited well-sustained release properties and markedly enhanced the cytotoxicity of 5-FU against HCT116/L-OHP or HCT8/VCR MDR cells in two-dimensional (2D) and its parental cells in 3D collagen gel culture with twofold to threefold decrease in the IC50 values, as demonstrated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Hoechst/propidium iodide staining and flow cytometry analysis. Furthermore, the possible mechanism was explored by high-performance liquid chromatography and rhodamine 123 accumulation experiment. Overall, the results demonstrated that 5-FU/NPs increase intracellular concentration of 5-FU and enhance its anticancer efficiency by inducing apoptosis. It was suggested that this novel NPs are a promising carrier to decrease toxic of 5-FU and has the potential to reverse the forms of both intrinsic and acquired drug resistance in 2D and 3D cultures. PMID:24523221

Shi, Wei-Bin; Le, Van-Minh; Gu, Chun-Hua; Zheng, Yuan-Hong; Lang, Mei-Dong; Lu, Yan-Hua; Liu, Jian-Wen

2014-04-01

192

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

193

Cytochalasin B inhibits the proliferation of human glioma U251 cells through cell cycle arrest and apoptosis.  

PubMed

Cytochalasin B (CB) is known to inhibit a number of cancer types, but its effects on gliomas are unknown. We examined the in vitro effects of CB on the proliferation of human glioma U251 cells, as well as determined its mechanism of action. Cell proliferation was determined using CCK-8. The effect of CB on U251 cell morphology was observed under a transmission electron microscope. Cell cycle distribution was assessed using propidium iodine and Giemsa staining, and cell apoptosis was determined by annexin V-fluorescein isothiocyanate/propidium iodide. Cell cycle-related proteins were determined by Western blot. CB effectively inhibited U251 cell proliferation in a dose- and time-dependent manner. The 24, 48, 72, and 96 h IC50 values were 6.41 x 10(-2), 9.76 x 10(-4), 2.57 x 10(-5), and 2.08 x 10(-5) M, respectively. CB increased the proportion of cells in the G2/M phase in a dose-dependent manner, thus increasing the mitotic index and decreasing cdc2 and cyclin B1 protein levels. CB induced morphological changes in the cytoskeleton. Additionally, 10(-5) M CB induced apoptosis in 23.4 ± 0.5% of U251 cells (P < 0.05 vs control group). Caspase-3, -8, and -9 activities were increased after CB treatment. CB inhibited U251 glioma cell proliferation by damaging the microfilament structure. CB also induced glioma cell apoptosis, suggesting that it may be an effective therapeutic agent against gliomas. PMID:25526201

Tong, Z G; Liu, N; Song, H S; Li, J Q; Jiang, J; Zhu, J Y; Qi, J P

2014-01-01

194

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

PubMed Central

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

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

2013-01-01

195

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

PubMed

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

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

2014-12-01

196

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

197

The Cellular Basis for Biocide-Induced Fluorescein Hyperfluorescence in Mammalian Cell Culture  

PubMed Central

Clinical examination of the ocular surface is commonly carried out after application of sodium fluorescein in both veterinary and medical practice by assessing the resulting ‘staining’. Although localized intensely stained regions of the cornea frequently occur after exposure to ‘adverse’ clinical stimuli, the cell biology underlying this staining is unknown, including whether intense fluorescein staining indicates the presence of damaged cells. Ocular exposure to certain contact lens multipurpose solutions (MPS) gives rise to intense fluorescein staining referred to as solution induced corneal staining (SICS), and we have made use of this phenomenon with Vero and L929 cell culture models to investigate the fundamental biology of fluorescein interactions with cells. We found that all cells take up fluorescein, however a sub-population internalize much higher levels, giving rise to brightly staining ‘hyperfluorescent’ cells within the treated cultures, which contain fluorescein throughout the cell cytoplasm and nucleus. The numbers of these hyperfluorescent cells are significantly increased after exposure to MPS associated with SICS. Surprisingly, hyperfluorescent cells did not show higher levels of staining with propidium iodide, a marker of lysed cells. Consistently, treatment with the cytolytic toxin benzalkonium chloride resulted in almost all cells staining with propidium iodide, and the complete abolition of fluorescein hyperfluorescence. Finally we found that internalization of fluorescein and its loss from treated cells both require cellular activity, as both processes were halted after incubation at 4°C. We conclude that fluorescein hyperfluorescence can be replicated in three diverse cell cultures, and is increased by MPS-treatment, as occurs clinically. The process involves the concentration of fluorescein by a sub-population of cells that are active, and does not occur in lysed cells. Our data suggest that corneal staining in the clinic reflects active living cells, and is not directly caused by dead cells being produced in response to adverse clinical stimuli. PMID:24489650

Bakkar, May M.; Hardaker, Luke; March, Peter; Morgan, Philip B.; Maldonado-Codina, Carole; Dobson, Curtis B.

2014-01-01

198

The cellular basis for biocide-induced fluorescein hyperfluorescence in mammalian cell culture.  

PubMed

Clinical examination of the ocular surface is commonly carried out after application of sodium fluorescein in both veterinary and medical practice by assessing the resulting 'staining'. Although localized intensely stained regions of the cornea frequently occur after exposure to 'adverse' clinical stimuli, the cell biology underlying this staining is unknown, including whether intense fluorescein staining indicates the presence of damaged cells. Ocular exposure to certain contact lens multipurpose solutions (MPS) gives rise to intense fluorescein staining referred to as solution induced corneal staining (SICS), and we have made use of this phenomenon with Vero and L929 cell culture models to investigate the fundamental biology of fluorescein interactions with cells. We found that all cells take up fluorescein, however a sub-population internalize much higher levels, giving rise to brightly staining 'hyperfluorescent' cells within the treated cultures, which contain fluorescein throughout the cell cytoplasm and nucleus. The numbers of these hyperfluorescent cells are significantly increased after exposure to MPS associated with SICS. Surprisingly, hyperfluorescent cells did not show higher levels of staining with propidium iodide, a marker of lysed cells. Consistently, treatment with the cytolytic toxin benzalkonium chloride resulted in almost all cells staining with propidium iodide, and the complete abolition of fluorescein hyperfluorescence. Finally we found that internalization of fluorescein and its loss from treated cells both require cellular activity, as both processes were halted after incubation at 4 °C. We conclude that fluorescein hyperfluorescence can be replicated in three diverse cell cultures, and is increased by MPS-treatment, as occurs clinically. The process involves the concentration of fluorescein by a sub-population of cells that are active, and does not occur in lysed cells. Our data suggest that corneal staining in the clinic reflects active living cells, and is not directly caused by dead cells being produced in response to adverse clinical stimuli. PMID:24489650

Bakkar, May M; Hardaker, Luke; March, Peter; Morgan, Philip B; Maldonado-Codina, Carole; Dobson, Curtis B

2014-01-01

199

Manumycin induces apoptosis in prostate cancer cells  

PubMed Central

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

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

2014-01-01

200

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

201

Purification of murine pulmonary type II cells for flow cytometric cell cycle analysis.  

PubMed

Mice are widely used as animal models for in vivo lung disease. Despite this fact, few methods exist for isolation of type II pneumocytes from mouse lung, limiting the study of alveolar epithelial characteristics in these models. This study investigated several methods for labeling murine lung cell suspensions for flow cytometric identification and sorting of type II pneumocytes. Crude lung cell suspensions were prepared after intratracheal instillation of Dispase and were labeled using phosphine alone or in combination with Helix pomatia lectin, Maclura pomifera lectin, or anti-murine-CD32. Crude cell suspensions yielded 17.4 million cells per animal with 19.5% type II pneumocytes by Pap staining. Ultrastructural evaluation of the sorted cell pellets (1-1.5 million cells each) demonstrated optimal type II cell purity in preparations labeled with phosphine and anti-CD32 (94.3% type II cells, 0.4% macrophages, 2.8% Clara cells, and 2.5% other). Nuclear suspensions appropriate for cell cycle analysis were produced by sorting the type II cells directly into hypotonic propidium iodide, and these preparations clearly demonstrated a substantial increase in S-phase type II cells during proliferative repair of BHT-induced acute lung injury. PMID:7621777

Harrison, J H; Porretta, C P; Leming, K

1995-01-01

202

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

203

In vitro approaches to evaluate palytoxin-induced toxicity and cell death in intestinal cells.  

PubMed

Palytoxin isolated from the genus Palythoa is the most potent marine toxin known. The aim of the present study was to quantify palytoxin-induced cellular injury in the human intestinal cell line Caco-2. Cellular damage was measured by evaluating cell proliferation, cell membrane permeability, cell morphology and apoptotic markers. Furthermore, changes in F-actin were studied after exposure of cells to increasing amounts of palytoxin. The results show that cell proliferation decreased in a concentration-dependent manner with a mean IC(50) value of about 0.1 nM. A noticeable increase of cell detachment correlated with cell rounding and F-actin depolymerization was observed in palytoxin-treated cells. Moreover LDH was released from the cells in a dose and time dependent manner, although under these conditions there was no propidium iodide uptake. On the other hand, palytoxin impaired mitochondrial activity but other apoptotic markers, such as DNA fragmentation or caspases activation, were not observed. The results obtained in this paper suggest that the effects of palytoxin in Caco-2 cells were very potent and unspecific, since a primary necrosis and a secondary apoptosis seem to occur under these conditions. PMID:17604342

Valverde, I; Lago, J; Vieites, J M; Cabado, A G

2008-04-01

204

Effects of exogenous agmatine in human leukemia HMC-1 and HL-60 cells on proliferation, polyamine metabolism and cell cycle.  

PubMed

Impairment of agmatine homeostasis is involved in the regulation of cell proliferation in malignant solid tumors. The present study aimed at analyzing the relevance of agmatine homeostasis in pathophysiology of human leukemia. Proliferation of the human leukemia cells HMC-1 and HL-60 was determined in the absence or presence of agmatine. Apoptosis and cell cycle distribution was investigated by determination of caspase-3 activity and/or flow cytometry after staining with propidium iodide. Expression analysis was performed by qPCR and by a microarray genechip. Exogenous agmatine inhibited proliferation of both HMC-1 and HL-60 cells. The antiproliferative effect was due to interference of agmatine with the cell cycle with no evident signs of apoptosis. Comparative analysis of expression of mRNA in untreated HMC-1 cells and in non-leukemic human mast cells revealed a much lower expression of agmatinase and diamine oxidase in HMC-1 cells indicating a significantly reduced agmatine catabolism in the leukemic cells. At the mRNA level, inhibition of proliferation of both cell lines by agmatine was associated with cell type-specific alterations of the expression of enzymes of the polyamine pathway. The present results point to a significant role of agmatine homeostasis in the (patho)physiology of cell proliferation of leukemic cells, at least in HMC-1 and HL-60 cells, that may serve as a potential target for an adjuvant therapy in the treatment of human leukemia. PMID:21236489

Haenisch, Britta; Bönisch, Heinz; Cichon, Sven; Allam, Jean-Pierre; Novak, Natalija; Molderings, Gerhard J

2011-09-01

205

Gap junctional communication promotes apoptosis in a connexin-type-dependent manner  

PubMed Central

Gap junctions (GJs) have been described to modulate cell death and survival. It still remains unclear whether this effect requires functional GJ channels or depends on channel-independent effects of connexins (Cx), the constituents of GJs. Therefore, we analysed the apoptotic response to streptonigrin (SN, intrinsic apoptotic pathway) or to ?-Fas (extrinsic apoptotic pathway) in HeLa cells expressing Cx43 as compared with empty vector-transfected (CTL) cells. Apoptosis assessed by annexin V-fluorescein isothiocyanate/propidium iodide staining was significantly higher in HeLa-Cx43 compared with HeLa-CTL cells. Moreover, the cleavage of caspase-7 or Parp occurred earlier in HeLa-Cx43 than in HeLa-CTL cells. Comparative analysis of the effect of two further (endothelial) Cx (Cx37 and Cx40) on apoptosis revealed that apoptosis was highest in HeLa-Cx43 and lowest in HeLa-Cx37 cells, and correlated with the GJ permeability (assessed by spreading of a GJ-permeable dye and locally induced Ca2+ signals). Pharmacologic inhibition of GJ formation in HeLa-Cx43 cells reduced apoptosis significantly. The role of GJ communication was further analysed by the expression of truncated Cx43 proteins with and without channel-forming capacity. Activation of caspases was higher in cells expressing the channel-building part (HeLa-Cx43NT-GFP) than in cells expressing the channel-incompetent C-terminal part of Cx43 (HeLa-Cx43CT-GFP) only. A hemichannel-dependent release and, hence, paracrine effect of proapoptotic signals could be excluded since the addition of a peptide (Pep)-blocking Cx43-dependent hemichannels (but not GJs) did not reduce apoptosis in HeLa-Cx43 cells. Treatment with SN resulted in a significant higher increase of the intracellular free Ca2+ concentration in HeLa-Cx43 and HeLa-Cx43NT-GFP cells compared with HeLa-CTL or HeLa-Cx43CT-GFP cells, suggesting that Ca2+ or a Ca2+-releasing agent could play a signalling role. Blocking of inositol triphosphate receptors reduced the SN-induced Ca2+ increase as well as the increase in apoptosis. Our observations suggest that Cx43 and Cx40 but not Cx37 promote apoptosis via gap junctional transfer of pro-apoptotic signals between cells. PMID:23579271

Kameritsch, P; Khandoga, N; Pohl, U; Pogoda, K

2013-01-01

206

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

PubMed

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

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

2014-11-01

207

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

208

Tendon cells in vivo form a three dimensional network of cell processes linked by gap junctions.  

PubMed Central

Tendons respond to mechanical load by modifying their extracellular matrix. The cells therefore sense mechanical load and coordinate an appropriate response to it. We show that tendon cells have the potential to communicate with one another via cell processes and gap junctions and thus could use direct cell/cell communication to detect and/or coordinate their load responses. Unfixed cryosections of adult rat digital flexor tendons were stained with the fluorescent membrane dye DiI to demonstrate cell shape. Similar sections were immunolabelled with monoclonal antibodies to rat connexin 32 or connexin 43 to demonstrate gap junctions and counterstained with propidium iodide to show nuclei, or the membrane stain DiOC7 to show cell membranes. Sections were examined with a laser scanning confocal microscope and 3-dimensional reconstructions were prepared from optical section series to demonstrate cell shape and the position of connexin immunolabel. Cells had a complex interconnected morphology with gap junctions at points of contact with other cells. Cell bodies contained the nucleus and extended broad flat lateral cell processes that enclosed collagen bundles and interacted with similar processes from adjacent cells. They also had long thin longitudinal processes interacting with the cell process network further along the tendon. Connexin 43 occurred where cell processes met and between cell bodies, whereas connexin 32 was only found between cell bodies. The results indicate the presence of a 3-dimensional communicating network of cell processes within tendons. The intimate relationship between cell processes and collagen fibril bundles suggests that the cell process network could be involved in load sensing and coordination of response to load. The presence of 2 different types of connexins suggests that there could be at least 2 distinct communicating networks. Images Fig. 1 A-F Fig. 2 A-F Fig. 3 A,B PMID:8982835

McNeilly, C M; Banes, A J; Benjamin, M; Ralphs, J R

1996-01-01

209

Synchronization of mammalian cells and nuclei by centrifugal elutriation.  

PubMed

Synchronized populations of large numbers of cells can be obtained by centrifugal elutriation on the basis of sedimentation properties of small round particles, with minimal perturbation of cellular functions. The physical characteristics of cell size and sedimentation velocity are operative in the technique of centrifugal elutriation also known as counterstreaming centrifugation. The elutriator is an advanced device for increasing the sedimentation rate to yield enhanced resolution of cell separation. A random population of cells is introduced into the elutriation chamber of an elutriator rotor running in a specially designed centrifuge. By increasing step by step the flow rate of the elutriation fluid, successive populations of relatively homogeneous cell size can be removed from the elutriation chamber and used as synchronized subpopulations. For cell synchronization by centrifugal elutriation early log S phase cell populations are most suitable where most of the cells are in G1 and S phase (>80%). Protocols for the synchronization of nuclei of murine pre-B cells and high-resolution centrifugal elutriation of CHO cells are given. The verification of purity and cell cycle positions of cells in elutriated fractions includes the measurement of DNA synthesis by [(3)H]-thymidine incorporation and DNA content by propidium iodide flow cytometry. PMID:21755439

Banfalvi, Gaspar

2011-01-01

210

K562 cells display different vulnerability to H2 O2 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 (H2 O2 ) 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 H2 O2 -induced oxidative stress at 500??M and above. PMID:25181960

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

2015-02-01

211

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

PubMed Central

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

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

2010-01-01

212

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

213

Effect of Semax peptide on survival of cultured rat pheochromocytoma cells during oxidative stress.  

PubMed

We studied the effects of Semax (antiinsulin peptide with neuroprotective effect) on the survival of cultured rat pheochromocytoma cell after oxidative stress induced by short-term incubation with hydrogen peroxide. Studies with fluorescent dyes propidium iodide and Hoechst 33258 showed that cell incubation with hydrogen peroxide led to the formation of damaged cells with characteristic signs of necrosis. Semax dose-dependently reduced the number of cells damaged by oxidative stress. The efficiency of Semax depended on the time of its addition to the culture medium. The results suggest that the neuroprotective effect of Semax in ischemic stroke can be due to its capacity to protect neurons from damage caused by oxidative stress. PMID:12802399

Safarova, E R; Shram, S I; Zolotarev, Yu A; Myasoedov, N F

2003-03-01

214

Inhibition of Autophagy Potentiates Atorvastatin-Induced Apoptotic Cell Death in Human Bladder Cancer Cells in Vitro  

PubMed Central

Statins are cholesterol reduction agents that exhibit anti-cancer activity in several human cancers. Because autophagy is a crucial survival mechanism for cancer cells under stress conditions, cooperative inhibition of autophagy acts synergistically with other anti-cancer drugs. Thus, this study investigates whether combined treatment of atorvastatin and autophagy inhibitors results in enhancing the cytotoxic effects of atorvastatin, upon human bladder cancer cells, T24 and J82, in vitro. To measure cell viability, we performed the EZ-Cytox cell viability assay. We examined apoptosis by flow cytometry using annexin-V/propidium iodide (PI and western blot using procaspase-3 and poly (ADP-ribose) polymerase (PARP) antibodies. To examine autophagy activation, we evaluated the co-localization of LC3 and LysoTracker by immunocytochemistry, as well as the expression of LC3 and p62/sequestosome-1 (SQSTM1) by western blot. In addition, we assessed the survival and proliferation of T24 and J82 cells by a clonogenic assay. We found that atorvastatin reduced the cell viability of T24 and J82 cells via apoptotic cell death and induced autophagy activation, shown by the co-localization of LC3 and LysoTracker. Moreover, pharmacologic inhibition of autophagy significantly enhanced atorvastatin-induced apoptosis in T24 and J82 cells. In sum, inhibition of autophagy potentiates atorvastatin-induced apoptotic cell death in human bladder cancer cells in vitro, providing a potential therapeutic approach to treat bladder cancer. PMID:24815071

Kang, Minyong; Jeong, Chang Wook; Ku, Ja Hyeon; Kwak, Cheol; Kim, Hyeon Hoe

2014-01-01

215

Inhibition of autophagy potentiates atorvastatin-induced apoptotic cell death in human bladder cancer cells in vitro.  

PubMed

Statins are cholesterol reduction agents that exhibit anti-cancer activity in several human cancers. Because autophagy is a crucial survival mechanism for cancer cells under stress conditions, cooperative inhibition of autophagy acts synergistically with other anti-cancer drugs. Thus, this study investigates whether combined treatment of atorvastatin and autophagy inhibitors results in enhancing the cytotoxic effects of atorvastatin, upon human bladder cancer cells, T24 and J82, in vitro. To measure cell viability, we performed the EZ-Cytox cell viability assay. We examined apoptosis by flow cytometry using annexin-V/propidium iodide (PI and western blot using procaspase-3 and poly (ADP-ribose) polymerase (PARP) antibodies. To examine autophagy activation, we evaluated the co-localization of LC3 and LysoTracker by immunocytochemistry, as well as the expression of LC3 and p62/sequestosome-1 (SQSTM1) by western blot. In addition, we assessed the survival and proliferation of T24 and J82 cells by a clonogenic assay. We found that atorvastatin reduced the cell viability of T24 and J82 cells via apoptotic cell death and induced autophagy activation, shown by the co-localization of LC3 and LysoTracker. Moreover, pharmacologic inhibition of autophagy significantly enhanced atorvastatin-induced apoptosis in T24 and J82 cells. In sum, inhibition of autophagy potentiates atorvastatin-induced apoptotic cell death in human bladder cancer cells in vitro, providing a potential therapeutic approach to treat bladder cancer. PMID:24815071

Kang, Minyong; Jeong, Chang Wook; Ku, Ja Hyeon; Kwak, Choel; Kim, Hyeon Hoe

2014-01-01

216

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

217

Caspofungin Kills Candida albicans by Causing both Cellular Apoptosis and Necrosis  

PubMed Central

Caspofungin exerts candidacidal activity by inhibiting cell wall (1,3)-?-d-glucan synthesis. We investigated the physiologic mechanisms of caspofungin-induced Candida albicans cell death. Apoptosis (programmed cell death) and necrosis were studied after C. albicans SC5314 cells were exposed to caspofungin at 0.06, 0.125, and 0.5 ?g/ml (0.5×, 1×, and 4× the MIC, respectively) for 3 h. Caspofungin at 0.125 and 0.5 ?g/ml reduced cellular viability by >50%, as measured by colony counts and methylene blue exclusion. Apoptosis and necrosis were demonstrated by annexin V and propidium iodide staining for phosphatidylserine externalization and loss of membrane integrity, respectively. At all concentrations of caspofungin, 20 to 25% and 5 to 7% of C. albicans cells exhibited early apoptosis and late apoptosis/necrosis, respectively (P value was not significant [NS]). Necrosis, on the other hand, was significantly greater at 0.125 (43%) and 0.5 (48%) ?g/ml than at 0.06 ?g/ml (26%) (P values of 0.003 and 0.003, respectively). The induction of apoptosis at concentrations less than or equal to the MIC was corroborated by dihydrorhodamine 123 (DHR-123) and dihydroethidium (DHE) staining (reactive oxygen species production), JC-1 staining (mitochondrial membrane potential dissipation), and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and 4?,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining (DNA damage and nuclear fragmentation). Moreover, electron microscopy of cells exposed to 0.125 ?g/ml of caspofungin showed hallmark apoptotic features like chromatin margination and condensation and nuclear blebs. Apoptosis was associated with metacaspase 1 activation, as demonstrated by D2R staining. Caspofungin exerts activity against C. albicans by directly killing cells (resulting in necrosis) and causing others to undergo programmed cell death (apoptosis). Apoptosis is initiated at subinhibitory concentrations, suggesting that strategies to target this process may augment the benefits of antifungal agents. PMID:23114781

Hao, Binghua; Cheng, Shaoji; Nguyen, M. Hong

2013-01-01

218

Inhibition of VDAC1 prevents Ca²?-mediated oxidative stress and apoptosis induced by 5-aminolevulinic acid mediated sonodynamic therapy in THP-1 macrophages.  

PubMed

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

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

2014-12-01

219

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

SciTech Connect

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

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

1982-06-01

220

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

PubMed Central

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

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

2014-01-01

221

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

222

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

223

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

224

Active Targeting to Osteosarcoma Cells and Apoptotic Cell Death Induction by the Novel Lectin Eucheuma serra Agglutinin Isolated from a Marine Red Alga  

PubMed Central

Previously, we demonstrated that the novel lectin Eucheuma serra agglutinin from a marine red alga (ESA) induces apoptotic cell death in carcinoma. We now find that ESA induces apoptosis also in the case of sarcoma cells. First, propidium iodide assays with OST cells and LM8 cells showed a decrease in cell viability after addition of ESA. With 50??g/ml ESA, the viabilities after 24 hours decreased to 54.7 ± 11.4% in the case of OST cells and to 41.7 ± 12.3% for LM8 cells. Second, using fluorescently labeled ESA and flow cytometric and fluorescence microscopic measurements, it could be shown that ESA does not bind to cells that were treated with glycosidases, indicating importance of the carbohydrate chains on the surface of the cells for efficient ESA-cell interactions. Third, Span 80 vesicles with surface-bound ESA as active targeting ligand were shown to display sarcoma cell binding activity, leading to apoptosis and complete OST cell death after 48 hours at 2??g/ml ESA. The findings indicate that Span 80 vesicles with surface-bound ESA are a potentially useful drug delivery system not only for the treatment of carcinoma but also for the treatment of osteosarcoma. PMID:23346404

Hayashi, Keita; Walde, Peter; Miyazaki, Tatsuhiko; Sakayama, Kenshi; Nakamura, Atsushi; Kameda, Kenji; Masuda, Seizo; Umakoshi, Hiroshi; Kato, Keiichi

2012-01-01

225

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

PubMed Central

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

2013-01-01

226

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

227

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

228

Cytotoxicity of Voriconazole on Cultured Human Corneal Endothelial Cells?  

PubMed Central

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

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

2011-01-01

229

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

230

In vitro effects of Sutherlandia frutescens water extracts on cell numbers, morphology, cell cycle progression and cell death in a tumorigenic and a non-tumorigenic epithelial breast cell line.  

PubMed

Sutherlandia frutescens is a South African herb traditionally used for internal cancers, diabetes, a variety of inflammatory conditions and recently to improve the overall health in cancer and HIV/AIDS patients. The in vitro effects of S. frutescens extracts were evaluated on cell numbers, morphology, cell cycle progression and cell death. Dose-dependent studies (2-10 mg/ml) revealed a decrease in malignant cell numbers when compared to their controls. S. frutescens extracts (10 mg/ml) decreased cell growth in a statistically significantly manner to 26% and 49% (P<0.001) in human breast adenocarcinoma (MCF-7) and human non-tumorigenic epithelial mammary gland cells (MCF-12A) respectively after 72 h of exposure. Cell density was significantly compromised and hypercondensed chromatin, cytoplasmic shrinking, membrane blebbing and apoptotic bodies were more pronounced in the MCF-7 cell line. Both S. frutescens-treated cell lines exhibited and increased tendency for acridine orange staining, suggesting increased lysosomal and/or autophagy activity. Flow cytometry showed an increase in the sub G(1) apoptotic fraction and an S phase arrest in both the 5 mg/ml and 10 mg/ml S. frutescens-treated cells. S. frutescens induced an increase in apoptosis in both cell lines as detected by Annexin V and propidium iodide flow cytometric measurement. At 10 mg/ml, late stages of apoptosis were more prominent in MCF-7 S. frutescens-treated cells when compared to the MCF-12A cells. Transmission electron microscopy revealed hallmarks of increased vacuolarization and hypercondensed chromatin, suggesting autophagic and apoptotic processes. The preliminary study demonstrates that S. frutescens water extracts exert a differential action mechanism in non-tumorigenic MCF-12A cells when compared to tumorigenic MCF-7 cells, warranting future studies on this multi-purpose medicinal plant in southern Africa. PMID:19527821

Stander, Andre; Marais, Sumari; Stivaktas, Voula; Vorster, Christiaan; Albrecht, Carl; Lottering, Mona-Liza; Joubert, Annie M

2009-07-01

231

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

232

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

233

Redirection of Human Cancer Cells upon the Interaction with the Regenerating Mouse Mammary Gland Microenvironment  

PubMed Central

Tumorigenesis is often described as a result of accumulated mutations that lead to growth advantage and clonal expansion of mutated cells. There is evidence in the literature that cancer cells are influenced by the microenvironment. Our previous studies demonstrated that the mouse mammary gland is capable of redirecting mouse cells of non-mammary origins as well as Mouse Mammary Tumor Virus (MMTV)-neu transformed cells toward normal mammary epithelial cell fate during gland regeneration. Interestingly, the malignant phenotype of MMTV-neu transformed cells was suppressed during serial transplantation experiments. Here, we discuss our studies that demonstrated the potential of the regenerating mouse mammary gland to redirect cancer cells of different species into a functional tumor-free mammary epithelial cell progeny. Immunochemistry for human specific CD133, mitochondria, cytokeratins as well as milk proteins and FISH for human specific probe identified human epithelial cell progeny in ducts, lobules, and secretory acini. Fluorescent In Situ Hybridization (FISH) for human centromeric DNA and FACS analysis of propidium iodine staining excluded the possibility of mouse-human cell fusion. To our knowledge this is the first evidence that human cancer cells of embryonic or somatic origins respond to developmental signals generated by the mouse mammary gland microenvironment during gland regeneration in vivo. PMID:24709643

Rosenfield, Sonia M.; Smith, Gilbert H.

2013-01-01

234

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

235

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

PubMed Central

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

2014-01-01

236

Paraquat induces apoptosis of cultured rat cortical cells.  

PubMed

Paraquat (PQ; 1,1'-dimethyl-4,4'-bipyridinium dichloride) is widely used as a universal herbicide. Although systemic treatment with PQ gives rise to the highest level of the herbicide in the cerebral cortex, our knowledge of its effects in this brain region is very limited. We took advantage of rat cortical cell cultures to analyze how PQ affects cortical neurons. Lactate dehydrogenase (LDH) assay and propidium iodide (PI) staining showed that PQ was cytotoxic to cortical neurons with an IC50 on the third day after treatment of approximately 10 microM. PQ-treated cells had shrunken soma with condensed nuclei and disintegrated dendrites, typical signs of apoptosis. Immunocytochemistry of 8-day in vitro (DIV) cells one day after PQ treatment with anti-phospho-H2AX antibody showed that the average number of punctae per nucleus had increased several-fold, indicating substantial DNA fragmentation. Furthermore, double-staining of 7.5 DIV cultures (50 microM PQ) with PI and an antibody against annexin V (AN), an impermeable plasma protein which specifically binds to phosphatidylserine (PS), showed that the percentages of AN(+)/PI(-) cells had also increased several-fold, pointing to considerable movement of PS from the inner to the outer leaflet of the plasma membrane. Taken together, our data indicate that PQ induces apoptosis in cortical cell cultures. PMID:15055535

Kim, Sung Jin; Kim, Jang Eok; Moon, Il Soo

2004-02-29

237

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

238

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

PubMed Central

Autophagy is essential for prolonging yeast survival during nutrient deprivation; however, this report shows that some autophagy proteins may also be accelerating population death in those conditions. While leucine starvation caused YCA1-mediated apoptosis characterized by increased annexin V staining, nitrogen deprivation triggered necrotic death characterized by increased propidium iodide uptake. Although a ?atg8 strain died faster than its parental strain during nitrogen starvation, this mutant died slower than its parent during leucine starvation. Conversely, a ?atg11 strain died slower than its parent during nitrogen starvation, but faster during leucine starvation. Curiously, although GFP-Atg8 complemented the ?atg8 mutation, this protein made ATG8 cells more sensitive to nitrogen starvation, and less sensitive to leucine starvation. These results were difficult to explain if autophagy only extended life but could be an indication that a second form of autophagy could concurrently facilitate either apoptotic or necrotic cell death. PMID:22361650

Dziedzic, Slawomir A.; Caplan, Allan B.

2012-01-01

239

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

PubMed

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

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

2015-02-01

240

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

241

Determination of cell cycle phases in live B16 melanoma cells using IRMS.  

PubMed

The knowledge of cell cycle phase distribution is of paramount importance for understanding cellular behaviour under normal and stressed growth conditions. This task is usually assessed using Flow Cytometry (FC) or immunohistochemistry. Here we report on the use of FTIR microspectroscopy in Microfluidic Devices (MD-IRMS) as an alternative technique for studying cell cycle distribution in live cells. Asynchronous, S- and G0-synchronized B16 mouse melanoma cells were studied by running parallel experiments based on MD-IRMS and FC using Propidium Iodide (PI) staining. MD-IRMS experiments have been done using silicon-modified BaF2 devices, where the thin silicon layer prevents BaF2 dissolution without affecting the transparency of the material and therefore enabling a better assessment of the Phosphate I (PhI) and II (PhII) bands. Hierarchical Cluster Analysis (HCA) of cellular microspectra in the 1300-1000 cm(-1) region pointed out a distribution of cells among clusters, which is in good agreement with FC results among G0/G1, S and G2/M phases. The differentiation is mostly driven by the intensity of PhI and PhII bands. In particular, PhI almost doubles from the G0/G1 to G2/M phase, in agreement with the trend followed by nucleic acids during cellular progression. MD-IRMS is then proposed as a powerful method for the in situ determination of the cell cycle stage of an individual cell, without any labelling or staining, which gives the advantage of possibly monitoring specific cellular responses to several types of stimuli by clearly separating the spectral signatures related to the cellular response from those of cells that are normally progressing. PMID:23662303

Bedolla, Diana E; Kenig, Saša; Mitri, Elisa; Ferraris, Paolo; Marcello, Alessandro; Grenci, Gianluca; Vaccari, Lisa

2013-07-21

242

Cell-death assessment by fluorescent and nonfluorescent cytosolic and nuclear staining techniques.  

PubMed

Apoptosis, a genetically programmed cellular event leads to biochemical and morphological changes in cells. Alterations in DNA caused by several factors affect nucleus and ultimately the entire cell leading to compromised function of the organ and organism. DNA, a master regulator of the cellular events, is an important biomolecule with regards to cell growth, cell death, cell migration and cell differentiation. It is therefore imperative to develop the staining techniques that may lead to visualize the changes in nucleus where DNA is housed, to comprehend the cellular pathophysiology. Over the years a number of nuclear staining techniques such as propidium iodide, Hoechst-33342, 4', 6-diamidino-2-phenylindole (DAPI), Acridine orange-Ethidium bromide staining, among others have been developed to assess the changes in DNA. Some nonnuclear staining techniques such as Annexin-V staining, which although does not stain DNA, but helps to identify the events that result from DNA alteration and leads to initiation of apoptotic cell death. In this review, we have briefly discussed some of the most commonly used fluorescent and nonfluorescent staining techniques that identify apoptotic changes in cell, DNA and the nucleus. These techniques help in differentiating several cellular and nuclear phenotypes that result from DNA damage and have been identified as specific to necrosis or early and late apoptosis as well as scores of other nuclear deformities occurring inside the cells. PMID:24831993

Atale, N; Gupta, S; Yadav, U C S; Rani, V

2014-07-01

243

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

244

Human anterior lens capsule serving as a substrate for human trabecular meshwork cells cultivation.  

PubMed

To investigate if human anterior lens capsule is a suitable substrate for the culture of primary human trabecular meshwork (HTM) cells. Trabecular meshwork cells derived from four human donors were seeded on anterior lens capsules that were prepared from the lenses of donor eyes. Cell morphology and viability were examined at 1, 3, 5 and 7 days. Cell viability was measured based on a two-colour fluorescence assay (membrane-impermeable propidium iodide and membrane permeable Hoechst 33342). Immunocytochemistry studied Zonula occludens-1 (ZO-1), vimentin, tissue transglutaminase (tTgase) and Na(+)/K(+)-adenosine triphosphatase (Na(+)/K(+)-ATPase). Morphology of the cultivated cells followed a typical model while their viability was > 95% in all cases. ZO-1 was found at the cell boundaries of the HTM-AC complex. Vimentin was located at the lateral membranes of the HTM cells. Na(+)/K(+)-ATPase was found at the basolateral membrane of the HTM cells. tTgase was also identified. Anterior lens capsule can be considered as a suitable alternative substrate for cultivation of HTM cells and assist the expansion of existing knowledge about glaucoma pathophysiology and therapy. PMID:22836959

Kopsachilis, Nikolaos; Tsaousis, Konstantinos T; Tsinopoulos, Ioannis T; Kruse, Friedrich E; Welge-Lüssen, Ulrich

2013-09-01

245

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

PubMed

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

246

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

SciTech Connect

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

Watson, A.J.; Klaniecki, J.; Hanson, C.V. (Oncogen Corporation, Seattle, WA (USA))

1990-04-01

247

Flow-cytometric determination of intracellular pH, esterase activity and cell volume in human leukemic cell lines following in vitro incubation with cytostatic drugs.  

PubMed

A recently developed flow cytometric assay method using patient tumor cells allows the determination not only of their sensitivity to cytostatic drugs but also of biochemical and biophysical parameters after treatment, such as esterase concentration and intracellular pH of the living cells. DNA-content of the dead cells and cell volume of living and dead cells. The T-cell lines CEM, Molt4, Jurkat, the B-cell lines RPMI1788, Daudi, Raji and the promyelocytic line HL60 were incubated with: cytosine arabinoside (ara-C), L-asparaginase, daunorubicin, vincristine and prednisone for 48 h. Living cells then stained with esterase and pH-dye 1,4-diacetoxy-2,3-dicyanobenzene (ADB) and dead cells with DNA-dye propidium-iodide (PI). The esterase concentration, an index of metabolic activity, decreased in the T-cell lines under the influence of ara-C, daunorubicin and vincristine, whereas in the B-cell lines smaller changes in esterase concentration were observed (P less than 0.001). A decrease in intracellular pH was seen in the ara-C and daunorubicin-incubated cells Molt4, CEM and HL60, whereas in the B-cell lines no significant change in intracellular pH was found. In all lines except Jurkat the cell volume of the surviving cells increased under the influence of certain drugs (primarily ara-C and daunorubicin); B-cell lines showed a greater swelling than T-cell lines (P = 0.001). PMID:2486910

Neubauer, A; Valet, G; Huhn, D

1989-12-01

248

HSP70 inhibition by 2-phenylethynesulfonamide induces lysosomal cathepsin D release and immunogenic cell death in primary effusion lymphoma.  

PubMed

Heat-shock protein (HSP) 70 is aberrantly expressed in different malignancies and has a cancer-specific cell-protective effect. As such, it has emerged as a promising target for anticancer therapy. In this study, the effect of the HSP70-specific inhibitor (PES), also Pifitrin-?, on primary effusion lymphoma (PEL) cell viability was analyzed. PES treatment induced a dose- and time-dependent cytotoxic effect in BC3 and BCBL1 PEL cells by inducing lysosome membrane permeabilization, relocation of cathepsin D in the cytosol, Bid cleavage, mitochondrial depolarization with release and nuclear translocation of apoptosis-activating factor. The PES-induced cell death in PEL cells was characterized by the appearance of Annexin-V/propidium iodide double-positive cells from the early times of treatment, indicating the occurrence of an additional type of cell death other than apoptosis, which, accordingly, was not efficiently prevented by the pan-caspase inhibitor Z-VAD-fmk. Conversely, PES-induced cell death was robustly reduced by pepstatin A, which inhibits Bid and caspase 8 processing. In addition, PES was responsible for a block of the autophagic process in PEL cells. Finally, we found that PES-induced cell death has immunogenic potential being able to induce dendritic cell activation. PMID:23868063

Granato, M; Lacconi, V; Peddis, M; Lotti, L V; Di Renzo, L; Renzo, L D; Gonnella, R; Santarelli, R; Trivedi, P; Frati, L; D'Orazi, G; Faggioni, A; Cirone, M

2013-01-01

249

Peripheral neural cell sensitivity to mTHPC-mediated photodynamic therapy in a 3D in vitro model  

PubMed Central

Background: The effect of photodynamic therapy (PDT) on neural cells is important when tumours are within or adjacent to the nervous system. The purpose of this study was to investigate PDT using the photosensitiser, meta-tetrahydroxyphenyl chlorin (mTHPC), on rat neurons and satellite glia, compared with human adenocarcinoma cells (MCF-7). Methods: Fluorescence microscopy confirmed that mTHPC was incorporated into all three cell types. Sensitivity of cells exposed to mTHPC-PDT (0–10??g?ml–1) was determined in a novel 3-dimensional collagen gel culture system. Cell death was quantified using propidium iodide and cell types were distinguished using immunocytochemistry. In some cases, neuron survival was confirmed by measuring subsequent neurite growth in monolayer culture. Results: MCF-7s and satellite glia were significantly more sensitive to PDT than neurons. Importantly, 4??g?ml–1 mTHPC-PDT caused no significant neuron death compared with untreated controls but was sufficient to elicit substantial cell death in the other cell types. Initially, treatment reduced neurite length; neurons then extended neurites equivalent to those of untreated controls. The protocol was validated using hypericin (0–3??g?ml–1), which caused neuron death equivalent to other cell types. Conclusion: Neurons in culture can survive mTHPC-PDT under conditions sufficient to kill tumour cells and other nervous system cells. PMID:19638975

Wright, K E; Liniker, E; Loizidou, M; Moore, C; MacRobert, A J; Phillips, J B

2009-01-01

250

Natural killer cells induce eosinophil activation and apoptosis.  

PubMed

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

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

2014-01-01

251

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

PubMed

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

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

2014-10-01

252

Salidroside inhibits endogenous hydrogen peroxide induced cytotoxicity of endothelial cells.  

PubMed

Salidroside, a phenylpropanoid glycoside isolated from Rhodiola rosea L., shows potent antioxidant property. Herein, we investigated the protective effects of salidroside against hydrogen peroxide (H2O2)-induced oxidative damage in human endothelial cells (EVC-304). EVC-304 cells were incubated in the presence or absence of low steady states of H2O2 (3-4?µM) generated by glucose oxidase (GOX) with or without salidroside. 3(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH) assays were performed, together with Hoechst 33258 staining and flow cytometric analysis using Annexin-V and propidium iodide (PI) label. The results indicated that salidroside pretreatment attenuated endogenous H2O2 induced apoptotic cell death in EVC-304 cells in a dose-dependent pattern. Furthermore, Western blot data revealed that salidroside inhibited activation of caspase-3, 9 and cleavage of poly(ADP-ribose) polymerase (PARP) induced by endogenous H2O2. It also decreased the expression of Bax and rescued the balance of pro- and anti-apoptotic proteins. All these results demonstrated that salidroside may present a potential therapy for oxidative stress in cardiovascular and cerebrovascular diseases. PMID:23965749

Zhao, Xingyu; Jin, Lianhai; Shen, Nan; Xu, Bin; Zhang, Wei; Zhu, Hongli; Luo, Zhengli

2013-01-01

253

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

254

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

255

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

256

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

NASA Astrophysics Data System (ADS)

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

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

2011-12-01

257

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

258

Overexpression of human arginine decarboxylase rescues human mesenchymal stem cells against H?O? toxicity through cell survival protein activation.  

PubMed

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

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

2013-03-01

259

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

PubMed

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

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

2013-01-01

260

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

261

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

262

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

PubMed

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

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

2011-04-01

263

Cytostatic and apoptosis-inducing activity of boswellic acids toward malignant cell lines in vitro.  

PubMed

Boswellic acids from frankincense were indentified as the active compounds which inhibit leukotriene biosynthesis, 5-lipoxygenase and exert antiproliferative activity toward a variety of malignant cells. Because of the relevance for the clinical application, we tested the ethanolic extract of Boswellia serrata gum resin containing a defined amount of boswellic acids for its cytotoxic, cytostatic and apoptotic activity on five leukemia (HL-60, K 562, U937, MOLT-4, THP-1) and two brain tumor (LN-18, LN-229) cell lines by WST-1 assay and flow cytometry. The Boswellia serrata extract induced dose-dependent antiproliferative effects on all human malignant cells tested with GI50 values (extract concentration producing 50% cell growth inhibition) between 57.0 and 124.1 micrograms/ml. In three haematological cell lines (K562, U937, MOLT-4) the effect of total extract expressed in GI50 was 2.8-, 3.3- and 2.3-times more potent (p < 0.05) than pure 3-O-acetyl-11-keto-beta-boswellic acid (AKBA). Morphological changes after 24-27 hours and the detection of apoptotic cells by AnnexinV-binding and/or by the detection of propidium iodide-labelled DNA with flow cytometry, confirmed the apoptotic cell death. The results of this study suggest the effectiveness of Boswellia serrata extract with defined content of boswellic acids. PMID:12530009

Hostanska, Katarina; Daum, Gerhard; Saller, Reinhard

2002-01-01

264

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.

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

2015-01-01

265

Intracellular calcium and survival of tadpole forebrain cells in anoxia.  

PubMed

The frog brain survives hypoxia with a slow loss of energy charge and ion homeostasis. Because hypoxic death in most neurons is associated with increases in intracellular calcium ([Ca2+]i), we examined the relationship between [Ca2+]i and survival of a mixed population of isolated cells from the forebrain of North American bullfrog Rana catesbeiana tadpoles. Forebrain cells from stage V-XV tadpoles were isolated by enzymatic digestion and loaded with one of three different calcium indicators (Fura-2, Fura 2-FF and BTC) to provide estimates of [Ca2+]i accurate at low and high [Ca2+]i. Propidium iodide (PI) fluorescence was used as an indicator of cell viability. Cells were exposed to anoxia (100% N2) and measurements of [Ca2+]i and cell survival made from 1 h to 18 h. Intracellular [Ca2+] increased significantly after 3-6 h anoxia (P<0.05), regardless of the type of Ca2+ indicator used; however, there were substantial differences in the measurements of [Ca2+]i with the different indicators, reflecting their varying affinities for Ca2+. Resting [Ca2+]i was approximately 50 nmol l(-1) and increased to about 9-30 micromol l(-1) after 4-6 h anoxia. The significant increase in [Ca2+]i during anoxia was not associated with significant increases in cell death, with 85-95% survival over this time period. Cells exposed to anoxia for 18 h, or those made anoxic for 4-6 and reoxygenated for 12 h to 16 h, had survival rates greater than 70%, but survival was significantly less than normoxic controls. These results indicate that large increases in [Ca2+]i are not necessarily associated with hypoxic cell death in vertebrate brain cells. PMID:15695760

Hedrick, Michael S; Fahlman, Christian S; Bickler, Philip E

2005-02-01

266

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

267

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

PubMed Central

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

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

2013-01-01

268

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

PubMed

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

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

2014-03-15

269

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

270

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

271

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

272

Triptolide induces s phase arrest and apoptosis in gallbladder cancer cells.  

PubMed

Gallbladder carcinoma is the most common malignancy of the biliary tract, with a very low 5-year survival rate and extremely poor prognosis. Thus, new effective treatments and drugs are urgently needed for the treatment of this malignancy. In this study, for the first time we investigated the effects of triptolide on gallbladder cancer cells and identified the mechanisms underlying its potential anticancer effects. The MTT assay showed that triptolide decreased cell viability in a dose- and time-dependent manner. The results of the colony formation assay indicated that triptolide strongly suppressed colony formation ability in GBC-SD and SGC-996 cells. Flow cytometric analysis revealed that triptolide induced S phase arrest in gallbladder cancer cells. In addition, triptolide induced apoptosis, as shown by the results of annexin V/propidium iodide double-staining and Hoechst 33342 staining. Furthermore, triptolide decreased mitochondrial membrane potential (??m) in a dose-dependent manner. Finally, western blot analysis of triptolide-treated cells revealed the activation of caspase-3, caspase-9, PARP, and Bcl-2; this result demonstrated that triptolide induced apoptosis in gallbladder cancer cells by regulating apoptosis-related protein expression, and suggests that triptolide may be a promising drug to treat gallbladder carcinoma. PMID:24566325

Hu, Yun-Ping; Tan, Zhu-Jun; Wu, Xiang-Song; Liu, Tian-Yu; Jiang, Lin; Bao, Run-Fa; Shu, Yi-Jun; Li, Mao-Lan; Weng, Hao; Ding, Qian; Tao, Feng; Liu, Ying-Bin

2014-01-01

273

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

PubMed

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

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

2014-01-01

274

Chemical compositions and antiproliferation activities of the chloroform fraction from Pyropolyporus fomentarius in K562 cells.  

PubMed

Pyropolyporus fomentarius, a fungus of the polyporaceae family, has been used in the treatment of various diseases, such as gastroenteric disorder, hepatocirrhosis, oral ulcer, inflammation, and several cancers. This study was conducted to investigate the compositions and cell growth inhibition effects of P. fomentarius chloroform (CHCl3) fraction and to clarify the possible mechanisms. Gas chromatography-mass spectrometry analysis was performed to investigate the composition of the P. fomentarius CHCl3 fraction. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell membrane damage was evaluated with a scanning electron microscope and flow cytometry following propidium iodide and bis-(1,3-dibarbituric acid)-trimethine oxanol staining. Apoptosis was analyzed using annexin V-PE/7-amino-actinomycin D (7-AAD) staining. Generation of intracellular calcium ion (Ca(2+)), reactive oxygen species (ROS), and changes of mitochondrial membrane potential (?? m) were detected by flow cytometry using fluo 3-acetoxymethyl ester, 2',7'-dichlorofluorescin-diacetate, and rhodamine 123. Our obtained data indicate that P. fomentarius CHCl3 fraction could inhibit proliferation of K562 cells depending on both the dosage and the incubation time, cause cell membrane damage, influence intracellular [Ca(2+)]i variation, promote the yield of ROS, decrease the level of ?? m, and initiate the apoptotic response in K562 cells. PMID:25403175

Zhang, Y; Wang, P; Xiao, Y; Wang, X; Yang, S; Liu, Q

2014-11-17

275

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

PubMed

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

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

2014-03-01

276

Rapid isolation of integrin rich multipotent stem cell pool and reconstruction of mouse epidermis equivalent.  

PubMed

We describe here epidermis reconstruction using multipotent mouse epidermal stem cells (EpSCs) enriched from keratinocyte isolates exploting exclusively the stem cell-adhesive property. This method excluded flowcytometry and was swift. Percent enrichment was measured by the uptake of Propidium iodide and Hoechst-33342 dye using flowcytometry to determine EpSCs yield. The sorted cells were characterized by analysis of stem cell markers using immunocytochemistry and immunoblotting techniques. Epidermis was reconstructed using the identified seeding density of EpSCs and the airlift tissue culture. Histology of natural vs reconstructed mammalian epidermis was also compared. Results showed a radical improvement of near 99% in the yield of integrin overexpressing EpSCs. The enriched EpSCs tested positive for biomarkers namely cytokeratin K-15 and, K-14, p63, beta-1-integrin, CD34 and could be passaged for longer durations. Adhesion sorted cells reconstructed the epidermis. The process of tissue reconstruction was faster using the adhesion sorted cells than the FACS sorted EpSCs. The product bioengineered using multipotent EpSCs was histologically similar to normal epidermis. Features like strata basalae, spinosum, granulosum, and corneum were alike real epidermis. The reconstructed epidermis displayed normal homeostasis, which can be considered an approximating actual product for investigative dermatology, toxicology, therapeutic research, regenerative medicine, and tissue engineering. PMID:24660112

Kumar, Sushil; Poojan, Shiv; Verma, Vikas; Verma, Mukesh K; Lohani, Mohatashim

2014-01-01

277

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

PubMed

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

2015-03-01

278

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

279

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

280

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

PubMed Central

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

Brudzynski, Katrina; Sjaarda, Calvin

2014-01-01

281

Different concentrations of 17?-estradiol modulates apoptosis induced by interleukin-1? in rat annulus fibrosus cells.  

PubMed

Interleukin?1? (IL?1?) is a pleiotropic cytokine that mediates inflammatory and cell death activities. IL?1? has been previously reported to induce apoptosis of intervertebral disc (IVD) cells in IVD degeneration. Accumulating data have suggested that post?menopausal women have a high incidence of IVD degeneration. It has therefore been proposed that estrogen may have a close association with IVD degeneration. Whether estrogen is able to protect IVD cells from apoptosis remains unclear. The present study aimed to examine whether 17??estradiol (17??E2) inhibited IL?1??induced apoptosis of rat annulus fibrosus (AF) cells. Additionally, the dose?response effect of 17??E2 on cell apoptosis was investigated. AF cells were isolated from male Sprague Dawley rats and cultured in complete medium. Following approximately two weeks, the AF cells were treated with IL?1? (75 ng/ml) for 24 h, with a pretreatment of 17??E2 for 1 h. Apoptosis of AF cells was analyzed by annexin V/propidium iodide binding assay and morphological changes, together with an assessment of caspase?3 activity. Cell viability of the AF cells was determined by MTT assay. The level of apoptosis and caspase?3 activity in the AF cells was increased whereas the cell viability was decreased following treatment with IL?1? (75 ng/ml), as compared with the control group. This effect was reversed by pretreatment with 17??E2, in a dose?dependent manner. The protective effect of 17??E2 was abolished by estrogen receptor antagonist ICI182, 780. These results indicate that 17??E2 protects rat AF cells from apoptosis induced by IL?1?, in a dose?dependent manner. PMID:25175856

Wang, Haiying; Ding, Wenyuan; Yang, Dalong; Gu, Tixin; Yang, Sidong; Bai, Zhilong

2014-11-01

282

Novel estradiol analogue induces apoptosis and autophagy in esophageal carcinoma cells.  

PubMed

Cancer is the second leading cause of death in South Africa. The critical role that microtubules play in cell division makes them an ideal target for the development of chemotherapeutic drugs that prevent the hyperproliferation of cancer cells. The new in silico-designed estradiol analogue 2-ethyl-3-O-sulfamoylestra-1,3,5(10)16-tetraene (ESE-16) was investigated in terms of its in vitro antiproliferative effects on the esophageal carcinoma SNO cell line at a concentration of 0.18 ?M and an exposure time of 24 h. Polarization-optical differential interference contrast and triple fluorescent staining (propidium iodide, Hoechst 33342 and acridine orange) revealed a decrease in cell density, metaphase arrest, and the occurrence of apoptotic bodies in the ESE-16-treated cells when compared to relevant controls. Treated cells also showed an increase in the presence of acidic vacuoles and lysosomes, suggesting the occurrence of autophagic processes. Cell death via autophagy was confirmed using the Cyto-ID autophagy detection kit and the aggresome detection assay. Results showed an increase in autophagic vacuole and aggresome formation in ESE-16 treated cells, confirming the induction of cell death via autophagy. Cell cycle progression demonstrated an increase in the sub-G1 fraction (indicative of the presence of apoptosis). In addition, a reduction in mitochondrial membrane potential was also observed, which suggests the involvement of apoptotic cell death induced by ESE-16 via the intrinsic apoptotic pathway. In this study, it was demonstrated that ESE-16 induces cell death via both autophagy and apoptosis in esophageal carcinoma cells. This study paves the way for future investigation into the role of ESE-16 in ex vivo and in vivo studies as a possible anticancer agent. PMID:24563014

Wolmarans, Elize; Mqoco, Thandi V; Stander, Andre; Nkandeu, Sandra D; Sippel, Katherine; McKenna, Robert; Joubert, Annie

2014-03-01

283

Phytosphingosine kills Candida albicans by disrupting its cell membrane.  

PubMed

The mechanism of action of phytosphingosine (PHS), a member of the sphingosine family which has candidacidal activity when added externally, was investigated. Previously, it has been reported that the fungicidal activity of PHS is based on the induction of caspase-independent apoptosis. In contrast, we found that addition of PHS causes a direct permeabilization of the plasma membrane of yeast, highlighted by the influx of the membrane probe propidium iodide, and the efflux of small molecules (i.e., adenine nucleotides) as well as large cellular constituents such as proteins. Freeze-fracture electron microscopy revealed that PHS treatment causes severe damage of the plasma membrane of the cell, which seems to have lost its integrity completely. We also found that PHS reverts the azide-induced insensitivity to histatin 5 (Hst5) of Candida albicans. In a previous study, we had found that the decreased sensitivity to Hst5 of energy-depleted cells is due to rigidification of the plasma membrane, which could be reverted by the membrane fluidizer benzyl alcohol. In line with the increased membrane permeabilization and ultrastructural damage, this reversal of the azide-induced insensitivity by PHS also points to a direct interaction between PHS and the cytoplasmic membrane of C. albicans. PMID:19919184

Veerman, Enno C I; Valentijn-Benz, Marianne; van't Hof, Wim; Nazmi, Kamran; van Marle, Jan; Amerongen, Arie V Nieuw

2010-01-01

284

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

285

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

286

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

287

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

PubMed

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

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

2013-12-01

288

Enhanced cell adhesion to silicone implant material through plasma surface modification.  

PubMed

Silicone implant material is widely used in the field of plastic surgery. Despite its benefits the lack of biocompatibility this material still represents a major problem. Due to the surface characteristics of silicone, protein adsorption and cell adhesion on this polymeric material is rather low. The aim of this study was to create a stable collagen I surface coating on silicone implants via glow-discharge plasma treatment in order to enhance cell affinity and biocompatibility of the material. Non-plasma treated, collagen coated and conventional silicone samples (non-plasma treated, non-coated) served as controls. After plasma treatment the change of surface free energy was evaluated by drop-shape analysis. The quality of the collagen coating was analysed by electron microscopy and Time-Of-Flight Secondary Ion Mass Spectrometry. For biocompatibility tests mouse fibroblasts 3T3 were cultivated on the different silicone surfaces and stained with calcein-AM and propidium iodine to evaluate cell viability and adherence. Analysis of the different surfaces revealed a significant increase in surface free energy after plasma pre-treatment. As a consequence, collagen coating could only be achieved on the plasma activated silicone samples. The in vitro tests showed that the collagen coating led to a significant increase in cell adhesion and cell viability. PMID:19641852

Hauser, J; Zietlow, J; Köller, M; Esenwein, S A; Halfmann, H; Awakowicz, P; Steinau, H U

2009-12-01

289

A shift to 50°C provokes death in distinct ways for glucose- and oleate-grown cells of Yarrowia lipolytica.  

PubMed

Based on the observation that shocks provoked by heat or amphiphilic compounds present some similarities, this work aims at studying whether cells grown on oleate (amphiphilic pre-stress) acquire a tolerance to heat shock. In rich media, changing glucose for oleate significantly enhanced the cell resistance to the shock, however, cells grown on a minimal oleate medium lost their ability to grow on agar with the same kinetic than glucose-grown cells (more than 7-log decrease in 18 min compared with 3-log for oleate-grown cells). Despite this difference in kinetics, the sequence of events was similar for oleate-grown cells maintained at 50°C with a (1) loss of ability to form colonies at 27°C, (2) loss of membrane integrity and (3) lysis (observed only for some minimal-oleate-grown cells). Glucose-grown cells underwent different changes. Their membranes, which were less fluid, lost their integrity as well and cells were rapidly inactivated. But, surprisingly, their nuclear DNA was not stained by propidium iodide and other cationic fluorescent DNA-specific probes but became stainable by hydrophobic ones. Moreover, they underwent a dramatic increase in membrane viscosity. The evolution of lipid bodies during the heat shock depended also on the growth medium. In glucose-grown cells, they seemed to coalesce with the nuclear membrane whereas for oleate-grown cells, they coalesced together forming big droplets which could be released in the medium. In some rare cases of oleate-grown cells, lipid bodies were fragmented and occupied all the cell volume. These results show that heat triggers programmed cell death with uncommon hallmarks for glucose-grown cells and necrosis for methyl-oleate-grown cells. PMID:21863313

Ta, Thi Minh Ngoc; Cao-Hoang, Lan; Romero-Guido, Cynthia; Lourdin, Morgane; Phan-Thi, Hanh; Goudot, Sébastien; Marechal, Pierre-André; Waché, Yves

2012-03-01

290

Perifosine induces protective autophagy and upregulation of ATG5 in human chronic myelogenous leukemia cells in vitro  

PubMed Central

Aim: The efficacy of the Akt inhibitor perifosine against chronic myeloid leukemia (CML) cells and its mechanisms of action are unknown. In this study, the cytotoxic effects of perifosine on CML and acute myeloid leukemia (AML) cell lines were compared to elucidate the mechanisms underlying the differences. Methods: Human AML cell lines Kasumi-1 and HL-60, and the CML cell line K562 were used. Cell viability was quantitated using MTT assay. Apoptosis was determined using Annexin V-FITC/propidium iodide and Hoechst staining, which were followed by flow cytometry and fluorescence microscopy analysis, respectively. Caspase pathway activation and the expression of autophagy-related genes were examined using Western blot. Autophagy was studied using electron microscopy, the acridine orange staining method, and GFP-LC3 was examined with fluorescence microscopy. Results: In contrast to AML cell lines, the CML cell lines K562 and K562/G (an imatinib-insensitive CML cell line) were resistant to perifosine (2.5–20 ?mol/L) in respect to inhibiting cell growth and inducing apoptosis. Perifosine (2.5, 5, and 10 ?mol/L) inhibited Akt and its phosphorylation in AML cells, but not in CML cells. Treatment with perifosine (20 ?mol/L) resulted in autophagy in CML cells as shown by the increased formation of acidic vesicular organelles and the accumulation of LC3-II. Treatment of CML cells with perifosine (5, 10, and 20 ?mol/L) dose-dependently upregulated AGT5, but not Beclin 1 at the protein level. Furthermore, inhibition of autophagy by chloroquine (40 nmol/L) significantly suppressed the cell growth and induced apoptosis in CML cells treated with perifosine (20 ?mol/L). Conclusion: Our results show that CML cell lines were resistant to the Akt inhibitor perifosine in vitro, which is due to perifosine-induced protective autophagy and upregulation of ATG5. PMID:22407228

Tong, Yin; Liu, Yan-yan; You, Liang-shun; Qian, Wen-bin

2012-01-01

291

Selective tumor cell killing by triptolide in p53 wild-type and p53 mutant ovarian carcinomas.  

PubMed

Triptolide is a traditional Chinese medicinal herb-derived antineoplastic agent. However, its antitumor activity against gynecologic carcinomas has not yet been well described. It is the purpose of this article to investigate the effect and mechanism of triptolide in human ovarian cancer using both A2780 (p53 wild) and OVCAR-3 (p53 mutated) cells. Our results showed that triptolide exerted a potent inhibitory effect on the growth and proliferation of both cell lines in a dose- and time-dependent manner and that the effect was independent of the expression of p53. In contrast, triptolide had only a marginal cytotoxicity in noncancerous ovary cells, lung fibroblast cells, and macrophage cells, indicating differential inhibitory effects of the drug on cell growth between ovarian cancer cells and normal tissue cells. Exposure of the ovarian cancer cells to triptolide induced apoptosis, as evaluated by annexin V/propidium iodide-labeled flow cytometry. Triptolide-induced apoptosis was accompanied by cytochrome c release and caspase-3 activation and was associated with downregulation of Bcl-2 and upregulation of Bax. Cell cycle analysis demonstrated that treatment with triptolide induced cell cycle S phase arrest in A2780 cells and G2/M phase arrest in OVCAR-3 cells. Further detection by Western blotting revealed that the cell cycle arrest by triptolide in both cell lines occurred in concert with increased expression of p21(CIP1/WAF1). This study shows that triptolide selectively kills ovarian cancer cells with different p53 status predominantly through regulating the coordinate and dynamic cellular processes of proliferation and apoptosis, thereby making it a promising chemotherapeutic agent against a broad spectrum of ovarian carcinomas. PMID:24880464

Wu, Jianyuan; Li, Qingdi Quentin; Zhou, Huiping; Lu, Yinying; Li, Jueli M; Ma, Yao; Wang, Li; Fu, Tingting; Gong, Xingjiang; Weintraub, Michael; Wu, Shuangchan; Ding, Hong

2014-07-01

292

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

PubMed Central

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

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

2013-01-01

293

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

NASA Astrophysics Data System (ADS)

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

Ramírez, Gustavo; Edwards, Katrina

2014-05-01

294

Media Effects on Nitrosomonas Europaea Monochloramine Disinfection Kinetics Using Propidium Monoazide Quantitative Real-time PCR  

EPA Science Inventory

Monochloramine use as a secondary disinfectant in the United States is predicted to increase to 57% of all surface and 7% of all ground water systems. With monochloramine addition, there is a risk of nitrification in the distribution system by ammonia-oxidizing bacteria (AOB). Ni...

295

Media Effects on Nitrosomonas Europaea Monochloramine Disinfection Kinetics Using Propidium Monoazide Quantitative Real-time PCR  

EPA Science Inventory

Monochloramine use as a secondary disinfectant in the United States is predicted to increase to 57% of all surface and 7% of all ground water systems. With monochloramine addition, there is a risk of nitrification in the distribution system by ammonia-oxidizing bacteria (AOB). Ba...

296

Media effects on Nitrosomonas Europaea Monochloramine Disinfection Kinetics using Propidium Monoazide Quantitative Real-time PCR  

EPA Science Inventory

Monochloramine use as a secondary disinfectant in the United States is predicted to increase to 57% of all surface and 7% of all ground water systems. With monochloramine addition, there is a risk of nitrification in the distribution system by ammonia-oxidizing bacteria (AOB). Ba...

297

Removal of Free Extracellular DNA from Environmental Samples by Ethidium Monoazide and Propidium Monoazide  

Microsoft Academic Search

For pathogen risk assessment and hygienic safety control in anaerobic digesters, various culture-based microbiological meth- ods are in use. However, with the application of classical methods, a number of problems arise: long cultivation times for some mi- croorganisms, the complexity of anaerobic cultivation, and time- consuming lab work (enrichment of selected organisms, selective cultivation, and subsequent systematic differentiation). Alterna- tively,

Andreas O. Wagner; Cornelia Malin; Brigitte A. Knapp; Paul Illmer

2008-01-01

298

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

299

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

300

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

PubMed Central

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

Samarghandian, Saeed; Shabestari, Mahmoud M

2013-01-01

301

Neuropeptide Y receptors activation protects rat retinal neural cells against necrotic and apoptotic cell death induced by glutamate.  

PubMed

It has been claimed that glutamate excitotoxicity might have a role in the pathogenesis of several retinal degenerative diseases, including glaucoma and diabetic retinopathy. Neuropeptide Y (NPY) has neuroprotective properties against excitotoxicity in the hippocampus, through the activation of Y1, Y2 and/or Y5 receptors. The principal objective of this study is to investigate the potential protective role of NPY against glutamate-induced toxicity in rat retinal cells (in vitro and in an animal model), unraveling the NPY receptors and intracellular mechanisms involved. Rat retinal neural cell cultures were prepared from newborn Wistar rats (P3-P5) and exposed to glutamate (500 ?M) for 24 h. Necrotic cell death was evaluated by propidium iodide (PI) assay and apoptotic cell death using TUNEL and caspase-3 assays. The cell types present in culture were identified by immunocytochemistry. The involvement of NPY receptors was assessed using selective agonists and antagonists. Pre-treatment of cells with NPY (100 nM) inhibited both necrotic cell death (PI-positive cells) and apoptotic cell death (TUNEL-positive cells and caspase 3-positive cells) triggered by glutamate, with the neurons being the cells most strongly affected. The activation of NPY Y2, Y4 and Y5 receptors inhibited necrotic cell death, while apoptotic cell death was only prevented by the activation of NPY Y5 receptor. Moreover, NPY neuroprotective effect was mediated by the activation of PKA and p38K. In the animal model, NPY (2.35 nmol) was intravitreally injected 2 h before glutamate (500 nmol) injection into the vitreous. The protective role of NPY was assessed 24 h after glutamate (or saline) injection by TUNEL assay and Brn3a (marker of ganglion cells) immunohistochemistry. NPY inhibited the increase in the number of TUNEL-positive cells and the decrease in the number of Brn3a-positive cells induced by glutamate. In conclusion, NPY and NPY receptors can be considered potential targets to treat retinal degenerative diseases, such as glaucoma and diabetic retinopathy. PMID:23681231

Santos-Carvalho, A; Elvas, F; Alvaro, A R; Ambrósio, A F; Cavadas, C

2013-01-01

302

Neuropeptide Y receptors activation protects rat retinal neural cells against necrotic and apoptotic cell death induced by glutamate  

PubMed Central

It has been claimed that glutamate excitotoxicity might have a role in the pathogenesis of several retinal degenerative diseases, including glaucoma and diabetic retinopathy. Neuropeptide Y (NPY) has neuroprotective properties against excitotoxicity in the hippocampus, through the activation of Y1, Y2 and/or Y5 receptors. The principal objective of this study is to investigate the potential protective role of NPY against glutamate-induced toxicity in rat retinal cells (in vitro and in an animal model), unraveling the NPY receptors and intracellular mechanisms involved. Rat retinal neural cell cultures were prepared from newborn Wistar rats (P3-P5) and exposed to glutamate (500??M) for 24?h. Necrotic cell death was evaluated by propidium iodide (PI) assay and apoptotic cell death using TUNEL and caspase-3 assays. The cell types present in culture were identified by immunocytochemistry. The involvement of NPY receptors was assessed using selective agonists and antagonists. Pre-treatment of cells with NPY (100?nM) inhibited both necrotic cell death (PI-positive cells) and apoptotic cell death (TUNEL-positive cells and caspase 3-positive cells) triggered by glutamate, with the neurons being the cells most strongly affected. The activation of NPY Y2, Y4 and Y5 receptors inhibited necrotic cell death, while apoptotic cell death was only prevented by the activation of NPY Y5 receptor. Moreover, NPY neuroprotective effect was mediated by the activation of PKA and p38K. In the animal model, NPY (2.35?nmol) was intravitreally injected 2?h before glutamate (500?nmol) injection into the vitreous. The protective role of NPY was assessed 24?h after glutamate (or saline) injection by TUNEL assay and Brn3a (marker of ganglion cells) immunohistochemistry. NPY inhibited the increase in the number of TUNEL-positive cells and the decrease in the number of Brn3a-positive cells induced by glutamate. In conclusion, NPY and NPY receptors can be considered potential targets to treat retinal degenerative diseases, such as glaucoma and diabetic retinopathy. PMID:23681231

Santos-Carvalho, A; Elvas, F; Álvaro, A R; Ambrósio, A F; Cavadas, C

2013-01-01

303

Photic Injury to Cultured RPE Varies Among Individual Cells in Proportion to Their Endogenous Lipofuscin Content as Modulated by Their Melanosome Content  

PubMed Central

Purpose. We determined whether photic stress differentially impairs organelle motility of RPE lipofuscin and melanin granules, whether lethal photic stress kills cells in proportion to lipofuscin abundance, and whether killing is modulated by melanosome content. Methods. Motility of endogenous lipofuscin and melanosome granules within the same human RPE cells in primary culture was quantified by real-time imaging during sublethal blue light irradiation. Cell death during lethal irradiation was quantified by dynamic imaging of the onset of nuclear propidium iodide fluorescence. Analyzed were individual cells containing different amounts of autofluorescent lipofuscin, or similar amounts of lipofuscin and a varying content of phagocytized porcine melanosomes, or phagocytized black latex beads (control for light absorbance). Results. Lipofuscin granules and melanosomes showed motility slowing with mild irradiation, but slowing was greater for lipofuscin. On lethal irradiation, cell death was earlier in cells with higher lipofuscin content, but delayed by the copresence of melanosomes. Delayed death did not occur with black beads, suggesting that melanosome protection was due to properties of the biological granule, not simple screening. Conclusions. Greater organelle motility slowing of the more photoreactive lipofuscin granule compared to melanosomes suggests that lipofuscin mediates mild photic injury within RPE cells. With lethal light stress endogenous lipofuscin mediates killing, but the effect is cell autonomous and modulated by coincident melanosome content. Developing methods to quantify the frequency of individual cells with combined high lipofuscin and low melanosome content may have value for predicting the photic stress susceptibility of the RPE monolayer in situ. PMID:25034597

Zareba, Mariusz; Skumatz, Christine M. B.; Sarna, Tadeusz J.; Burke, Janice M.

2014-01-01

304

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

305

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

PubMed Central

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

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

2011-01-01

306

Enhancement of Raman Light Scattering in Dye-Labeled Rat Glioma Cells by Langmuir-Blodgett CNT-Bundles Arranged on Metal-Containing Conducting Polymer Film  

E-print Network

We have fabricated layered nanocomposite consisting of a nanoporous anodic alumina sublayer (AOA), an ultrathin metal-containing polymer Langmuir-Blodgett (LB) film coating AOA, and multi-walled carbon nanotube (MCNT) - bundles which are arranged on the LB-film. MCNTs were preliminarily chemically modified by carboxyl groups and functionalized by stearic acid. We have experimentally observed an enhancement of Raman light scattering on surface plasmons in the LB-monolayers. This enhancement is due to charge and energy transfer. We demonstrate that propidium iodide (PI) fluorescence is quenched by the MCNT-bundles. A method of two-dimensional system imaging based on the MCNT-enhanced Raman spectroscopy has been proposed. This method has been applied to visualize focal adhesion sites on membranes of living PI-labeled rat glioma cells.

Egorov, A S; Grushevskaya, H V; Krot, V I; Krylova, N G; Lipnevich, I V; Orekhovskaya, T I; Shulitsky, B G

2015-01-01

307

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

308

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

309

Endoplasmic reticulum stress sensitizes human esophageal cancer cell to radiation  

PubMed Central

AIM: To investigate the role of endoplasmic reticulum (ER) stress in cancer radiotherapy and its molecular mechanism. METHODS: Tunicamycin (TM) was applied to induce ER stress in human esophageal cancer cell line EC109, and the radiosensitization effects were detected by acute cell death and clonogenic survival assay. Cell cycle arrest induced by TM was determined by flow cytometric analysis after the cellular DNA content was labeled with propidium iodide. Apoptosis of EC109 cells induced by TM was detected by annexin V staining and Western blotting of caspase-3 and its substrate poly ADP-ribose polymerase. Autophagic response was determined by acridine orange (AO) staining and Western blotting of microtubule-associated protein-1 light chain-3 (LC3) and autophagy related gene 5 (ATG5). In order to test the biological function of autophagy, specific inhibitor or Beclin-1 knockdown was used to inhibit autophagy, and its effect on cell apoptosis was thus detected. Additionally, involvement of the phosphatidylinositol-3 kinase (PI3K)/Akt/mammalian target of the rapamycin (mTOR) pathway was also detected by Western blotting. Finally, male nude mice inoculated subcutaneously with EC109 cells were used to confirm cell model observations. RESULTS: Our results showed that TM treatment enhanced cell death and reduced the colony survival fraction induced by ionizing radiation (IR), which suggested an obvious radiosensitization effect of TM. Moreover, TM and IR combination treatment led to a significant increase of G2/M phase and apoptotic cells, compared with IR alone. We also observed an increase of AO positive cells, and the protein level of LC3-II and ATG5 was induced by TM treatment, which suggested an autophagic response in EC109 cells. However, inhibition of autophagy by using a chemical inhibitor or Beclin-1 silencing led to increased cell apoptosis and decreased cell viability, which suggested a cytoprotective role of autophagy in stressed EC109 cells. Furthermore, TM treatment also activated mTORC1, and in turn reduced Akt phosphorylation, which suggested the PI3K/Akt/mTOR signal pathway was involved in the TM-induced autophagic response in EC109 cells. Tumor xenograft results also showed synergistic retarded tumor growth by TM treatment and IR, as well as the involvement of the PI3K/Akt/mTOR pathway. CONCLUSION: Our data showed that TM treatment sensitized human esophageal cancer cells to radiation via apoptosis and autophagy both in vitro and in vivo. PMID:23555162

Pang, Xue-Li; He, Gang; Liu, Yang-Bo; Wang, Yan; Zhang, Bo

2013-01-01

310

Plumbagin inhibits cell growth and potentiates apoptosis in human gastric cancer cells in vitro through the NF-?B signaling pathway  

PubMed Central

Aim: To investigate the effects and underlying mechanisms of plumbagin, a naphthoquinone derived from medicinal plant Plumbago zeylanica, on human gastric cancer (GC) cells. Methods: Human gastric cancer cell lines SGC-7901, MKN-28, and AGS were used. The cell viability was examined using CCK-8 viability assay. Cell proliferation rate was determined using both clonogenic assay and EdU incorporation assay. Apoptosis was detected via Annexin V/propidium iodide double-labeled flow cytometry. Western blotting was used to assess the expression of both NF-?B-regulated gene products and TNF-?-induced activation of p65, I?B?, and IKK. The intracellular location of NF-?B p65 was detected using confocal microscopy. Results: Plumbagin (2.5–40 ?mol/L) concentration-dependently reduced the viability of the GC cells. The IC50 value of plumbagin in SGC-7901, MKN-28, and AGS cells was 19.12, 13.64, and 10.12 ?mol/L, respectively. The compound (5–20 ?mol/L) concentration-dependently induced apoptosis of SGC-7901 cells, and potentiated the sensitivity of SGC-7901 cells to chemotherapeutic agents TNF-?and cisplatin. The compound (10 ?mol/L) downregulated the expression of NF-?B-regulated gene products, including IAP1, XIAP, Bcl-2, Bcl-xL, tumor factor (TF), and VEGF. In addition to inhibition of NF-?B p65 nuclear translocation, the compound also suppressed TNF-?-induced phosphorylation of p65 and IKK, and the degradation of I?B?. Conclusion: Plumbagin inhibits cell growth and potentiates apoptosis in human GC cells through the NF-?B pathway. PMID:22231395

Li, Jing; Shen, Lin; Lu, Fu-rong; Qin, You; Chen, Rui; Li, Jia; Li, Yan; Zhan, Han-zi; He, Yuan-qiao

2012-01-01

311

Glypican and Biglycan in the Nuclei of Neurons and Glioma Cells: Presence of Functional Nuclear Localization Signals and Dynamic Changes in Glypican During the Cell Cycle  

PubMed Central

We have investigated the expression patterns and subcellular localization in nervous tissue of glypican, a major glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan that is predominantly synthesized by neurons, and of biglycan, a small, leucine-rich chondroitin sulfate proteoglycan. By laser scanning confocal microscopy of rat central nervous tissue and C6 glioma cells, we found that a significant portion of the glypican and biglycan immunoreactivity colocalized with nuclear staining by propidium iodide and was also seen in isolated nuclei. In certain regions, staining was selective, insofar as glypican and biglycan immunoreactivity in the nucleus was seen predominantly in a subpopulation of large spinal cord neurons. The amino acid sequences of both proteoglycans contain potential nuclear localization signals, and these were demonstrated to be functional based on their ability to target ?-galactosidase fusion proteins to the nuclei of transfected 293 cells. Nuclear localization of glypican ?-galactosidase or Fc fusion proteins in transfected 293 cells and C6 glioma cells was greatly reduced or abolished after mutation of the basic amino acids or deletion of the sequence containing the nuclear localization signal, and no nuclear staining was seen in the case of heparan sulfate and chondroitin sulfate proteoglycans that do not possess a nuclear localization signal, such as syndecan-3 or decorin (which is closely related in structure to biglycan). Transfection of COS-1 cells with an epitope-tagged glypican cDNA demonstrated transport of the full-length proteoglycan to the nucleus, and there are also dynamic changes in the pattern of glypican immunoreactivity in the nucleus of C6 cells both during cell division and correlated with different phases of the cell cycle. Our data therefore suggest that in certain cells and central nervous system regions, glypican and biglycan may be involved in the regulation of cell division and survival by directly participating in nuclear processes. PMID:9362504

Liang, Yu; Häring, Monika; Roughley, Peter J.; Margolis, Renée K.; Margolis, Richard U.

1997-01-01

312

[Flow cytometric analysis using live/dead staining for yeast cells which were autoclaved and treated with various disinfectants].  

PubMed

Flow cytometric analysis using live/dead staining dyes was applied to discriminate yeast cells treated with autoclave and various disinfectants. One fluorogenic dye, propidium iodide cannot leak into live cells with intact membranes, whereas another fluorogenic dye, thiazole orange is permeant and can enter all cells, live and dead. Thus a combination of these two dyes can classify cell populations into live, injured and dead cells. When the yeast cells were autoclaved at 121 degrees C for 5 min, almost all the cells were classified as being dead, but some yeast strains contained a little injured cells (up to 8.5%). It was also demonstrated that none of the yeast cells autoclaved could produce a visible colony on the agar plate. The 70% ethylalcohol was the most effective disinfectant tested. Almost all the yeast cells treated were classified as being dead, and no visible colony appeared after agar plate incubation. The cell suspensions of yeast treated with 0.5% chlorhexidine contained injured cells with >95%, but no visible colony appeared. Whereas, the numbers of live yeast cells after treatment with 0.025% benzalkonium chloride were less than detectable counts (<1.0%) by flow cytometry, however, visible colonies appeared on the agar plate with 10(1) to 3.6 x 10(3) colony forming units/ml. With these results, it became apparent that the combination with fluorogenic dyes and flow cytometry can provide a reliable test method to discriminate dead and/or injured cells from the reproductive live yeast cells, and is applicable for further studies. However, it should be noted that minimum detectable levels are sometimes far different between the standard colony forming assay and the flow cytometric determination. PMID:17441466

Tokashiki, Yoshino T; Tanokuchi, Yuhko; Nakasone, Isamu; Yamane, Nobuhisa

2007-03-01

313

p-Cresol Affects Reactive Oxygen Species Generation, Cell Cycle Arrest, Cytotoxicity and Inflammation/Atherosclerosis-Related Modulators Production in Endothelial Cells and Mononuclear Cells  

PubMed Central

Aims Cresols are present in antiseptics, coal tar, some resins, pesticides, and industrial solvents. Cresol intoxication leads to hepatic injury due to coagulopathy as well as disturbance of hepatic circulation in fatal cases. Patients with uremia suffer from cardiovascular complications, such as atherosclerosis, thrombosis, hemolysis, and bleeding, which may be partly due to p-cresol toxicity and its effects on vascular endothelial and mononuclear cells. Given the role of reactive oxygen species (ROS) and inflammation in vascular thrombosis, the objective of this study was to evaluate the effect of p-cresol on endothelial and mononuclear cells. Methods EA.hy926 (EAHY) endothelial cells and U937 cells were exposed to different concentrations of p-cresol. Cytotoxicity was evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5 -diphenyltetrazolium bromide (MTT) assay and trypan blue dye exclusion technique, respectively. Cell cycle distribution was analyzed by propidium iodide flow cytometry. Endothelial cell migration was studied by wound closure assay. ROS level was measured by 2?,7?-dichlorofluorescein diacetate (DCF) fluorescence flow cytometry. Prostaglandin F2? (PGF2?), plasminogen activator inhibitor-1 (PAI-1), soluble urokinase plasminogen activator receptor (suPAR), and uPA production were determined by Enzyme-linked immunosorbant assay (ELISA). Results Exposure to 100–500 µM p-cresol decreased EAHY cell number by 30–61%. P-cresol also decreased the viability of U937 mononuclear cells. The inhibition of EAHY and U937 cell growth by p-cresol was related to induction of S-phase cell cycle arrest. Closure of endothelial wounds was inhibited by p-cresol (>100 µM). P-cresol (>50 µM) also stimulated ROS production in U937 cells and EAHY cells but to a lesser extent. Moreover, p-cresol markedly stimulated PAI-1 and suPAR, but not PGF2?, and uPA production in EAHY cells. Conclusions p-Cresol may contribute to atherosclerosis and thrombosis in patients with uremia and cresol intoxication possibly due to induction of ROS, endothelial/mononuclear cell damage and production of inflammation/atherosclerosis-related molecules. PMID:25517907

Chan, Chiu-Po; Yeung, Sin-Yuet; Hsien, Hsiang-Chi; Lin, Bor-Ru; Yeh, Chien-Yang; Tseng, Wan-Yu; Tseng, Shui-Kuan; Jeng, Jiiang-Huei

2014-01-01

314

Luteolin acts as a radiosensitizer in non?small cell lung cancer cells by enhancing apoptotic cell death through activation of a p38/ROS/caspase cascade.  

PubMed

To improve radiation therapy, the development of effective radiosensitizer is required. Fifty percent inhibitory concentration (IC50) values of 3',4',5',7'?tetrahydroxyflavone (luteolin) against NCI?H460 and ?H1299 non?small cell lung cancer (NSCLC) cells were determined using 3?(4,5?dimethylthiazol?2?yl)?2,5?diphenyltetrazolium bromide (MTT) assays. Radiosensitizing activity was assessed in vitro by treating cells with luteolin prior to irradiation of ??ionizing radiation (IR), and performing cell count and clonogenic assays. Cell signaling pathways involved in the radiosensitizing effects of luteolin were examined using propidium iodide (PI) uptake, reactive oxygen species (ROS) detection and immunoblot assays, with or without specific chemical inhibitors. Apoptotic cell death was confirmed by PI uptake and immunoblot assays. In vivo radiosensitizing activity was tested using an NCI?H460 cell xenograft model in nude mice. Tumor size was measured and apoptosis was determined with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in animals in four treatment groups: mock?treated control, luteolin only, IR only, and luteolin plus IR. Treatment with luteolin or IR induced NSCLC cell death in vitro, but the combination of luteolin pre?treatment and IR was more effective than either agent alone, yielding dose enhancement ratios (DERs) of 1.22 and 1.35 for NCI?H460 and ?H1299 cells, respectively. Combined treatment with luteolin and IR enhanced apoptotic cell death in association with downregulation of B?cell lymphoma 2 (Bcl?2) and activation of caspase?3, ?8, and ?9; it also induced phosphorylation of p38 mitogen?activated protein kinase (MAPK) and ROS accumulation. Inhibition of p38 MAPK decreased ROS production, and inhibition of either p38 MAPK or ROS production attenuated apoptotic cell death and activation of caspase?8 and ?9. In a xenograft model, tumor growth was delayed by 21.8 days in the luteolin/IR combination group compared with controls, and apoptotic cell death was increased. The enhancement factor of the luteolin and IR combination was 1.83. Collectively, these findings indicate that luteolin acts as a radiosensitizer by enhancing apoptotic cell death through activation of a p38/ROS/caspase cascade. PMID:25586525

Cho, Hyun-Ji; Ahn, Kwang-Chul; Choi, Jae Yeon; Hwang, Sang-Gu; Kim, Wun-Jae; Um, Hong-Duck; Park, Jong Kuk

2015-03-01

315

Endothelial cell Blood cells  

E-print Network

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

Wilmers, Chris

316

Endoplasmic reticulum calcium release potentiates the ER stress and cell death caused by an oxidative stress in MCF-7 cells.  

PubMed

Increase in cytosolic calcium concentration ([Ca2+](c)), release of endoplasmic reticulum (ER) calcium ([Ca2+](er)) and ER stress have been proposed to be involved in oxidative toxicity. Nevertheless, their relative involvements in the processes leading to cell death are not well defined. In this study, we investigated whether oxidative stress generated during ascorbate-driven menadione redox cycling (Asc/Men) could trigger these three events, and, if so, whether they contributed to Asc/Men cytoxicity in MCF-7 cells. Using microspectrofluorimetry, we demonstrated that Asc/Men-generated oxidative stress was associated with a slow and moderate increase in [Ca2+](c), largely preceding permeation of propidium iodide, and thus cell death. Asc/Men treatment was shown to partially deplete ER calcium stores after 90 min (decrease by 45% compared to control). This event was associated with ER stress activation, as shown by analysis of eIF2 phosphorylation and expression of the molecular chaperone GRP94. Thapsigargin (TG) was then used to study the effect of complete [Ca2+](er) emptying during the oxidative stress generated by Asc/Men. Surprisingly, the combination of TG and Asc/Men increased ER stress to a level considerably higher than that observed for either treatment alone, suggesting that [Ca2+](er) release alone is not sufficient to explain ER stress activation during oxidative stress. Finally, TG-mediated [Ca2+](er) release largely potentiated ER stress, DNA fragmentation and cell death caused by Asc/Men, supporting a role of ER stress in the process of Asc/Men cytotoxicity. Taken together, our results highlight the involvement of ER stress and [Ca2+](er) decrease in the process of oxidative stress-induced cell death in MCF-7 cells. PMID:20006589

Dejeans, Nicolas; Tajeddine, Nicolas; Beck, Raphaël; Verrax, Julien; Taper, Henryk; Gailly, Philippe; Calderon, Pedro Buc

2010-05-01

317

CFTR modulates programmed cell death by decreasing intracellular pH in Chinese hamster lung fibroblasts.  

PubMed

To study the potential influence of cystic fibrosis conductance regulator (CFTR) on intracellular pH regulation during apoptosis induction, we used PS120 Chinese hamster lung fibroblasts devoid of the Na(+)/H(+) exchanger (NHE1 isoform) transfected with constructs, allowing the expression of CFTR and/or NHE1. Kinetics of lovastatin-induced apoptosis were measured by orcein staining, double staining with Hoechst-33258, propidium iodide, DNA fragmentation, and annexin V labeling. In PS120 control cells, the percentage of apoptotic cells after 40 h of lovastatin treatment was 23 +/- 3%, whereas in PS120 CFTR-transfected cells, this percentage was 40 +/- 4%. In PS120 NHE1 cells, the transfection with CFTR did not modify the percentage of apoptotic cells after 40 h (control: 19 +/- 3%, n = 8; CFTR: 17 +/- 1%, n = 8), indicating that blocking intracellular acidification by overexpressing the Na(+)/H(+) exchanger inhibited the enhancement of apoptosis induced by CFTR. In all cell lines, the initial pH values were identical (pH = 7.46 +/- 0.04, n = 9), and treatment with lovastatin led to intracellular acidification. However, the pH value after 40 h was lower in PS120 CFTR-transfected cells (pH = 6.85 +/- 0.02, n = 10) than in PS120 cells (pH = 7.15 +/- 0.03, n = 10). To further investigate the origin of this increased intracellular acidification observed in CFTR-transfected cells, the activity of the DIDS-inhibitable Cl(-)/HCO exchanger was studied. 8-Bromoadenosine 3',5'-cyclic monophosphate incubation resulted in Cl(-)/HCO exchanger activation in PS120 CFTR-transfected cells but had no effect on PS120 cells. Together, our results suggest that CFTR can enhance apoptosis in Chinese hamster lung fibroblasts, probably due to the modulation of the Cl(-)/HCO exchanger, resulting in a more efficient intracellular acidification. PMID:11502558

Barrière, H; Poujeol, C; Tauc, M; Blasi, J M; Counillon, L; Poujeol, P

2001-09-01

318

Effect of Paris saponin I on radiosensitivity in a gefitinib-resistant lung adenocarcinoma cell line.  

PubMed

Previous studies have observed that Paris saponin I (PSI) exerts a wide range of pharmacological activities, including cytotoxic activity against a number of malignancies, such as non-small cell lung cancers. The present study aimed to investigate the radiosensitization of PSI treatment on a gefitinib-resistant lung adenocarcinoma cell line, PC-9-ZD, and its possible mechanism. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay was used to determine the growth inhibition effect of PSI. A clonogenic assay was performed to determine the radiosensitizing effect of PSI treatment on the PC-9-ZD cell line. A single-hit multi-target model was used to plot survival curves and calculate sensitizing enhancement ratios. The cell cycle was analyzed by flow cytometry and cell apoptosis was analyzed with fluorescein-isothiocyanate-Annexin V/propidium iodide and Hoechst staining. The expression levels of the proteins were detected by western blotting. There was a significant reduction observed in the proliferation of the PC-9-ZD cell lines that were treated with PSI. PSI enhanced the radiosensitivity of the PC-9-ZD cells with a sensitization enhancement ratio of 1.77. Furthermore, PSI induced G2/M arrest and apoptosis of the irradiated PC-9-ZD cells. Notably, B-cell lymphoma 2 (Bcl-2) was downregulated, and caspase-3, Bcl-2-like protein 4 (Bax) and cyclin-dependent kinase inhibitor 1 (P21(waf1/cip1)) were upregulated by the PSI treatment. The present study showed that PSI treatment exhibited potent radiosensitivity against gefitinib-resistant PC-9-ZD cells in vitro. This radiosensitivity was associated with cell cycle arrest at the G2/M phase, and apoptosis via an increase in caspase-3, Bax and P21(waf1/cip1) as well as a decrease in Bcl-2 production. PMID:24932289

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

2014-06-01

319

Relation of Cumulus Cell Status with Single Oocyte Maturity, Fertilization Capability and Patient Age  

PubMed Central

Background The production of competent oocytes depends on a bi-directional communication between the oocyte and cumulus cells. The goal of this study was to determine whether simple parameters monitored in cumulus cells from individual human oocytes have any predictive value, and thus correlate with clinically relevant parameters. Methods 97 cumulus-oocyte complexes were recovered from 31 patients undergoing ICSI treatment. After the oocytes were denuded, cumulus cell density from individual oocytes was determined. Cells were probed for viability using propidium iodide and for apoptosis by Annexin V staining or by monitoring caspase activity. These parameters were correlated with oocyte status, fertilization ability and patient age (?29 years old and ?30 years old). All variables were checked for normal distribution and then compared by Kruskal-Wallis, Mann-Whitney or one-way ANOVA tests. Results Mature oocytes were surrounded by more cumulus cells (16073±2595, p = 0.026), which were also more viable and less apoptotic than atretic or degenerated oocytes. Mature oocytes that fertilized had higher caspase activity in the surrounding cumulus cells than those that did not fertilize. Younger patients presented lower cumulus cells density (8882±2380 vs. 15036±2143 cells; p = 0.034); and cumulus cells had higher apoptosis levels in younger patients than older ones (6775.5±1831.6 RLU vs. 2591±46.5 RLU, p = 0.002 for caspase activity). Conclusion The data suggests that high density and apoptosis of cumulus cells are promising parameters to indirectly predict individual oocyte status. Although more studies and a larger data set are needed, cumulus cells presented the potential to be used as simple predictors of female fertility and/or ovarian ageing. PMID:24696155

Lourenço, Bárbara; Sousa, Ana Paula; Almeida-Santos, Teresa; Ramalho-Santos, João

2014-01-01

320

Production of savinase and population viability of Bacillus clausii during high-cell-density fed-batch cultivations.  

PubMed

The growth and product formation of a Savinase-producing Bacillus clausii were investigated in high-cell-density fed-batch cultivations with both linear and exponential feed profiles. The highest specific productivity of Savinase was observed shortly after the end of the initial batch phase for all feed profiles applied and, in addition, there was a time-dependent decrease in specific productivity. The specific glucose uptake rate increased with time for constant specific growth rate indicating that the maintenance requirements increased with time, possibly due to a decreasing K(+) concentration. The physiological state of the cells was monitored during the cultivations using a flow cytometry assay based on the permeability of the cell membrane to propidium iodide. In the latter parts of the fed-batch cultures with a linear feed profile, a large portion of the cell population was found to have a permeable membrane, indicating a large percentage of dead cells. By assuming that only cells with a nonpermeable membrane contributed to growth and product formation, the physiological properties of this subpopulation were calculated. PMID:12783490

Christiansen, Torben; Michaelsen, Søren; Wümpelmann, Mogens; Nielsen, Jens

2003-08-01

321

Crocin Effects on Human Myeloma Cells Regarding Intracellular Redox State, DNA Fragmentation, and Apoptosis or Necrosis Profile  

PubMed Central

Background: Well-documented studies reported several pharmacological properties for crocin, the active compound of Crocus sativus, such as its antitumor, radical scavenging, antidepressant, and memory-enhancing effects. Objectives: We aimed to evaluate the possible cytotoxic activity of crocin on B lymphocytes in human myeloma (U266 cell line) after 24- and 48-hour treatment. Materials and Methods: For this purpose, cell viability was determined by the colorimetric MTT assay and cell death pattern was evaluated using Annexin V-FITC/propidium iodide (PI) apoptosis detection kit. ROS (reactive oxygen species) production and DNA fragmentation were assessed using 2?,7?-dichlorofluorescein diacetate (DCFH-DA) kit and PI staining, respectively. Results: The highest concentration of crocin significantly decreased ROS production after 48 hours of treatment. However, crocin had no effect on the expression level of HSP (Heat shock protein). Additionally, its administration caused a mild decline in cell viability and a mild increase in the population of DNA fragmented cells as well as apoptosis. Conclusions: In our study, no prominent effect was seen; therefore, in order to have a better perspective of crocin activity against cancerous cell lines, further studies are highly recommended.

Rezaee, Ramin; Jamialahmadi, Khadijeh; Riahi Zanjani, Bamdad; Mahmoudi, Mahmoud; Abnous, Khalil; Zamani Taghizadeh Rabe, Shahrzad; Tabasi, Nafiseh; Zali, Marjan; Rezaee, Marjan; Amin, Bahareh; Karimi, Gholamreza

2014-01-01

322

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

PubMed

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

Patel, Pinaki B; Thakkar, Vasudev R

2014-01-01

323

Novel Drug Delivery System Based on Docetaxel-Loaded Nanocapsules as a Therapeutic Strategy Against Breast Cancer Cells  

PubMed Central

In the field of cancer therapy, lipid nanocapsules based on a core-shell structure are promising vehicles for the delivery of hydrophobic drugs such as docetaxel. The main aim of this work was to evaluate whether docetaxel-loaded lipid nanocapsules improved the anti-tumor effect of free docetaxel in breast cancer cells. Three docetaxel-loaded lipid nanocapsules were synthesized by solvent displacement method. Cytotoxic assays were evaluated in breast carcinoma (MCF-7) cells treated by the sulforhodamine B colorimetric method. Cell cycle was studied by flow cytometry and Annexin V-FITC, and apoptosis was evaluated by using propidium iodide assays. The anti-proliferative effect of docetaxel appeared much earlier when the drug was encapsulated in lipid nanoparticles than when it was free. Docetaxel-loaded lipid nanocapsules significantly enhanced the decrease in IC50 rate, and the treated cells evidenced apoptosis and a premature progression of the cell cycle from G(1) to G(2)-M phase. The chemotherapeutic effect of free docetaxel on breast cancer cells is improved by its encapsulation in lipid nanocapsules. This approach has the potential to overcome some major limitations of conventional chemotherapy and may be a promising strategy for future applications in breast cancer therapy. PMID:22606019

Sánchez-Moreno, Paola; Boulaiz, Houria; Ortega-Vinuesa, Juan Luis; Peula-García, José Manuel; Aránega, Antonia

2012-01-01

324

Cell adhesive and growth behavior on electrospun nanofibrous scaffolds by designed multifunctional composites.  

PubMed

Nanostructured biocomposite scaffolds of poly(l-lactide) (PLLA) blended with collagen (coll) or hydroxyapatite (HA), or both for tissue engineering application, were fabricated by electrospinning. The electrospun scaffolds were characterized for the morphology, chemical and tensile properties by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), water contact angle (WCA), Fourier transform infrared (FTIR) measurement, and tensile testing. Electrospun biocomposite scaffolds of PLLA and collagen or (and) HA in the diameter range of 200-700 nm mimic the nanoscale structure of the extracellular matrix (ECM) with a well-interconnection pore network structure. The presence of collagen in the scaffolds increased their hydrophility, and enhanced cell attachment and proliferation, while HA improved the tensile properties of the scaffolds. The biocompatibility of the electrospun scaffolds and the viability of contacting cells were evaluated by 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) nuclear staining and by fluorescein diacetate (FDA) and propidium iodide (PI) double staining methods. The results support the conclusion that 293T cells grew well on composite scaffolds. Compared with pure PLLA scaffolds a greater density of viable cells was seen on the composites, especially the PLLA/HA/collagen scaffolds. PMID:21227659

Cao, Ding; Wu, Yi-Pan; Fu, Zhi-Feng; Tian, Yuan; Li, Cong-Ju; Gao, Chun-Ying; Chen, Zhong-Liang; Feng, Xi-Zeng

2011-05-01

325

Neuroprotective effects of bovine colostrum on intracerebral hemorrhage-induced apoptotic neuronal cell death in rats?  

PubMed Central

Brain cell death after intracerebral hemorrhage may be mediated in part by an apoptotic mechanism. Colostrum is the first milk produced by mammals for their young. It plays an important role in protection and development by providing various antibodies, growth factors and nutrients, and has been used for various diseases in many countries. In the present study, we investigated the anti-apoptotic effects of bovine colostrum using organotypic hippocampal slice cultures and an intracerebral hemorrhage animal model. We performed densitometric measurements of propidium iodide uptake, a step-down avoidance task, Nissl staining, and caspase-3 immunohistochemistry. The present results revealed that colostrum treatment significantly suppressed N-methyl-D-aspartic acid-induced neuronal cell death in the rat hippocampus. Moreover, colostrum treatment improved short-term memory by suppressing hemorrhage-induced apoptotic neuronal cell death and decreasing the volume of the lesion induced by intracerebral hemorrhage in the rat hippocampus. These results suggest that colostrum may have a beneficial role in recovering brain function following hemorrhagic stroke by suppressing apoptotic cell death.

Kim, Sung Eun; Ko, Il Gyu; Shin, Mal Soon; Kim, Chang Ju; Ko, Young Gwan; Cho, Hanjin

2012-01-01

326

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

PubMed

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

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

2014-05-01

327

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

PubMed

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

Marczak, Agnieszka; Bukowska, Barbara; Rogalska, Aneta

2014-01-01

328

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

PubMed

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

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

2012-01-01

329

Progress in understanding preferential detection of live cells using viability dyes in combination with DNA amplification.  

PubMed

The ideal scenario in most applications of microbial diagnostics is that only viable cells are detected. Bacteria were traditionally considered viable when they could be cultured, whereas today's viability concept tends to be alternatively based on the presence of some form of metabolic activity, a positive energy status, responsiveness, detection of RNA transcripts that tend to degrade rapidly after cell death, or of an intact membrane. The latter criterion, although conservative, was the focus of one of the most successful recent approaches to detect viable cells in combination with DNA amplification techniques. The technology is based on sample treatment with the photoactivatable, and cell membrane impermeant, nucleic acid intercalating dyes ethidium monoazide (EMA) or propidium monoazide (PMA) followed by light exposure prior to extraction of DNA and amplification. Light activation of DNA-bound dye molecules results in irreversible DNA modification and subsequent inhibition of its amplification. Sample pretreatment with viability dyes has so far been mainly used in combination with PCR (leading to the term viability PCR, v-PCR), and increasingly with isothermal amplification method. The principle is not limited to bacteria, but has also successfully been applied to fungi, protozoa and viruses. Despite the success of the method, some practical limitations have been identified, especially when applied to environmental samples. In part they can be minimized by choice of experimental parameters and conditions adequate for a particular sample. This review summarizes current knowledge and presents aspects which are important when designing experiments employing viability dyes. PMID:22940102

Fittipaldi, Mariana; Nocker, Andreas; Codony, Francesc

2012-11-01

330

Enhanced apoptotic effects of dihydroartemisinin-aggregated gelatin and hyaluronan nanoparticles on human lung cancer cells.  

PubMed

Recent studies suggest that dihydroartemisinin (DHA), a derivative of artemisinin isolated from the traditional Chinese herb Artemisia annua L., has anticancer properties. Due to poor water solubility, poor oral activity, and a short plasma half-life, large doses of DHA have to be injected to achieve the necessary bioavailability. This study examined increasing DHA bioavailability by encapsulating DHA within gelatin (GEL) or hyaluronan (HA) nanoparticles via an electrostatic field system. Observations from transmission electron microscopy show that DHA in GEL and HA nanoparticles formed GEL/DHA and HA/DHA aggregates that were approximately 30-40 nm in diameter. The entrapment efficiencies for DHA were approximately 13 and 35% for the GEL/DHA and HA/DHA aggregates, respectively. The proliferation of A549 cells was inhibited by the GEL/DHA and HA/DHA aggregates. Fluorescent annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining displayed low background staining with annexin V-FITC or PI on DHA-untreated cells. In contrast, annexin V-FITC and PI stains dramatically increased when the cells were incubated with GEL/DHA and HA/DHA aggregates. These results suggest that DHA-aggregated GEL and HA nanoparticles exhibit higher anticancer proliferation activities than DHA alone in A549 cells most likely due to the greater aqueous dispersion after hydrophilic GEL or HA nanoparticles aggregation. These results demonstrate that DHA can aggregate with nanoparticles in an electrostatic field environment to form DHA nanosized aggregates. PMID:24039154

Sun, Qian; Teong, Benjamin; Chen, I-Fen; Chang, Shwu Jen; Gao, Jimin; Kuo, Shyh-Ming

2014-04-01

331

Immunological approach in the evaluation of regional lymph nodes of patients with squamous cell carcinoma of the head and neck.  

PubMed

In cancer, regional lymph node (LN) cells are one of the first components of the immune system to have contact with tumor cells or their products. Therefore, the phenotype and functional properties of hematopoietic cells present within the tumor-draining LN are important to understanding their role in the control of malignant cells. Based on the locoregional metastatic behavior of squamous cell carcinoma of the head and neck (SCCH&N) region, we analyzed tumor-draining lymph nodes from SCCH&N patients to obtain insights into regional tumor immunity. Using a three-color fluorescent labeling technique, surface antigen expression was visualized in mononuclear cells of lymph nodes that were obtained from head and neck cancer patients and compared to mononuclear cells of normal lymph nodes. Cell cycle analyses were performed using propidium iodide. Proliferation after phytohemagglutinin stimulation was measured by a sodium tetrazolium-based assay. LN histology was correlated with flow cytometric findings. Regional lymph nodes of head and neck cancer patients undergo morphologic and functional changes. Flow cytometry revealed a decrease in CD8(+) T cells and in some lymph nodes the presence of second or third populations of larger cells with distinct size and granularity that expressed both T (gammadelta/alphabeta) and different natural killer cell markers. Moreover, cell cycle analyses and proliferation assays showed a diminished response to mitogenic stimuli. These changes were found in both metastatic and hyperplastic lymph nodes from head and neck cancer patients; however, no alterations were found in control lymph nodes or peripheral blood mononuclear cells from noncancer patients. The immune alterations detected in lymphocytes present within the draining lymph nodes of head and neck cancer patients may improve our understanding of how tumor cells escape host immunosurveillance. However, this dysfunction in local draining lymph nodes may not be detected systemically. PMID:11781066

Verastegui, Emma; Morales, Rocio; Barrera, José Luis; Müeller, Anja; Guzman, Beatriz; Meneses, Abelardo; Alfaro, Guillermo

2002-01-01

332

Maxadilan Prevents Apoptosis in iPS Cells and Shows No Effects on the Pluripotent State or Karyotype  

PubMed Central

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a structurally endogenous peptide with many biological roles. Maxadilan, a 61-amino acid vasodilatory peptide, specifically activates the PACAP type I receptor (PAC1). Although PAC1 has been identified in embryonic stem cells, little is known about its presence or effects in human induced pluripotent stem (iPS) cells. In the present study, we investigated the expression of PAC1 in human iPS cells by reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analysis. To study the physiological effects mediated by PAC1, we evaluated the role of maxadilan in preventing apoptotic cell death induced by ultraviolet C (UVC). After exposure to UVC, the iPS cells showed a marked reduction in cell viability and a parallel increase of apoptotic cells, as demonstrated by WST-8 analysis, annexin V/propidium iodide (PI) analysis and the terminal transferase dUTP nick end labeling (TUNEL) assay. The addition of 30 nM of maxadilan dramatically increased iPS cell viability and reduced the percentage of apoptotic cells. The anti-apoptotic effects of maxadilan were correlated to the downregulation of caspase-3 and caspase-9. Concomitantly, immunofluorescence, western blot analysis, real-time quantitative polymerase chain reaction (RT-qPCR) analysis and in vitro differentiation results showed that maxadilan did not affect the pluripotent state of iPS cells. Moreover, karyotype analysis showed that maxadilan did not affect the karyotype of iPS cells. In summary, these results demonstrate that PAC1 is present in iPS cells and that maxadilan effectively protects iPS cells against UVC-induced apoptotic cell death while not affecting the pluripotent state or karyotype. PMID:22457805

Zhao, Zhiyi; Yu, Rongjie; Yang, Jiayin; Liu, Xiaofei; Tan, Meihua; Li, HongYang; Chen, Jiansu

2012-01-01

333

Cell cycle regulatory factors in juxta-tumoral renal parenchyma.  

PubMed

The aim of this study was to evaluate regulatory cell cycle factors in juxta-tumoral renal parenchyma in order to obtain information regarding early primary changes occurred in normal renal cells. Specimens of juxta-tumoral renal parenchyma were harvested from the tumoral kidney in 10 patients with no history of treatment before surgery. The expression of p53, Bcl-2, Rb and PCNA was studied by immunohistochemical methods in paraffin-embedded tissues. The apoptotic status was evaluated by flow-cytometry analysis following propidium iodide incorporation. The p53 protein expression was recognized in most of the cases (80%) with different intensities. High intensity apoptotic process detected in juxta-tumoral parenchyma seemed to be p53 dependent and well correlated with the low Bcl-2 expression. 70% of cases were Rb positive. In this type of tissue Rb has only an anti-proliferative and anti-tumoral role. PCNA was present in half of the cases being low expressed due to the tissue regenerating mechanism. Our data suggest that the high intensity of programmed cell death in this type of tissue is supported by the status of cell regulatory factors that control this process. Previous studies have demonstrated that healthy renal tissue has neither apoptosis nor mitotic activity. Juxta-tumoral renal tissue is also displaying normal morphology and DNA content (diploidy) but the microenvironmental status induced by the tumor presence prompts cells to choose death rather than malignant transformation. Further studies are necessary to emphasize if these results have a clinical relevance for the outcome of therapeutical approaches in renal carcinomas. PMID:16286997

Petru?c?, Daniela Nicoleta; Petrescu, Amelia; Vrabie, Camelia; Niculescu, L; Jinga, V; Diaconu, Carmen; Bra?oveanu, Lorelei

2005-01-01

334

Synergistic effects of IAP inhibitor LCL161 and paclitaxel on hepatocellular carcinoma cells.  

PubMed

Inhibitor of Apoptosis Proteins (IAPs) are key regulators of apoptosis in hepatocellular carcinoma (HCC) and their expression is negatively correlated with patient survival. LCL161 is a small molecule inhibitor of IAPs that has potent antitumour activity in a range of solid tumours. In HCC, response to LCL161 therapy has shown to be mediated by Bcl-2 expression. In this study, we aim to determine whether LCL161 has any therapeutic potential in HCC. Protein expression was determined by Western blot. Cell proliferation was determined by Cell Proliferation ELISA and BrdU colorimetric assays. Apoptosis was determined by Annexin V assay. Cell cycle analysis was performed by staining cells with propidium iodide and analysed in a FACScan. Automated Cell Counter and phase contrast microscopy were used to determine the cell viability. We have found that LCL161 targets (cIAP1, cIAP2 and XIAP) were up-regulated in HCC tumours. Both high Bcl-2 expressing HuH7 cells and low Bcl-2 expressing SNU423 cells showed strong resistance to LCL161 therapy with significant effects on both apoptosis and cell viability only evident at LCL161 concentrations of ?100?M. At these doses there was significant inhibition of IAP targets, however there was also significant inhibition of off-target proteins including pERK and pJNK suggesting apoptosis caused by drug toxicity. However, when used in combination with paclitaxel in HuH7 and SNU423 cells, LCL161 had significant antiproliferative effects at doses as low as 2?M and this was independent of Bcl-2 expression. Thus, LCL161 may be a useful agent in combination with paclitaxel to treat liver tumours. PMID:24976294

Tian, Aiping; Wilson, George S; Lie, Stefanus; Wu, Guang; Hu, Zenan; Hebbard, Lionel; Duan, Wei; George, Jacob; Qiao, Liang

2014-09-01

335

A simple mechanistic way to increase the survival of Mammalian cells during processing for dry storage.  

PubMed

Recently, there has been considerable interest in developing processing methods that enable storage of cells in a dry state. Most of these studies describe cell viability following processing as a function of the final moisture content reached or the duration of drying. Recently, a cumulative osmotic stress model has been proposed, which takes both final moisture content and duration of drying into consideration in an effort to account for the effects of cumulative processing stresses. The present study demonstrates the applicability of this approach and elucidates a simple mechanistic technique to reduce cumulative osmotic stress during processing. Mouse macrophage cells (J774) were exposed to increasing concentrations of trehalose-containing 0.33×?phosphate-buffered saline (PBS) by step-changing the extracellular solution in 2 increments of 0.7?Osm, using only trehalose as the additive solute. Three minutes was provided for equilibration prior to drying in a traditional low-humidity chamber. The data were compared with that of cells dried directly in isotonic 0.2?M trehalose in 0.33× PBS. Following dehydration, cells were rehydrated and viability was assessed 45?min postrehydration using a combination of trypan blue staining for membrane integrity of detached cells and calcein AM-propidium iodide fluorescence assay for live-dead staining of the attached cells. Cells that were preprocessed to higher trehalose concentrations in step changes prior to drying had higher viability scores at comparable final moisture levels when compared with cells dried in iso-osmotic solution, up to a limit of ?8?Osm, at which point cells processed by both methods approached zero viability. PMID:24845939

Chakraborty, Nilay; Biswas, Debasree; Elliott, Gloria D

2010-06-01

336

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

PubMed Central

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

Hossain, Md. Zakir; Kleve, Maurice G

2011-01-01

337