Comparison of tetrazolium colorimetric and [3H]-uridine assays for in vitro chemosensitivity testing.

PubMed

Ford, C H; Richardson, V J; Tsaltas, G

1989-01-01

We have routinely used a [3H]-uridine microplate assay for assessing chemosensitivity. A colorimetric assay with the advantages of safety, cost and simplicity has previously been described and relies on the ability of living cells to reduce a soluble tetrazolium salt, 3-4,5-dimethylthiazol-2,5-diphenyl-tetrazolium bromide (MMT), into an insoluble formazan precipitate. We compared the chemosensitivity of 14 human tumour cell lines of colonic, lung and cervical carcinoma origin to doxorubicin, vindesine or vindesine immunoconjugates in both the [3H]-uridine assay and a modified MTT assay to evaluate whether we could change to the non-radiolabelled method. Correlation between the concentration of drug causing 50% inhibition of cell growth (IC50) for these agents between the two assays was very poor. However, taking account of recent reports in the literature, we modified the MTT assay by removing serum-containing medium and using dimethyl sulphoxide to solubilise the formazan precipitate. This considerably improved the correlation between the assays for doxorubicin (r = 0.871; P = 0.001) and vindesine (r = 0.981; P less than 0.001). Our data indicates that the MTT assay can be used to replace the [3H]-uridine assay for chemosensitivity screening, but further modifications are necessary to improve the sensitivity and decrease the problem of cell loss after washing, which was noted with some adherent cell lines.

A sensitive WST-8-based bioassay for PEGylated granulocyte colony stimulating factor using the NFS-60 cell line.

PubMed

Tiwari, Krishna; Wavdhane, Madan; Haque, Shafiul; Govender, Thavendran; Kruger, Hendrik G; Mishra, Maheshwari K; Chandra, Ramesh; Tiwari, Dileep

2015-06-01

Granulocyte colony stimulating factor (G-CSF) has been commonly used to treat neutropenia caused by chemotherapy, radiotherapy, and organ transplants. Improved in vitro efficacy of G-CSF has already been observed by conjugating it to polyethylene glycol (PEG). The in vivo bioassay using tetrazolium dye with the NFS-60 cell line has been recommended for G-CSF but no such monographs are available for PEGylated G-CSF in pharmacopeias. In the present study, the assay recommended for G-CSF was evaluated for its suitability to PEGylated G-CSF. The generally used MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]-based assay was compared with a bioassay employing a water-soluble tetrazolium dye, WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium], using NFS-60 cells at a concentration of 7 × 10(5) cells/ml against 800 IU/ml of PEGylated G-CSF at 24, 48, 72, and 72 h time points to determine the efficacy of PEGylated G-CSF. Further, the optimized WST-8 dye-based assay was used to test the potency of various commercially available PEGylated G-CSF preparations. The results demonstrated enhanced sensitivity of the WST-8-based assay over the conventional MTS-based assay for determining the potency of PEGylated G-CSF using the NFS-60 cell line. Our study demonstrates the potential application of WST-8-based bioassays for other biotherapeutic proteins of human and veterinary interest.

Lysozyme activity and nitroblue-tetrazolium reduction in leukaemic cells

PubMed Central

Catovsky, D.; Galton, D. A. G.

1973-01-01

The cytochemical methods for lysozyme and nitroblue-tetrazolium reduction have been used to study the blast cells of acute myeloid leukaemia. Both proved useful in characterizing the cases with predominant monocytic differentiation. The demonstration of lysozyme activity helped to define two main groups: (a) with predominantly lysozyme-negative cells (myeloblastic-promyelocytic), and (b) with considerable numbers of positive cells (monoblastic-monocytic). In addition this test was also of value in the differentiation of other leukaemic disorders. Reduction of nitroblue-tetrazolium was also a feature of monocytic differentiation. The combination of these two methods with those for myeloperoxidase and non-specific esterase activity contributes to the cytological characterization of acute myeloid leukaemia. Images PMID:4511938

Enhanced tetrazolium violet reduction of Salmonella spp. by magnesium addition to the culture media.

PubMed

Junillon, Thomas; Morand, Lucie; Flandrois, Jean Pierre

2014-09-01

Tetrazolium salts (TTZ), such as tetrazolium violet (TV), have been widely used for microbiological studies. The formation of the colored formazan product due to bacterial reduction of the uncolored reagent is extensively exploited to stain cells or colonies in agar or on filters. But an important toxic effect of tetrazolium salts on bacteria exists that limits their use at high concentrations, impairing the efficient staining of the colonies. This is especially the case for Salmonella spp. where we observed, using a classic photometric approach and mathematical modeling of the growth, an important impact of tetrazolium violet on the apparent growth rate below the inhibitory concentration. In this study, we demonstrate that adding magnesium to the medium in the presence of TV leads to a significant increase in the apparent growth rate. Moreover, when higher TV concentrations are used which lead to total inhibition of Salmonella strains, magnesium addition to the culture media allows growth and TV reduction. This effect of magnesium may allow the use of higher TTZ concentrations in liquid growth media and enhance bacteria detection capabilities. Copyright © 2014 Elsevier Ltd. All rights reserved.

Mammalian cell line-based bioassays for toxicological evaluation of landfill leachate treated by Pseudomonas sp. ISTDF1.

PubMed

Ghosh, Pooja; Das, Mihir Tanay; Thakur, Indu Shekhar

2014-01-01

Landfill leachate has become a serious environmental concern because of the presence of many hazardous compounds which even at trace levels are a threat to human health and environment. Therefore, it is important to assess the toxicity of leachate generated and discharge it conforming to the safety standards. The present work examined the efficiency of an earlier reported Pseudomonas sp. strain ISTDF1 for detoxification of leachate collected from Okhla landfill site (New Delhi, India). GC-MS analysis performed after treatment showed the removal of compounds like alpha-limonene diepoxide, brominated dioxin-2-one, Bisphenol A, nitromusk, phthalate derivative, and nitrobenzene originally found in untreated leachate. ICP-AES analysis for heavy metals also showed reduction in concentrations of Zn, Cd, Cr, Fe, Ni, and Pb bringing them within the limit of safety discharge. Methyl tetrazolium (MTT) assay for cytotoxicity, alkaline comet assay for genotoxicity, and 7-ethoxyresorufin-O-deethylase (EROD) assay for dioxin-like behavior were carried out in human hepato-carcinoma cell line HepG2 to evaluate the toxic potential of treated and untreated leachates. The bacterium reduced toxicity as shown by 2.5-fold reduction of MTT EC50 value, 7-fold reduction in Olive Tail Moment, and 2.8-fold reduction in EROD induction after 240 h of bacterial treatment.

Theranostic reduction-sensitive gemcitabine prodrug micelles for near-infrared imaging and pancreatic cancer therapy

NASA Astrophysics Data System (ADS)

Han, Haijie; Wang, Haibo; Chen, Yangjun; Li, Zuhong; Wang, Yin; Jin, Qiao; Ji, Jian

2015-12-01

A biodegradable and reduction-cleavable gemcitabine (GEM) polymeric prodrug with in vivo near-infrared (NIR) imaging ability was reported. This theranostic GEM prodrug PEG-b-[PLA-co-PMAC-graft-(IR820-co-GEM)] was synthesized by ring-opening polymerization and ``click'' reaction. The as-prepared reduction-sensitive prodrug could self-assemble into prodrug micelles in aqueous solution confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). In vitro drug release studies showed that these prodrug micelles were able to release GEM in an intracellular-mimicking reductive environment. These prodrug micelles could be effectively internalized by BxPC-3 pancreatic cancer cells, which were observed by confocal laser scanning microscopy (CLSM). Meanwhile, a methyl thiazolyl tetrazolium (MTT) assay demonstrated that this prodrug exhibited high cytotoxicity against BxPC-3 cells. The in vivo whole-animal near-infrared (NIR) imaging results showed that these prodrug micelles could be effectively accumulated in tumor tissue and had a longer blood circulation time than IR820-COOH. The endogenous reduction-sensitive gemcitabine prodrug micelles with the in vivo NIR imaging ability might have great potential in image-guided pancreatic cancer therapy.A biodegradable and reduction-cleavable gemcitabine (GEM) polymeric prodrug with in vivo near-infrared (NIR) imaging ability was reported. This theranostic GEM prodrug PEG-b-[PLA-co-PMAC-graft-(IR820-co-GEM)] was synthesized by ring-opening polymerization and ``click'' reaction. The as-prepared reduction-sensitive prodrug could self-assemble into prodrug micelles in aqueous solution confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM). In vitro drug release studies showed that these prodrug micelles were able to release GEM in an intracellular-mimicking reductive environment. These prodrug micelles could be effectively internalized by BxPC-3 pancreatic cancer cells, which were observed by confocal laser scanning microscopy (CLSM). Meanwhile, a methyl thiazolyl tetrazolium (MTT) assay demonstrated that this prodrug exhibited high cytotoxicity against BxPC-3 cells. The in vivo whole-animal near-infrared (NIR) imaging results showed that these prodrug micelles could be effectively accumulated in tumor tissue and had a longer blood circulation time than IR820-COOH. The endogenous reduction-sensitive gemcitabine prodrug micelles with the in vivo NIR imaging ability might have great potential in image-guided pancreatic cancer therapy. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06734k

Non-destructive monitoring of viability in an ex vivo organ culture model of osteochondral tissue.

PubMed

Elson, K M; Fox, N; Tipper, J L; Kirkham, J; Hall, R M; Fisher, J; Ingham, E

2015-06-30

Organ culture is an increasingly important tool in research, with advantages over monolayer cell culture due to the inherent natural environment of tissues. Successful organ cultures must retain cell viability. The aim of this study was to produce viable and non-viable osteochondral organ cultures, to assess the accumulation of soluble markers in the conditioned medium for predicting tissue viability. Porcine femoral osteochondral plugs were cultured for 20 days, with the addition of Triton X-100 on day 6 (to induce necrosis), camptothecin (to induce apoptosis) or no toxic additives. Tissue viability was assessed by the tissue destructive XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide tetrazolium salt) assay method and LIVE/DEAD® staining of the cartilage at days 0, 6 and 20. Tissue structure was assessed by histological evaluation using haematoxylin & eosin and safranin O. Conditioned medium was assessed every 3-4 days for glucose depletion, and levels of lactate dehydrogenase (LDH), alkaline phosphatase (AP), glycosaminoglycans (GAGs), and matrix metalloproteinase (MMP)-2 and MMP-9. Necrotic cultures immediately showed a reduction in glucose consumption, and an immediate increase in LDH, GAG, MMP-2 and MMP-9 levels. Apoptotic cultures showed a delayed reduction in glucose consumption and delayed increase in LDH, a small rise in MMP-2 and MMP-9, but no significant effect on GAGs released into the conditioned medium. The data showed that tissue viability could be monitored by assessing the conditioned medium for the aforementioned markers, negating the need for tissue destructive assays. Physiologically relevant whole- or part-joint organ culture models, necessary for research and pre-clinical assessment of therapies, could be monitored this way, reducing the need to sacrifice tissues to determine viability, and hence reducing the sample numbers necessary.

Colorimetric micro-assay for accelerated screening of mould inhibitors

Treesearch

Carol A. Clausen; Vina W. Yang

2013-01-01

Since current standard laboratory methods are time-consuming macro-assays that rely on subjective visual ratings of mould growth, rapid and quantitative laboratory methods are needed to screen potential mould inhibitors for use in and on cellulose-based products. A colorimetric micro-assay has been developed that uses XTT tetrazolium salt to enzymatically assess...

Colorimetric susceptibility testing for Aspergillus fumigatus: comparison of menadione-augmented 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and Alamar blue tests.

PubMed Central

Jahn, B; Stüben, A; Bhakdi, S

1996-01-01

Two colorimetric methods that use Alamar Blue or 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) for assaying the in vitro activities of antifungal agents have been described. We report that both tests performed similarly when the antifungal activity of amphotericin B against Candida albicans was determined. However, only the MTT test generated interpretable data when Aspergillus fumigatus was used. PMID:8818910

Accelerated Colorimetric Micro-assay for Screening Mold Inhibitors

Treesearch

Carol A. Clausen; Vina W. Yang

2014-01-01

Rapid quantitative laboratory test methods are needed to screen potential antifungal agents. Existing laboratory test methods are relatively time consuming, may require specialized test equipment and rely on subjective visual ratings. A quantitative, colorimetric micro-assay has been developed that uses XTT tetrazolium salt to metabolically assess mold spore...

Tetrazolium Reduction-Malachite Green Method for Assessing the Viability of Filamentous Bacteria in Activated Sludge

PubMed Central

Bitton, Gabriel; Koopman, Ben

1982-01-01

A method was developed to assess the activity of filamentous bacteria in activated sludge. It involves the incubation of activated sludge with 2(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride followed by staining with malachite green. Both cells and 2(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride-formazan crystals can be observed in prepared specimens by using bright-field microscopy. This procedure allowed us to distinguish between inactive and actively metabolizing filaments after chlorine application to control the bulking of activated sludge. Images PMID:16345999

Biofilm Formation by Pseudomonas Species Onto Graphene Oxide-TiO2 Nanocomposite-Coated Catheters: In vitro Analysis

NASA Astrophysics Data System (ADS)

Deb, Ananya; Vimala, R.

The present study focuses on the development of an in vitro model system for biofilm growth by Pseudomonas aerouginosa onto small discs of foley catheter. Catheter disc used for the study was coated with graphene oxide-titanium oxide composite (GO-TiO2) and titanium oxide (TiO2) and characterized through XRD, UV-visible spectroscopy. Morphological analysis was done by scanning electron microscopy (SEM). The biofilm formed on the catheter surface was quantified by crystal violet (CV) staining method and a colorimetric assay (MTT assay) which involves the reduction of tetrazolium salt. The catheter coated with GO-TiO2 showed reduced biofilm growth in comparison to the TiO2-coated and uncoated catheter, thus indicating that it could be successfully used in coating biomedical devices to prevent biofilm formation which is a major cause of nosocomial infection.

The microculture tetrazolium assay (MTA): another colorimetric method of testing Plasmodium falciparum chemosensitivity.

PubMed

Delhaes, L; Lazaro, J E; Gay, F; Thellier, M; Danis, M

1999-01-01

Malarial lactate dehydrogenase (LDH), which uses 3-acetyl pyridine adenine dinucleotide as coenzyme in a reaction leading to the formation of pyruvate from L-lactate, may be used to study the susceptibility of Plasmodium falciparum to a drug in vitro. Several methods to determine the activity of this enzyme are available. One, the colorimetric method of Makler and colleagues, was modified slightly, by using sodium-2,3-bis-[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5 - carboxanilide (XTT) and following the reaction by measuring the optical density at 450 nm. Using two, culture-adapted strains of P. falciparum, this LDH assay was compared with the unmodified Makler's assay and with the isotopic microtest based on the incorporation of tritium-labelled hypoxanthine. Fresh, clinical P. falciparum isolates were also tested in the presence of several drugs, including chloroquine, mefloquine, quinine, halofantrine, atovaquone and qinghaosu derivatives. The results of the three assays were correlated for all the drugs tested except atovaquone. The two enzymatic assays are non-radioactive, rapid, reliable, inexpensive to perform and semi-automatic. However, they do require an initial parasitaemia of 2% with a haematocrit of 1.8%.

Development of a high-throughput assay for rapid screening of butanologenic strains.

PubMed

Agu, Chidozie Victor; Lai, Stella M; Ujor, Victor; Biswas, Pradip K; Jones, Andy; Gopalan, Venkat; Ezeji, Thaddeus Chukwuemeka

2018-02-21

We report a Thermotoga hypogea (Th) alcohol dehydrogenase (ADH)-dependent spectrophotometric assay for quantifying the amount of butanol in growth media, an advance that will facilitate rapid high-throughput screening of hypo- and hyper-butanol-producing strains of solventogenic Clostridium species. While a colorimetric nitroblue tetrazolium chloride-based assay for quantitating butanol in acetone-butanol-ethanol (ABE) fermentation broth has been described previously, we determined that Saccharomyces cerevisiae (Sc) ADH used in this earlier study exhibits approximately 13-fold lower catalytic efficiency towards butanol than ethanol. Any Sc ADH-dependent assay for primary quantitation of butanol in an ethanol-butanol mixture is therefore subject to "ethanol interference". To circumvent this limitation and better facilitate identification of hyper-butanol-producing Clostridia, we searched the literature for native ADHs that preferentially utilize butanol over ethanol and identified Th ADH as a candidate. Indeed, recombinant Th ADH exhibited a 6-fold higher catalytic efficiency with butanol than ethanol, as measured using the reduction of NADP + to NADPH that accompanies alcohol oxidation. Moreover, the assay sensitivity was not affected by the presence of acetone, acetic acid or butyric acid (typical ABE fermentation products). We broadened the utility of our assay by adapting it to a high-throughput microtiter plate-based format, and piloted it successfully in an ongoing metabolic engineering initiative.

Development of an XTT tetrazolium salt-based assay for detection of specific hyperthermia sensitizers in a high-flux screening programme.

PubMed

Lechpammer, S; Asea, A; Mallick, R; Zhong, R; Sherman, M Y; Calderwood, S K

2002-01-01

It is now possible to search for new drugs using high-throughput screening of chemical libraries accumulated over the past few years. To detect potential new hyperthermia sensitizers, we are screening for chemical inhibitors of thermotolerance. For the screening of a large chemical library, a rapid and simple assay based on the XTT-tetrazolium salt with the addition of intermediate electron acceptor, phenazine methosulphate (PMS) as a promoter, was developed. It was found that the sensitivity of the XTT/PMS assay is sufficient for assessing thermal cell killing and thermotolerance, although it was highly dependent on cell number and type. When the formazan assay system was challenged with the bioflavonoid drug quercetin (up to 25mm) and validated against the clonogenic cell survival assay, significant decreases in thermotolerant cell viability were observed, directly reflecting inhibition of thermotolerance. Although short-term assays can, in some instances, underestimate overall cell killing, the dose dependency of inhibition of thermotolerance by quercetin recorded in this study by clonogenic and XTT/PMS assays was similar. Application of the XTT/PMS assay in chemical library screening was highly effective in differentiating potential thermotolerance inhibitors from both chemicals with lack of efficacy and from toxic compounds. Taken together, these results show that the XTT/PMS assay, when carried out under careful conditions, is well suited for primary high-flux screen of many thousands of compounds, thus opening up new areas for discovery of hyperthermia sensitizers.

A novel, colorimetric neutralization assay for measuring antibodies to influenza viruses.

PubMed

Lehtoranta, Liisa; Villberg, Anja; Santanen, Riitta; Ziegler, Thedi

2009-08-01

A colorimetric cell proliferation assay for measuring neutralizing antibodies to influenza viruses in human sera is described. Following a 90-min incubation, the serum-virus mixture was transferred to Madin-Darby canine kidney cells cultured in 96-well plates. After further incubation for three days, a tetrazolium salt was added to the wells. Cellular mitochondrial dehydrogenases cleave the tetrazolium salt to formazan, and the resulting color change is read by a spectrophotometer. The absorbance values correlate directly to the number of viable cells in the assay well and thus also to the neutralizing activity of influenza-specific antibodies present in the serum. With the few hands-on manipulations required, this assay allows simultaneous testing of a considerable number of sera, offers opportunities for automation, and is suitable for use under biosafety level-3 conditions. The test was used to study the antibody response after the administration of seasonal, inactivated, trivalent influenza vaccine. Antibody titers determined by the neutralization test in pre- and post-vaccination serum pairs were compared with those obtained by the hemagglutination inhibition assay. The neutralization test yielded higher pre- and post-vaccination titers and a larger number of significant increases in post-vaccination antibody titer than the hemagglutination inhibition test. This new test format could serve as a valuable laboratory tool for influenza vaccine studies.

Toxicity and biocompatibility profile of 3D bone scaffold developed by Universitas Indonesia: A preliminary study

NASA Astrophysics Data System (ADS)

Rahyussalim A., J.; Kurniawati, T.; Aprilya, D.; Anggraini, R.; Ramahdita, Ghiska; Whulanza, Yudan

2017-02-01

Scaffold as a biomaterial must fulfill some requirements to be safely implanted to the human body. Toxicity and biocompatibility test are needed to evaluate scaffold material in mediating cell proliferation and differentiation, secreting extracelullar matrix and carrying biomolecular signals for cell communication. An in vitro study with mesenchymal stem cells consisted of direct contact test and indirect contact test using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay was conducted on 4 scaffolds made of poly-L-lactic acid (PLA), polyvinyl alcohol (PVA), and hydroxyapatite-poly (vinyl alcohol) composite. There were cells-substrate adhesion impairment, morphological changes, cell death and reduction in cell proliferation seen at 2nd and 6th day in most tested scaffold. Cell count result at day-6 showed proliferation inhibition of more than 50% cell death (inhibition value >50) in all tested scaffold. In MTT assay, two scaffolds were proven non-toxic. In conclusion, various scaffold materials showed different toxicity effect. The toxicity and biocompatibility profile in this study is a preliminary data for further research aiming to use those local-made scaffolds to fill human bone defect in various needs.

Tecoma sambucifolia: anti-inflammatory and antinociceptive activities, and 'in vitro' toxicity of extracts of the 'huarumo' of peruvian incas.

PubMed

Alguacil, L F; Galán de Mera, A; Gómez, J; Llinares, F; Morales, L; Muñoz-Mingarro, M D; Pozuelo, J M; Vicente Orellana, J A

2000-06-01

Aqueous and alcoholic extracts of pods and flowers of Tecoma sambucifolia H.B.K. (Bignoniaceae) ('huarumo') were analysed to determine their anti-inflammatory activity (carrageenan-induced edema test), antinociceptive activity (acetic acid writhing test) and 'in vitro' toxicity in Chinese hamster ovary cells, human hepatome cells and human larynx epidermal carcinoma cells. The cytotoxic effects of both extracts were evaluated by two endpoint systems: neutral red uptake assay and tetrazolium assay. The results showed that all extracts have anti-inflammatory and antinociceptive activity, but the highest potency is that of the alcoholic extracts. There were significant differences in cytotoxicity between extracts and among the response of cells to them. The highest cytotoxicity was noted with the alcoholic extract, and the human hepatome cell line was the most sensitive, especially to the alcoholic extract of flowers. The aqueous pod extract appeared to have the best pharmaco-toxicological profile, since it provided a significant reduction of both pain and inflammation together with the lowest cytotoxicity.

Graphene Synthesis & Graphene/Polymer Nanocomposites

NASA Astrophysics Data System (ADS)

Liao, Ken-Hsuan

We successfully developed a novel, fast, hydrazine-free, high-yield method for producing single-layered graphene. Graphene sheets were formed from graphite oxide by reduction with de-ionized water at 130 ºC. Over 65% of the sheets are single graphene layers. A dehydration reaction of exfoliated graphene oxide was utilized to reduce oxygen and transform C-C bonds from sp3 to sp2. The reduction appears to occur in large uniform interconnected oxygen-free patches so that despite the presence of residual oxygen the sp2 carbon bonds formed on the sheets are sufficient to provide electronic properties comparable to reduced graphene sheets obtained using other methods. Cytotoxicity of aqueous graphene was investigated with Dr. Yu-Shen Lin by measuring mitochondrial activity in adherent human skin fibroblasts using two assays. The methyl-thiazolyl-diphenyl-tetrazolium bromide (MTT) assay, a typical nanotoxicity assay, fails to predict the toxicity of graphene oxide and graphene toxicity because of the spontaneous reduction of MTT by graphene and graphene oxide, resulting in a false positive signal. An appropriate alternate assessment, using the water soluble tetrazolium salt (WST-8) assay, reveals that the compacted graphene sheets are more damaging to mammalian fibroblasts than the less densely packed graphene oxide. Clearly, the toxicity of graphene and graphene oxide depends on the exposure environment (i.e. whether or not aggregation occurs) and mode of interaction with cells (i.e. suspension versus adherent cell types). Ultralow percolation concentration of 0.15 wt% graphene, as determined by surface resistance and modulus, was observed from in situ polymerized thermally reduced graphene (TRG)/ poly-urethane-acrylate (PUA) nanocomposite. A homogeneous dispersion of TRG in PUA was revealed by TEM images. The aspect ratio of dispersed TRG, calculated from percolation concentration and modulus, was found to be equivalent to the reported aspect ratio of single-layered free standing TRG. This indicates TRG is mono-layer-dispersed in the matrix polymer. How graphene/polymer nanocomposite glass transition temperatures ( Tg) vary was investigated in this study. We measured Tg in PMMA. We used isotactic PMMA (i-PMMA) and syndiotactic-rich atactic PMMA (a-PMMA) to make TRG/PMMA nanocomposites using solvent blending and in situ polymerization in order to investigate the stereo-regularity and processing effects on the Tg. A T g increase was found in i-PMMA and in situ PMMA but not in a-PMMA. The results can be explained by the thin film confinement effect of polymer. We attribute the Tg increase to both a higher interaction density and a stronger hydrogen bonding at the interfaces. We have studied the elastic modulus of graphene oxide with various oxygen content. We used in situ AFM nano-indentation to measure the influence of oxygen on the elastic modulus of graphene oxide with various carbon/oxygen (C/O) ratios. The results show that chemical reduction (lower oxygen contents) decreases the elastic modulus of graphene oxide. We speculate that chemical reduction of oxygen atoms of epoxy groups on graphene oxide surface removes the bridging effect between carbon atoms, which leads to more flexible sheets. (Abstract shortened by UMI.).

The response characteristics of tetrazolium violet solutions to gamma irradiation

NASA Astrophysics Data System (ADS)

Emi-Reynolds, G.; Kovács, András; Fletcher, J. J.

2007-08-01

The dosimetry characteristics of various solutions of tetrazolium violet, TV, (2,5-diphenyl-3-(1-naphthyl)-2H-tetrazolium chloride) to gamma irradiation are reported. The optical absorption spectra of these solutions show peaks between 400 and 600 nm with a shoulder at around 550 nm. The dose response of the optical absorbance values of aqueous and aqueous-alcoholic solutions containing different concentrations of TV was measured in the 250 Gy up to 75 kGy dose range. The formation of formazan product was observed due to radiolytic reduction in both solutions. Its formation was found more pronounced in N 2-saturated as well as in alkaline solutions. The results indicate that the 1 mM TV solution can be used for food irradiation and medical sterilization dosimetry at gamma irradiation facilities.

INFLUENCE OF SEASONAL FACTORS ON OYSTER HEMOCYTE KILLING OF VIBRIO PARAHEMOLYTICUS

EPA Science Inventory

Seasonal variation of cellular defenses of oyster Crassostrea virginica against Vibrio parahaemolyticus were examined from June 1997 to December 1998 using a recently developed bactericidal assay that utilizes a tetrazolium dye. Mean hemocyte numbers, plasma lysozyme, and P. mari...

Metabolism in the Uncultivated Giant Sulfide-Oxidizing Bacterium Thiomargarita Namibiensis Assayed Using a Redox-Sensitive Dye

NASA Astrophysics Data System (ADS)

Bailey, J.; Flood, B.; Ricci, E.

2014-12-01

The colorless sulfur bacteria are non-photosynthetic chemolithotrophs that live at interfaces between nitrate, or oxygen, and hydrogen sulfide. In sulfidic settings such as cold seeps and oxygen minimum zones, these bacteria are thought to constitute a critical node in the geochemical cycling of carbon, sulfur, nitrogen, and phosphorous. Many of these bacteria remain uncultivated and their metabolisms and physiologies are incompletely understood. Thiomargarita namibiensis is the largest of these sulfur bacteria, with individual cells reaching millimetric diameters. Despite the current inability to maintain a Thiomargarita culture in the lab, their large size allows for individual cells to be followed in time course experiments. Here we report on the novel use of a tetrazolium-based dye that measures the flux of NADH production from catabolic pathways via a colorimetric response. Staining with this dye allows for metabolism to be detected, even in the absence of observable cell division. When coupled to microscopy, this approach also allows for metabolism in Thiomargaritato be differentiated from that of epibionts or contaminants in xenic samples. The results of our tetrazolium dye-based assay suggests that Thiomargarita is the most metabolically versatile under anoxic conditions where it appears capable of using acetate, succinate, formate, thiosulfate, citrate, thiotaurine, hydrogen sulfide, and perhaps hydrogen as electron donors. Under hypoxic conditions, staining results suggest the utilization of acetate, citrate, and hydrogen sulfide. Cells incubated under oxic conditions showed the weakest tetrazolium staining response, and then only to hydrogen sulfide and questionably succinate. These initial results using a redox sensitive dye suggest that Thiomargarita is most metabolically versatile under anaerobic and hypoxic conditions. The results of this assay can be further evaluated using molecular approaches such as transcriptomics, as well as provide cultivation strategies.

Copolymerization of Glycolide and ɛ-Caprolactone Using 12-Aminolauric Acid Modified Montmorillonite

NASA Astrophysics Data System (ADS)

Gallos, HAV; Reyes, LQ

2017-09-01

Poly(glycolide-co-ɛ-caprolactone) (PGLYCL) nanocomposites were prepared by copolymerization glycolide (GLY) and ɛ-caprolactone (ɛ-CL) in the presence of varying loadings 12-aminolauric acid (12-ALA)-modified montmorillonite. Copolymerization was successfully achieved based on the increase in polymer molecular weight after the reaction determined by gel permeation chromatography (GPC). The amount of the poly(glycolide) block and poly(ɛ-caprolactone) block units in the copolymer, identified by proton nuclear magnetic resonance (1H-NMR) spectroscopy, suggested that the increase in organo-clay loading cause a reduction GLYL: ɛ-CLL ratio. The arrangement of the monomers in the polymer products was elucidated to have an ABA triblock structure, where PCL block is the central block and the PGLY is found at both end of the copolymer. The presence of intercalated and exfoliated silicates in the nanocomposites were observed by x-ray diffraction (XRD) analysis. The biocompatibility of the nanocomposites with NCTC 292 mouse normal fibroblast was high relative to untreated cell cultures using tetrazolium bromide (MTT)-dye reduction assay.

Disinfection of Streptococcus mutans biofilm by a non-thermal atmospheric plasma brush

NASA Astrophysics Data System (ADS)

Hong, Qing; Dong, Xiaoqing; Chen, Meng; Xu, Yuanxi; Sun, Hongmin; Hong, Liang; Wang, Yong; Yu, Qingsong

2016-07-01

This study investigated the argon plasma treatment effect on disinfecting dental biofilm by using an atmospheric pressure plasma brush. Streptococcus mutans biofilms were developed for 3 days on the surfaces of hydroxyapatite (HA) discs, which were used to simulate human tooth enamel. After plasma treatment, cell viability in the S. mutans biofilms was characterized by using 3-(4,5-dimethylazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and confocal laser scanning microscopy (CLSM). Compared with the untreated control group, about 90% bacterial reduction in the biofilms was observed after 1 min plasma treatment. Scanning electron microscopy (SEM) examination indicated severe cell damages occurred on the top surface of the plasma treated biofilms. Confocal laser scanning microscopy (CLSM) showed that plasma treatment was effective as deep as 20 µm into the biofilms. When combined with antibiotic treatment using 0.2% chlorhexidine digluconate solution, the plasma treatment became more effective and over 96% bacterial reduction was observed with 1 min plasma treatment.

Polo-like kinase 1, a new therapeutic target in hepatocellular carcinoma

PubMed Central

Mok, Wei Chuen; Wasser, Shanthi; Tan, Theresa; Lim, Seng Gee

2012-01-01

AIM: To investigate the role of polo-like kinase 1 (PLK1) as a therapeutic target for hepatocellular carcinoma (HCC). METHODS: PLK1 gene expression was evaluated in HCC tissue and HCC cell lines. Gene knockdown with short-interfering RNA (siRNA) was used to study PLK1 gene and protein expression using real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, and cell proliferation using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium (MTS) and bromodeoxyuridine (BrdU) assays. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and caspase-inhibition assay. Huh-7 cells were transplanted into nude mice and co-cultured with PLK1 siRNA or control siRNA, and tumor progression was compared with controls. RESULTS: RT-PCR showed that PLK1 was overexpressed 12-fold in tumor samples compared with controls, and also was overexpressed in Huh-7 cells. siRNA against PLK1 showed a reduction in PLK1 gene and protein expression of up to 96% in Huh-7 cells, and a reduction in cell proliferation by 68% and 92% in MTS and BrdU cell proliferation assays, respectively. There was a 3-fold increase in apoptosis events, and TUNEL staining and caspase-3 assays suggested that this was caspase-independent. The pan-caspase inhibitor Z-VAD-FMK was unable to rescue the apoptotic cells. Immnofluorescence co-localized endonuclease-G to fragmented chromosomes, implicating it in apoptosis. Huh-7 cells transplanted subcutaneously into nude mice showed tumor regression in siPLK1-treated mice, but not in controls. CONCLUSION: Knockdown of PLK1 overexpression in HCC was shown to be a potential therapeutic target, leading to apoptosis through the endonuclease-G pathway. PMID:22826617

Taraxinic acid, a hydrolysate of sesquiterpene lactone glycoside from the Taraxacum coreanum NAKAI, induces the differentiation of human acute promyelocytic leukemia HL-60 cells.

PubMed

Choi, Jung-Hye; Shin, Kyung-Min; Kim, Na-Young; Hong, Jung-Pyo; Lee, Yong Sup; Kim, Hyoung Ja; Park, Hee-Juhn; Lee, Kyung-Tae

2002-11-01

The present work was performed to elucidate the active moiety of a sesquiterpene lactone, taraxinic acid-1'-O-beta-D-glucopyranoside (1). from Taraxacum coreanum NAKAI on the cytotoxicity of various cancer cells. Based on enzymatic hydrolysis and MTT assay, the active moiety should be attributed to the aglycone taraxinic acid (1a). rather than the glycoside (1). Taraxinic acid exhibited potent antiproliferative activity against human leukemia-derived HL-60. In addition, this compound was found to be a potent inducer of HL-60 cell differentiation as assessed by a nitroblue tetrazolium reduction test, esterase activity assay, phagocytic activity assay, morphology change, and expression of CD 14 and CD 66 b surface antigens. These results suggest that taraxinic acid induces the differentiation of human leukemia cells to monocyte/macrophage lineage. Moreover, the expression level of c-myc was down-regulated during taraxinic acid-dependent HL-60 cell differentiation, whereas p21(CIP1) and p27(KIP1) were up-regulated. Taken together, our results suggest that taraxinic acid may have potential as a therapeutic agent in human leukemia.

Investigation of Total Phenolic and Flavonoid Contents, and Evaluation of Antimicrobial and Antioxidant Activities from Baeckea frutescens Extracts

NASA Astrophysics Data System (ADS)

Nisa, K.; Nurhayati, S.; Apriyana, W.; Indrianingsih, A. W.

2017-12-01

Baeckea frutescens L. is a medicinal plant endemic to the tropical area and it has been used by locals for topical and oral ailments. This study investigated total phenolic and flavonoid contents and also evaluated in vitro antimicrobial and antioxidant activities of of Baeckea frutescens crude extracts. These extracts were assessed for their antibacterial activities against strains of Escherichia coli, Staphylococcus aureus, Salmonella thypii, and Pseudomonas aureginosa by the broth micro-dilution methods using a modified tetrazolium-based colorimetric assay (3-(4, 5-dimethylthiazol)-2, 5-diphenyl tetrazolium bromide (MTT) assay). Baeckea frutescens crude extracts were also tested against the stable DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) free-radical. The results indicated that Baeckea frutescens water and ethanol extracts possesed remarkable antibacterial activity with the minimum inhibitory concentration less than 100 μg/ml against Escherichia coli and Salmonella thypi. On the evaluation of the antioxidant activity via DPPH assay, Baeckea frutescens ethanol extracts exhibited a good antioxidant activity with IC50 less than 50 μg/ml and Baeckea frutescens water extracts showed a moderate antioxidant activity with IC50 less than 100 μg/ml.

Neuroprotective effects of water-soluble Ganoderma lucidum polysaccharides on cerebral ischemic injury in rats.

PubMed

Zhou, Zi-Yi; Tang, Yu-Ping; Xiang, Jun; Wua, Pin; Jin, Hui-Ming; Wang, Zhong; Mori, Masao; Cai, Ding-Fang

2010-08-19

To investigate the neuroprotective effects of water-soluble Ganoderma lucidum polysaccharides (GLPS) on cerebral ischemic injury in rats, and to explore the involved mechanisms. Two models [middle cerebral artery occlusion (MCAO) in Sprague-Dawley (SD) rats and oxygen and glucose deprivation (OGD) in primary cultured rat cortical neurons] were employed to mimic ischemia-reperfusion (I/R) damage, in vivo and in vitro, respectively. Cerebral infarct area was measured by tetrazolium staining, and neurological functional deficits were assessed at 24h after I/R. Neuronal apoptosis was studied by Nissl staining and DNA fragmentation assay. Neuronal injury was assessed by morphological examination using phase-contrast microscopy and quantified by measuring the amount of lactate dehydrogenase (LDH) leakage, cell viability was measured by sodium 3'-1- (phenylaminocarbonyl)-3, 4-tetrazolium-bis (4-methoxy-6-nitro) benzene sulfonic acid (XTT) reduction. Neuronal apoptosis was determined by flow cytometry, and electron microscopy was used to study morphological changes of neurons. Caspase-3, -8 and -9 activation and Bcl-2, Bax protein expression were determined by western blot analysis. Oral administration of GLPS (100, 200 and 400mg/kg) significantly reduced cerebral infarct area, attenuated neurological functional deficits, and reduced neuronal apoptosis in ischemic cortex. In OGD model, GLSP (0.1, 1 and 10 microg/ml) effectively reduced neuronal cell death and relieved cell injury. Moreover, GLPS decreased the percentage of apoptotic neurons, relieved neuronal morphological damage, suppressed overexpression of active caspases-3, -8 and -9 and Bax, and inhibited the reduction of Bcl-2 expression. Our findings indicate that GLPS protects against cerebral ischemic injury by inhibiting apoptosis by downregulating caspase-3 activation and modulating the Bcl-2/Bax ratio. (c) 2010 Elsevier Ireland Ltd. All rights reserved.

A simple and rapid method for optical visualization and quantification of bacteria on textiles

PubMed Central

Stiefel, Philipp; Schneider, Jana; Amberg, Caroline; Maniura-Weber, Katharina; Ren, Qun

2016-01-01

To prevent bacterial contamination on textiles and the associated undesired effects different biocidal coatings have been investigated and applied. However, due to health and environmental concerns anti-adhesive coatings preventing the binding of bacteria would be favored. To develop such anti-adhesive coatings simple assays for reliable and fast screening are beneficial. Here an easy-to-handle, robust and rapid assay to assess bacteria on textiles utilizing a tetrazolium salt was reported. The assay allowed direct eye visualization of the color change of the textiles containing bacteria, facilitating fast screening. Quantification of the adhered bacteria could be done by generating standard curves which correlate the staining intensity to cell numbers. An additional advantage of the described assay is that with the same detection method anti-adhesive and biocidal effects can be investigated. The method was applied to different coatings, using Pseudomonas aeruginosa and Staphylococcus aureus as model organisms. The detection limit was found to be between 2.5 * 106 and 9.4 * 108 for P. aeruginosa and between 1 * 106 and 3.3 * 108 for S. aureus. The anti-adhesive coating PLUMA was demonstrated to reduce bacterial adhesion without killing them, whereas the biocidal coating TH22-27 caused a clear reduction in the number of viable cells. PMID:28004762

U.S. Army Medical Bioengineering Research and Development Laboratory Annual Progress Report FY 1985. Volume 1

DTIC Science & Technology

1985-10-01

heterotrophic carbon dioxide fixa- tion. An assay for T2 toxin using the yeast Cryptococcus luteolus was evaluated as a rapid screening device for toxic...Bioassay for Mycotoxins Using Cryptococcus luteolus with Tetrazolium Salts." Poster session at the American Soc. for Microbiol. Annual Meeting, March

Targeted antitumoral dehydrocrotonin nanoparticles with L-ascorbic acid 6-stearate.

PubMed

Frungillo, Lucas; Martins, Dorival; Teixeira, Sérgio; Anazetti, Maristela Conti; Melo, Patrícia da Silva; Durán, Nelson

2009-12-01

Tumoral cells are known to have a higher ascorbic acid uptake than normal cells. Therefore, the aim of this study was to obtain polymeric nanoparticles containing the antitumoral compound trans-dehydrocrotonin (DHC) functionalized with L-ascorbic acid 6-stearate (AAS) to specifically target this system tumoral cells. Nanoparticle suspensions (NP-AAS-DHC) were prepared by the nanoprecipitation method. The systems were characterized for AAS presence by thin-layer chromatography and for drug loading (81-88%) by UV-Vis spectroscopy. To further characterize these systems, in vitro release kinetics, size distribution (100-140 nm) and Zeta potential by photon-correlation spectroscopic method were used. In vitro toxicity against HL60 cells was evaluated by tetrazolium reduction and Trypan blue exclusion assays. Cell death by apoptosis was quantified and characterized by flow cytometry and caspase activity. Zeta potential analyses showed that the system has a negatively charged outer surface and also indicate that AAS is incorporated on the external surface of the nanoparticles. In vitro release kinetics assay showed that DHC loaded in nanoparticles had sustained release behavior. In vitro toxicity assays showed that NP-AAS-DHC suspension was more effective as an antitumoral than free DHC or NP-DHC and increased apoptosis induction by receptor-mediated pathway. 2009 Wiley-Liss, Inc. and the American Pharmacists Association

Protective effects of anti-ricin A-chain RNA aptamer against ricin toxicity

PubMed Central

Fan, Shaoan; Wu, Feng; Martiniuk, Frank; Hale, Martha L; Ellington, Andrew D; Tchou-Wong, Kam-Meng

2008-01-01

AIM: To investigate the therapeutic potential of an RNA ligand (aptamer) specific for the catalytic ricin A-chain (RTA), the protective effects of a 31-nucleotide RNA aptamer (31RA), which formed a high affinity complex with RTA, against ricin-induced toxicity in cell-based luciferase translation and cell cytotoxicity assays were evaluated. METHODS: To test the therapeutic potential of anti-RTA aptamers in Chinese hamster ovary (CHO) AA8 cells stably transfected with a tetracycline regulatable promoter, ricin ribotoxicity was measured using luciferase and ricin-induced cytotoxicity was ascertained by MTS cell proliferation assay with tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]. RESULTS: Inhibition of protein synthesis by ricin in CHO AA8 cells resulted in diminished luciferase activity and treatment with polyclonal antibody against deglycosylated RTA (dgA) neutralized the inhibitory effects of ricin on luciferase activity and protected against ricin-induced cytotoxicity as measured by MTS assay. The 31RA anti-RTA aptamer inhibited the translation of luciferase mRNA in cell-free reticulocyte translation assay. 31RA aptamer also partially neutralized the inhibitory effects of ricin on luciferase activity and partially protected against ricin-induced cytotoxicity in CHO AA8 cells. CONCLUSION: We have shown that anti-RTA RNA aptamer can protect against ricin ribotoxicity in cell-based luciferase and cell cytotoxicity assays. Hence, RNA aptamer that inhibits RTA enzymatic activity represents a novel class of nucleic acid inhibitor that has the potential to be developed as a therapeutic agent for the treatment of ricin intoxication. PMID:19009652

Biocompatibility of crystalline opal nanoparticles.

PubMed

Hernández-Ortiz, Marlen; Acosta-Torres, Laura S; Hernández-Padrón, Genoveva; Mendieta, Alicia I; Bernal, Rodolfo; Cruz-Vázquez, Catalina; Castaño, Victor M

2012-10-22

Silica nanoparticles are being developed as a host of biomedical and biotechnological applications. For this reason, there are more studies about biocompatibility of silica with amorphous and crystalline structure. Except hydrated silica (opal), despite is presents directly and indirectly in humans. Two sizes of crystalline opal nanoparticles were investigated in this work under criteria of toxicology. In particular, cytotoxic and genotoxic effects caused by opal nanoparticles (80 and 120 nm) were evaluated in cultured mouse cells via a set of bioassays, methylthiazolyldiphenyl-tetrazolium-bromide (MTT) and 5-bromo-2'-deoxyuridine (BrdU). 3T3-NIH cells were incubated for 24 and 72 h in contact with nanocrystalline opal particles, not presented significant statistically difference in the results of cytotoxicity. Genotoxicity tests of crystalline opal nanoparticles were performed by the BrdU assay on the same cultured cells for 24 h incubation. The reduction of BrdU-incorporated cells indicates that nanocrystalline opal exposure did not caused unrepairable damage DNA. There is no relationship between that particles size and MTT reduction, as well as BrdU incorporation, such that the opal particles did not induce cytotoxic effect and genotoxicity in cultured mouse cells.

Diterpenes from Xylopia langsdorffiana inhibit cell growth and induce differentiation in human leukemia cells.

PubMed

Castello Branco, Marianna V S; Anazetti, Maristella C; Silva, Marcelo S; Tavares, Josean F; Diniz, Margareth F F Melo; Frungillo, Lucas; Haun, Marcela; Melo, Patrícia S

2009-01-01

Two new diterpenes were isolated from stems and leaves of Xylopia langsdorffiana, ent-atisane-7alpha,16alpha-diol (xylodiol) and ent-7alpha-acetoxytrachyloban-18-oic acid (trachylobane), along with the known 8(17),12E,14-labdatrien-18-oic acid (labdane). We investigated their antitumour effects on HL60, U937 and K562 human leukemia cell lines. We found that xylodiol was the most potent diterpene in inhibiting cell proliferation of HL60, U937 and K562 cells, with mean IC50 values of 90, 80 and 50 microM, respectively. Based on the nitroblue tetrazolium (NBT) reduction assay, all the diterpenes were found to induce terminal differentiation in HL60 and K562 cells, with xylodiol being the most effective. NBT reduction was increased by almost 120% after 12 h exposure of HL60 cells to xylodiol at a concentration lower than the IC50 (50 microM). Thus, xylodiol inhibited human leukemia cell growth in vitro partly by inducing cell differentiation, and merits further studies to examine its mechanism of action as a potential antitumoural agent.

Cytotoxicity assessment of antibiofouling compounds and by-products in marine bivalve cell cultures.

PubMed

Domart-Coulon, I; Auzoux-Bordenave, S; Doumenc, D; Khalanski, M

2000-06-01

Short-term primary cell cultures were derived from adult marine bivalve tissues: the heart of oyster Crassostrea gigas and the gill of clam Ruditapes decussatus. These cultures were used as experimental in vitro models to assess the acute cytotoxicity of an organic molluscicide, Mexel-432, used in antibiofouling treatments in industrial cooling water systems. A microplate cell viability assay, based on the enzymatic reduction of tetrazolium dye (MTT) in living bivalve cells, was adapted to test the cytotoxicity of this compound: in both in vitro models, toxicity thresholds of Mexel-432 were compared to those determined in vivo with classic acute toxicity tests. The clam gill cell model was also used to assess the cytotoxicity of by-products of chlorination, a major strategy of biofouling control in the marine environment. The applications and limits of these new in vitro models for monitoring aquatic pollutants were discussed, in reference with the standardized Microtox test.

Dammarane triterpene saponin from Bacopa monniera as the superoxide inhibitor in polymorphonuclear cells.

PubMed

Pawar, R; Gopalakrishnan, C; Bhutani, K K

2001-11-01

The hydroalcoholic extract of the whole plant of Bacopa monniera Wettst. (Scrophulariaceae), exhibited an inhibitory effect on superoxide released from polymorphonuclear (PMN) cells in the nitroblue tetrazolium (NBT) assay. The major saponin bacoside A(3) was found to be responsible for this effect in the herb. This compound showed 85, 91.66, 91.66, and 83 % inhibitions of NBT reduction at the concentrations of 200, 100, 50, and 25 microg/ml, respectively, with an IC(50) value of 10.22 microg/ml. These inhibitory effects were compared with those of the standard positive controls, quercetin and ascorbic acid with IC(50) of 111 and 14.16 microg/ml, respectively. Another major saponin bacopasaponin C was found to be much less potent as compared to bacoside A(3) whereas the remaining two mixtures of saponins were found to be inactive.

[In vitro activity of matrine against Candida albicans biofilms].

PubMed

Wu, Lan; Zhou, Zeng-tong; Zhou, Yong-mei; Wang, Hai-yan; Shi, Lin-jun

2009-08-01

To establish a model of Candida albicans biofilms and to examine the effect of matrine on C.albicans biofilms and ultrastructure. C. albicans collection strain ATCC76615 was obtained and propagated. Biofilms were formed in 96-well microtiter plates. Antifungal susceptibility testing of C. albicans biofilms were assessed with the tetrazolium salt (XTT) reduction assay. Confocal laser scanning microscopy (CLSM) and dead/live fluorescent staining technique were combined to detect the effects of Matrine on preformed C. albican biofilms' composition and ultrastructure. Matrine was active against different growth stages (early,middle,mature) of biofilms; The bioactivity and drug-resistance of C. albican biofilm increased with culturing time. CLSM showed that C. albicans biofilms were inhibited and growth were predominantly composed of yeast cells and pseudohyphae. This study demonstrates that Matrine has potent activity against C.albicans biofilms in vitro and potential therapeutic implication for biofilm-associated candidal infections.

Synthesis, characterization, antibacterial activity, SOD mimic and interaction with DNA of drug based copper(II) complexes

NASA Astrophysics Data System (ADS)

Patel, Mohan N.; Dosi, Promise A.; Bhatt, Bhupesh S.; Thakkar, Vasudev R.

2011-02-01

Novel metal complexes of the second-generation quinolone antibacterial agent enrofloxacin with copper(II) and neutral bidentate ligands have been prepared and characterized with elemental analysis reflectance, IR and mass spectroscopy. Complexes have been screened for their in-vitro antibacterial activity against two Gram (+ve)Staphylococcus aureus, Bacillus subtilis, and three Gram (-ve)Serratia marcescens, Escherichia coli and Pseudomonas aeruginosa organisms using the double dilution technique. The binding of this complex with CT-DNA has been investigated by absorption titration, salt effect and viscosity measurements. Binding constant is ranging from 1.3 × 10 4-3.7 × 10 4. The cleavage ability of complexes has been assessed by gel electrophoresis using pUC19 DNA. The catalytic activity of the copper(II) complexes towards the superoxide anion (O 2rad -) dismutation was assayed by their ability to inhibit the reduction of nitroblue tetrazolium (NBT).

Nutritional Status, DNA Damage, and Tumor Pathology

DTIC Science & Technology

2006-08-01

tetrazolium bromide (MTT), 4.8 mM EDTA, 1 mg/ml bovine serum albumin (BSA), 50 µg/ml (U) alcohol dehydrogenase (yeast; 507 U/mg prot), and 1.9 mM phenazine ...NADP-specific isocitrate dehydrogenase (porcine heart; 26-29 U/mg prot), and 1.9 mM phenazine ethosulfate. After incubation of assays for NAD or

Combinatorial cytotoxic effects of Curcuma longa and Zingiber officinale on the PC-3M prostate cancer cell line

PubMed Central

Kurapati, Kesava Rao V.; Samikkannu, Thangavel; Kadiyala, Dakshayani B.; Zainulabedin, Saiyed M.; Gandhi, Nimisha; Sathaye, Sadhana S.; Indap, Manohar A.; Boukli, Nawal; Rodriguez, Jose W.; Nair, Madhavan P.N.

2015-01-01

Background Many plant-derived products exhibit potent chemopreventive activity against animal tumor models as well as rodent and human cancer cell lines. They have low side effects and toxicity and presumably modulate the factors that are critical for cell proliferation, differentiation, senescence and apoptosis. The present study investigates the effects of some medicinal plant extracts from generally recognized as safe plants that may be useful in the prevention and treatment of cancer. Methods Clonogenic assays using logarithmically-growing cells were performed to test the effect. The cytotoxic effects of Curcuma longa and Zingiber officinale were studied using sulforhodamine B assay, tetrazolium dye assay, colony morphology and microscopic analysis. Results Out of the 13 lyophilized plant-derived extracts evaluated for growth-inhibitory effects on the PC-3M prostate cancer cell line, two extracts derived from C. longa and Z. officinale showed significant inhibitory effects on colony-forming ability. The individual and augmentative effects of these two extracts were tested for their narrow range effective lower concentration on PC-3M in clonogenic assays. At relatively lower concentrations, C. longa showed significant inhibition of colony formation in clonogenic assays; whereas at same concentrations Z. officinale showed only moderate inhibitory effects. However, when both the agents were tested together at the same concentrations, the combined effects were much more significant than their individual ones. On normal prostate epithelial cells both C. longa and Z. officinale had similar effects but at a lower magnitude. These observations were confirmed by several cytotoxicity assays involving the morphological appearance of the colonies, microscopic observations, per cent inhibition in comparison to control by sulforhodamine B and tetrazolium dye assay. Conclusions From these observations, it was concluded that the combined effects of C. longa and Z. officinale are much greater than their individual effects, suggesting the role of multiple components and their synergistic mode of actions to elicit stronger beneficial effects. PMID:23072849

Utility of the nitroblue tetrazolium reduction test for assessment of reactive oxygen species production by seminal leukocytes and spermatozoa.

PubMed

Esfandiari, Navid; Sharma, Rakesh K; Saleh, Ramadan A; Thomas, Anthony J; Agarwal, Ashok

2003-01-01

The purpose of this study was to evaluate the ability of spermatozoa and leukocytes in semen to produce reactive oxygen species (ROS) by using nitroblue tetrazolium (NBT) staining and to examine the association between NBT staining and levels of ROS as measured by chemiluminescence. Twenty-one infertility patients (leukocytospermia; n = 8; nonleukocytospermia, n = 13) and 9 healthy donors were included. Standard semen analysis and density gradient centrifugation were performed to test NBT staining, ROS, and total antioxidant capacity. A ROS-total antioxidant capacity (ROS-TAC) score was calculated by using principal component analysis. In the leukocytospermic group, after separation on a density gradient, the percentage of NBT-positive staining was significantly higher in sperm suspensions contaminated with leukocytes (median [25th, 75th percentiles]; 70% [61%, 79%]) compared to the nonleukocytospermic group (14.5% [9%, 25.5%]; P =.03) and donors (7% [3%, 11%]; P =.02), respectively. A strong positive correlation was seen between levels of ROS in whole ejaculates and NBT-positive staining in leukocytes (r = 0.59; P <.0006) and in leukocyte fractions (r = 0.72; P <.0001) after density gradient separation. Similarly, ROS was positively correlated with excessive cytoplasmic retention in spermatozoa from whole ejaculates and abnormal spermatozoa after separation on density gradients (r = 0.72; P <.0001). The ROS-TAC score was inversely correlated with NBT staining in leukocytes in whole ejaculates (r = -0.960, P <.0007) and in both leukocyte fractions (r = -0.39; P <.04) and spermatozoa with cytoplasmic retention (r = -0.38; P <.04). Our results indicate that the NBT reduction test can be used to assess the contribution of seminal leukocytes and defective spermatozoa towards ROS generation in semen. Levels of ROS assessed by chemiluminescence assay are strongly correlated with the results of NBT staining.

Nano-silymarin provides protection against γ-radiation-induced oxidative stress in cultured human embryonic kidney cells.

PubMed

Adhikari, Manish; Arora, Rajesh

2015-10-01

Radiation can produce biological damage, mainly oxidative stress, via production of free radicals, including reactive oxygen species (ROS). Nanoparticles are of interest as radioprotective agents, particularly due to their high solubility and bioavailability. Silymarin is a hepatoprotective agent but has poor oral bioavailability. Silymarin was formulated as a nanoemulsion with the aim of improving its bioavailability and therapeutic efficacy. In the present study, we evaluated self-nanoemulsifying drug delivery systems (SNEDDS) formulated with surfactants and co-surfactants. Nano-silymarin was characterized by estimating % transmittance, globule size, and polydispersity index, and by transmission electron microscopy (TEM). The nano-silymarin obtained was in the range of 3-8nm diameter. With regard to DNA damage, measured by a plasmid relaxation assay, maximum protection was obtained at 10μg/mL. Cytotoxicity of nano-silymarin to human embryonic kidney (HEK) cells was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Protective efficacy against γ-radiation was assessed by reduction in micronucleus frequency and ROS generation, using the 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) assay. Radiation-induced apoptosis was estimated by microscopic analysis and cell-cycle estimation. Nano-silymarin was radioprotective, supporting the possibility of developing new approaches to radiation protection via nanotechnology. Copyright © 2015 Elsevier B.V. All rights reserved.

Biocompatibility of crystalline opal nanoparticles

PubMed Central

2012-01-01

Background Silica nanoparticles are being developed as a host of biomedical and biotechnological applications. For this reason, there are more studies about biocompatibility of silica with amorphous and crystalline structure. Except hydrated silica (opal), despite is presents directly and indirectly in humans. Two sizes of crystalline opal nanoparticles were investigated in this work under criteria of toxicology. Methods In particular, cytotoxic and genotoxic effects caused by opal nanoparticles (80 and 120 nm) were evaluated in cultured mouse cells via a set of bioassays, methylthiazolyldiphenyl-tetrazolium-bromide (MTT) and 5-bromo-2′-deoxyuridine (BrdU). Results 3T3-NIH cells were incubated for 24 and 72 h in contact with nanocrystalline opal particles, not presented significant statistically difference in the results of cytotoxicity. Genotoxicity tests of crystalline opal nanoparticles were performed by the BrdU assay on the same cultured cells for 24 h incubation. The reduction of BrdU-incorporated cells indicates that nanocrystalline opal exposure did not caused unrepairable damage DNA. Conclusions There is no relationship between that particles size and MTT reduction, as well as BrdU incorporation, such that the opal particles did not induce cytotoxic effect and genotoxicity in cultured mouse cells. PMID:23088559

Evaluation of biocompatibility of various ceramic powders with human fibroblasts in vitro.

PubMed

Li, J; Liu, Y; Hermansson, L; Söremark, R

1993-01-01

Cell reaction to powders of ceramics was studied in vitro. Cultured human fibroblasts were exposed to different types of ceramic powders: zirconia (ZP), alumina (A), tricalcium phosphate (TCP) and hydroxyapatite (HA), at various concentrations. The cell viability at the different exposure times was measured by the colony formation (expressed as colony forming efficiency, CFE), neutral red uptake (NR) and colorimetric tetrazolium (MTT) reduction. Alumina and hydroxyapatite showed no cytotoxic effects at studied doses (1-500 mug/ml) while zirconia and tricalcium phosphate inhibited cell viability, with 50% of CFE reduction at the concentration of about 50 mug/ml. In order to study the cytotoxic mechanism of zirconia powder, two further experiments were included, viz. the cellular response to the sintered zirconia ceramic powders (CZP) which were obtained by crushing the sintered ceramic material; and the measurement of the degradation of zirconia ceramic plate in the different solutions, i.e., either in saline or in 0.02 M lactic acid (pH 2.72). Similar cell reactions were obtained for the CZP and ZP by using MTT and NR assays. Slow releases of ions from zirconia ceramic plate, yttrium in both solutions and zirconium and yttrium in lactic acid, were detected.

Glycation-assisted synthesized gold nanoparticles inhibit growth of bone cancer cells.

PubMed

Rahim, Moniba; Iram, Sana; Khan, Mohd Sajid; Khan, M Salman; Shukla, Ankur R; Srivastava, A K; Ahmad, Saheem

2014-05-01

This study presents a novel approach to synthesize glycogenic gold nanoparticles (glycogenic GNps) capped with glycated products (Schiff's base, Heyns products, fructosylamine etc.). These glycogenic GNps have been found to be active against human osteosarcoma cell line (Saos-2) with an IC50 of 0.187 mM, while the normal human embryonic lung cell line (L-132) remained unaffected up to 1mM concentration. The size of glycogenic GNps can also be controlled by varying the time of incubation of gold solution. Glycation reactions involving a combination of fructose and HSA (Human Serum Albumin) were found to be effective in the reduction of gold to glycogenic GNps whereas glucose in combination with HSA did not result in the reduction of gold. The progress of the reaction was followed using UV-visible spectroscopy and NBT (Nitroblue tetrazolium) assay. The glycogenic GNps were found to be spherical in shape with an average size of 24.3 nm, in a stable emulsion. These GNps were characterized using UV-visible spectroscopy, zeta potential analysis, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Copyright © 2013 Elsevier B.V. All rights reserved.

Comparison of Spectrophotometric and Visual Readings of NCCLS Method and Evaluation of a Colorimetric Method Based on Reduction of a Soluble Tetrazolium Salt, 2,3-Bis {2-Methoxy-4-Nitro-5-[(Sulfenylamino) Carbonyl]-2H- Tetrazolium-Hydroxide}, for Antifungal Susceptibility Testing of Aspergillus Species

PubMed Central

Meletiadis, Joseph; Mouton, Johan W.; Meis, Jacques F. G. M.; Bouman, Bianca A.; Donnelly, Peter J.; Verweij, Paul E.

2001-01-01

The susceptibilities of 25 clinical isolates of various Aspergillus species (Aspergillus fumigatus, A. flavus, A. terreus, A. ustus, and A. nidulans) to itraconazole (ITC) and amphotericin B (AMB) were determined using the standard proposed by NCCLS for antifungal susceptibility testing of filamentous fungi, a modification of this method using spectrophotometric readings, and a colorimetric method using the tetrazolium salt 2,3-bis {2-methoxy-4-nitro-5-[(sulfenylamino) carbonyl]-2H-tetrazolium-hydroxide} (XTT). Five MIC end points for ITC (MIC-0, no visible growth or ≤5% the growth control value [GC]; MIC-1, slight growth or 6 to 25% the GC; MIC-2, prominent reduction in growth or 26 to 50% the GC; MIC-3, slight reduction in growth or 51 to 75% the GC; and MIC-4, no reduction in growth or 76 to 100% the GC) and one for AMB (MIC-0) were determined visually by four observers and spectrophotometrically. The intraexperimental (between the observers) and interexperimental (between the experiments) levels of agreement of the NCCLS and XTT methods exceeded 95% for MIC-0 of AMB and MIC-0 and MIC-1 of ITC. The MIC-2 of ITC showed lower reproducibility, although spectrophotometric reading and/or incubation for 48 h increased the interexperimental reproducibility from 85 to >93%. Between visual and spectrophotometric readings, high levels of agreement were found for AMB (≈97%) and MIC-1 (≈92%) and MIC-2 (≈88%) of ITC. Poor agreement was found for MIC-0 of ITC (51% after 24 h), since the spectrophotometric readings resulted in higher MIC-0 values than the visual readings. The agreement was increased to 98% by shifting the threshold level of MIC-0 from 5 to 10% relative optical density and by establishing an optical density of greater than 0.1 for the GC as the validation criterion. No statistically significant differences were found between the NCCLS method and the XTT method, with the levels of agreement exceeding 97% for MIC-0 of AMB and 83% for MIC-0, MIC-1, and MIC-2 of ITC. The XTT method and spectrophotometric readings can increase the sensitivity and the precision, respectively, of in vitro susceptibility testing of Aspergillus species. PMID:11724829

Zymographic differentiation of [NiFe]-Hydrogenases 1, 2 and 3 of Escherichia coli K-12

PubMed Central

2012-01-01

Background When grown under anaerobic conditions, Escherichia coli K-12 is able to synthesize three active [NiFe]-hydrogenases (Hyd1-3). Two of these hydrogenases are respiratory enzymes catalysing hydrogen oxidation, whereby Hyd-1 is oxygen-tolerant and Hyd-2 is considered a standard oxygen-sensitive hydrogenase. Hyd-3, together with formate dehydrogenase H (Fdh-H), forms the formate hydrogenlyase (FHL) complex, which is responsible for H2 evolution by intact cells. Hydrogen oxidation activity can be assayed for all three hydrogenases using benzyl viologen (BV; Eo′ = -360 mV) as an artificial electron acceptor; however ascribing activities to specific isoenzymes is not trivial. Previously, an in-gel assay could differentiate Hyd-1 and Hyd-2, while Hyd-3 had long been considered too unstable to be visualized on such native gels. This study identifies conditions allowing differentiation of all three enzymes using simple in-gel zymographic assays. Results Using a modified in-gel assay hydrogen-dependent BV reduction catalyzed by Hyd-3 has been described for the first time. High hydrogen concentrations facilitated visualization of Hyd-3 activity. The activity was membrane-associated and although not essential for visualization of Hyd-3, the activity was maximal in the presence of a functional Fdh-H enzyme. Furthermore, through the use of nitroblue tetrazolium (NBT; Eo′ = -80 mV) it was demonstrated that Hyd-1 reduces this redox dye in a hydrogen-dependent manner, while neither Hyd-2 nor Hyd-3 could couple hydrogen oxidation to NBT reduction. Hydrogen-dependent reduction of NBT was also catalysed by an oxygen-sensitive variant of Hyd-1 that had a supernumerary cysteine residue at position 19 of the small subunit substituted for glycine. This finding suggests that tolerance toward oxygen is not the main determinant that governs electron donation to more redox-positive electron acceptors such as NBT. Conclusions The utilization of particular electron acceptors at different hydrogen concentrations and redox potentials correlates with the known physiological functions of the respective hydrogenase. The ability to rapidly distinguish between oxygen-tolerant and standard [NiFe]-hydrogenases provides a facile new screen for the discovery of novel enzymes. A reliable assay for Hyd-3 will reinvigorate studies on the characterisation of the hydrogen-evolving FHL complex. PMID:22769583

Evaluation of the antimicrobial activity and cytotoxicity of phytogenic gold nanoparticles

NASA Astrophysics Data System (ADS)

Sreekanth, T. V. M.; Nagajyothi, P. C.; Supraja, N.; Prasad, T. N. V. K. V.

2015-06-01

Among the nanoscale materials, noble metal nanoparticles have been attracting the scientific community due to their unique properties and selectivity in biological applications. In the present investigation, gold nanoparticles (AuNPs) were synthesized using rhizome extract of Dioscorea batatas through a simple, clean, inexpensive and eco-friendly method. Treating 1 mM chloroauric acid (HAuCl4) with the rhizome extract at 50 °C resulted in the formation of AuNPs. The reduction of AuNPs was observed by the color change of the solution from colorless to dark red wine. The synthesized nanoparticles were characterized using the techniques UV-Vis spectrophotometers, Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy and transmission electron microscopy. Green synthesized AuNPs were found to be toxic against gram-positive and gram-negative bacteria in liquid media. MTT (dimethyl thiazolyl diphenyl tetrazolium salt) assay showed 21.5 % cell inhibition in lower concentration (0.2 mM) and >50 % cell inhibition after 48 h exposure at higher concentrations (0.8-1 mM).

Antiplasmodial activity of extracts of Tridax procumbens and Phyllanthus amarus in in vitro Plasmodium falciparum culture systems.

PubMed

Appiah-Opong, R; Nyarko, A K; Dodoo, D; Gyang, F N; Koram, K A; Ayisi, N K

2011-12-01

Aqueous extracts of Tridax procumbens (TP) (Compositae) and Phyllanthus amarus (PA) (Euphorbiaceae) are used in traditional medicine in Ghana to treat malaria. Previous studies have demonstrated the anti-trypanosoma, anti-bacterial and anti-HIV effects of TP and PA. To assess the antiplasmodial activity of extracts of TP and PA. Aqueous extracts of TP and PA were prepared. A portion of each was freeze-dried and the remaining extracted sequentially with ethyl acetate and chloroform. Ethanolic extracts were also prepared. The antiplasmodial activity of the extracts was assessed with the 3H-hypoxanthine assay using chloroquine-resistant (Dd2) Plasmodium falciparum parasites. Chloroquine was used as the reference drug. The modified tetrazolium-based colorimetric assay was also used to evaluate the red blood cell (RBC)-protective/antiplasmodial activities and cytotoxicities of the extracts. Results showed that TP and PA have antiplasmodial activities. The aqueous and ethanolic extracts of PA were the most active, yielding EC50 values of 34.9 µg/ml and 31.2 µg/ml, respectively in the tetrazolium-based assay. The TP and PA produced and IC50 values of 24.8 µg/ml and 11.7 µg/ml, respectively in the hypoxanthine assay. Protection of human RBCs against P. falciparum damage by the extracts highly correlated with their antiplasmodial activities. None of the extracts, within the concentration range (1.9-500 µg/ml) studied produced any overt toxicity to human RBCs. The results indicate that both PA and TP have activities against chloroquine-resistant P. falciparum (Dd2) parasites. The antiplasmodial principles extracted into water and ethanol but not chloroform or ethyl acetate.

A comparative study of three cytotoxicity test methods for nanomaterials using sodium lauryl sulfate.

PubMed

Kwon, Jae-Sung; Kim, Kwang-Mahn; Kim, Kyoung-Nam

2014-10-01

The biocompatibility evaluation of nanomaterials is essential for their medical diagnostic and therapeutic usage, where a cytotoxicity test is the simplest form of biocompatibility evaluation. Three methods have been commonly used in previous studies for the cytotoxicity testing of nanomaterials: trypan blue exclusion, colorimetric assay using water soluble tetrazolium (WST), and imaging under a microscope following calcein AM/ethidium homodimer-1 staining. However, there has yet to be a study to compare each method. Therefore, in this study three methods were compared using the standard reference material of sodium lauryl sulfate (SLS). Each method of the cytotoxicity test was carried out using mouse fibroblasts of L-929 exposed to different concentrations of SLS. Compared to the gold standard trypan blue exclusion test, both colorimetric assay using water soluble tetrazolium (WST) and imaging under microscope with calcein AM/ethidium homodimer-1 staining showed results that were not statistically different. Also, each method exhibited various advantages and disadvantages, which included the need of equipment, time taken for the experiment, and provision of additional information such as cell morphology. Therefore, this study concludes that all three methods of cytotoxicity testing may be valid, though careful consideration will be needed when selecting tests with regard to time, finances, and the amount of information required by the researcher(s).

Immunostimulatory acivity of Calophyllum brasiliense, Ipomoea pes-caprae and Matayba elaeagnoides demonstrated by human peripheral blood mononuclear cells proliferation.

PubMed

Philippi, Marina Elisa; Duarte, Bruna Momm; Da Silva, Carolina Vieira; De Souza, Michel Thomaz; Niero, Rivaldo; Cechinel Filho, Valdir; Bueno, Edneia Casagranda

2010-01-01

This study evaluates the effect of methanol extracts of three Brazilian medicinal plants on in vitro proliferation of human mononuclear cells. Lymphoproliferation assay was carried out by incubating human peripheral blood mononuclear cells from healthy donors (1 x 10(6) cells/mL) with extracts of Calophyllum brasiliense (roots), Ipomoea pes-caprae (whole plant) and Matayba elaeagnoides (bark), both at 10, 50, 100 and 200 microg/mL, alone or with phytohemagglutinin (PHA, 5 microg/mL), in 96-well microplates at 37 degrees C with 5% CO2, for 72 h. The quantification of cell proliferation assay was performed by blue tetrazolium (MTT) reduction with reading at 540 nm. Cells incubated with only the culture medium were used as negative control for cell proliferation, while the positive control consisted of cells and PHA. The results suggest that the extracts of all three studied plants induce T lymphocyte proliferation. I. pes-caprae showed immunostimulatory activity three times higher than the C. brasiliense extract, while that of the M. elaeagnoides extract was 1.5 times higher. The results demonstrate immunostimulatory effects of these three plants, therefore the continuity of these studies is recommended, in order to determine the active principles.

Antibacterial Activity of Azadirachta indica, Pongamia pinnata, Psidium guajava, and Mangifera indica and their mechanism of action against Streptococcus mutans.

PubMed

Bodiba, Dikonketso Cathrine; Prasad, Preety; Srivastava, Ajay; Crampton, Brigdet; Lall, Namrita Sharan

2018-01-01

Curative plants have reportedly been used to make chewing sticks/toothbrushes intended for the treatment of oral diseases. The in vitro antibacterial activities of Azadirachta indica , Pongamia pinnata , Psidium guajava , and Mangifera indica were evaluated against Streptococcus mutans , along with the cytotoxicity and antioxidant and synergistic potentials. The effect of M. indica on the expression of crucial virulence genes spaP and gtfB of S. mutans was determined. The antibacterial activity was determined using a modified microdilution method. The antioxidant potential was evaluated using diphenyl picrylhydrazyl (DPPH), Griess reagent, and nitroblue tetrazolium calorimetric assays. The synergistic activity was investigated using a modified checkerboard method, while the cytotoxicity was determined according to a cell proliferation 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt assay. Reverse transcription was the chosen method for determining the difference in expression of the spaP and gtfB genes after treatment with the plant sample. M. indica and A. indica had the highest antibacterial activity at concentrations of 0.3 mg/ml and 6.25 mg/ml, respectively. A. indica had the best free radical scavenging of DPPH, exhibiting 50% inhibition at 28.72 μg/ml; while M. indica showed better superoxide scavenging potential than the positive control quercetin. Both M. indica and A. indica had adequate activity against the nitric oxide-free radical (12.87 and 18.89 μg/ml, respectively). M. indica selectively reduced the expression of the gtfB gene, indicating a mechanism involving Glucotranferases, specifically targeting bacterial attachment. Mangifera indica and Azadirachta indica had very good antibacterial activity against Streptococcus mutans and moderate toxicity against Vero cells M. indica had the best antioxidant capacity overall M. indica reduced the expression of gtfB gene at 0.5 mg/ml. Abbreviations used : AA: Ascorbic acid; BHI: Brain-heart infusion; CHX: Chlorhexidine; DPPH: Diphenyl picrylhydrazyl; DMSO: Dimethlysulfoxide; NBT: Nitroblue tetrazolium; NO: Nitric oxide.

S14 as a Therapeutic Target in Breast Cancer

DTIC Science & Technology

2005-08-01

dimethylthiazol-2yl)-5-(3-carboxymethyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (Promega). Oxidation of MTS by viable mitochondria yields a...potential mediators of the observed superinduction. The amplified signal could not be attributed to progestin induction of PPAR Gamma Coactivator-113 (PGC-lf3...quantitation of siRNA transfection efficiency, verification of localization of the siRNA to the interior of the cells, using more than one siRNA, and the

Biocompatibility Assessment of Novel Bioresorbable Alloys Mg-Zn-Se and Mg-Zn-Cu for Endovascular Applications: In- Vitro Studies.

PubMed

Persaud-Sharma, Dharam; Budiansky, Noah; McGoron, Anthony J

2013-01-01

Previous studies have shown that using biodegradable magnesium alloys such as Mg-Zn and Mg-Zn-Al possess the appropriate mechanical properties and biocompatibility to serve in a multitude of biological applications ranging from endovascular to orthopedic and fixation devices. The objective of this study was to evaluate the biocompatibility of novel as-cast magnesium alloys Mg-1Zn-1Cu wt.% and Mg-1Zn-1Se wt.% as potential implantable biomedical materials, and compare their biologically effective properties to a binary Mg-Zn alloy. The cytotoxicity of these experimental alloys was evaluated using a tetrazolium based- MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and a lactate dehydrogenase membrane integrity assay (LDH). The MTS assay was performed on extract solutions obtained from a 30-day period of alloy immersion and agitation in simulated body fluid to evaluate the major degradation products eluted from the alloy materials. Human foreskin fibroblast cell growth on the experimental magnesium alloys was evaluated for a 72 hour period, and cell death was quantified by measuring lactate dehydrogenase concentrations. Both Mg-Zn-Se and Mg-Zn-Cu alloys exhibit low cytotoxicity levels which are suitable for biomaterial applications. The Mg-Zn-Cu alloy was found to completely degrade within 72 hours, resulting in lower human foreskin fibroblast cell viability. The Mg-Zn-Se alloy was shown to be less cytotoxic than both the Mg-Zn-Cu and Mg-Zn alloys.

Biocompatibility Assessment of Novel Bioresorbable Alloys Mg-Zn-Se and Mg-Zn-Cu for Endovascular Applications: In- Vitro Studies

PubMed Central

Budiansky, Noah; McGoron, Anthony J.

2013-01-01

Previous studies have shown that using biodegradable magnesium alloys such as Mg-Zn and Mg-Zn-Al possess the appropriate mechanical properties and biocompatibility to serve in a multitude of biological applications ranging from endovascular to orthopedic and fixation devices. The objective of this study was to evaluate the biocompatibility of novel as-cast magnesium alloys Mg-1Zn-1Cu wt.% and Mg-1Zn-1Se wt.% as potential implantable biomedical materials, and compare their biologically effective properties to a binary Mg-Zn alloy. The cytotoxicity of these experimental alloys was evaluated using a tetrazolium based- MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay and a lactate dehydrogenase membrane integrity assay (LDH). The MTS assay was performed on extract solutions obtained from a 30-day period of alloy immersion and agitation in simulated body fluid to evaluate the major degradation products eluted from the alloy materials. Human foreskin fibroblast cell growth on the experimental magnesium alloys was evaluated for a 72 hour period, and cell death was quantified by measuring lactate dehydrogenase concentrations. Both Mg-Zn-Se and Mg-Zn-Cu alloys exhibit low cytotoxicity levels which are suitable for biomaterial applications. The Mg-Zn-Cu alloy was found to completely degrade within 72 hours, resulting in lower human foreskin fibroblast cell viability. The Mg-Zn-Se alloy was shown to be less cytotoxic than both the Mg-Zn-Cu and Mg-Zn alloys. PMID:24058329

MTS dye based colorimetric CTLL-2 cell proliferation assay for product release and stability monitoring of interleukin-15: assay qualification, standardization and statistical analysis.

PubMed

Soman, Gopalan; Yang, Xiaoyi; Jiang, Hengguang; Giardina, Steve; Vyas, Vinay; Mitra, George; Yovandich, Jason; Creekmore, Stephen P; Waldmann, Thomas A; Quiñones, Octavio; Alvord, W Gregory

2009-08-31

A colorimetric cell proliferation assay using soluble tetrazolium salt [(CellTiter 96(R) Aqueous One Solution) cell proliferation reagent, containing the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) and an electron coupling reagent phenazine ethosulfate], was optimized and qualified for quantitative determination of IL-15 dependent CTLL-2 cell proliferation activity. An in-house recombinant Human (rHu)IL-15 reference lot was standardized (IU/mg) against an international reference standard. Specificity of the assay for IL-15 was documented by illustrating the ability of neutralizing anti-IL-15 antibodies to block the product specific CTLL-2 cell proliferation and the lack of blocking effect with anti-IL-2 antibodies. Under the defined assay conditions, the linear dose-response concentration range was between 0.04 and 0.17ng/ml of the rHuIL-15 produced in-house and 0.5-3.0IU/ml for the international standard. Statistical analysis of the data was performed with the use of scripts written in the R Statistical Language and Environment utilizing a four-parameter logistic regression fit analysis procedure. The overall variation in the ED(50) values for the in-house reference standard from 55 independent estimates performed over the period of 1year was 12.3% of the average. Excellent intra-plate and within-day/inter-plate consistency was observed for all four parameter estimates in the model. Different preparations of rHuIL-15 showed excellent intra-plate consistency in the parameter estimates corresponding to the lower and upper asymptotes as well as to the 'slope' factor at the mid-point. The ED(50) values showed statistically significant differences for different lots and for control versus stressed samples. Three R-scripts improve data analysis capabilities allowing one to describe assay variations, to draw inferences between data sets from formal statistical tests, and to set up improved assay acceptance criteria based on comparability and consistency in the four parameters of the model. The assay is precise, accurate and robust and can be fully validated. Applications of the assay were established including process development support, release of the rHuIL-15 product for pre-clinical and clinical studies, and for monitoring storage stability.

Rapid screening of potential autophagic inductor agents using mammalian cell lines.

PubMed

Martins, Waleska K; Severino, Divinomar; Souza, Cleidiane; Stolf, Beatriz S; Baptista, Maurício S

2013-06-01

Recent progress in understanding the molecular basis of autophagy has demonstrated its importance in several areas of human health. Affordable screening techniques with higher sensitivity and specificity to identify autophagy are, however, needed to move the field forward. In fact, only laborious and/or expensive methodologies such as electron microscopy, dye-staining of autophagic vesicles, and LC3-II immunoblotting or immunoassaying are available for autophagy identification. Aiming to fulfill this technical gap, we describe here the association of three widely used assays to determine cell viability - Crystal Violet staining (CVS), 3-[4, 5-dimethylthiaolyl]-2, 5-diphenyl-tetrazolium bromide (MTT) reduction, and neutral red uptake (NRU) - to predict autophagic cell death in vitro. The conceptual framework of the method is the superior uptake of NR in cells engaging in autophagy. NRU was then weighted by the average of MTT reduction and CVS allowing the calculation of autophagic arbitrary units (AAU), a numeric variable that correlated specifically with the autophagic cell death. The proposed strategy is very useful for drug discovery, allowing the investigation of potential autophagic inductor agents through a rapid screening using mammalian cell lines B16-F10, HaCaT, HeLa, MES-SA, and MES-SA/Dx5 in a unique single microplate. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Preliminary studies on the activities of spin traps as scavengers of free radicals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ogunbiyi, P.O.; Washington, I.

1991-03-15

The spin trapping agents, N-t-Butyl-a-phenyl-nitrone (PBN) and 5,5-Dimethyl-1-pyroline-N-oxide (DMPO) have been used to investigate the primary free radicals involved in various tissue injuries. Also, PBN and DMPO can provide some protection against free radical-induced lung injuries. However, their therapeutic potentials as free radical scavengers remained unexamined. In this study, the effects of PBN and DMPO on guinea pig lung microsomal lipid peroxidation were investigated using thiobarbituric acid-reactive substance assay. Superoxide anions (O{sup 2}{minus}) were generated in an enzymatic and a non-enzymatic system. PBN and DMPO each, significantly inhibited NADPH-stimulated lipid peroxidation irrespective of the presence of Fe{sup 3+}. Cytochrome cmore » reduction by the enzymatic and nitro blue tetrazolium reduction by the non-enzymatic O{sup 2}{minus} generating systems were both inhibited by PBN and DMPO as well as superoxide dismutase and dimethyl sulfoxide when compared with the controls. The spin traps exhibited lower potencies in these systems than the reference compounds, SOD and DMSO, which are well established as O{sup 2}{minus} and hydroxyl radical scavengers respectively. Results demonstrate the free radical scavenging properties of PBN and DMPO. This is an indication of their possible usefulness as antioxidants.« less

Comparative analysis of quantitative methodologies for Vibrionaceae biofilms.

PubMed

Chavez-Dozal, Alba A; Nourabadi, Neda; Erken, Martina; McDougald, Diane; Nishiguchi, Michele K

2016-11-01

Multiple symbiotic and free-living Vibrio spp. grow as a form of microbial community known as a biofilm. In the laboratory, methods to quantify Vibrio biofilm mass include crystal violet staining, direct colony-forming unit (CFU) counting, dry biofilm cell mass measurement, and observation of development of wrinkled colonies. Another approach for bacterial biofilms also involves the use of tetrazolium (XTT) assays (used widely in studies of fungi) that are an appropriate measure of metabolic activity and vitality of cells within the biofilm matrix. This study systematically tested five techniques, among which the XTT assay and wrinkled colony measurement provided the most reproducible, accurate, and efficient methods for the quantitative estimation of Vibrionaceae biofilms.

Effect of prostaglandin E2 on cytotoxic activity and granzyme A protease release by murine adherent IL-2 activated killer cells.

PubMed

Vaillier, D; Daculsi, R; Gualde, N

1994-04-01

The effects of prostaglandin E2 (PGE2) have been studied on a highly purified population of murine IL-2 activated killer cells obtained by selecting plastic-adherent splenocytes (AK cells) after incubation with high doses of recombinant IL-2. AK cells were highly cytotoxic for YAC-1 target cells. The cytotoxic activity was detectable at one hour after initiation of the cytotoxic assay and then increased with time. Cytotoxic activity of AK cells was inhibited by the addition of PGE2 or forskolin during the cytotoxic assay. When AK cells were generated in the presence of PGE2, the yielding cytotoxic activity was lower than the one expressed by "regular" AK cells but were insensitive to the inhibitory effect of PGE2 even if their lytic capability was still suppressed by forskolin. The presence of PGE2 during the AK cell culture had no effect on the cellular proliferation. Moreover, using tetrazolium-based colorimetric assay which reflects the cellular activation, it was observed that AK cells cultured in presence of PGE2 had an increased capacity to cleave the tetrazolium salt to formazan. Since the cytotoxic activity of killer cells is related to expression of serine esterase enzymes we evaluated the effects of PGE2 on serine esterase (Granzyme A) release after one hour of incubation of AK cells either alone or in presence of PGE2, YAC-1 cells or both. We observed that (i) AK cells spontaneously release granzyme A, (ii) the level of granzyme A was significantly increased when AK cells were incubated either with YAC-1 cells or PGE2 but did not change when YAC-1 cells and PGE2 were both associated with AK cells.

Comparison between the Standardized Clinical and Laboratory Standards Institute M38-A2 Method and a 2,3-Bis(2-Methoxy-4-Nitro-5-[(Sulphenylamino)Carbonyl]-2H-Tetrazolium Hydroxide- Based Method for Testing Antifungal Susceptibility of Dermatophytes ▿

PubMed Central

Shehata, Atef S.; Mukherjee, Pranab K.; Ghannoum, Mahmoud A.

2008-01-01

In this study, we determined the utility of a 2,3-bis(2-methoxy-4-nitro-5-[(sulfenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT)-based assay for determining antifungal susceptibilities of dermatophytes to terbinafine, ciclopirox, and voriconazole in comparison to the Clinical and Laboratory Standards Institute (CLSI) M38-A2 method. Forty-eight dermatophyte isolates, including Trichophyton rubrum (n = 15), Trichophyton mentagrophytes (n = 7), Trichophyton tonsurans (n = 11), and Epidermophyton floccosum (n = 13), and two quality control strains, were tested. In the XTT-based method, MICs were determined spectrophotometrically at 490 nm after addition of XTT and menadione. For the CLSI method, the MICs were determined visually. With T. rubrum, the XTT assay revealed MIC ranges of 0.004 to >64 μg/ml, 0.125 to 0.25 μg/ml, and 0.008 to 0.025 μg/ml for terbinafine, ciclopirox, and voriconazole, respectively. Similar MIC ranges were obtained against T. rubrum by using the CLSI method. Additionally, when tested with T. mentagrophytes, T. tonsurans, and E. floccosum isolates, the XTT and CLSI methods resulted in comparable MIC ranges. Both methods revealed similar lowest drug concentrations that inhibited 90% of the isolates for the majority of tested drug-dermatophyte combinations. The levels of agreement within 1 dilution between both methods were as follows: 100% with terbinafine, 97.8% with ciclopirox, and 89.1% with voriconazole. However, the agreement within 2 dilutions between these two methods was 100% for all tested drugs. Our results revealed that the XTT assay can be a useful tool for antifungal susceptibility testing of dermatophytes. PMID:18832129

The protective effect of Malva sylvestris on rat kidney damaged by vanadium

PubMed Central

2011-01-01

Background The protective effect of the common mallow (Malva sylvestris) decoction on renal damages in rats induced by ammonium metavanadate poisoning was evaluated. On the one hand, vanadium toxicity is associated to the production of reactive oxygen species, causing a lipid peroxidation and an alteration in the enzymatic antioxidant defence. On the other hand, many medicinal plants are known to possess antioxidant and radical scavenging properties, thanks to the presence of flavonoids. These properties were confirmed in Malva sylvestris by two separate methods; namely, the Diphenyl-2-picrylhydrazyl assay and the Nitroblue Tetrazolium reduction assay. Results In 80 rats exposed to ammonium metavanadate (0.24 mmol/kg body weight in drinking water) for 90 days, lipid peroxidation levels and superoxide dismutase, catalase and glutathione peroxidase activities were measured in kidney. A significant increase in the formation of free radicals and antioxidant enzyme activities was noticed. In addition, a histological examination of kidney revealed a structural deterioration of the renal cortical capsules and a shrinking of the Bowman space. In animals intoxicated by metavanadate but also given a Malva sylvestris decoction (0.2 g dry mallow/kg body weight), no such pathologic features were observed: lipid peroxidation levels, antioxidant enzyme activities and histological features appeared normal as compared to control rats. Conclusion Malva sylvestris is proved to have a high antioxidative potential thanks to its richness in phenolic compounds. PMID:21513564

Antimicrobial effects of Piper hispidum extract, fractions and chalcones against Candida albicans and Staphylococcus aureus.

PubMed

Costa, G M; Endo, E H; Cortez, D A G; Nakamura, T U; Nakamura, C V; Dias Filho, B P

2016-09-01

Three chalcones, 2'-hydroxy-4,4',6'-trimethoxychalcone, 2'-hydroxy-4,4',6'-tetramethoxychalcone, and 3,2'-dihydroxy-4,4',6'-trimethoxychalcone, were isolated from the leaves of Piper hispidum in a bioguided fractionation of crude extract. The antimicrobial activity of crude extract of P. hispidum leaves was determined against bacteria Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus and yeasts Candida albicans, C. parapsilosis and C. tropicalis. Fractions and chalcones were tested against C. albicans and S. aureus. The checkerboard assay was performed to assess synergic interactions between extract and antifungal drugs, and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay was used to evaluate anti-biofilm effects of extract. The extract was active against yeasts, S. aureus and B. subtilis with MIC values between 15.6 and 62.5μg/mL. Synergistic effects of extract associated with fluconazole and nystatin were observed against C. albicans, with fractional inhibitory concentration indices of 0.37 and 0.24, respectively. The extract was also effective against C. albicans and S. aureus biofilm cells at concentrations of 62.5 and 200μg/mL, respectively. Thus, P. hispidum may be a possible source of bioactive substances with antimicrobial properties. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Trypanocidal activity of a new pterocarpan and other secondary metabolites of plants from Northeastern Brazil flora.

PubMed

Vieira, Nashira Campos; Espíndola, Laila Salmen; Santana, Jaime Martins; Veras, Maria Leopoldina; Pessoa, Otília Deusdênia Loiola; Pinheiro, Sávio Moita; de Araújo, Renata Mendonça; Lima, Mary Anne Sousa; Silveira, Edilberto Rocha

2008-02-15

Two hundred fifteen compounds isolated from plants of Northeastern Brazil flora have been assayed against epimastigote forms of Trypanosoma cruzi, using the tetrazolium salt MTT as an alternative method. Eight compounds belonging to four different species: Harpalyce brasiliana (Fabaceae), Acnistus arborescens and Physalis angulata (Solanaceae), and Cordia globosa (Boraginaceae) showed significant activity. Among them, a novel and a known pterocarpan, a chalcone, four withasteroids, and a meroterpene benzoquinone were the represented chemical classes.

In vitro Toxicity and Inflammatory Response Induced by Copper Nanoparticles in Rat Alveolar Macrophages

DTIC Science & Technology

2008-03-01

the cupric form is most prevalent (Linder and Hazegh-Azam, 1996:797S). Cupric compounds are blue-green in color and highly soluble in water...reduced clearance of particles due to macrophage damage after exposure to various doses of titanium dioxide ( TiO2 )(5, 50, and 250 mg/m3) (Warheit, et al...viability. The assay measures cell viability when the tetrazolium compound is bioreduced by viable cells to a colored formazan product (see Figure 3

Degradation of diesel oil by immobilized Candida tropicalis and biofilm formed on gravels.

PubMed

Chandran, Preethy; Das, Nilanjana

2011-11-01

The performance of diesel oil degradation by Candida tropicalis immobilized on various conventional matrices (sodium alginate, carboxyl methyl cellulose, chitosan) and biowaste materials (wheat bran, sawdust, peanut hull powder) was investigated using the method of entrapment and physical adsorption. The yeast species immobilized in wheat bran showed enhanced efficiency in degrading diesel oil (98%) compared to free cells culture (80%) over a period of 7 days. Copious amount of exopolysaccharides were also produced in the presence of diesel oil. The biofilm forming ability of C. tropicalis on PVC strips was evaluated using XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) reduction assay and monitored by scanning electron microscopy and atomic force microscopy. Yeast biofilm formed on gravels showed 97% degradation of diesel oil over a period of 10 days. The potential use of the biofilms for preparing trickling filters (gravel particles), for attenuating hydrocarbons in oily liquid wastes before their disposal in the open environment is suggested and discussed. This is the first successful attempt for 'artificially' establishing hydrocarbon degrading yeast biofilm on solid substrates.

Removal of Zn(II) from electroplating effluent using yeast biofilm formed on gravels: batch and column studies

PubMed Central

2014-01-01

Background Present study deals with the removal of Zn(II) ions from effluent using yeast biofilm formed on gravels. Methods The biofilm forming ability of Candida rugosa and Cryptococcus laurentii was evaluated using XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) reduction assay and monitored by scanning electron microscopy (SEM), and Confocal laser scanning microscopy (CLSM). Copious amount of extracellular polymeric substances (EPS) produced by yeast species was quantified and characterized by Fourier transform infrared spectroscopy (FT-IR). Results Yeast biofilm formed on gravels by C. rugosa and C. laurentii showed 88% and 74.2% removal of Zn(II) ions respectively in batch mode. In column mode, removal of Zn(II) ions from real effluent was found to be 95.29% by C. rugosa biofilm formed on gravels. Conclusion The results of the present study showed that there is a scope to develop a cost effective method for the efficient removal of Zn(II) from effluent using gravels coated with yeast biofilm. PMID:24397917

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goven, A.J.; Fitzpatrick, L.C.; Venables, B.J.

Understanding the toxic potential and mechanisms of action of environmental xenobiotics is fundamental for assessing risk to public and environmental health. Current established protocols with earthworms focus primarily on defining the lethal effects of chemicals associated with soil contamination. Development of sublethal assays, until recently, has been largely ignored. Here the authors develop rationale for use of earthworms as a model organism for comprehensive assessment of risks to higher wildlife from contaminated soils and hazardous waste sites. They present a panel of lethal (LC/LD50`s) and sublethal measurement endpoint biomarkers, developed within the framework of the National Toxicology Program`s tiered immunotoxicitymore » protocol for mice and according to published criteria for good measurement endpoints, that represent sensitive phylogenetically-conserved processes. Specifically the authors discuss immunosuppressive effects of terrestrial heavy metal and organic contamination on the innate, nonspecific and specific immune responses of earthworm, Lumbricus terrestris, coelomocytes in terms of total and differential cell counts, lysozyme activity, nitroblue tetrazolium dye reduction, phagocytic activity and secretary rosette formation. Findings indicate that sensitive phylogenetically conserved immune responses present in invertebrates can be used to assess or predict risk to wildlife from contaminated soils.« less

Structure-based pharmacophore design and virtual screening for novel potential inhibitors of epidermal growth factor receptor as an approach to breast cancer chemotherapy.

PubMed

Mahernia, Shabnam; Hassanzadeh, Malihe; Sharifi, Niusha; Mehravi, Bita; Paytam, Fariba; Adib, Mehdi; Amanlou, Massoud

2018-02-01

Cancer cells are described with features of uncontrolled growth, invasion and metastasis. The epidermal growth factor receptor subfamily of tyrosine kinases (EGFR-TK) plays a crucial regulatory role in the control of cellular proliferation and progression of various cancers. Therefore, its inhibition might lead to the discovery of a new generation of anticancer drugs. In the present study, structure-based pharmacophore modeling, molecular docking and molecular dynamics simulations were applied to identify potential hits, which exhibited good inhibition on the proliferation of MCF-7 breast cancer cell line and favorable binding interactions on EGFR-TK. Selected compounds were examined for their anticancer activity against the Michigan Cancer Foundation-7 (MCF-7) breast cancer cell line which overexpresses EGFR using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay. Compounds 1 and 2, with an isoindoline-1-one core, induced significant inhibition of breast cancer cells proliferation with IC[Formula: see text] values 327 and 370 nM, respectively.

Use of a stainless steel washer platform to study Acinetobacter baumannii adhesion and biofilm formation on abiotic surfaces.

PubMed

Orsinger-Jacobsen, Samantha J; Patel, Shenan S; Vellozzi, Ernestine M; Gialanella, Phillip; Nimrichter, Leonardo; Miranda, Kildare; Martinez, Luis R

2013-12-01

Acinetobacter baumannii is a frequent cause of hospital-acquired pneumonia, and has recently increased in incidence as the causative agent of severe disease in troops wounded in Afghanistan and Iraq. Clinical approaches are limited since A. baumannii strains isolated from patients are extremely resistant to current antimicrobials. A. baumannii can survive desiccation and during outbreaks has been recovered from various sites in the patients' environment. To better understand its prevalence in hospital settings, we used a stainless steel washer (SSW) platform to investigate A. baumannii biofilm formation on abiotic surfaces. Scanning electron microscopy demonstrated that A. baumannii forms strong biofilms on stainless steel surfaces. This platform was combined with a colorimetric 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay to observe the metabolic activity of bacterial cells, and to facilitate the manipulation and comparison of multiple A. baumannii clinical strains. A strong correlation between XTT and c.f.u. assays was demonstrated. To complement the cell viability assays, A. baumannii biofilm mass was measured by crystal violet staining. Furthermore, the effect of commonly used disinfectants and environmental stressors on A. baumannii biofilms and planktonic cells was compared and characterized. Biofilms on SSWs were significantly more resistant than their planktonic counterparts, providing additional evidence that may allow us to understand the high prevalence of this microbe in hospital settings. Our results validate that SSWs are a simple, versatile and innovative method to study A. baumannii biofilms in vitro.

Use of a stainless steel washer platform to study Acinetobacter baumannii adhesion and biofilm formation on abiotic surfaces

PubMed Central

Orsinger-Jacobsen, Samantha J.; Patel, Shenan S.; Vellozzi, Ernestine M.; Gialanella, Phillip; Nimrichter, Leonardo; Miranda, Kildare

2013-01-01

Acinetobacter baumannii is a frequent cause of hospital-acquired pneumonia, and has recently increased in incidence as the causative agent of severe disease in troops wounded in Afghanistan and Iraq. Clinical approaches are limited since A. baumannii strains isolated from patients are extremely resistant to current antimicrobials. A. baumannii can survive desiccation and during outbreaks has been recovered from various sites in the patients’ environment. To better understand its prevalence in hospital settings, we used a stainless steel washer (SSW) platform to investigate A. baumannii biofilm formation on abiotic surfaces. Scanning electron microscopy demonstrated that A. baumannii forms strong biofilms on stainless steel surfaces. This platform was combined with a colorimetric 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay to observe the metabolic activity of bacterial cells, and to facilitate the manipulation and comparison of multiple A. baumannii clinical strains. A strong correlation between XTT and c.f.u. assays was demonstrated. To complement the cell viability assays, A. baumannii biofilm mass was measured by crystal violet staining. Furthermore, the effect of commonly used disinfectants and environmental stressors on A. baumannii biofilms and planktonic cells was compared and characterized. Biofilms on SSWs were significantly more resistant than their planktonic counterparts, providing additional evidence that may allow us to understand the high prevalence of this microbe in hospital settings. Our results validate that SSWs are a simple, versatile and innovative method to study A. baumannii biofilms in vitro. PMID:24025603

Inhibitory efficacy of geraniol on biofilm formation and development of adaptive resistance in Staphylococcus epidermidis RP62A.

PubMed

Kannappan, Arunachalam; Sivaranjani, Murugesan; Srinivasan, Ramanathan; Rathna, Janarthanam; Pandian, Shunmugiah Karutha; Ravi, Arumugam Veera

2017-10-01

The current study has been designed to delineate the efficacy of geraniol (GE) on biofilm formation in Staphylococcus epidermidis as well as the effect of subinhibitory concentrations of GE on the development of adaptive resistance. Biofilm biomass quantification assay was performed to evaluate the antibiofilm activity of GE against S. epidermidis. Microscopic observation of biofilms and extracellular polymeric substance (EPS), slime and cell surface hydrophobicity (CSH) production were also studied to support the antibiofilm potential of GE. In addition, S. epidermidis was examined for its adaptive resistance development upon continuous exposure of GE at its subinhibitory concentrations.Results/Key findings. The MIC of GE against S. epidermidis was 512 µg ml -1 . Without hampering the growth of the pathogen, GE at its sub-MICs (50, 100, 150 and 200 µg ml -1 ) exhibited a dose-dependent increase in antibiofilm activity. The minimal biofilm inhibitory concentration (MBIC) of GE was found to be 200 µg ml -1 with a maximum biofilm inhibition of 85 %. Disintegrated biofilm architecture, reduced EPS, slime and CSH production validated the antibiofilm efficacy of GE. Although the action of GE on preformed biofilm is limited, a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay and live/dead cell staining method revealed reduction in the viability (47 %) of biofilm inhabitants at 2×MIC concentration. Sequential exposure of S. epidermidis to the sub-MICs of GE resulted in poor development of adaptive resistance with diminished biofilm formation. The present study highlights the potential of GE as a suitable candidate for the control of biofilm-mediated S. epidermidis infections.

Effects of Starvation on Physiological Activity and Chlorine Disinfection Resistance in Escherichia coli O157:H7

PubMed Central

Lisle, John T.; Broadaway, Susan C.; Prescott, Annette M.; Pyle, Barry H.; Fricker, Colin; McFeters, Gordon A.

1998-01-01

Escherichia coli O157:H7 can persist for days to weeks in microcosms simulating natural conditions. In this study, we used a suite of fluorescent, in situ stains and probes to assess the influence of starvation on physiological activity based on membrane potential (rhodamine 123 assay), membrane integrity (LIVE/DEAD BacLight kit), respiratory activity (5-cyano-2,3-di-4-tolyl-tetrazolium chloride assay), intracellular esterase activity (ScanRDI assay), and 16S rRNA content. Growth-dependent assays were also used to assess substrate responsiveness (direct viable count [DVC] assay), ATP activity (MicroStar assay), and culturability (R2A agar assay). In addition, resistance to chlorine disinfection was assessed. After 14 days of starvation, the DVC values decreased, while the values in all other assays remained relatively constant and equivalent to each other. Chlorine resistance progressively increased through the starvation period. After 29 days of starvation, there was no significant difference in chlorine resistance between control cultures that had not been exposed to the disinfectant and cultures that had been exposed. This study demonstrates that E. coli O157:H7 adapts to starvation conditions by developing a chlorine resistance phenotype. PMID:9835545

Effects of starvation on physiological activity and chlorine disinfection resistance in Escherichia coli O157:H7

NASA Technical Reports Server (NTRS)

Lisle, J. T.; Broadaway, S. C.; Prescott, A. M.; Pyle, B. H.; Fricker, C.; McFeters, G. A.

1998-01-01

Escherichia coli O157:H7 can persist for days to weeks in microcosms simulating natural conditions. In this study, we used a suite of fluorescent, in situ stains and probes to assess the influence of starvation on physiological activity based on membrane potential (rhodamine 123 assay), membrane integrity (LIVE/DEAD BacLight kit), respiratory activity (5-cyano-2,3-di-4-tolyl-tetrazolium chloride assay), intracellular esterase activity (ScanRDI assay), and 16S rRNA content. Growth-dependent assays were also used to assess substrate responsiveness (direct viable count [DVC] assay), ATP activity (MicroStar assay), and culturability (R2A agar assay). In addition, resistance to chlorine disinfection was assessed. After 14 days of starvation, the DVC values decreased, while the values in all other assays remained relatively constant and equivalent to each other. Chlorine resistance progressively increased through the starvation period. After 29 days of starvation, there was no significant difference in chlorine resistance between control cultures that had not been exposed to the disinfectant and cultures that had been exposed. This study demonstrates that E. coli O157:H7 adapts to starvation conditions by developing a chlorine resistance phenotype.

Antibacterial Activity of Azadirachta indica, Pongamia pinnata, Psidium guajava, and Mangifera indica and their mechanism of action against Streptococcus mutans

PubMed Central

Bodiba, Dikonketso Cathrine; Prasad, Preety; Srivastava, Ajay; Crampton, Brigdet; Lall, Namrita Sharan

2018-01-01

Background: Curative plants have reportedly been used to make chewing sticks/toothbrushes intended for the treatment of oral diseases. Objective: The in vitro antibacterial activities of Azadirachta indica, Pongamia pinnata, Psidium guajava, and Mangifera indica were evaluated against Streptococcus mutans, along with the cytotoxicity and antioxidant and synergistic potentials. The effect of M. indica on the expression of crucial virulence genes spaP and gtfB of S. mutans was determined. Materials and Methods: The antibacterial activity was determined using a modified microdilution method. The antioxidant potential was evaluated using diphenyl picrylhydrazyl (DPPH), Griess reagent, and nitroblue tetrazolium calorimetric assays. The synergistic activity was investigated using a modified checkerboard method, while the cytotoxicity was determined according to a cell proliferation 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt assay. Reverse transcription was the chosen method for determining the difference in expression of the spaP and gtfB genes after treatment with the plant sample. Results: M. indica and A. indica had the highest antibacterial activity at concentrations of 0.3 mg/ml and 6.25 mg/ml, respectively. A. indica had the best free radical scavenging of DPPH, exhibiting 50% inhibition at 28.72 μg/ml; while M. indica showed better superoxide scavenging potential than the positive control quercetin. Both M. indica and A. indica had adequate activity against the nitric oxide-free radical (12.87 and 18.89 μg/ml, respectively). M. indica selectively reduced the expression of the gtfB gene, indicating a mechanism involving Glucotranferases, specifically targeting bacterial attachment. SUMMARY Mangifera indica and Azadirachta indica had very good antibacterial activity against Streptococcus mutans and moderate toxicity against Vero cellsM. indica had the best antioxidant capacity overallM. indica reduced the expression of gtfB gene at 0.5 mg/ml. Abbreviations used: AA: Ascorbic acid; BHI: Brain–heart infusion; CHX: Chlorhexidine; DPPH: Diphenyl picrylhydrazyl; DMSO: Dimethlysulfoxide; NBT: Nitroblue tetrazolium; NO: Nitric oxide; PMID:29576705

The in vitro anti-viral potential of Setarud (IMOD™), a commercial herbal medicine with protective activity against acquired immune deficiency syndrome in clinical trials

PubMed Central

Zabihollahi, Rezvan; Namazi, Rahele; Aghasadeghi, Mohammad Reze; Esfahani, Azar Farhang; Sadat, Seyed Mehdi; Modarressi, Mohammad Hossein

2012-01-01

Objectives: Setarud (IMOD™) is a herbal medicine with beneficial effect for patients suffering Human immunodeficiency virus (HIV) infection and has been approved for IV (intra venues) injection. The beneficial effect of IMOD administration for acquired immune deficiency syndrome (AIDS) patient has been proved in previous clinical trials. Here the in vitro inhibitory effect of IMOD against HIV-1, Herpes simplex virus (HSV) and murine leukemia viruses (MLV) was evaluated. Materials and Methods: HIV single cycle replication and HSV plaque reduction assays were used to evaluate the anti-viral effect. The level of HIV replication was monitored by p24 capture Enzyme-linked immunosorbent assay (ELISA). The single round infection [with green fluorescent protein (GFP) reporter MLV and HIV], virucidal and time-of-additions (HSV) assays were utilized to determine the mode of anti-viral activity. The toxicity of IMOD for cells was monitored by XTT (sodium 3_-[1 (phenylaminocarbonyl)- 3,4-tetrazolium]-bis (4-methoxy-6-nitro)benzene sulfonic acid) cell proliferation assay kit. Results: IMOD inhibited 50% of HIV-1 and HSV replication (IC50) at 6.5 × 10-4 and 4.3 × 10-3V/V concentrations, respectively. The IC50 value against HIV-1 and MLV infection were 6 × 10-4V/V and 4.9 × 10-4V/V. Virucidal assay showed that IMOD reduces the potency of HIV and HSV particles to 41 and 54% of control, respectively. Time-of-addition study revealed that IMOD inhibits the replication of HSV at a stage after penetration of virions to the target cells. Conclusions: Data from this study indicate that IMOD has significant anti-viral activity against HIV, HSV and MLV. Setarud could be subjected to further investigation after isolation of the constituents and determination of the toxic components. PMID:23087503

Test of Antifibrotic Drugs in a Cellular Model of Fibrosis Based on Muscle-Derived Fibroblasts from Duchenne Muscular Dystrophy Patients.

PubMed

Zanotti, Simona; Mora, Marina

2018-01-01

An in vitro model of muscle fibrosis, based on the use of primary human fibroblasts isolated from muscle biopsies of patients affected by Duchenne muscular dystrophies (DMD) and cultivated in monolayer and 3D conditions, is used to test the potential antifibrotic activity of pirfenidone (PFD). This in vitro model may be usefully also to evaluate the toxicity and efficacy of other candidate molecules for the treatment of fibrosis. The drug toxicity is evaluated using a colorimetric assay based on the conversion of tetrazolium salt (MTT) to insoluble formazan, while the effect of the drug on cell proliferation is measured with the bromodeoxyuridine incorporation assay. The efficacy of the drug is evaluated in fibroblast monolayers by quantitating synthesis and deposition of intracellular collagen with a spectrophotometric picrosirius red-based assay, and by quantitating cell migration using a "scratch" assay. The efficacy of PFD as antifibrotic drug is also evaluated in a 3D fibroblast model by measuring diameters and number of nodules.

Cytotoxicities and genotoxicities of cements based on calcium silicate and of dental formocresol.

PubMed

Ko, Hyunjung; Jeong, Youngdan; Kim, Miri

2017-03-01

Increasing interest is being paid to the toxicities of dental materials. The purpose of this study was to determine the cytotoxicities and genotoxicities of endodontic compounds to Chinese hamster ovary (CHO-K1) reproductive cells. Cultured CHO-K1 cells were treated with dental formocresol, two types of calcium hydroxide paste, and two types of mineral trioxide aggregate cement for 24h. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was performed on each culture, and the micronucleus frequency was determined by performing a micronucleus assay. Alkaline comet assay and γ-H2AX immunofluorescence assay were used to detect DNA damage. Out of the five materials tested, only dental formocresol significantly increased DNA damage. The mineral trioxide aggregate cements based on calcium silicate were not found to be potentially genotoxic. The data suggest that dental formocresol should not be recommended for use in vital pulp therapy on young teeth. Copyright © 2017 Elsevier B.V. All rights reserved.

Antioxidant and antigenotoxic activities in Acacia salicina extracts and its protective role against DNA strand scission induced by hydroxyl radical.

PubMed

Chatti, Ines Bouhlel; Boubaker, Jihed; Skandrani, Ines; Bhouri, Wissem; Ghedira, Kamel; Chekir Ghedira, Leila

2011-08-01

The antioxidant potency of Acacia salicina extracts was investigated. Total antioxidant capacity was determined using an ABTS(+) assay. Superoxide radical scavenging was measured using riboflavin-light-nitro blue tetrazolium (NBT) assay. In addition, the content of phenols, total flavonoids and sterols were measured in the tested extracts. The petroleum ether exhibited a potent scavenging activity toward ABTS radical cations. Whereas, chloroform extract showed the highest activity against superoxides radicals and was also able to protect pKS plasmid DNA against hydroxyl radicals induced DNA damages. The antimutagenicity of these extracts was assayed using the Ames assay against Salmonella typhimurium TA98 and S. typhimurium TA 1535 tester strains at different concentrations. These extracts decreased significantly the mutagenecity induced by sodium azide (SA) and 4-nitro-o-phenylenediamine (NOP). The antioxidant and antimutagenecity activities exhibited by A. salicina depended on the chemical composition of the tested extracts. Copyright © 2011 Elsevier Ltd. All rights reserved.

Clonogenicity of human leukemic cells protected from cell-lethal agents by heat shock protein 70

PubMed Central

Bases, Robert

2005-01-01

Pretreatment of human leukemia THP-1 cells with heat shock protein Hsp70 (Hsp70) protected them from the cell-lethal effects of the topoisomerase II inhibitor, lucanthone and from ionizing radiation. Cell viability was scored in clonogenic assays of single cells grown in liquid medium containing 0.5% methyl cellulose. Colonies were observed and rapidly scored after staining with the tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide. The frequency of abasic sites in the deoxyribonucleic acid (DNA) of THP-1 cells was reduced when these cells were treated with Hsp70. Hsp70 is presumed to have protected the cells by promoting repair of cell DNA, in agreement with previous studies that showed that Hsp70 enhanced base excision repair by purified enzymes. The shoulders of radiation dose-response curves were enhanced by pretreatment of cells with Hsp70 and, importantly, were reduced when cells were transfected with ribonucleic acid designed to silence Hsp70. Hsp70 influenced repair of sublethal damage after radiation. PMID:15832946

Pathogenic features and characteristics of food borne pathogens biofilm: Biomass, viability and matrix.

PubMed

Lin, Shiqi; Yang, Ling; Chen, Gu; Li, Bing; Chen, Dingqiang; Li, Lin; Xu, Zhenbo

2017-10-01

Biofilm is a ubiquitous growth pattern of bacterial species survival but is notorious for its threat on public health and food contamination. Extensive studies of the biofilm structure, formation, quantification, quorum sensing system and underlying control strategies have been reported during the past decades. Insightful elucidation of the pathogenic features and characteristic of bacterial biofilm can facilitate in devising appropriate control strategies for biofilm eradication. Therefore, this review mainly summarized the pathogenic features of biofilms from food borne microorganisms, including the biomass (which could be quantified using crystal violet and fluorogenic dye Syto9 assays), viability (which could be determined by tetrazolium salts, fluorescein diacetate, resazurin staining and alamar blue assays) and matrix (which are commonly detected by dimethyl methylene blue and wheat germ agglutinin assays). In addition, three features were further compared with its particular benefits in specific application. Copyright © 2017 Elsevier Ltd. All rights reserved.

Addition of DNase Improves the In Vitro Activity of Antifungal Drugs against Candida albicans Biofilms

PubMed Central

Martins, Margarida; Henriques, Mariana; Lopez-Ribot, José L.; Oliveira, Rosário

2011-01-01

SUMMARY Background Cells within Candida albicans biofilms display decreased susceptibility to most clinically used antifungal agents. We recently demonstrated that extracellular DNA (eDNA) plays an important role in biofilm integrity, as a component of the biofilm matrix. Objective To gain insight into the contributions of eDNA to C. albicans biofilms antifungal susceptibility by the investigation of the impact of the combined use of deoxyribonuclease I (DNase) and antifungals to treat biofilms. Methods C. albicans biofilms were formed using a simple and reproducible 96-well plate-based method. The activity of the combined use of 0.13 mg l−1 DNase and antifungals was estimated by the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay, and total viable counts. Results and Conclusions Here we report the improved efficacy of amphotericin B when in combination with DNase against C. albicans biofilms, as assessed by XTT readings and viable counts. Furthermore, although DNase increased the efficacy of caspofungin in the reduction of mitochondrial activity, no changes were observed in terms of culturable cells. DNase did not affect biofilm cells susceptibility to fluconazole. This work suggests that agents that target processes affecting the biofilm structural integrity may have potential use as adjuvants of a catheter–lock therapy. PMID:21668524

Piqueria trinervia as a source of metabolites against Giardia intestinalis.

PubMed

Rufino-González, Yadira; Ponce-Macotela, Martha; Jiménez-Estrada, Manuel; Jiménez-Fragoso, Candy N; Palencia, Guadalupe; Sansón-Romero, Gabriel; Anzo-Osorio, Anahi; Martínez-Gordillo, Mario N

2017-12-01

Piqueria trinervia Cav. (Asteraceae) is a plant species with a long history in traditional medicine to cure diarrhoea and other digestive disorders. The study investigates the antigiardial activity of piquerol, trinervinol, red oil and two fractions (F1 and F2) from P. trinervia. P. trinervia was collected in the Ajusco in Mexico City. Aerial parts were ground and mixed with water to obtain the extract, which was treated with dichloromethane to isolate piquerol and trinervinol (P & T). Remnants were the red oil, fractions 1 and 2 (RO, F1 & F2). Trophozoites of Giardia intestinalis were treated with P, T, RO, F1 and F2 at different concentrations (0.78-200 μg/mL) for 48 h. Antigiardial activity was measured using the methylene blue reduction, and the cytotoxicity assayed on human fibroblasts and Vero cells by reduction of tetrazolium salts. Trinervinol and piquerol showed antigiardial activity with an IC 50  = 2.03 and 2.42 μg/mL, and IC 90  = 13.03 and 8.74 μg/mL, respectively. The concentrations of trinervinol (CC 50  = 590 μg/mL) and piquerol (CC 50  = 501 μg/mL) were not cytotoxic to human fibroblasts. Compounds from P. trinervia showed antigiardial activity; to enhance this activity, piquerol and trinervinol can be chemically modified.

Chi-square analysis of the reduction of ATP levels in L-02 hepatocytes by hexavalent chromium.

PubMed

Yuan, Yang; Peng, Li; Gong-Hua, Hu; Lu, Dai; Xia-Li, Zhong; Yu, Zhou; Cai-Gao, Zhong

2012-06-01

This study explored the reduction of adenosine triphosphate (ATP) levels in L-02 hepatocytes by hexavalent chromium (Cr(VI)) using chi-square analysis. Cells were treated with 2, 4, 8, 16, or 32 μM Cr(VI) for 12, 24, or 36 h. Methyl thiazolyl tetrazolium (MTT) experiments and measurements of intracellular ATP levels were performed by spectrophotometry or bioluminescence assays following Cr(VI) treatment. The chi-square test was used to determine the difference between cell survival rate and ATP levels. For the chi-square analysis, the results of the MTT or ATP experiments were transformed into a relative ratio with respect to the control (%). The relative ATP levels increased at 12 h, decreased at 24 h, and increased slightly again at 36 h following 4, 8, 16, 32 μM Cr(VI) treatment, corresponding to a "V-shaped" curve. Furthermore, the results of the chi-square analysis demonstrated a significant difference of the ATP level in the 32-μM Cr(VI) group (P < 0.05). The results suggest that the chi-square test can be applied to analyze the interference effects of Cr(VI) on ATP levels in L-02 hepatocytes. The decreased ATP levels at 24 h indicated disruption of mitochondrial energy metabolism and the slight increase of ATP levels at 36 h indicated partial recovery of mitochondrial function or activated glycolysis in L-02 hepatocytes.

Isolation of furocoumarins from bergamot fruits as HL-60 differentiation-inducing compounds.

PubMed

Kawaii, S; Tomono, Y; Katase, E; Ogawa, K; Yano, M

1999-10-01

The HL-60 differentiation-inducing compounds in bergamot fruits were isolated with column chromatography and identified as bergamottin, bergapten, and citropten by (1)H and (13)C NMR. Their HL-60 differentiation-inducing activity was measured by examining nitro blue tetrazolium (NBT) reducing, nonspecific acid esterase (NSE), specific esterase (SE), and phagocytic activities, and bergamottin showed the strongest activity among the coumarins isolated from bergamot fruits. The structure-activity relationship obtained from HL-60 differentiation assay suggests that hydrophobicity of furocoumarins is correlated with their activity.

Polyphosphoester-Camptothecin Prodrug with Reduction-Response Prepared via Michael Addition Polymerization and Click Reaction.

PubMed

Du, Xueqiong; Sun, Yue; Zhang, Mingzu; He, Jinlin; Ni, Peihong

2017-04-26

Polyphosphoesters (PPEs), as potential candidates for biocompatible and biodegradable polymers, play an important role in material science. Various synthetic methods have been employed in the preparation of PPEs such as polycondensation, polyaddition, ring-opening polymerization, and olefin metathesis polymerization. In this study, a series of linear PPEs has been prepared via one-step Michael addition polymerization. Subsequently, camptothecin (CPT) derivatives containing disulfide bonds and azido groups were linked onto the side chain of the PPE through Cu(I)-catalyzed azidealkyne cyclo-addition "click" chemistry to yield a reduction-responsive polymeric prodrug P(EAEP-PPA)-g-ss-CPT. The chemical structures were characterized by nuclear magnetic resonance spectroscopy, gel permeation chromatography, Fourier transform infrared, ultraviolet-visible spectrophotometer, and high performance liquid chromatograph analyses, respectively. The amphiphilic prodrug could self-assemble into micelles in aqueous solution. The average particle size and morphology of the prodrug micelles were measured by dynamic light scattering and transmission electron microscopy, respectively. The results of size change under different conditions indicate that the micelles possess a favorable stability in physiological conditions and can be degraded in reductive medium. Moreover, the studies of in vitro drug release behavior confirm the reduction-responsive degradation of the prodrug micelles. A methyl thiazolyl tetrazolium assay verifies the good biocompatibility of P(EAEP-PPA) not only for normal cells, but also for tumor cells. The results of cytotoxicity and the intracellular uptake about prodrug micelles further demonstrate that the prodrug micelles can efficiently release CPT into 4T1 or HepG2 cells to inhibit the cell proliferation. All these results show that the polyphosphoester-based prodrug can be used for triggered drug delivery system in cancer treatment.

In vitro synergism between berberine and miconazole against planktonic and biofilm Candida cultures.

PubMed

Wei, Guo-Xian; Xu, Xin; Wu, Christine D

2011-06-01

To investigate the antimycotic activity of the plant alkaloid berberine (BBR), alone and in combination with antifungal azoles, against planktonic and biofilm Candida cultures. The minimum inhibitory concentrations (MICs) of BBR, miconazole (MCZ), and fluconazole (FLC) towards Candida albicans, Candida glabrata, Candida kefyr, Candida krusei, Candida parapsilosis, and Candida tropicalis were determined by a microdilution method. For C. albicans, the synergistic effects of BBR combined with MCZ or FLC were examined in a paper disc agar diffusion assay and checkerboard microdilution assay. The effect of the BBR/MCZ combination was further investigated in a C. albicans biofilm formation model with a dual-chamber flow cell. The effect on metabolic activity of biofilm cells was established using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT)/menadione. Berberine inhibited the growth of various Candida species (MICs 0.98-31.25mg/L) in the following order of susceptibility: C. krusei > C. kefyr > C. glabrata > C. tropicalis > C. parapsilosis and C. albicans. Synergism between BBR and MCZ or FLC was observed in the disc diffusion assay as well as in suspension showing an FIC index <0.5 (∑FIC=0.19). Whilst neither BBR (16 mg/L) nor MCZ (0.8 mg/L) alone significantly inhibited biofilm formation of C. albicans, their combination reduced biofilm formation by >91% after 24 h, as established from the reduction in surface area coverage (P<0.01). The BBR/MCZ combination also exhibited synergy against the metabolic activity of pre-formed C. albicans biofilms in polystyrene microtiter plates (∑FIC=0.25). Berberine exhibits synergistic effects with commonly used antimycotic drugs against C. albicans, either in planktonic or in biofilm growth phases. Published by Elsevier Ltd.

Metallic ions released from stainless steel, nickel-free, and titanium orthodontic alloys: toxicity and DNA damage.

PubMed

Ortiz, Antonio José; Fernández, Esther; Vicente, Ascensión; Calvo, José L; Ortiz, Clara

2011-09-01

The aims of this study were to determine the amounts of metallic ions that stainless steel, nickel-free, and titanium alloys release to a culture medium, and to evaluate the cellular viability and DNA damage of cultivated human fibroblasts with those mediums. The metals were extracted from 10 samples (each consisting of 4 buccal tubes and 20 brackets) of the 3 orthodontic alloys that were submerged for 30 days in minimum essential medium. Next, the determination of metals was performed by using inductively coupled plasma mass spectrometry, cellular viability was assessed by using the tetrazolium reduction assay (MTT assay) (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide), and DNA damage was determined with the Comet assay. The metals measured in all the samples were Ti(47), Cr(52), Mn(55), Co(59), Ni(60), Mo(92), Fe(56), Cu(63), Zn(66), As(75), Se(78), Cd(111), and Pb(208). The cellular viability of the cultured fibroblasts incubated for 7 days with minimum essential medium, with the stainless steel alloy submerged, was close to 0%. Moreover, high concentrations of titanium, chromium, manganese, cobalt, nickel, molybdenum, iron, copper, and zinc were detected. The nickel-free alloy released lower amounts of ions to the medium. The greatest damage in the cellular DNA, measured as the olive moment, was also produced by the stainless steel alloy followed by the nickel-free alloy. Conversely, the titanium alloy had an increased cellular viability and did not damage the cellular DNA, as compared with the control values. The titanium brackets and tubes are the most biocompatible of the 3 alloys studied. Copyright © 2011 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.

Antioxidant effects of gamma-oryzanol on human prostate cancer cells.

PubMed

Klongpityapong, Papavadee; Supabphol, Roongtawan; Supabphol, Athikom

2013-01-01

To assess the antioxidant effects of gamma-oryzanol on human prostate cancer cells. Cytotoxic activity of gamma-oryzanol on human DU145 and PC3 prostate cancer cells was determined by proliferation assay using 3-(4, 5-dimethylthiazol, 2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) reagent. mRNA levels of genes involved in the intracellular antioxidant system, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glutathione reductase (GSR) were determined by reverse transcription-polymerase chain reaction (RT-PCR). Cancer cell lysates were used to measure lipid peroxidation using thiobarbituric acid reactive substance (TBARS). Glutathione contents of the cell lysates were estimated by the reaction between sulfhydryl group of 5, 5'-dithio (bis) nitrobenzoic acid (DTNB) to produce a yellow- color of 5-thio-2-nitrobenzoic acid using colorimetric assay. Catalase activity was also analysed by examining peroxidative function. Protein concentration was estimated by Bradford's assay. All concentrations of gamma-oryzanol, 0.1-2.0mg/ml, significantly inhibited cell growth in a dose- and time-dependent fashion in both prostate cancer cell lines, DU145 and PC3. Gene expression of catalase in DU145 and PC3 exposed to gamma-orizanol at 0.5mg/ml for 14 days was down regulated, while mRNA of GPX was also down regulated in PC3. The MDA and glutathione levels including catalase activity in the cell lysates of DU145 and PC3 treated with gamma-oryzanol 0.1 and 0.5mg/ml were generally decreased. This study highlighted effects of gamma-oryzanol via the down-regulation of antioxidant genes, catalase and GPX, not cytotoxic roles. This might be interesting for adjuvant chemotherapy to make prostate cancer cells more sensitive to free radicals. It might be useful for the reduction of cytotoxic agents and cancer chemoprevention.

The effect of bovine lactoferrin and lactoferricin B on the ability of feline calicivirus (a norovirus surrogate) and poliovirus to infect cell cultures.

PubMed

McCann, K B; Lee, A; Wan, J; Roginski, H; Coventry, M J

2003-01-01

To characterize the effect of bovine lactoferrin and lactoferricin B against feline calicivirus (FCV), a norovirus surrogate and poliovirus (PV), as models for enteric viruses. Crandell-Reese feline kidney (CRFK) cells were used for the propagation of FCV and monkey embryo kidney (MEK) cells for PV. The assays included visual assessment of cell lines for cytopathic effects and determination of the percentage cell death using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium] dye reduction assay. Incubation of bovine lactoferrin with CRFK cells either prior to or together with FCV inoculation substantially reduced FCV infection. In contrast, the interference of lactoferrin with the infection of cells with PV was demonstrated only when lactoferrin was present with cell lines and virus for the entire assay period. Using indirect immunofluorescence, lactoferrin was detected on the surface of both CRFK and MEK cells, suggesting that the interference of viral infection may be attributed to lactoferrin binding to the surfaces of susceptible cells, thereby preventing the attachment of the virus particles. Lactoferricin B, a cationic antimicrobial peptide derived from the N-terminal domain of bovine lactoferrin, reduced FCV but not PV infection. Lactoferrin was shown to interfere with the infection of cells for both FCV and PV. However, lactoferricin B showed no interference of infection with PV and interference with infection for FCV required the presence of lactoferricin B together with the cell line and virus. An in vitro basis is provided for the effects of bovine lactoferrin and lactoferricin B in moderating food-borne infections of enteric viruses.

Cytotoxicity of exfoliated transition-metal dichalcogenides (MoS2 , WS2 , and WSe2 ) is lower than that of graphene and its analogues.

PubMed

Teo, Wei Zhe; Chng, Elaine Lay Khim; Sofer, Zdeněk; Pumera, Martin

2014-07-28

Studies involving transition-metal dichalcogenides (TMDs) have been around for many decades and in recent years, many were focused on using TMDs to synthesize inorganic analogues of carbon nanotubes, fullerene, as well as graphene and its derivatives with the ultimate aim of employing these materials into consumer products. In view of this rising trend, we investigated the cytotoxicity of three common exfoliated TMDs (exTMDs), namely MoS2 , WS2 , and WSe2 , and compared their toxicological effects with graphene oxides and halogenated graphenes to find out whether these inorganic analogues of graphenes and derivatives would show improved biocompatibility. Based on the cell viability assessments using methylthiazolyldiphenyl-tetrazolium bromide (MTT) and water-soluble tetrazolium salt (WST-8) assays on human lung carcinoma epithelial cells (A549) following a 24 h exposure to varying concentrations of the three exTMDs, it was concluded that MoS2 and WS2 nanosheets induced very low cytotoxicity to A549 cells, even at high concentrations. On the other hand, WSe2 exhibited dose-dependent toxicological effects on A549 cells, reducing cell viability to 31.8 % at the maximum concentration of 400 μg mL(-1) ; the higher cytotoxicity displayed by WSe2 might be linked to the identity of the chalcogen. In comparison with graphene oxides and halogenated graphenes, MoS2 and WS2 were much less hazardous, whereas WSe2 showed similar degree of cytotoxicity. Future in-depth studies should be built upon this first work on the in vitro cytotoxicity of MoS2 and WS2 to ensure that they do not pose acute toxicity. Lastly, nanomaterial-induced interference control experiments revealed that exTMDs were capable of reacting with MTT assay viability markers in the absence of cells, but not with WST-8 assay. This suggests that the MTT assay is not suitable for measuring the cytotoxicity of exTMDs because inflated results will be obtained, giving false impressions that the materials are less toxic. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

In vitro inhibitory activities of magnolol against Candida spp.

PubMed

Zhou, Peiru; Fu, Jingya; Hua, Hong; Liu, Xiaosong

2017-01-01

Candida spp. cause various infections involving the skin, mucosa, deep tissues, and even life-threatening candidemia. They are regarded as an important pathogen of nosocomial bloodstream infection, with a high mortality rate. As a result of prolonged exposure to azoles, the therapeutic failure associated with azoles resistance has become a serious challenge in clinical situations. Therefore, novel, alternative antifungals are required urgently. In the present study, the CLSI M-27A broth microdilution method and the 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay were used to evaluate the antifungal effects of magnolol against various standard Candida strains in planktonic mode and biofilm formation, respectively. The antifungal activity of magnolol was demonstrated in planktonic C. albicans and non-albicans Candida species, especially fluconazole-resistant Candida krusei , with the minimum inhibitory concentrations ranging from 10 to 40 μg/mL. The BMIC 90 (minimum concentration with 90% Candida biofilm inhibited) values of magnolol ranged from 20 to 160 μg/mL, whereas the BMIC 90 values of fluconazole were more than 128 μg/mL. As an alternative and broad-spectrum antifungal agent, magnolol might be of benefit to the treatment of refractory Candida infection.

The anti-biofouling behavior of high voltage pulse electric field (HPEF) mediated by carbon fiber composite coating in seawater.

PubMed

Feng, Tiantian; Wu, Jinyi; Chai, Ke; Yang, Pengpeng

2018-04-25

One of the most important research areas in the marine industry is to investigate new and effective anti-biofouling technologies. In this study, high voltage pulse electric field (HPEF) mediated by carbon fiber (CF) composite coating was utilized to prevent the fouling of bacteria, microalgae and barnacle larvae in seawater. The plate count, 2, 3, 5-triphenyl-tetrazolium chloride (TTC) reduction assay and neutral red (NR) staining and larval motility detection showed that the inactivation rates were at the highest levels, which reached 99.1%, 99.9%, 99.7%, 98.7% and 85% respectively for Pseudomonas sp., Vibrio sp., iron bacteria, Navicula sp. and the second stage nauplii of Balanus reticulatus, under the HPEF with 19 kV pulse amplitude, 23.15 kHz frequency and 0.5 duty cycle. The field-emission scanning electron microscopy (FE-SEM) of Navicula sp. revealed that the HPEF brought about the cell lysis and the cell organic matter release on the coating, which could be the mechanism of the inactivation by the HPEF. Additionally, the FE-SEM and Raman spectroscopy indicated that the HPEF hardly damaged the coating. Copyright © 2018 Elsevier B.V. All rights reserved.

A strong protective action of guttiferone-A, a naturally occurring prenylated benzophenone, against iron-induced neuronal cell damage.

PubMed

Figueredo, Yanier Núñez; García-Pupo, Laura; Cuesta Rubio, Osmany; Delgado Hernández, René; Naal, Zeki; Curti, Carlos; Pardo Andreu, Gilberto L

2011-01-01

Guttiferone-A (GA) is a natural occurring polyisoprenylated benzophenone with several reported pharmacological actions. We have assessed the protective action of GA on iron-induced neuronal cell damage by employing the PC12 cell line and primary culture of rat cortical neurons (PCRCN). A strong protection by GA, assessed by the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carbox-anilide (XTT) assay, was revealed, with IC(50) values <1 µM. GA also inhibited Fe(3+)-ascorbate reduction, iron-induced oxidative degradation of 2-deoxiribose, and iron-induced lipid peroxidation in rat brain homogenate, as well as stimulated oxygen consumption by Fe(2+) autoxidation. Absorption spectra and cyclic voltammograms of GA-Fe(2+)/Fe(3+) complexes suggest the formation of a transient charge transfer complex between Fe(2+) and GA, accelerating Fe(2+) oxidation. The more stable Fe(3+) complex with GA would be unable to participate in Fenton-Haber Weiss-type reactions and the propagation phase of lipid peroxidation. The results show a potential of GA against neuronal diseases associated with iron-induced oxidative stress.

Superior anticancer activity is demonstrated by total extract of Curcuma longa L. as opposed to individual curcuminoids separated by centrifugal partition chromatography.

PubMed

Kukula-Koch, Wirginia; Grabarska, Aneta; Łuszczki, Jarogniew; Czernicka, Lidia; Nowosadzka, Ewa; Gumbarewicz, Ewelina; Jarząb, Agata; Audo, Gregoire; Upadhyay, Shakti; Głowniak, Kazimierz; Stepulak, Andrzej

2018-05-01

Three curcuminoids: bisdemethoxycurcumin, demethoxycurcumin, and curcumin from turmeric were successfully separated by a high capacity solvent system composed of heptane: chloroform: methanol: water mixture (5: 6: 3: 2 v/v/v/v) tailored for centrifugal partition chromatographs at K-values of 0.504, 1.057, 1.644, respectively. These three ferulic acid derivatives obtained at a purity rate exceeding 95% were analysed by an HPLC-MS spectrometer. Turmeric extract inhibited the proliferation/viability of A549 human lung cancer, HT29 colon cancer, and T98G glioblastoma cell lines in (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay (MTT). Single curcuminoids significantly decreased the viability/proliferation of lung cancer cells in a dose-dependent manner. However, total extract displayed the superior anticancer activity in the investigated cell lines. Crude extract in combination with cisplatin augmented the decrease in the viability of cancer cells compared with single compound treatment in A549 lung cancer cells. Total extract of Curcuma longa could be regarded as being more effective against lung cancer cells in vitro than its separated compounds. Copyright © 2018 John Wiley & Sons, Ltd.

The anti-biofilm activity of lemongrass (Cymbopogon flexuosus) and grapefruit (Citrus paradisi) essential oils against five strains of Staphylococcus aureus.

PubMed

Adukwu, E C; Allen, S C H; Phillips, C A

2012-11-01

To determine the sensitivity of five strains of Staphylococcus aureus to five essential oils (EOs) and to investigate the anti-biofilm activity of lemongrass and grapefruit EOs. Antimicrobial susceptibility screening was carried out using the disk diffusion method. All of the strains tested were susceptible to lemongrass, grapefruit, bergamot and lime EOs with zones of inhibition varying from 2·85 to 8·60 cm although they were resistant to lemon EO. Lemongrass EO inhibited biofilm formation at 0·125% (v/v) as measured by colorimetric assay and at 0·25% (v/v) no metabolic activity was observed as determined by 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction. Grapefruit EO did not show any anti-biofilm activity. Following exposure to lemongrass EO extensive disruption to Staph. aureus biofilms was shown under scanning electron microscopy. In comparison to the other EOs tested, lemongrass exhibited the most effective antimicrobial and anti-biofilm activity. The effect of lemongrass EO highlights its potential against antibiotic resistant Staph. aureus in the healthcare environment. © 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

Enhancement of caffeic acid phenethyl ester on all-trans retinoic acid-induced differentiation in human leukemia HL-60 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuo, H.-C.; Kuo, W.-H.; Lee, Y.-J.

2006-10-01

All-trans retinoic acid (ATRA) induces complete remission in a high proportion of patients with acute promyelocytic leukemia (APL); however, the response is sometimes very slow. Furthermore, relapse and resistance to treatment often occur despite continued treatment with ATRA. Thereafter, combination treatment strategies have been suggested to circumvent these problems. The present study demonstrates that caffeic acid phenethyl ester (CAPE), a major component of honeybee propolis, enhanced ATRA-induced granulocytic differentiation in HL-60, a human promyelocytic cell line. The differentiation was assessed by Wright-Giemsa stain, nitroblue tetrazolium reduction, and membrane differentiation marker CD11b. In addition, CAPE enhanced ATRA-induced cell cycle arrest atmore » the G1 phase by decreasing the association of cdk2-cyclin E complex. Finally, it was demonstrated that CAPE promoted the ATRA-mediated nuclear transcription activation of RAR{alpha} assessed by EMSA assay and enhanced the expression of target genes including RAR{alpha}, C/EBP{epsilon}, and p21 protein resulting in the differentiation development of leukemia. It is suggested that CAPE possesses the potential to enhance the efficiency of ATRA in the differentiation therapy of APL.« less

Aspartame induces angiogenesis in vitro and in vivo models.

PubMed

Yesildal, F; Aydin, F N; Deveci, S; Tekin, S; Aydin, I; Mammadov, R; Fermanli, O; Avcu, F; Acikel, C H; Ozgurtas, T

2015-03-01

Angiogenesis is the process of generating new blood vessels from preexisting vessels and is considered essential in many pathological conditions. The purpose of the present study is to evaluate the effect of aspartame on angiogenesis in vivo chick chorioallantoic membrane (CAM) and wound-healing models as well as in vitro 2,3-bis-2H-tetrazolium-5-carboxanilide (XTT) and tube formation assays. In CAM assay, aspartame increased angiogenesis in a concentration-dependent manner. Compared with the control group, aspartame has significantly increased vessel proliferation (p < 0.001). In addition, in vivo rat model of skin wound-healing study showed that aspartame group had better healing than control group, and this was statistically significant at p < 0.05. There was a slight proliferative effect of aspartame on human umbilical vein endothelial cells on XTT assay in vitro, but it was not statistically significant; and there was no antiangiogenic effect of aspartame on tube formation assay in vitro. These results provide evidence that aspartame induces angiogenesis in vitro and in vivo; so regular use may have undesirable effect on susceptible cases. © The Author(s) 2015.

Fabrication, Characterization, and Evaluation of Bionanocomposites Based on Natural Polymers and Antibiotics for Wound Healing Applications.

PubMed

Rădulescu, Marius; Holban, Alina Maria; Mogoantă, Laurențiu; Bălşeanu, Tudor-Adrian; Mogoșanu, George Dan; Savu, Diana; Popescu, Roxana Cristina; Fufă, Oana; Grumezescu, Alexandru Mihai; Bezirtzoglou, Eugenia; Lazar, Veronica; Chifiriuc, Mariana Carmen

2016-06-10

The aim of our research activity was to obtain a biocompatible nanostructured composite based on naturally derived biopolymers (chitin and sodium alginate) loaded with commercial antibiotics (either Cefuroxime or Cefepime) with dual functions, namely promoting wound healing and assuring the local delivery of the loaded antibiotic. Compositional, structural, and morphological evaluations were performed by using the thermogravimetric analysis (TGA), scanning electron microscopy (SEM), and fourier transform infrared spectroscopy (FTIR) analytical techniques. In order to quantitatively and qualitatively evaluate the biocompatibility of the obtained composites, we performed the tetrazolium-salt (MTT) and agar diffusion in vitro assays on the L929 cell line. The evaluation of antimicrobial potential was evaluated by the viable cell count assay on strains belonging to two clinically relevant bacterial species (i.e., Escherichia coli and Staphylococcus aureus).

A porphyrin-based metal-organic framework as a pH-responsive drug carrier

NASA Astrophysics Data System (ADS)

Lin, Wenxin; Hu, Quan; Jiang, Ke; Yang, Yanyu; Yang, Yu; Cui, Yuanjing; Qian, Guodong

2016-05-01

A low cytotoxic porphyrin-based metal-organic framework (MOF) PCN-221, which exhibited high PC12 cell viability via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay, was selected as an oral drug carrier. Methotrexate (MTX) was chosen as the model drug molecule which was absorbed into inner pores and channels of MOFs by diffusion. PCN-221 showed high drug loading and sustained release behavior under physiological environment without "burst effect". The controlled pH-responsive release of drugs by PCN-221 revealed its promising application in oral drug delivery.

Protective effect of Aronia melanocarpa fruit juice in a model of cisplatin-induced cytotoxicity in vitro.

PubMed

Valcheva-Kuzmanova, Stefka V; Beronova, Anna B; Momekov, Georgi Tz

2013-01-01

The aim of the present study was to investigate the protective potential of Aronia melanocarpa fruit juice in a model of cisplatin-induced cytotoxicity in the human embryonal kidney cell line HEK293T. The cellular viability was assessed using the MTT-dye reduction assay based on the reduction of the yellow tetrazolium dye MTT to a violet formazan product via the mitochondrial succinate dehydrogenase in viable cells. Cisplatin was applied in various concentrations either alone or after a 24-hour pretreatment of the cells with Aronia melanocarpa fruit juice at 0.1 and 0.05 mg/ml. The half maximal inhibitory concentrations (IC50 values) were derived from the concentration-response curves to cisplatin. Applied alone, the anticancer drug caused a prominent decrease of cellular viability with IC50 8.3 +/- 1.1 microM. The juice proved to significantly ameliorate the in vitro cytotoxicity of the platinum drug, in a concentration-dependent manner. The pretreatment of the cells with Aronia melanocarpa fruit juice resulted in a significant increase (p < 0.001) of IC50 for cisplatin to 25.1 +/- 2.7 microM (at 0.05 mg/ml) and 34.4 +/- 3.4 microM (at 0.1 mg/ml), respectively. The protective effect of Aronia melanocarpa fruit juice observed in this study is most probably due to its well appreciated antioxidant activity as oxidative stress plays a central role in the toxic effects of cisplatin.

Protein kinase C-beta inhibition induces apoptosis and inhibits cell cycle progression in acquired immunodeficiency syndrome-related non-hodgkin lymphoma cells.

PubMed

Saba, Nakhle S; Levy, Laura S

2012-01-01

Acquired immunodeficiency syndrome (AIDS)-related non-Hodgkin lymphoma (NHL) constitutes an aggressive variety of lymphomas characterized by increased extranodal involvement, relapse rate, and resistance to chemotherapy. Protein kinase C-beta (PKCβ) targeting showed promising results in preclinical and clinical studies involving a wide variety of cancers, but studies describing the role of PKCβ in AIDS-NHL are primitive if not lacking. In the present study, 3 AIDS-NHL cell lines were examined: 2F7 (AIDS-Burkitt lymphoma), BCBL-1 (AIDS-primary effusion lymphoma), and UMCL01-101 (AIDS-diffuse large B-cell lymphoma). Immunoblot analysis demonstrated expression of PKCβ1 and PKCβ2 in 2F7 and UMCL01-101 cells, and PKCβ1 alone in BCBL-1 cells. The viability of 2F7 and BCBL-1 cells decreased significantly in the presence of PKCβ-selective inhibitor at half-maximal inhibitory concentration of 14 and 15 μmol/L, respectively, as measured by tetrazolium dye reduction assay. In contrast, UMCL01-101 cells were relatively resistant. As determined using flow cytometric deoxynucleotidyl transferase dUTP nick-end labeling assay with propidium iodide staining, the responsiveness of sensitive cells was associated with apoptotic induction and cell cycle inhibition. Protein kinase C-beta-selective inhibition was observed not to affect AKT phosphorylation but to induce a rapid and sustained reduction in the phosphorylation of glycogen synthase kinase-3 beta, ribosomal protein S6, and mammalian target of rapamycin in sensitive cell lines. The results indicate that PKCβ plays an important role in AIDS-related NHL survival and suggest that PKCβ targeting should be considered in a broader spectrum of NHL. The observations in BCBL-1 were unexpected in the absence of PKCβ2 expression and implicate PKCβ1 as a regulator in those cells.

[Detection method for the ability of hemp (Cannabis sativa L.) seed germination by the use of 2,3,5-triphenyl-2H-tetrazolium chloride (TTC)].

PubMed

Ogata, Jun; Kikura-Hanajiri, Ruri; Yoshimatsu, Kayo; Kiuchi, Fumiyuki; Goda, Yukihiro

2008-11-01

Cannabis plants show a high Delta9-tetrahydrocannabinol content and are used as a psychoactive drug. Therefore the cultivation of hemp and its possession are prohibited by law in Japan. Meanwhile, Cannabis seeds have been used as a component of shichimi-togarashi (a Japanese spice), bird feed, or a crude drug (mashinin). To exclude the possibility of germination, it is officially noticed that hemp seeds must be killed. However, the number of violators has increased in recent years. To judge the ability of seed germination, a germination test is performed. However, the test requires several days and thus has not been used for on-site inspection. In this study, we developed a rapid detection method to determine the ability of Cannabis seeds to germinate using 2,3,5-triphenyl-2H-tetrazolium chloride (TTC). The principle of the assay is as follows. The endogenous respiratory enzymes in hemp seeds convert added colorless TTC into red 1,3,5-triphenylformazan. Consequently, a living embryo is stained red, while red does not appear in the dead seeds. The reaction was active over a pH range of 8.0-9.0, and the optimum activity was found from 40 to 50 degrees C. Under the optimum conditions, we were able to determine the ability of seeds to germinate based on the presence of color within 20 min. Since this method is rapid and simple, it is applicable to on-site inspections. In addition, it could be used as an alternative technique to the germination test, because erroneous decisions is cannot occur under the assay principle.

Antimycobacterial and cytotoxic activity of selected medicinal plant extracts

PubMed Central

Nguta, Joseph M.; Appiah-Opong, Regina; Nyarko, Alexander K.; Yeboah-Manu, Dorothy; Addo, Phyllis G.A.; Otchere, Isaac; Kissi-Twum, Abena

2016-01-01

Ethnopharmacological relevance Tuberculosis (TB) caused by Mycobacterium tuberculosis remains an ongoing threat to human health. Several medicinal plants are used traditionally to treat tuberculosis in Ghana. The current study was designed to investigate the antimycobacterial activity and cytotoxicity of crude extracts from five selected medicinal plants. Material and methods The microplate alamar blue assay (MABA) was used for antimycobacterial studies while the CellTiter 96® AQueous Assay, which is composed of solutions of a novel tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent (phenazine methosulfate) PMS, was used for cytotoxic studies. Correlation coefficients were used to compare the activity of crude extracts against nonpathogenic strains and the pathogenic Mycobacterium tuberculosis subsp.tuberculosis. Results Results of the MIC determinations indicated that all the crude extracts were active on all the three tested mycobacterial strains. Minimum inhibitory concentration values as low as 156.3 µg/mL against M. tuberculosis; Strain H37Ra (ATCC® 25,177™) were recorded from the leaves of Solanum torvum Sw. (Solanaceae). Cytotoxicity of the extracts varied, and the leaves from S. torvum had the most promising selectivity index. Activity against M. tuberculosis; Strain H37Ra was the best predictor of activity against pathogenic Mycobacterium tuberculosis subsp.tuberculosis (correlation coefficient=0.8). Conclusion The overall results of the present study provide supportive data on the use of some medicinal plants for tuberculosis treatment. The leaves of Solanum torvum are a potential source of anti-TB natural products and deserve further investigations to develop novel anti-TB agents against sensitive and drug resistant strains of M. tuberculosis. PMID:26875647

Bactericidal activity of juvenile chinook salmon macrophages against Aeromonas salmonicida after exposure to live or heat-killed Renibacterium salmoninarum or to soluble proteins produced by R. salmoninarum

USGS Publications Warehouse

Siegel, D.C.; Congleton, J.L.

1997-01-01

Macrophages isolated from the anterior kidney of juvenile chinook salmon Oncorhynchus tshawytscha in 96-well microtiter plates were exposed for 72 h to 0, 105, or 106 live or heat-killed Renibacterium salmoninarum cells per well or to 0, 0.1, 1.0, or 10 ??g/mL of R. salmoninarum soluble proteins. After treatment, the bactericidal activity of the macrophages against Aerornonas salmonicida was determined by a colorimetric assay based on the reduction of the tetrazolium dye MTT to formazan by viable bacteria. The MTT assay was modified to allow estimation of the percentage of bacteria killed by reference to a standard curve relating the number of bacteria added to microtiter wells to absorbance by formazan at 600 nm. The live and heat-killed R. salmoninarum treatments significantly (P < 0.001) increased killing of A. salmonicida by chinook salmon macrophages. In each of the five trials, significantly (P < 0.05) greater increases in killing occurred after exposure to 105 R. salmoninarum cells than to 106 R. salmoninarum cells per well. In contrast, treatment of macrophages with 10 ??g/mL R. salmoninarum soluble proteins significantly (P < 0.001) decreased killing of A. salmonicida, but treatment with lower doses did not. These results show that the bactericidal activity of chinook salmon macrophages is stimulated by exposure to R. salmoninarum cells at lower dose levels but inhibited by exposure to R. salmoninarum cells or soluble proteins at higher dose levels.

Hepatoprotective properties in the rat of Mitracarpus scaber (Rubiaceae).

PubMed

Germanò, M P; Sanogo, R; Costa, C; Fulco, R; D'Angelo, V; Torre, E A; Viscomi, M G; De Pasquale, R

1999-06-01

The effect of Mitracarpus scaber on carbon tetrachloride-induced acute liver damage in the rat has been evaluated. Results showed that treatment with Mitracarpus scaber decoction resulted in significant hepatoprotection against CCl4-induced liver injury both in-vivo and in-vitro. In-vivo, Mitracarpus scaber pretreatment reduced levels of serum glutamate-oxalate-transaminase (P < 0.01 for 250, 500 and 1000 mg kg(-1)) and serum glutamate-pyruvate-transaminase (P < 0.05 for 250 mg kg(-1) and P < 0.01 for 1000 mg kg(-1)) previously increased by administration of CCl4. In-vitro results indicated that addition to the culture medium of Mitracarpus scaber extracts significantly reduced glutamate-oxalate-transaminase (P < 0.05 for 100 microg mL(-1) and P < 0.01 for 10 and 1000 microg mL(-1)) and lactate dehydrogenase activity (P < 0.05 for 10 microg mL(-1)). Mitracarpus treatment also resulted in a good ( > 93%) survival rate for the CCl4-intoxicated hepatocytes as demonstrated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Moreover, as in the in-vitro assay, Mitracarpus scaber had radical-scavenging properties, shown by its reaction with the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical (EC50, the extract concentration resulting in a 50% reduction in the absorbance of DPPH blank solution, = 41.64+/-1.5 microg mL(-1)). The results of this study showed that Mitracarpus scaber had antihepatotoxic potential, a finding which supports the validity of traditional usage of this drug in Mali for the treatment of liver diseases.

A new paper-based analytical device for detection of Glucose-6-phosphate dehydrogenase deficiency.

PubMed

Kaewarsa, Phuritat; Laiwattanapaisal, Wanida; Palasuwan, Attakorn; Palasuwan, Duangdao

2017-03-01

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a genetic haemolytic disorder. Most persons with G6PD deficiency are asymptomatic, but exposure to oxidant drugs, such as the anti-malarial drug primaquine, may induce haemolysis, which is commonly found in Asian countries. A reliable test is necessary for diagnosing the deficiency to prevent an acute haemolytic crisis. This study proposes a novel quantitative method to detect G6PD deficiency using paper-based analytical devices (G6PDD-PAD). Wax printing was utilized for fabricating circular reaction zone patterns in paper. The colorimetric assay is based on the formation of formazan via a reduction of tetra-nitro blue tetrazolium (TNBT) by the G6PD enzyme on G6PDD-PAD. Detection was achieved by capturing the colour using a desktop scanner and the colour intensity was analysed with Adobe Photoshop C56. The results showed that the G6PD activity analysed by G6PDD-PAD was highly correlated with the standard biochemical assay (SBA) (r 2 =0.87, p<0.01). Moreover, good agreement by Bland-Altman bias plot was demonstrated between G6PDD-PAD and the SBA (mean bias 1.4 IU/gHb). The detection limit was 0 IU/gHb of G6PD activity. This study demonstrates the feasibility of using G6PDD-PAD. This simple, low-cost test ($0.1/test) should be useful for diagnosing G6PD deficiency in resource-limited settings. Copyright © 2016 Elsevier B.V. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vieira, J.M.B.D., E-mail: jmanya@terra.com.br; Laboratorio de Biologia de Anaerobios, IMPPG, UFRJ, Rio de Janeiro; Seabra, S.H.

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

[Effect of smoking on neutrophil oxidative metabolism].

PubMed

Zasimauskas, Darius; Zekonis, Gediminas

2008-01-01

Alterations in neutrophil function by tobacco products may play a central role in the pathogenesis of periodontal diseases and several smoking-related systemic diseases. The aim of the study was to evaluate the effect of smoking on neutrophil oxidative metabolism. The study included 17 smoking men free of systemic diseases who were referred for treatment of various odontological diseases to outpatient department of Kaunas University of Medicine Hospital. The age of subjects varied from 22 to 43 years. All subjects answered the questions about smoking habits. Clinical examination included assessment of oral hygiene status according to the OHI-s index and periodontal status according to Russell and Ramfjord indices. To evaluate the oxidative metabolism of neutrophils, luminol- and liucigenin-dependent chemiluminescence and nitroblue tetrazolium test were used. After smoking, extracellular liucigenin-dependent chemiluminescence response was higher as compared to the response before smoking, but total (intra- and extracellular) luminol-dependent chemiluminescence response was the same both before and after smoking. Exposure of neutrophils to smoking caused a significant increase in nitroblue tetrazolium reduction. The release of reactive oxygen species in neutrophils exposed to smoking may alter the pathogenic processes in periodontal diseases.

New visible and selective DNA staining method in gels with tetrazolium salts.

PubMed

Paredes, Aaron J; Naranjo-Palma, Tatiana; Alfaro-Valdés, Hilda M; Barriga, Andrés; Babul, Jorge; Wilson, Christian A M

2017-01-15

DNA staining in gels has historically been carried out using silver staining and fluorescent dyes like ethidium bromide and SYBR Green I (SGI). Using fluorescent dyes allows recovery of the analyte, but requires instruments such as a transilluminator or fluorimeter to visualize the DNA. Here we described a new and simple method that allows DNA visualization to the naked eye by generating a colored precipitate. It works by soaking the acrylamide or agarose DNA gel in SGI and nitro blue tetrazolium (NBT) solution that, when exposed to sunlight, produces a purple insoluble formazan precipitate that remains in the gel after exposure to light. A calibration curve made with a DNA standard established a detection limit of approximately 180 pg/band at 500 bp. Selectivity of this assay was determined using different biomolecules, demonstrating a high selectivity for DNA. Integrity and functionality of the DNA recovered from gels was determined by enzymatic cutting with a restriction enzyme and by transforming competent cells after the different staining methods, respectively. Our method showed the best performance among the dyes employed. Based on its specificity, low cost and its adequacy for field work, this new methodology has enormous potential benefits to research and industry. Copyright © 2016 Elsevier Inc. All rights reserved.

Synergistic effects of ICI 182,780 on the cytotoxicity of cisplatin in cervical carcinoma cell lines.

PubMed

García-López, Patricia; Rodríguez-Dorantes, Mauricio; Pérez-Cárdenas, Enrique; Cerbón, Marco; Mohar-Betancourt, Alejandro

2004-06-01

We investigated the ability of the novel pure antiestrogen ICI 182,780 to modulate the cytotoxic effects of cisplatin in several cervical cancer cell lines. The effect of cisplatin alone and cisplatin combined with ICI 182,780 on cellular death was studied using an assay based on a tetrazolium dye (sodium 3'-[1-(phenylamino-carbonyl)-3,4-tetrazolium], XTT). Before and after treatment with ICI 182,780, expression of the estrogen and progesterone receptor genes were assessed by a reverse transcriptase polymerase chain reaction (RT-PCR). Cell-cycle modifications after combined treatment with cisplatin and ICI 182,780 were studied by flow cytometry. Analysis of the data by the isobologram method showed that the combination of ICI 182,780 and cisplatin produced a synergistic antiproliferative effect in cervical cancer cells. The effect of ICI 182,780 on the cytotoxicity of cisplatin could be mediated, at least partially, by inhibition of estrogen and progesterone gene expression and by arresting the cell cycle at the G(2)/M phase. Our results suggest that ICI 182,780 can improve the efficacy of cisplatin in cancer cells and that this antihormonal drug therapy may be a useful candidate for further evaluation in combination with antineoplastic drugs, particularly cisplatin, in the treatment of cancer.

Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo

PubMed Central

Oliveira, R.J.; Mantovani, M.S.; da Silva, A.F.; Pesarini, J.R.; Mauro, M.O.; Ribeiro, L.R.

2014-01-01

The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero. PMID:24714812

Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo.

PubMed

Oliveira, R J; Mantovani, M S; Silva, A F da; Pesarini, J R; Mauro, M O; Ribeiro, L R

2014-04-01

The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero.

Development of a high-throughput colorimetric Zika virus infection assay.

PubMed

Müller, Janis A; Harms, Mirja; Schubert, Axel; Mayer, Benjamin; Jansen, Stephanie; Herbeuval, Jean-Philippe; Michel, Detlef; Mertens, Thomas; Vapalahti, Olli; Schmidt-Chanasit, Jonas; Münch, Jan

2017-04-01

Zika virus (ZIKV) is an emerging pathogen that causes congenital infections which may result in birth defects, such as microcephaly. Currently, no approved treatment or vaccination is available. ZIKV can be readily detected in cell culture where virally infected cells are normally stained by specific antibodies. As ZIKV regularly causes a cytopathic effect, we were wondering whether this viral property can be used to quantitatively determine viral infectivity. We here describe the use of an 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide-(MTT)-based cell viability assay that allows to determine ZIKV-induced cell death. We show that this colorimetric assay quantifies ZIKV infection over a broad range of viral dilutions in both monkey and human cells. It allows to determine inhibitory activities of antivirals that block ZIKV or to define the neutralizing antibody titers of ZIKV antisera. This MTT-based ZIKV detection assay can be evaluated by naked eye or computational tools, has a broad linear range, does not require large equipment or costly reagents, and thus represents a promising alternative to antibody-based assays, in particular in resource-poor settings. We propose to use this simple, fast, and cheap method for quantification of ZIKV neutralizing antibodies and testing of antiviral compounds.

Measurement of microbial activity in soil by colorimetric observation of in situ dye reduction: an approach to detection of extraterrestrial life

PubMed Central

Crawford, Ronald L; Paszczynski, Andrzej; Lang, Qingyong; Erwin, Daniel P; Allenbach, Lisa; Corti, Giancarlo; Anderson, Tony J; Cheng, I Francis; Wai, Chien; Barnes, Bruce; Wells, Richard; Assefi, Touraj; Mojarradi, Mohammad

2002-01-01

Background Detecting microbial life in extraterrestrial locations is a goal of space exploration because of ecological and health concerns about possible contamination of other planets with earthly organisms, and vice versa. Previously we suggested a method for life detection based on the fact that living entities require a continual input of energy accessed through coupled oxidations and reductions (an electron transport chain). We demonstrated using earthly soils that the identification of extracted components of electron transport chains is useful for remote detection of a chemical signature of life. The instrument package developed used supercritical carbon dioxide for soil extraction, followed by chromatography or electrophoresis to separate extracted compounds, with final detection by voltammetry and tandem mass-spectrometry. Results Here we used Earth-derived soils to develop a related life detection system based on direct observation of a biological redox signature. We measured the ability of soil microbial communities to reduce artificial electron acceptors. Living organisms in pure culture and those naturally found in soil were shown to reduce 2,3-dichlorophenol indophenol (DCIP) and the tetrazolium dye 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT). Uninoculated or sterilized controls did not reduce the dyes. A soil from Antarctica that was determined by chemical signature and DNA analysis to be sterile also did not reduce the dyes. Conclusion Observation of dye reduction, supplemented with extraction and identification of only a few specific signature redox-active biochemicals such as porphyrins or quinones, provides a simplified means to detect a signature of life in the soils of other planets or their moons. PMID:12150716

Targeted and controlled drug delivery system loading artersunate for effective chemotherapy on CD44 overexpressing cancer cells.

PubMed

Tran, Tuan Hiep; Nguyen, Tuan Duc; Van Nguyen, Han; Nguyen, Hanh Thuy; Kim, Jong Oh; Yong, Chul Soon; Nguyen, Chien Ngoc

2016-05-01

Poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles with negative surface charge were reversed to positive by cationic surfactant-DDAB before being coated with an anionic polymer, hyaluronic acid, to improve their site-specific intracellular delivery against CD44 receptor overexpressing cancer cells. Incorporating artesunate (ART)-a promising anticancer drug into PLGA/HA nanoparticles, is expected not only to overcome its poor aqueous solubility and stability but also enhance the activities. The obtained particles were characterized by dynamic light scattering, zeta potential measurements, and transmission electron microscopy (TEM). Cancer cell internalization of the NPs was evaluated by flow cytometry and cytotoxicity of the NPs was tested by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay. PLGA/HA nanoparticles showed greater extent of cellular uptake to SCC-7 and MCF-7 cells, indicating their affinity with CD44 receptor-mediated endocytosis. Almost 60 % of ART was released into the outer media after 48 h. In vitro fluorescence sorting demonstrated that PLGA/HA had highly efficient targeting and accumulation into CD44 receptor overexpression cells. The significant reduction in cell viability as well as greater induction of apoptosis suggested a potential in anticancer therapy of ART loaded PLGA/HA.

An active lifestyle induces positive antioxidant enzyme modulation in peripheral blood mononuclear cells of overweight/obese postmenopausal women.

PubMed

Farinha, Juliano Boufleur; De Carvalho, Nélson Rodrigues; Steckling, Flávia Mariel; De Vargas, Liziane Da Silva; Courtes, Aline Alves; Stefanello, Sílvio Terra; Martins, Caroline Curry; Bresciani, Guilherme; Dos Santos, Daniela Lopes; Soares, Félix Alexandre Antunes

2015-01-15

The aim of this study was to investigate the effects of an active lifestyle on mitochondrial functioning, viability, bioenergetics, and redox status markers in peripheral blood mononuclear cells (PBMC) of overweight/ obese postmenopausal women. We performed a cross-sectional study with postmenopausal women aged 45–64 years and body mass index N 25 kg/m2, divided into physically active (n = 23) and sedentary (n = 12) groups. Mitochondria functioning and viability, bioenergetics and redox status parameters were assessed in PBMC with spectrophotometric and fluorometric assays. No differences were found in the enzyme activity of complexes I and II of the electron transport chain (ETC), mitochondrial superoxide dismutase (MnSOD) activity, methyl-tetrazolium reduction levels and reduced glutathione and oxidized glutathione levels between the groups. However, the physically active group presented higher levels of reactive oxygen species (ROS) (P= 0.04) and increased catalase (CAT) (P= 0.029), total (P= 0.011) and cytosolic SOD (CuZnSOD) (P= 0.009) activities. An active lifestyle that includes aerobic exercise for at least 30 min, three times per week may improve antioxidant enzyme activities in PBMC in overweight/obese postmenopausal women, without changes in the activity of the ETC enzymes. However, this low intensity physical activity is not able to induce relevant mitochondrial adaptations.

Antitumor and antiviral activity of Colombian medicinal plant extracts.

PubMed

Betancur-Galvis, L; Saez, J; Granados, H; Salazar, A; Ossa, J

1999-01-01

Extracts of nine species of plants traditionally used in Colombia for the treatment of a variety of diseases were tested in vitro for their potential antitumor (cytotoxicity) and antiherpetic activity. MTT (Tetrazolium blue) and Neutral Red colorimetric assays were used to evaluate the reduction of viability of cell cultures in presence and absence of the extracts. MTT was also used to evaluate the effects of the extracts on the lytic activity of herpes simplex virus type 2 (HSV-2). The 50% cytotoxic concentration (CC50) and the 50% inhibitory concentration of the viral effect (EC50) for each extract were calculated by linear regression analysis. Extracts from Annona muricata, A. cherimolia and Rollinia membranacea, known for their cytotoxicity were used as positive controls. Likewise, acyclovir and heparin were used as positive controls of antiherpetic activity. Methanolic extract from Annona sp. on HEp-2 cells presented a CC50 value at 72 hr of 49.6x10(3)mg/ml. Neither of the other extracts examined showed a significant cytotoxicity. The aqueous extract from Beta vulgaris, the ethanol extract from Callisia grasilis and the methanol extract Annona sp. showed some antiherpetic activity with acceptable therapeutic indexes (the ratio of CC50 to EC50). These species are good candidates for further activity-monitored fractionation to identify active principles.

THE LOCALIZATION OF ENZYME ACTIVITIES IN THE RAT BRAIN

PubMed Central

Becker, Norwin H.; Goldfischer, Sidney; Shin, Woo-Yung; Novikoff, Alex B.

1960-01-01

Studies with rat brain illustrate the usefulness of formol-calcium-fixed tissue for studying both enzymatic "chemoarchitectonics" and intracellular organelles. Unembedded frozen sections and polyvinyl alcohol-embedded sections may be used to demonstrate the activities of DPNH-tetrazolium reductase localized in mitochondria and ergastoplasm, TPNH-tetrazolium reductase localized in mitochondria, ATPase (and/or apyrase or ADPase) in cell membranes, and acid phosphatase in lysosomes.1 Among the observations recorded are: (1) the presence of lysosomes in all cells of the brain; (2) the presence of numerous large lysosomes near the nuclei of capillary endothelial cells; (3) a polarized arrangement of large lysosomes in epithelial cells of the ependyma and choroid plexus; (4) the presence of ATPase activity in the cell membranes of some neurons; (5) the presence of either an apyrase or combination of ATPase and ADPase in the cell membranes of neuroglia and capillaries; (6) the presence of both DPNH- and TPNH-tetrazolium reductase activities in neuroglia; (7) the presence of DPNH- and TPNH-tetrazolium reductase activities in mitochondria and of DPNH-tetrazolium reductase activity in Nissl substance. The possible functional significance of these localizations is briefly discussed, as is their relation to "quantitative histochemistry" data available in the literature. PMID:13688468

The localization of enzyme activities in the rat brain.

PubMed

BECKER, N H; GOLDFISCHER, S; SHIN, W Y; NOVIKOFF, A B

1960-12-01

Studies with rat brain illustrate the usefulness of formol-calcium-fixed tissue for studying both enzymatic "chemoarchitectonics" and intracellular organelles. Unembedded frozen sections and polyvinyl alcohol-embedded sections may be used to demonstrate the activities of DPNH-tetrazolium reductase localized in mitochondria and ergastoplasm, TPNH-tetrazolium reductase localized in mitochondria, ATPase (and/or apyrase or ADPase) in cell membranes, and acid phosphatase in lysosomes.(1) Among the observations recorded are: (1) the presence of lysosomes in all cells of the brain; (2) the presence of numerous large lysosomes near the nuclei of capillary endothelial cells; (3) a polarized arrangement of large lysosomes in epithelial cells of the ependyma and choroid plexus; (4) the presence of ATPase activity in the cell membranes of some neurons; (5) the presence of either an apyrase or combination of ATPase and ADPase in the cell membranes of neuroglia and capillaries; (6) the presence of both DPNH- and TPNH-tetrazolium reductase activities in neuroglia; (7) the presence of DPNH- and TPNH-tetrazolium reductase activities in mitochondria and of DPNH-tetrazolium reductase activity in Nissl substance. The possible functional significance of these localizations is briefly discussed, as is their relation to "quantitative histochemistry" data available in the literature.

In-vivo neutrophil migration and nitroblue tetrazolium reduction in sickle cell disease.

PubMed

Akinyanju, O O

1985-01-01

In order to determine the contribution of neutrophil malfunction to the phenomenon of enhanced susceptibility of sickle cell disease patients to bacterial infection, the in-vivo neutrophil migration capacity in 23 sickle cell patients and in 14 normal controls; and the neutrophil reduction of nitroblue tetrazolium dye in 74 sickle cell patients and in 78 normal controls were studied. Secondarily the usefulness of the NBT test in distinguishing between osteomyelitis and uncomplicated bone pain was examined. No impairment of neutrophil migratory capacity was evident as no significant difference was observed between the mean migrated neutrophil count in the sickle cell subjects (1.99 X 10(9)/1) and that in normal controls (2.08 X 10(9)/1). The mean NBT scores were 19.9 +/- 8.9% in non-infected controls and 41.3 +/- 14.6% in infected controls (P less than 0.001). In sickle cell disease they were 23.6 +/- 6% in steady state subjects, 29.2 +/- 16.4% in sterile painful crises, 42.9 +/- 15% in non-osteomyelitic bacterial infection (P less than 0.001) and 18.9 +/- 4.2% during osteomyelitis. Thus all sickle cell subjects apart from those with osteomyelitis showed significant increases in the NBT scores during bacterial infection. The low score in sickle cell osteomyelitis is possibly associated with a relative neutrophil phagocytic defect which requires further elucidation. The NBT test was not useful in distinguishing uncomplicated painful crisis from early osteomyelitis in sickle cell disease.

PKI-587 and sorafenib alone and in combination on inhibition of liver cancer stem cell proliferation

PubMed Central

Gedaly, Roberto; Galuppo, Roberto; Musgrave, Yolanda; Angulo, Paul; Hundley, Jonathan; Shah, Malay; Daily, Michael F.; Chen, Changguo; Cohen, Donald A.; Spear, Brett T.; Evers, B. Mark

2015-01-01

Background Deregulated Ras/Raf/mitogen-activated protein kinase and PI3 K/AKT/mTOR signaling pathways are significant in hepatocellular carcinoma proliferation (HCC). In this study we evaluated differences in the antiproliferative effect of dual PI3 K/Akt/mTOR and Ras/Raf/mitogen-activated protein kinase inhibition of non liver cancer stem cell lines (PLC and HuH7) and liver cancer stem cell (LCSC) lines (CD133, CD44, CD24, and aldehyde dehydrogenase 1-positive cells). Materials and methods Flow cytometry was performed on the resulting tumors to identify the LCSC markers CD133, CD44, CD24, and aldehyde dehydrogenase 1. Methylthiazol tetrazolium assay was used to assess cellular proliferation. Finally, a Western blot assay was used to evaluate for inhibition of specific enzymes in these two signaling pathways. Results Using flow cytometry, we found that LCSC contain 64.4% CD133 + cells, 83.2% CD44 + cells, and 96.4% CD24 + cells. PKI-587 and sorafenib caused inhibiton of LCSC and HCC cell proliferation. PLC cells were more sensitive to PKI-587 than LCSC or Huh7 (P < 0.001). Interestingly, HuH7 cells were more sensitive to sorafenib than LCSC or PLC cells. Additionally, combination therapy with PKI-587 and sorafenib caused significantly more inhibition than monotherapy in HuH7, PLC, and LCSC. Using the methylthiazol tetrazolium assay, we found that the LCSC proliferation was inhibited with sorafenib monotherapy 39% at 5 μM (P < 0.001; n = 12) and 67% by PKI-587 at 0.1 μM (P = 0.002, n = 12) compared with control. The combination of PKI-587 and sorafenib, however, synergistically inhibited LCSC proliferation by 86% (P = 0.002; n = 12). Conclusions LCSC (CD133+, CD44+, CD24+) were able to develop very aggressive tumors with low cell concentrations at 4 to 6 wk. Cells CD133+, CD44+, CD24+ demonstrated at least moderate resistance to therapy in vitro. The combination of PKI-587 and sorafenib was better than either drug alone at inhibiting of LCSC and on HCC cell proliferation. PMID:23769634

PKI-587 and sorafenib alone and in combination on inhibition of liver cancer stem cell proliferation.

PubMed

Gedaly, Roberto; Galuppo, Roberto; Musgrave, Yolanda; Angulo, Paul; Hundley, Jonathan; Shah, Malay; Daily, Michael F; Chen, Changguo; Cohen, Donald A; Spear, Brett T; Evers, B Mark

2013-11-01

Deregulated Ras/Raf/mitogen-activated protein kinase and PI3 K/AKT/mTOR signaling pathways are significant in hepatocellular carcinoma proliferation (HCC). In this study we evaluated differences in the antiproliferative effect of dual PI3 K/Akt/mTOR and Ras/Raf/mitogen-activated protein kinase inhibition of non liver cancer stem cell lines (PLC and HuH7) and liver cancer stem cell (LCSC) lines (CD133, CD44, CD24, and aldehyde dehydrogenase 1-positive cells). Flow cytometry was performed on the resulting tumors to identify the LCSC markers CD133, CD44, CD24, and aldehyde dehydrogenase 1. Methylthiazol tetrazolium assay was used to assess cellular proliferation. Finally, a Western blot assay was used to evaluate for inhibition of specific enzymes in these two signaling pathways. Using flow cytometry, we found that LCSC contain 64.4% CD133 + cells, 83.2% CD44 + cells, and 96.4% CD24 + cells. PKI-587 and sorafenib caused inhibiton of LCSC and HCC cell proliferation. PLC cells were more sensitive to PKI-587 than LCSC or Huh7 (P < 0.001). Interestingly, HuH7 cells were more sensitive to sorafenib than LCSC or PLC cells. Additionally, combination therapy with PKI-587 and sorafenib caused significantly more inhibition than monotherapy in HuH7, PLC, and LCSC. Using the methylthiazol tetrazolium assay, we found that the LCSC proliferation was inhibited with sorafenib monotherapy 39% at 5 μM (P < 0.001; n = 12) and 67% by PKI-587 at 0.1 μM (P = 0.002, n = 12) compared with control. The combination of PKI-587 and sorafenib, however, synergistically inhibited LCSC proliferation by 86% (P = 0.002; n = 12). LCSC (CD133+, CD44+, CD24+) were able to develop very aggressive tumors with low cell concentrations at 4 to 6 wk. Cells CD133+, CD44+, CD24+, which demonstrated at least moderate resistance to therapy in vitro. The combination of PKI-587 and sorafenib was better than either drug alone at inhibiting of LCSC and on HCC cell proliferation. Copyright © 2013 Elsevier Inc. All rights reserved.

Antioxidant and Cytotoxic Effect of Barringtonia racemosa and Hibiscus sabdariffa Fruit Extracts in MCF-7 Human Breast Cancer Cell Line.

PubMed

Amran, Norliyana; Rani, Anis Najwa Abdul; Mahmud, Roziahanim; Yin, Khoo Boon

2016-01-01

The fruits of Barringtonia racemosa and Hibiscus sabdariffa have been used in the treatment of abscess, ulcer, cough, asthma, and diarrhea as traditional remedy. This study aims to evaluate cytotoxic effect of B. racemosa and H. sabdariffa methanol fruit extracts toward human breast cancer cell lines (MCF-7) and its antioxidant activities. Total antioxidant activities of extracts were assayed using 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH) and β-carotene bleaching assay. Content of phytochemicals, total flavonoid content (TFC), and total phenolic content (TPC) were determined using aluminum chloride colorimetric method and Folin-Ciocalteu's reagent, respectively. Cytotoxic activity in vitro was investigated through 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. B. racemosa extract exhibited high antioxidant activities compared to H. sabdariffa methanol fruit extracts in DPPH radical scavenging assay (inhibitory concentration [IC50] 15.26 ± 1.25 μg/mL) and ί-carotene bleaching assay (I% 98.13 ± 1.83%). B. racemosa also showed higher TPC (14.70 ± 1.05 mg gallic acid equivalents [GAE]/g) and TFC (130 ± 1.18 mg quercetin equivalents [QE]/g) compared to H. sabdariffa (3.80 ± 2.13 mg GAE/g and 40.75 ± 1.15 mg QE/g, respectively). In MTT assay, B. racemosa extract also showed a higher cytotoxic activity (IC50 57.61 ± 2.24 μg/mL) compared to H. sabdariffa. The present study indicated that phenolic and flavonoid compounds known for oxidizing activities indicated an important role among the contents of these plants extract. B. racemosa methanol extract have shown potent cytotoxic activity toward MCF-7. Following these promising results, further fractionation of the plant extract is underway to identify important phytochemical bioactives for the development of potential nutraceutical and pharmaceutical use. The phenolic and flavonoid compounds were present in B. racemosa and H. sabdariffa methanol extractsB. racemosa methanol extract was found to be potent antioxidant activityB. racemosa methanol extract have shown potent cytotoxic activity (IC50 57.61 ± 2.24 μg/mL) toward MCF-7The phenolic and flavonoid compounds may contribute to the antioxidant and cytotoxic activity of B. racemosa. Abbreviations Used: MCF-7: Human breast cancer cell lines, DMEM: Modified eagle medium, DPPH: 2,2'-diphenyl-1-picrylhydrazyl radical, TPC: Total phenolic content, Na2CO3: Sodium carbonate, GAE: Gallic acid equivalents, TFC: Total flavonoid content, NaNO2: Sodium nitrite, AlCl3: Aluminum chloride, NaOH: Sodium hydroxide, QE: Quercetin equivalents, MTT: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide, IC50: Inhibitory concentration, Analysis of variance, DLA: Dalton's lymphoma ascitic.

Shedding Light on the Oxygen Reduction Reaction Mechanism in Ether-Based Electrolyte Solutions: A Study Using Operando UV-Vis Spectroscopy.

PubMed

Hirshberg, Daniel; Sharon, Daniel; Afri, Michal; Lavi, Ronit; Frimer, Aryeh A; Metoki, Noa; Eliaz, Noam; Kwak, Won-Jin; Sun, Yang-Kook; Aurbach, Doron

2018-04-04

Using UV-vis spectroscopy in conjunction with various electrochemical techniques, we have developed a new effective operando methodology for investigating the oxygen reduction reactions (ORRs) and their mechanisms in nonaqueous solutions. We can follow the in situ formation and presence of superoxide moieties during ORR as a function of solvent, cations, anions, and additives in the solution. Thus, using operando UV-vis spectroscopy, we found evidence for the formation of superoxide radical anions during oxygen reduction in LiTFSI/diglyme electrolyte solutions. Nitro blue tetrazolium (NBT) was used to indicate the presence of superoxide moieties based on its unique spectral response. Indeed, the spectral response of NBT containing solutions undergoing ORR could provide a direct indication for the level of association of the Li cations with the electrolyte anions.

Study of a non-diffusing radiochromic gel dosimeter for 3D radiation dose imaging

NASA Astrophysics Data System (ADS)

Marsden, Craig Michael

2000-12-01

This thesis investigates the potential of a new radiation gel dosimeter, based on nitro-blue tetrazolium (NBTZ) suspended in a gelatin mold. Unlike all Fricke based gel dosimeters this dosimeter does not suffer from diffusive loss of image stability. Images are obtained by an optical tomography method. Nitro blue tetrazolium is a common biological indicator that when irradiated in an aqueous medium undergoes reduction to a highly colored formazan, which has an absorbance maximum at 525nm. Tetrazolium is water soluble while the formazan product is insoluble. The formazan product sticks to the gelatin matrix and the dose image is maintained for three months. Methods to maximize the sensitivity of the system were evaluated. It was found that a chemical detergent, Triton X-100, in combination with sodium formate, increased the dosimeter sensitivity significantly. An initial G-value of formazan production for a dosimeter composed of 1mM NBTZ, gelatin, and water was on the order of 0.2. The addition of Triton and formate produced a G-value in excess of 5.0. The effects of NBTZ, triton, formate, and gel concentration were all investigated. All the gels provided linear dose vs. absorbance plots for doses from 0 to >100 Gy. It was determined that gel concentration had minimal if any effect on sensitivity. Sensitivity increased slightly with increasing NBTZ concentration. Triton and formate individually and together provided moderate to large increases in dosimeter sensitivity. The dosimeter described in this work can provide stable 3D radiation dose images for all modalities of radiation therapy equipment. Methods to increase sensitivity are developed and discussed.

Synergistic activity of lysozyme and antifungal agents against Candida albicans biofilms on denture acrylic surfaces.

PubMed

Samaranayake, Y H; Cheung, B P K; Parahitiyawa, N; Seneviratne, C J; Yau, J Y Y; Yeung, K W S; Samaranayake, L P

2009-02-01

Denture related oral candidiasis is a recalcitrant fungal infection not easily resolved by topical antifungals. The antimycotic protein lysozyme, in saliva is an important host defense mechanism although its activity against Candida biofilms on denture acrylic has not been evaluated. (i) To establish a clinically relevant denture acrylic assay model to develop standardized Candida albicans biofilms, and (ii) assess the inhibitory effects of lysozyme alone and, the latter combined with antifungals (nystatin, amphotericin B, ketoconazole and 5-fluorocytosine) on sessile Candida cells and, finally (iii) to visualize the accompanying ultrastructural changes. The rotating-disc biofilm reactor was used to develop standardized 48 h Candida biofilms on acrylic discs in YNB/100 mM glucose medium and the biofilm metabolic activity was monitored using a tetrazolium reduction assay. The biofilm metabolic activity was similar in 18 identical denture acrylic discs (p<0.05) thus validating the rotating-disc biofilm model. Very low concentrations of lysozyme (6.25 microg/ml) significantly (p<0.01) inhibited Candida biofilm formation indicating that lysozyme may likely regulate intra-oral Candida biofilm development. Although 100 microg/ml lysozyme killed 45% of sessile Candida cells, further increasing its concentration (up to 240 microg/ml) had no such effect. Nystatin, amphotericin B, and ketoconazole in association with 100 microg/ml lysozyme exhibited effective synergistic killing of biofilm Candida in comparison to drug-free controls. Scanning electron and confocal scanning laser microscopy analysis confirmed the latter trends. Our results indicate that agents found in biological fluids such as lysozyme could be a safe adjunct to antifungals in future treatment strategies for recalcitrant candidal infections.

Candida species from oral cavity of HIV-infected children exhibit reduced virulence factors in the HAART era.

PubMed

Portela, Maristela Barbosa; Lima de Amorim, Elaine; Santos, Adrielle Mangabeira; Alexandre da Rocha Curvelo, José; de Oliveira Martins, Karol; Capillé, Cauli Lima; Maria de Araújo Soares, Rosangela; Barbosa de Araújo Castro, Gloria Fernanda

2017-01-01

This study aimed to assess, in vitro, the biofilm viability and the phospholipase and protease production of Candida spp. from the saliva of HIV infected children and healthy controls, and to correlate the results with the use of medical data. A total of 79 isolates were analyzed: 48 Candida albicans isolates (33/15) and 20 Candida parapsilosis sensu lato complex isolates (12/8) (from HIV/control patients, respectively), and 8 Candida krusei, 1 Candida tropicalis, 1 Candida dubliniensis and 1 Candida guilliermondii from HIV patients. The XTT (2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-Carboxanilide) reduction assay analyzed the biofilm viability. Phospholipase and protease assays were performed using the egg yolk and Bovine Serum Albumin agar plate methods, respectively. All isolates were able to form biofilm with cell viability. Quantitatively, Candida isolates from both groups presented a similar ability to form biofilm (p > 0.05). The biofilm viability activity was higher in C. albicans isolates than in non-albicans Candida isolates (p < 0.05) for both groups. Phospholipase activity was detected in 32 isolates (40.5%) and it was significantly higher in the HIV group (p = 0.006). Protease activity was detected in 66 isolates (84.8%) and most of them were relatively/very strong producers. No statistical association with medical data was found in the HIV group. Although Candida spp. isolates from HIV-positive children presented higher phospholipase production, in vitro they exhibited reduced virulence factors compared to isolates from healthy individuals. This finding may enlighten the role played by immunosuppression in the modulation of Candida virulence attributes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Activity and Phylogenetic Diversity of Bacterial Cells with High and Low Nucleic Acid Content and Electron Transport System Activity in an Upwelling Ecosystem

PubMed Central

Longnecker, K.; Sherr, B. F.; Sherr, E. B.

2005-01-01

We evaluated whether bacteria with higher cell-specific nucleic acid content (HNA) or an active electron transport system, i.e., positive for reduction of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), were responsible for the bulk of bacterioplankton metabolic activity. We also examined whether the phylogenetic diversity of HNA and CTC-positive cells differed from the diversity of Bacteria with low nucleic acid content (LNA). Bacterial assemblages were sampled both in eutrophic shelf waters and in mesotrophic offshore waters in the Oregon coastal upwelling region. Cytometrically sorted HNA, LNA, and CTC-positive cells were assayed for their cell-specific [3H]leucine incorporation rates. Phylogenetic diversity in sorted non-radioactively labeled samples was assayed using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes. Cell-specific rates of leucine incorporation of HNA and CTC-positive cells were on average only slightly greater than the cell-specific rates of LNA cells. HNA cells accounted for most bacterioplankton substrate incorporation due to high abundances, while the low abundances of CTC-positive cells resulted in only a small contribution by these cells to total bacterial activity. The proportion of the total bacterial leucine incorporation attributable to LNA cells was higher in offshore regions than in shelf waters. Sequence data obtained from DGGE bands showed broadly similar phylogenetic diversity across HNA, LNA, and CTC-positive cells, with between-sample and between-region variability in the distribution of phylotypes. Our results suggest that LNA bacteria are not substantially different from HNA bacteria in either cell-specific rates of substrate incorporation or phylogenetic composition and that they can be significant contributors to bacterial metabolism in the sea. PMID:16332746

Activity and phylogenetic diversity of bacterial cells with high and low nucleic acid content and electron transport system activity in an upwelling ecosystem.

PubMed

Longnecker, K; Sherr, B F; Sherr, E B

2005-12-01

We evaluated whether bacteria with higher cell-specific nucleic acid content (HNA) or an active electron transport system, i.e., positive for reduction of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), were responsible for the bulk of bacterioplankton metabolic activity. We also examined whether the phylogenetic diversity of HNA and CTC-positive cells differed from the diversity of Bacteria with low nucleic acid content (LNA). Bacterial assemblages were sampled both in eutrophic shelf waters and in mesotrophic offshore waters in the Oregon coastal upwelling region. Cytometrically sorted HNA, LNA, and CTC-positive cells were assayed for their cell-specific [3H]leucine incorporation rates. Phylogenetic diversity in sorted non-radioactively labeled samples was assayed using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes. Cell-specific rates of leucine incorporation of HNA and CTC-positive cells were on average only slightly greater than the cell-specific rates of LNA cells. HNA cells accounted for most bacterioplankton substrate incorporation due to high abundances, while the low abundances of CTC-positive cells resulted in only a small contribution by these cells to total bacterial activity. The proportion of the total bacterial leucine incorporation attributable to LNA cells was higher in offshore regions than in shelf waters. Sequence data obtained from DGGE bands showed broadly similar phylogenetic diversity across HNA, LNA, and CTC-positive cells, with between-sample and between-region variability in the distribution of phylotypes. Our results suggest that LNA bacteria are not substantially different from HNA bacteria in either cell-specific rates of substrate incorporation or phylogenetic composition and that they can be significant contributors to bacterial metabolism in the sea.

Proanthocyanidins polymeric tannin from Stryphnodendron adstringens are active against Candida albicans biofilms.

PubMed

Luiz, Raul Leal Faria; Vila, Taissa Vieira Machado; de Mello, João Carlos Palazzo; Nakamura, Celso Vataru; Rozental, Sonia; Ishida, Kelly

2015-03-19

Biofilm formation is important in Candida albicans pathogenesis and constitutes a mechanism of antifungal resistance. Thus, we evaluated the effect of proanthocyanidins polymer-rich fractions from Stryphnodendron adstringens (fraction F2 and subfraction F2.4) against C. albicans biofilms. Firstly, the antifungal activity of F2 and F2.4 against planktonic cells of Candida albicans (ATCC 10231) was determined using broth microdilution method. Anti-biofilm effect of F2 and F2.4 was evaluated during biofilm formation or on mature biofilm of C. albicans and compared with standard antifungals amphotericin B and fluconazole. Metabolic activity of sessile and dispersion cells from biofilms after antifungal treatments were measured using a tetrazolium reduction assay and the biofilm total biomass was quantified by crystal violet-based assay. Morphological alterations after treatments were observed using scanning electron microscopy. The anti-biofilm effect of F2 and F2.4 were comparable to standard antifungals (amphotericin B and fluconazole). F2 and F2.4 treatments reduced biofilm metabolic activity (in sessile and in dispersion cells) during biofilm formation, and in mature biofilms, unlike fluconazole, which only prevents the biofilm formation. Treatments with F2, F2.4 or fluconazole reduced biofilm biomass during biofilm formation, but not in mature biofilm. Amphotericin B presented higher inhibitory effect on biofilm formation and on mature biofilm of C. albicans. F2 and F2.4 treatments led to the appearance of dumbbell-shaped blastoconidia and of blastoconidia clusters in biofilms. Proanthocyanidins polymer-rich fractions from S. adstringens successfully inhibited C. albicans planktonic growth and biofilm development, and they represent a potential new agent for the treatment of biofilm-associated candidiasis.

Miltefosine is effective against Candida albicans and Fusarium oxysporum nail biofilms in vitro.

PubMed

Machado Vila, Taissa Vieira; Sousa Quintanilha, Natália; Rozental, Sonia

2015-11-01

Onychomycosis is a fungal nail infection that represents ∼50 % of all nail disease cases worldwide. Clinical treatment with standard antifungals frequently requires long-term systemic therapy to avoid chronic disease. Onychomycosis caused by non-dermatophyte moulds, such as Fusarium spp., and yeasts, such as Candida spp., is particularly difficult to treat, possibly due to the formation of drug-resistant fungal biofilms on affected areas. Here, we show that the alkylphospholipid miltefosine, used clinically against leishmaniasis and cutaneous breast metastases, has potent activity against biofilms of Fusarium oxysporum and Candida albicans formed on human nail fragments in vitro. Miltefosine activity was compared with that of commercially available antifungals in the treatment of biofilms at two distinct developmental phases: formation and maturation (pre-formed biofilms). Drug activity towards biofilms formed on nail fragments and on microplate surfaces (microdilution assays) was evaluated using XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assays, and drug effects on fingernail biofilms were analysed by scanning electron microscopy (SEM). For F. oxysporum, miltefosine at 8 μg ml- 1 inhibited biofilm formation by 93%, whilst 256 μg ml- 1 reduced the metabolic activity of pre-formed nail biofilms by 93%. Treatment with miltefosine at 1000 μg ml- 1 inhibited biofilm formation by 89% and reduced the metabolic activity of pre-formed C. albicans biofilms by 99%. SEM analyses of biofilms formed on fingernail fragments showed a clear reduction in biofilm biomass after miltefosine treatment, in agreement with XTT results. Our results show that miltefosine has potential as a therapeutic agent against onychomycosis and should be considered for in vivo efficacy studies, especially in topical formulations for refractory disease treatment.

The toxicity study of functionalized CNT from fermented tapioca on neuroblastoma cell

NASA Astrophysics Data System (ADS)

Nurulhuda, I.; Mazatulikhma, M. Z.; Alrokayan, S.; Khan, H.; Rusop, M.

2018-05-01

Carbon nanotubes known as one of the most interesting types of nanomaterials, especially use in application directly to cells. Somehow the use should take into consideration regarding the potential adverse impact on human health. Current study, the carbon nanotube was synthesized from fermented tapioca and functionalized with polyethylene glycol and directly test on the neuroblastoma cells in vitro. The toxicity effect on cells was assessed by 3(4, 5-dimethylthiazol-2-yl)-2, 5-tetrazolium bromide assays. It showed a dose-and time-dependent less toxic effect on functionalized carbon nanotube compared to non-functionalized. This leads us to the conclusion that functionalized carbon nanotube can be use for drug delivery in future.

Cytotoxic activity of ethanolic extract of the marine sponge Aaptos suberitoides against T47D cell

NASA Astrophysics Data System (ADS)

Nurhayati, Awik Puji Dyah; Prastiwi, Rarastoeti; Sukardiman, Wahyuningsih, Tri

2018-04-01

Aaptos suberitoides marine sponge produce many kinds of secondary metabolites. The purpose of this study were to examine the cytotoxic, proliferation inhibition and apoptosis induction of marine sponge A.suberitoides. The sponge was extracted with 96 % ethanol. Ethanol extract cytotoxicity assay were performed with MTT method (Microculture Tetrazolium) against to cell line of T47D. The proliferation inhibition were done by doubling time. The apoptosis induction by observing the treated cell morphology after staining with acrydine orange. The results show that cytotoxic activity of the ethanol extract was 153.109 µg/mL, inhibits cell proliferation cell lines of T47D at 24 hours of incubation and apoptosis induction.

Rhizome extracts of Curcuma zedoaria Rosc induce caspase dependant apoptosis via generation of reactive oxygen species in filarial parasite Setaria digitata in vitro.

PubMed

Senathilake, K S; Karunanayake, E H; Samarakoon, S R; Tennekoon, K H; de Silva, E D

2016-08-01

Human lymphatic filariasis (LF) is mainly caused by filarial parasite Wuchereria bancrofti and is the second leading cause of long term and permanent disability in tropical countries. To date, incapability to eliminate long lived adult parasites by current drugs remains the major challenge in the elimination of LF. Hence, in the current study, the efficacy of rhizome extracts of Curcuma zedoaria (a plant traditionally used in Sri Lanka in the management of LF) was evaluated as an effective filaricide in vitro. Sequential solvent extracts of C. zedoaria rhizomes were screened for in vitro antifilarial activity at 0.01-1 mg/mL concentrations by motility inhibition assay and 3-(4, 5 dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium bromide (MTT) reduction assay using cattle parasite Setaria digitata as a model organism. Exposure of parasites to hexane and chloroform extracts of C. zedoaria caused a dose dependant reduction in motility and viability of microfilariae (IC50 = 72.42 μg/mL for hexane extract, 191.14 μg/mL for chloroform extract) and adult parasites (IC50 = 77.07 μg/mL for hexane extract, 259.87 μg/mL for chloroform extract). Both extracts were less toxic to human peripheral blood mononuclear cells when compared to filariae. A dose dependant increase in caspase 3/CED 3 and a decrease in total protein content, cyclooxygenase (COX) and protein tyrosine phosphatase (PTP) activities were observed in adult parasites treated with hexane or chloroform extract. A significant degree of chromatin condensation and apoptotic body formation were also observed in these worms by Hoechst 33342 and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling (TUNEL) staining respectively. Dose dependant chromosomal DNA laddering was observed in treated adult worms but not in microfilariae in response to both extracts. Oxidative stress parameters such as reduction in reduced glutathione (GSH) levels and increase in glutathione s transferase (GST), superoxide dismutase (SOD) and catalase activities, increased reactive oxygen levels (ROS) and lipid peroxidation were also observed indicating that an apoptotic event is induced by reactive oxygen species. Copyright © 2016 Elsevier Inc. All rights reserved.

Multi-centre assessment of nitroblue tetrazolium reactivity in human semen as a potential marker of oxidative stress.

PubMed

Gosálvez, Jaime; Coppola, Lamberto; Fernández, Jose Luis; López-Fernández, Carmen; Góngora, Alfredo; Faundez, Ricardo; Kim, John; Sayme, Nabil; de la Casa, Moises; Santiso, Rebeca; Harrison, Keith; Agarwal, Ashok; Johnston, Stephen; Esteves, Sandro C

2017-05-01

The nitroblue tetrazolium (NBT) reaction as a tracer of oxidative stress was examined in 707 ejaculates from seven clinics. Semen was initially surveyed by classifying the NBT reaction using a pre-established rank for the Oxisperm® test based on three colourimetric levels: L1, low (n = 141 [20%]); L2, medium (n = 538 [76%]) and L3, high (n = 28 [4%]). L3 was indicative of a high level of superoxide anions. Halosperm® chromatin dispersion assay was used to analyse samples of ejaculates 30 min after ejaculation; no difference was found in DNA fragmentation of L1 or L3; L3 category semen samples incubated for 24 h at 37 o C showed a significantly faster rate (P < 0.001) of DNA damage than those in L1. The NBT reaction was further characterized in the ejaculates of 100 patients to determine the relative contribution of seminal plasma, spermatozoa, or both. Seminal plasma was the most significant fraction of •O 2 - localization, whereas sperm fractions generated detectable reactive oxygen species in only 32% of the ejaculates. Formazan precipitates were primarily associated with the sperm mid-piece and seminal leukocytes; however, not all spermatozoa stained positive to formazan and not all leukocytes presented with equivalent production of superoxide anions. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Polypropylene Biocomposites with Boron Nitride and Nanohydroxyapatite Reinforcements

PubMed Central

Chan, Kai Wang; Wong, Hoi Man; Yeung, Kelvin Wai Kwok; Tjong, Sie Chin

2015-01-01

In this study, we develop binary polypropylene (PP) composites with hexagonal boron nitride (hBN) nanoplatelets and ternary hybrids reinforced with hBN and nanohydroxyapatite (nHA). Filler hybridization is a sound approach to make novel nanocomposites with useful biological and mechanical properties. Tensile test, osteoblastic cell culture and dimethyl thiazolyl diphenyl tetrazolium (MTT) assay were employed to investigate the mechanical performance, bioactivity and biocompatibility of binary PP/hBN and ternary PP/hBN-nHA composites. The purpose is to prepare biocomposite nanomaterials with good mechanical properties and biocompatibility for replacing conventional polymer composites reinforced with large hydroxyapatite microparticles at a high loading of 40 vol%. Tensile test reveals that the elastic modulus of PP composites increases, while tensile elongation decreases with increasing hBN content. Hybridization of hBN with nHA further enhances elastic modulus of PP. The cell culture and MTT assay show that osteoblastic cells attach and proliferate on binary PP/hBN and ternary PP/hBN-20%nHA nanocomposites. PMID:28787984

Lidocaine inhibits the proliferation of lung cancer by regulating the expression of GOLT1A.

PubMed

Zhang, Lei; Hu, Rong; Cheng, Yanyong; Wu, Xiaoyang; Xi, Siwei; Sun, Yu; Jiang, Hong

2017-10-01

Lidocaine is the most commonly used local anaesthetic in clinical and can inhibit proliferation, suppress invasion and migration and induce apoptosis in human lung adenocarcinoma (LAD) cells. However, its specific downstream molecular mechanism is unclear. LAD cell lines, A549 and H1299 cells, were treated with lidocaine. The proliferation was evaluated by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) and bromodeoxyuridine (BrdU) assay. The expression level of related proteins was detected by real-time quantitative PCR (qPCR) and Western blot assay. The results indicated that lidocaine dose-dependently suppressed the proliferation of A549 and H1299 cells. In the LAD patients' samples, GOLT1A was upregulated and involved in the poor prognosis and higher grade malignancy. Additionally, GOLT1A mediates the function of lidocaine on repressing proliferation by regulating the cell cycle in A549 cells. Our findings suggest that lidocaine downregulates the GOLT1A expression to repress the proliferation of lung cancer cells. © 2017 John Wiley & Sons Ltd.

Atmospheric-pressure plasma jet characterization and applications on melanoma cancer treatment (B/16-F10)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mashayekh, Shahriar; Rajaee, Hajar; Hassan, Zuhir M.

2015-09-15

A new approach in medicine is the use of cold plasma for various applications such as sterilization blood coagulation and cancer cell treatment. In this paper, a pin-to-hole plasma jet for biological applications has been designed and manufactured and characterized. The characterization includes power consumption via Lissajous method, thermal behavior of atmospheric-pressure plasma jet by using Infra-red camera as a novel method and using Speicair software to determine vibrational and transitional temperatures, and optical emission spectroscopy to determine the generated species. Treatment of Melanoma cancer cells (B16/F10) was also implemented, and tetrazolium salt dye (MTT assay) and flow cytometry weremore » used to evaluate viability. Effect of ultraviolet photons on cancerous cells was also observed using an MgF{sub 2} crystal with MTT assay. Finally, in-vivo studies on C57 type mice were also done in order to have a better understanding of the effects in real conditions.« less

Determination of the optimum conditions for lung cancer cells treatment using cold atmospheric plasma

NASA Astrophysics Data System (ADS)

Akhlaghi, Morteza; Rajaei, Hajar; Mashayekh, Amir Shahriar; Shafiae, Mojtaba; Mahdikia, Hamed; Khani, Mohammadreza; Hassan, Zuhair Mohammad; Shokri, Babak

2016-10-01

Cold atmospheric plasmas (CAPs) can affect live cells and organisms due to the production of different reactive species. In this paper, the effects of various parameters of the CAP such as the treatment time, gas mixture, gas flow rate, applied voltage, and distance from the nozzle on the LL/2 lung cancer cell line have been studied. The probable effect of UV radiation has also been investigated using an MgF2 filter. Besides the cancerous cells, the 3T3 fibroblast cell line as a normal cell has been treated with the CAP. The Methylthiazol Tetrazolium assay showed that all parameters except the gas flow rate could impress effectively on the cancer cell viability. The cell proliferation seemed to be stopped after plasma treatment. The flow cytometry assay revealed that apoptosis and necrosis were appreciable. It was also found that treating time up to 2 min will not exert any effect on the normal cells.

Molecular docking study, synthesis and biological evaluation of Schiff bases as Hsp90 inhibitors.

PubMed

Dutta Gupta, Sayan; Snigdha, D; Mazaira, Gisela I; Galigniana, Mario D; Subrahmanyam, C V S; Gowrishankar, N L; Raghavendra, N M

2014-04-01

Heat shock protein 90 (Hsp90) is an emerging attractive target for the discovery of novel cancer therapeutic agents. Docking methods are powerful in silico tools for lead generation and optimization. In our mission to rationally develop novel effective small molecules against Hsp90, we predicted the potency of our designed compounds by Sybyl surflex Geom X docking method. The results of the above studies revealed that Schiff bases derived from 2,4-dihydroxy benzaldehyde/5-chloro-2,4-dihydroxy benzaldehyde demonstrated effective binding with the protein. Subsequently, a few of them were synthesized (1-10) and characterized by IR, (1)HNMR and mass spectral analysis. The synthesized molecules were evaluated for their potential to suppress Hsp90 ATPase activity by Malachite green assay. The anticancer studies were performed by 3-(4,5-dimethythiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay method. The software generated results was in satisfactory agreement with the evaluated biological activity. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Synergistic growth inhibition by sorafenib and vitamin K2 in human hepatocellular carcinoma cells.

PubMed

Zhang, Yafei; Zhang, Bicheng; Zhang, Anran; Zhao, Yong; Zhao, Jie; Liu, Jian; Gao, Jianfei; Fang, Dianchun; Rao, Zhiguo

2012-09-01

Sorafenib is an oral multikinase inhibitor that has been proven effective as a single-agent therapy in hepatocellular carcinoma, and there is a strong rationale for investigating its use in combination with other agents. Vitamin K2 is nearly non-toxic to humans and has been shown to inhibit the growth of hepatocellular carcinoma. In this study, we evaluated the effects of a combination of sorafenib and vitamin K2 on the growth of hepatocellular carcinoma cells. Flow cytometry, 3-(4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide) and nude mouse xenograft assays were used to examine the effects of sorafenib and vitamin K2 on the growth of hepatocellular carcinoma cells. Western blotting was used to elucidate the possible mechanisms underlying these effects. Assays for 3-(4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide) revealed a strong synergistic growth-inhibitory effect between sorafenib and vitamin K2. Flow cytometry showed an increase in cell cycle arrest and apoptosis after treatment with a combination of these two drugs at low concentrations. Sorafenib-mediated inhibition of extracellular signal-regulated kinase phosphorylation was promoted by vitamin K2, and downregulation of Mcl-1, which is required for sorafenib-induced apoptosis, was observed after combined treatment. Vitamin K2 also attenuated the downregulation of p21 expression induced by sorafenib, which may represent the mechanism by which vitamin K2 promotes the inhibitory effects of sorafenib on cell proliferation. Moreover, the combination of sorafenib and vitamin K2 significantly inhibited the growth of hepatocellular carcinoma xenografts in nude mice. Our results determined that combined treatment with sorafenib and vitamin K2 can work synergistically to inhibit the growth of hepatocellular carcinoma cells. This finding raises the possibility that this combined treatment strategy might be promising as a new therapy against hepatocellular carcinoma, especially for patients with poor liver tolerance.

Microelectrode Array-evaluation of Neurotoxic Effects of Magnesium as an Implantable Biomaterial

PubMed Central

Huang, Ting; Wang, Zhonghai; Wei, Lina; Kindy, Mark; Zheng, Yufeng; Xi, Tingfei; Gao, Bruce Z.

2016-01-01

Magnesium (Mg)-based biomaterials have shown great potential in clinical applications. However, the cytotoxic effects of excessive Mg2+ and the corrosion products from Mg-based biomaterials, particularly their effects on neurons, have been little studied. Although viability tests are most commonly used, a functional evaluation is critically needed. Here, both methyl thiazolyl tetrazolium (MTT) and lactate dehydrogenase (LDH) assays were used to test the effect of Mg2+ and Mg-extract solution on neuronal viability. Microelectrode arrays (MEAs), which provide long-term, real-time recording of extracellular electrophysiological signals of in vitro neuronal networks, were used to test for toxic effects. The minimum effective concentrations (ECmin) of Mg2+ from the MTT and LDH assays were 3 mmol/L and 100 mmol/L, respectively, while the ECmin obtained from the MEA assay was 0.1 mmol/L. MEA data revealed significant loss of neuronal network activity when the culture was exposed to 25% Mg-extract solution, a concentration that did not affect neuronal viability. For evaluating the biocompatibility of Mg-based biomaterials with neurons, MEA electrophysiological testing is a more precise method than basic cell-viability testing. PMID:27110081

Microelectrode Array-evaluation of Neurotoxic Effects of Magnesium as an Implantable Biomaterial.

PubMed

Huang, Ting; Wang, Zhonghai; Wei, Lina; Kindy, Mark; Zheng, Yufeng; Xi, Tingfei; Gao, Bruce Z

2016-01-01

Magnesium (Mg)-based biomaterials have shown great potential in clinical applications. However, the cytotoxic effects of excessive Mg 2+ and the corrosion products from Mg-based biomaterials, particularly their effects on neurons, have been little studied. Although viability tests are most commonly used, a functional evaluation is critically needed. Here, both methyl thiazolyl tetrazolium (MTT) and lactate dehydrogenase (LDH) assays were used to test the effect of Mg 2+ and Mg-extract solution on neuronal viability. Microelectrode arrays (MEAs), which provide long-term, real-time recording of extracellular electrophysiological signals of in vitro neuronal networks, were used to test for toxic effects. The minimum effective concentrations (EC min ) of Mg 2+ from the MTT and LDH assays were 3 mmol/L and 100 mmol/L, respectively, while the EC min obtained from the MEA assay was 0.1 mmol/L. MEA data revealed significant loss of neuronal network activity when the culture was exposed to 25% Mg-extract solution, a concentration that did not affect neuronal viability. For evaluating the biocompatibility of Mg-based biomaterials with neurons, MEA electrophysiological testing is a more precise method than basic cell-viability testing.

Assessment of anti-angiogenic and anti-tumoral potentials of Origanum onites L. essential oil.

PubMed

Bostancıoğlu, Rakibe Beklem; Kürkçüoğlu, Mine; Başer, Kemal Hüsnü Can; Koparal, Ayşe Tansu

2012-06-01

Medicinal plants and culinary herbs with anti-angiogenic and little toxicity properties have gained importance. Non-toxic anti-angiogenic phytochemicals are useful in combating cancer by preventing the formation of new blood vessels to support the tumor growth. We have investigated the essential oil of Origanum onites L. (OOEO), for a possible anti-angiogenic activity. OOEO was analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS). The anti-proliferative activities (by MTT assay, 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H-tetrazolium bromide), anti-angiogenic activities (by tube formation assay), cell migration inhibiting capability (migration assay) and apoptotic potential (DAPI staining) of OOEO were evaluated on rat adipose tissue endothelial cells (RATECs) and 5RP7 (c-H-ras transformed rat embryonic fibroblasts) cells. Our results revealed that OOEO could markedly inhibit cell viability and induced apoptosis of 5RP7 cells and also could block in vitro tube formation and migration of RATEC. These results imply that OOEO having anti-angiogenic activity might be useful in preventing angiogenesis-related diseases and in combating cancer. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

PubMed

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

2016-01-01

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

Water extract of Semecarpus parvifolia Thw. leaves inhibits cell proliferation and induces apoptosis on HEp-2 cells.

PubMed

Soysa, Preethi; Jayarthne, Panchima; Ranathunga, Imali

2018-03-05

Semecarpus parvifolia Thw is used as an ingredient of poly herbal decoctions to treat cancer in traditional medicine. The present study aims to investigate the antiproliferative activity on HEp 2 cells by the water extract of S. parvifolia leaves and to evaluate potential mechanisms. The plant extract was exposed to S. parvifolia for 24 hours and antiproliferative activity was quantified by Sulforhodamine B (SRB), 3-(4, 5-dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) and Lactate dehydrogenase (LDH) assays. Morphological changes were observed after staining cells with ethidium bromide/acridine orange (EB/AO) and Giemsa dye. Comet assay was performed to evaluate the DNA damage. The toxicity of the plant extract was determined by brine shrimp lethality assay. S. parvifolia leaves reduced the cell proliferation in a dose and time dependent manner. A two fold increase in NO level was observed at higher concentrations. Morphological changes characteristic to apoptosis were observed in light microscopy, Giemsa and EB/AO stained cells. Fragmented DNA further confirmed its capacity to induce apoptosis. No lethality was observed with brine shrimps. The results suggest that Semecarpus parvifolia Thw induces apoptosis in HEp-2 cells through a NO dependent pathway.

Toxicity of Nanoparticles and an Overview of Current Experimental Models.

PubMed

Bahadar, Haji; Maqbool, Faheem; Niaz, Kamal; Abdollahi, Mohammad

2016-01-01

Nanotechnology is a rapidly growing field having potential applications in many areas. Nanoparticles (NPs) have been studied for cell toxicity, immunotoxicity, and genotoxicity. Tetrazolium-based assays such as MTT, MTS, and WST-1 are used to determine cell viability. Cell inflammatory response induced by NPs is checked by measuring inflammatory biomarkers, such as IL-8, IL-6, and tumor necrosis factor, using ELISA. Lactate dehydrogenase (LDH) assay is used for cell membrane integrity. Different types of cell cultures, including cancer cell lines have been employed as in vitro toxicity models. It has been generally agreed that NPs interfere with either assay materials or with detection systems. So far, toxicity data generated by employing such models are conflicting and inconsistent. Therefore, on the basis of available experimental models, it may be difficult to judge and list some of the more valuable NPs as more toxic to biological systems and vice versa. Considering the potential applications of NPs in many fields and the growing apprehensions of FDA about the toxic potential of nanoproducts, it is the need of the hour to look for new internationally agreed free of bias toxicological models by focusing more on in vivo studies.

Cyclosporine A decreases the fluconazole minimum inhibitory concentration of Candida albicans clinical isolates but not biofilm formation and cell growth.

PubMed

Wibawa, T; Nurrokhman; Baly, I; Daeli, P R; Kartasasmita, G; Wijayanti, N

2015-03-01

Among the genus Candida, Candida albicans is the most abundant species in humans. One of the virulent factors of C. albicans is its ability to develop biofilm. Biofilm forming microbes are characterized by decreasing of its susceptibility to antibiotics and antifungal. The fungicidal effect of fluconazole may be enhanced by cyclosporine A in laboratory engineered C. albicans strains. The aim of this work is to analyze the synergistic effect of cyclosporine A with fluconazole in C. albicans clinical isolates and the effect of cycolsporine A alone in the biofilm formation. Six fluconazole resistant and six sensitive C. albicans clinical isolates were analyzed for its minimum inhibitory concentration (MICs), biofilm formation, and cell growths. A semi-quantitative XTT [2,3-bis(2-methoxy-4-nitro-5- sulfo-phenyl)-2H-tetrazolium-5-carboxanilide] reduction assay was conducted to measure the biofilm formation. Cyclosporine A has synergistic effect with fluconazole that was shown by decreasing MICs of both fluconazole resistant and sensitive C. albicans clinical isolates. However, cyclosporine A alone did not influence the biofilm formation and cell growth of both fluconazole resistant and sensitive C. albicans clinical isolates. These results indicated that cyclosporine A might be a promising candidate of adjuvant therapy for fluconazole against both fluconazole resistant and sensitive C. albicans clinical isolates.

Synthesis of Gold Mediated Biocompatible Nanocomposite of Lactone Enriched Fraction from Sahadevi (Vernonia cinerea Lees): An Assessment of Antimalarial Potential.

PubMed

Jyotshna; Shanker, Karuna; Khare, Puja; Tiwari, Nimisha; Mohanty, Shilpa; Bawankule, Dnyaneshwar U; Pal, Anirban

2016-01-01

Metals reduction into submicro/nano size through bhasma preparations for therapeutic use is well established in ancient traditional system of Indian medicines i.e. Ayurveda. Recently, nanotechnology has drawn the attention of researchers to develeope various size and shape nanoparicles / composite for number of applications.In this article, we report the enrichment of lactone enriched fraction (LEF) by liquid-liquid portioning of Vernonia cinerea metabolic extract and sysnthesis of mediated nano-gold composite (LEF-AuNPs) in single step process. The morphological characteristic based on transmission electron microscope (TEM) image analysis showed that LEF-AuNPs were predominantly nanopolygons and nanobots in shapes ranging from 50-200 nm in size. Abundance of phytochemicals in both LEF and LEF-AuNPs was dissimilar. In LEF, montanol- a diterpenoid, while in LEF-AuNPs, neophytadiene- a phytanes was the major compound. HPLC profile of relatively polar compounds also varied drastically. In-vitro biocompatibility, cytotoxicity [MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) based assay] and storage stabilitiy of LEF-AuNPs were evaluated. The moderate ability of LEF-AuNPs to restrict parasitaemia, extended mean survival time of mice infected with Plasmodium berghei and lack of any evident toxicity provides new opportunities for the safe delivery and applications of such nanocomposites in malaria therapy.

Cytotoxic potentials of biologically fabricated platinum nanoparticles from Streptomyces sp. on MCF-7 breast cancer cells.

PubMed

Baskaran, Balraj; Muthukumarasamy, Arulmozhi; Chidambaram, Siva; Sugumaran, Abimanyu; Ramachandran, Krithikadevi; Rasu Manimuthu, Thaneswari

2017-04-01

Biosynthesis of novel therapeutic nano-scale materials for biomedical and pharmaceutical applications has been enormously developed, since last decade. Herein, the authors report an ecological way of synthesising the platinum nanoparticles (PtNPs) using Streptomyces sp. for the first time . The produced PtNPs exhibited the face centred cubic system. The fourier transform infrared spectrum revealed the existence of amino acids in proteins which serves as an essential reductant for the formation of PtNPs. The spherical morphology of the PtNPs with an average size of 20-50 nm was observed from topographical images of atomic force microscopy and field emission scanning electron microscopy. The X-ray fluorescence spectrum confirms the presence of PtNPs with higher purity. The PtNPs size was further confirmed with transmission electron microscopy analysis and the particles were found to exist in the same size regime. Additionally, PtNPs showed the characteristic surface plasmon resonance peak at 262 nm. Dynamic light scattering studies report that 97.2% of particles were <100 nm, with an average particle diameter of about 45 nm. Furthermore, 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-tetrazolium assay based in vitro cytotoxicity analysis was conducted for the PtNPs, which showed the inhibitory concentration (IC 50 ) at 31.2 µg/ml against Michigan Cancer Foundation-7 breast cancer cells.

Protection of MES23.5 dopaminergic cells by obestatin is mediated by proliferative rather than anti-apoptotic action.

PubMed

Shen, Xiao-Li; Jia, Feng-Ju; Song, Ning; Xie, Jun-Xia; Jiang, Hong

2014-02-01

Obestatin is an endogenous peptide sharing a precursor with ghrelin. This study aims to investigate whether and how obestatin protects MES23.5 dopaminergic cells against 1-methyl-4-phenylpyridinium (MPP(+))-induced neurotoxicity. MES23.5 cells were pretreated with obestatin (10(-13)-10(-6) mol/L) for 20 min prior to incubation with 200 μmol/L MPP(+) for 12 or 24 h, or treated with obestatin alone (10(-13) to 10(-6) mol/L) for 0, 6, 12, and 24 h. The methyl thiazolyl tetrazolium (MTT) assay was used to measure cell viability. Flow cytometry was used to measure the caspase-3 activity and the mitochondrial transmembrane potential. Proliferating cell nuclear antigen (PCNA) protein levels were determined by Western blotting. Obestatin (10(-13) to 10(-7) mol/L) pretreatment blocked or even reversed the MPP(+)-induced reduction of viability in MES23.5 cells, but had no effect on MPP(+)-induced mitochondrial transmembrane potential collapse and caspase-3 activation. When applied alone, obestatin increased viability. Elevated PCNA levels occurred with 10(-7), 10(-9), 10(-11) and 10(-13) mol/L obestatin treatment for 12 h. The results suggest that the protective effects of obestatin against MPP(+) in MES23.5 cells are due to its proliferation-promoting rather than anti-apoptotic effects.

Effect of essential oils prepared from Thai culinary herbs on sessile Candida albicans cultures.

PubMed

Hovijitra, Ray S; Choonharuangdej, Suwan; Srithavaj, Theerathavaj

2016-01-01

Although medicinal herbs with fungicidal effects have been ubiquitously employed in traditional medicine, such effects of culinary herbs and spices still have to be elucidated. Therefore, it is noteworthy to determine the antifungal efficacy of some edible herbs used in Thai cuisine against sessile Candida albicans cultures, and to inquire if they can be further utilized as naturally-derived antifungals. Fourteen essential oils extracted from Thai culinary herbs and spices were tested for their antifungal activity against C. albicans using the agar disk diffusion method followed by broth micro-dilution method for the determination of minimum inhibitory concentration (MIC) and minimum fungicidal concentration. The oils with potent antifungal effects against planktonic fungi were then assessed for their effect against sessile fungus (adherent organisms and established biofilm culture). MIC of the oils against sessile C. albicans was evaluated by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide reduction assay. All selected culinary herbs and spices, except galangal, garlic, and turmeric, exhibited inhibitory effects on planktonic yeast cells. Cinnamon bark and sweet basil leaf essential oils exhibited potent fungicidal effect on planktonic and sessile fungus. Sessile MICs were 8-16 times higher than planktonic MICs. Consequently, both cinnamon bark and sweet basil leaf herbal oils seem to be highly effective anti-Candida choices. (J Oral Sci 58, 365-371, 2016).

Optimized delivery of skin keratinocytes by aerosolization and suspension in fibrin tissue adhesive.

PubMed

Harkin, Damien G; Dawson, Rebecca A; Upton, Zee

2006-01-01

Aerosolized suspensions of keratinocytes provide a potential therapy for wounds, but the effects of aerosolization on cell viability remain unclear. Likewise, little is known of the resulting cell distribution pattern and how this compares to the density required for epithelialization. The potential benefits of cospraying cells in the presence of fibrin adhesive are equally uncertain. Thus, in the present study we have optimized conditions for the aerosolization of cultured keratinocytes using a device (Tissomat) that supports the option for coapplication with fibrin (Tisseel). Cell viability was unaffected when sprayed at 10 psi, but a significant reduction in metabolic activity, as determined by the methylthiazoyldiphenol-tetrazolium assay, was observed at higher pressure. Bursts of 0.2 mL cell suspension (1.5x10(6)/mL) delivered from a height of 10 cm was sufficient to epithelialize an area of 10-15 cm2 within 7 days in vitro. Confluent areas corresponded to those with a density of 5,000-10,000 cells/cm2 at 24 hours. Optimal cell growth in Tisseel was achieved through dilution of fibrinogen (1-3 mg/mL) and thrombin (2-5 IU/mL). This optimized formulation eliminated fluid run-off postspraying and stimulated a twofold increase in cellular response. Therefore, our in vitro data supports the theory that aerosolized suspensions of keratinocytes in fibrin will benefit healing.

Differential effects of retinoic acid (RA) and N-(4-hydroxyphenyl) retinamide (4-HPR) on cell growth, induction of differentiation, and changes in p34cdc2, Bcl-2, and actin expression in the human promyelocytic HL-60 leukemic cells.

PubMed

Dipietrantonio, A; Hsieh, T C; Wu, J M

1996-07-25

Incubation of the HL-60 cells with 3 microM of RA and 4-HPR resulted in suppression of cell growth and decrease in cell viability. A significant percentage of the RA-treated cells also displayed differentiation towards neutrophils, as assayed by changes in nitroblue tetrazolium reduction (NBT) and alpha-naphthyl-acetate esterase (ANAE) activities, whereas the 4-HPR treated cells remained essentially undifferentiated. Flow cytometric analysis showed 4-HPR to cause partial cell arrest in the G2/M phase after a 3-day treatment and an additional G1 phase arrest after a 7-day treatment. With RA-treated cells, a reduction in the percentage of cells in the G1 phase was observed after 7 days of treatment. In 4-HPR-treated cells an extra peak, characteristic of cells undergoing apoptosis, was found in the cell cycle phase distribution analysis. Determination of specific protein expression changes by Western blot analysis showed that the p34cdc2 was down-regulated by both chemicals. Furthermore, RA induced bcl-2 but prevented the processing of actin, whereas 4-HPR had little effect on bcl-2 but increased the specific processing of actin. These results suggest that RA promotes neutrophil differentiation and the establishment of a semi apoptosis-resistant state, possibly through the overexpression of the bcl-2 gene. By contrast, 4-HPR may trigger apoptosis by inducing overall cyto-architectural changes and specific DNA fragmentation subsequent to increased turnover of the protein actin.

Radical scavenging activities of Rio Red grapefruits and Sour orange fruit extracts in different in vitro model systems.

PubMed

Jayaprakasha, G K; Girennavar, Basavaraj; Patil, Bhimanagouda S

2008-07-01

Antioxidant fractions from two different citrus species such as Rio Red (Citrus paradise Macf.) and Sour orange (Citrus aurantium L.) were extracted with five different polar solvents using Soxhlet type extractor. The total phenolic content of the extracts was determined by Folin-Ciocalteu method. Ethyl acetate extract of Rio Red and Sour orange was found to contain maximum phenolics. The dried fractions were screened for their antioxidant activity potential using in vitro model systems such as 1,1-diphenyl-2-picryl hydrazyl (DPPH), phosphomolybdenum method and nitroblue tetrazolium (NBT) reduction at different concentrations. The methanol:water (80:20) fraction of Rio Red showed the highest radical scavenging activity 42.5%, 77.8% and 92.1% at 250, 500 and 1000 ppm, respectively, while methanol:water (80:20) fraction of Sour orange showed the lowest radical scavenging activity at all the tested concentrations. All citrus fractions showed good antioxidant capacity by the formation of phosphomolybdenum complex at 200 ppm. In addition, superoxide radical scavenging activity was assayed using non-enzymatic (NADH/phenaxine methosulfate) superoxide generating system. All the extracts showed variable superoxide radical scavenging activity. Moreover, methanol:water (80:20) extract of Rio Red and methanol extract of Sour orange exhibited marked reducing power in potassium ferricyanide reduction method. The data obtained using above in vitro models clearly establish the antioxidant potential of citrus fruit extracts. However, comprehensive studies need to be conducted to ascertain the in vivo bioavailability, safety and efficacy of such extracts in experimental animals. To the best of our knowledge, this is the first report on antioxidant activity of different polar extracts from Rio Red and Sour oranges.

Enhancement of antibiotic activity by efflux inhibitors against multidrug resistant Mycobacterium tuberculosis clinical isolates from Brazil

PubMed Central

Coelho, Tatiane; Machado, Diana; Couto, Isabel; Maschmann, Raquel; Ramos, Daniela; von Groll, Andrea; Rossetti, Maria L.; Silva, Pedro A.; Viveiros, Miguel

2015-01-01

Drug resistant tuberculosis continues to increase and new approaches for its treatment are necessary. The identification of M. tuberculosis clinical isolates presenting efflux as part of their resistant phenotype has a major impact in tuberculosis treatment. In this work, we used a checkerboard procedure combined with the tetrazolium microplate-based assay (TEMA) to study single combinations between antituberculosis drugs and efflux inhibitors (EIs) against multidrug resistant M. tuberculosis clinical isolates using the fully susceptible strain H37Rv as reference. Efflux activity was studied on a real-time basis by a fluorometric method that uses ethidium bromide as efflux substrate. Quantification of efflux pump genes mRNA transcriptional levels were performed by RT-qPCR. The fractional inhibitory concentrations (FIC) indicated synergistic activity for the interactions between isoniazid, rifampicin, amikacin, ofloxacin, and ethidium bromide plus the EIs verapamil, thioridazine and chlorpromazine. The FICs ranged from 0.25, indicating a four-fold reduction on the MICs, to 0.015, 64-fold reduction. The detection of active efflux by real-time fluorometry showed that all strains presented intrinsic efflux activity that contributes to the overall resistance which can be inhibited in the presence of the EIs. The quantification of the mRNA levels of the most important efflux pump genes on these strains shows that they are intrinsically predisposed to expel toxic compounds as the exposure to subinhibitory concentrations of antibiotics were not necessary to increase the pump mRNA levels when compared with the non-exposed counterpart. The results obtained in this study confirm that the intrinsic efflux activity contributes to the overall resistance in multidrug resistant clinical isolates of M. tuberculosis and that the inhibition of efflux pumps by the EIs can enhance the clinical effect of antibiotics that are their substrates. PMID:25972842

Comparative Study of the Effects of Fluconazole and Voriconazole on Candida glabrata, Candida parapsilosis and Candida rugosa Biofilms.

PubMed

Madhavan, Priya; Jamal, Farida; Pei, Chong Pei; Othman, Fauziah; Karunanidhi, Arunkumar; Ng, Kee Peng

2018-06-01

Infections by non-albicans Candida species are a life-threatening condition, and formation of biofilms can lead to treatment failure in a clinical setting. This study was aimed to demonstrate the in vitro antibiofilm activity of fluconazole (FLU) and voriconazole (VOR) against C. glabrata, C. parapsilosis and C. rugosa with diverse antifungal susceptibilities to FLU and VOR. The antibiofilm activities of FLU and VOR in the form of suspension as well as pre-coatings were assessed by XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction assay. Morphological and intracellular changes exerted by the antifungal drugs on Candida cells were examined by scanning electron microscope (SEM) and transmission electron microscope (TEM). The results of the antibiofilm activities showed that FLU drug suspension was capable of killing C. parapsilosis and C. rugosa at minimum inhibitory concentrations (MICs) of 4× MIC FLU and 256× MIC FLU, respectively. While VOR MICs ranging from 2× to 32× were capable of killing the biofilms of all Candida spp tested. The antibiofilm activities of pre-coated FLU were able to kill the biofilms at ¼× MIC FLU and ½× MIC FLU for C. parapsilosis and C. rugosa strains, respectively. While pre-coated VOR was able to kill the biofilms, all three Candida sp at ½× MIC VOR. SEM and TEM examinations showed that FLU and VOR treatments exerted significant impact on Candida cell with various degrees of morphological changes. In conclusion, a fourfold reduction in MIC 50 of FLU and VOR towards ATCC strains of C. glabrata, C. rugosa and C. rugosa clinical strain was observed in this study.

Antifungal Efficacy of Caspofungin (MK-0991) in Experimental Pulmonary Aspergillosis in Persistently Neutropenic Rabbits: Pharmacokinetics, Drug Disposition, and Relationship to Galactomannan Antigenemia

PubMed Central

Petraitiene, Ruta; Petraitis, Vidmantas; Groll, Andreas H.; Sein, Tin; Schaufele, Robert L.; Francesconi, Andrea; Bacher, John; Avila, Nilo A.; Walsh, Thomas J.

2002-01-01

The antifungal efficacy, pharmacokinetics, and safety of caspofungin (CAS) were investigated in the treatment and prophylaxis of invasive pulmonary aspergillosis due to Aspergillus fumigatus in persistently neutropenic rabbits. Antifungal therapy consisted of 1, 3, or 6 mg of CAS/kg of body weight/day (CAS1, CAS3, and CAS6, respectively) or 1 mg of deoxycholate amphotericin B (AMB)/kg/day intravenously for 12 days starting 24 h after endotracheal inoculation. Prophylaxis (CAS1) was initiated 4 days before endotracheal inoculation. Rabbits treated with CAS had significant improvement in survival and reduction in organism-mediated pulmonary injury (OMPI) measured by pulmonary infarct score and total lung weight (P < 0.01). However, animals treated with CAS demonstrated a paradoxical trend toward increased residual fungal burden (log CFU per gram) and increased serum galactomannan antigen index (GMI) despite improved survival. Rabbits receiving prophylactic CAS1 also showed significant improvement in survival and reduction in OMPI (P < 0.01), but there was no effect on residual fungal burden. In vitro tetrazolium salt hyphal damage assays and histologic studies demonstrated that CAS had concentration- and dose-dependent effects on hyphal structural integrity. In parallel with a decline in GMI, AMB significantly reduced the pulmonary tissue burden of A. fumigatus (P ≤ 0.01). The CAS1, CAS3, and CAS6 dose regimens demonstrated dose-proportional exposure and maintained drug levels in plasma above the MIC for the entire 24-h dosing interval at doses that were ≥3 mg/kg/day. As serial galactomannan antigen levels may be used for therapeutic monitoring, one should be aware that profoundly neutropenic patients receiving echinocandins for aspergillosis might have persistent galactomannan antigenemia despite clinical improvement. CAS improved survival, reduced pulmonary injury, and caused dose-dependent hyphal damage but with no reduction in residual fungal burden or galactomannan antigenemia in persistently neutropenic rabbits with invasive pulmonary aspergillosis. PMID:11751105

Antioxidant and Cytotoxic Effect of Barringtonia racemosa and Hibiscus sabdariffa Fruit Extracts in MCF-7 Human Breast Cancer Cell Line

PubMed Central

Amran, Norliyana; Rani, Anis Najwa Abdul; Mahmud, Roziahanim; Yin, Khoo Boon

2016-01-01

Background: The fruits of Barringtonia racemosa and Hibiscus sabdariffa have been used in the treatment of abscess, ulcer, cough, asthma, and diarrhea as traditional remedy. Objective: This study aims to evaluate cytotoxic effect of B. racemosa and H. sabdariffa methanol fruit extracts toward human breast cancer cell lines (MCF-7) and its antioxidant activities. Materials and Methods: Total antioxidant activities of extracts were assayed using 2,2′-diphenyl-1-picrylhydrazyl radical (DPPH) and β-carotene bleaching assay. Content of phytochemicals, total flavonoid content (TFC), and total phenolic content (TPC) were determined using aluminum chloride colorimetric method and Folin–Ciocalteu's reagent, respectively. Cytotoxic activity in vitro was investigated through 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Results: B. racemosa extract exhibited high antioxidant activities compared to H. sabdariffa methanol fruit extracts in DPPH radical scavenging assay (inhibitory concentration [IC50] 15.26 ± 1.25 μg/mL) and ί-carotene bleaching assay (I% 98.13 ± 1.83%). B. racemosa also showed higher TPC (14.70 ± 1.05 mg gallic acid equivalents [GAE]/g) and TFC (130 ± 1.18 mg quercetin equivalents [QE]/g) compared to H. sabdariffa (3.80 ± 2.13 mg GAE/g and 40.75 ± 1.15 mg QE/g, respectively). In MTT assay, B. racemosa extract also showed a higher cytotoxic activity (IC50 57.61 ± 2.24 μg/mL) compared to H. sabdariffa. Conclusion: The present study indicated that phenolic and flavonoid compounds known for oxidizing activities indicated an important role among the contents of these plants extract. B. racemosa methanol extract have shown potent cytotoxic activity toward MCF-7. Following these promising results, further fractionation of the plant extract is underway to identify important phytochemical bioactives for the development of potential nutraceutical and pharmaceutical use. SUMMARY The phenolic and flavonoid compounds were present in B. racemosa and H. sabdariffa methanol extractsB. racemosa methanol extract was found to be potent antioxidant activityB. racemosa methanol extract have shown potent cytotoxic activity (IC50 57.61 ± 2.24 μg/mL) toward MCF-7The phenolic and flavonoid compounds may contribute to the antioxidant and cytotoxic activity of B. racemosa. Abbreviations Used: MCF-7: Human breast cancer cell lines, DMEM: Modified eagle medium, DPPH: 2,2’-diphenyl-1-picrylhydrazyl radical, TPC: Total phenolic content, Na2CO3: Sodium carbonate, GAE: Gallic acid equivalents, TFC: Total flavonoid content, NaNO2: Sodium nitrite, AlCl3: Aluminum chloride, NaOH: Sodium hydroxide, QE: Quercetin equivalents, MTT: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide, IC50: Inhibitory concentration, ANOVA: Analysis of variance, DLA: Dalton's lymphoma ascitic. PMID:26941539

Effect of leaching residual methyl methacrylate concentrations on in vitro cytotoxicity of heat polymerized denture base acrylic resin processed with different polymerization cycles

PubMed Central

BURAL, Canan; AKTAŞ, Esin; DENIZ, Günnur; ÜNLÜÇERÇI, Yeşim; BAYRAKTAR, Gülsen

2011-01-01

Objectives Residual methyl methacrylate (MMA) may leach from the acrylic resin denture bases and have adverse effects on the oral mucosa. This in vitro study evaluated and correlated the effect of the leaching residual MMA concentrations ([MMA]r) on in vitro cytotoxicity of L-929 fibroblasts. Material and Methods A total of 144 heat-polymerized acrylic resin specimens were fabricated using 4 different polymerization cycles: (1) at 74ºC for 9 h, (2) at 74ºC for 9 h and terminal boiling (at 100ºC) for 30 min, (3) at 74ºC for 9 h and terminal boiling for 3 h, (4) at 74ºC for 30 min and terminal boiling for 30 min. Specimens were eluted in a complete cell culture medium at 37ºC for 1, 2, 5 and 7 days. [MMA]r in eluates was measured using high-performance liquid chromatography. In vitro cytotoxicity of eluates on L-929 fibroblasts was evaluated by means of cell proliferation using a tetrazolium salt XTT (sodium 3´-[1-phenyl-aminocarbonyl)-3,4-tetrazolium]bis(4-methoxy-6-nitro)benzenesulphonic acid) assay. Differences in [MMA]r of eluates and cell proliferation values between polymerization cycles were statistically analyzed by Kruskal-Wallis, Friedman and Dunn's multiple comparison tests. The correlation between [MMA]r of eluates and cell proliferation was analyzed by Pearson's correlation test (p<0.05). Results [MMA]r was significantly (p≤0.001) higher in eluates of specimens polymerized with cycle without terminal boiling after elution of 1 and 2 days. Cell proliferation values for all cycles were significantly (p<0.01) lower in eluates of 1 day than those of 2 days. The correlation between [MMA]r and cell proliferation values was negative after all elution periods, showing significance (p<0.05) for elution of 1 and 2 days. MMA continued to leach from acrylic resin throughout 7 days and leaching concentrations markedly reduced after elution of 1 and 2 days. Conclusion Due to reduction of leaching residual MMA concentrations, use of terminal boiling in the polymerization process for at least 30 min and water storage of the heat-polymerized denture bases for at least 1 to 2 days before denture delivery is clinically recommended for minimizing the residual MMA and possible cytotoxic effects. PMID:21956586

Annonacin Exerts Antitumor Activity through Induction of Apoptosis and Extracellular Signal-regulated Kinase Inhibition

PubMed Central

Yap, Chee Voon; Subramaniam, Kavita S.; Khor, Sik Wey; Chung, Ivy

2017-01-01

Background: Endometrial cancer (EC) is the most common gynecologic malignancy in developed countries. Annonacin, a natural pure compound extracted from the seeds of Annona muricata, is a potential alternative therapeutic agent to treat EC. Objective: To study the antitumor activity of annonacin and its mechanism of action in EC cells (ECCs). Materials and Methods: Viability of ECCs treated with annonacin for 72 h was determined using methyl thiazolyl tetrazolium assay. The induction of cell cycle arrest and apoptotic cell death was evaluated using propidium iodide and annexin V-PE/7-AAD assay, respectively. DNA strand breaks were visualized using transferase dUTP nick end labeling assay, and the effects of annonacin on survival signaling were determined using western blotting. Results: Annonacin exhibited antiproliferative effects on EC cell lines (ECC-1 and HEC-1A) and primary cells (EC6-ept and EC14-ept) with EC50values ranging from 4.62 to 4.92 μg/ml. EC cells were shown arrested at G2/M phase after treated with 4 μg/ml of annonacin for 72 h. This led to a significant increase in apoptotic cell death (65.7%) in these cells when compared to vehicle-treated cells (P < 0.005). We further showed that annonacin-mediated apoptotic cell death was associated with an increase in caspase-3 cleavage and DNA fragmentation. Cell apoptosis was accompanied with downregulation of extracellular signal-regulated kinase survival protein expression and induction of G2/M cell cycle arrest. Conclusion: Annonacin may be a potential novel therapeutic agent for EC patients. SUMMARY We aimed to study the antitumor activity of annonacin and its mechanism of action in endometrial cancer cells. Annonacin exerted antiproliferation effects on both endometrial cancer cell lines and primary cells via induction of apoptosis and inhibition of extracellular signal-regulated kinase. Our data represented that annonacin could be an alternative therapeutic treatment to combat endometrial cancer. Abbreviations Used: 7-AAD: 7-Amino-Actinomycin, ATP: Adenosine diphosphate, BSA: Bovine serum albumin, DNA: Deoxyribonucleic acid, EC: Endometrial cancer, ECC-1: Endometrial cancer cell-1, EC50: Half maximal effective concentration, Ept: Epithelial, FBS: Fetal bovine serum, HEC-1A: Human endometrial carcinoma-1A, MTT: Methyl thiazolyl tetrazolium, NaCl: Sodium chloride, NADH: Nicotinamide adenine dinucleotide, RPMI 1640: Roswell Park Memorial Institute Medium, SDS: Sodium dodecyl sulfate PMID:29263632

Free radical scavenging activity and neuroprotective potentials of D138, one Cu(II)/Zn(II) Schiff-base complex derived from N,N'-bis(2-hydroxynaphthylmethylidene)-1,3-propanediamine.

PubMed

Wang, Che; Cai, Zheng-Xu; You, Zhong-Lu; Guo, Hui-Shu; Shang, De-Jing; Wang, Xiao-Ling; Zhang, Liang; Ma, Li-Jie; Tan, Jun; Le, Wei-Dong; Li, Song

2014-09-01

There is increasing evidence that free radicals play an important role in neuronal damages induced by diabetes mellitus or cerebral ischemia insults. Antioxidants with free radical scavenging activities have been shown to be beneficial and neuroprotective for these pathological conditions. Here, we report free radical scavenging activity and neuroprotective potential of D138, one copper(II)/zinc(II) Schiff-base complex derived from N,N'-2(2-hydroxynaphthylmethylidene)-1,3-propanediamine. The data from three in vitro assays, 2,2-diphenyl-1-picrylhydrazyl assay, nitro blue tetrazolium assay and hydroxyl radical scavenging assay, indicated that D138 presented a potent free radical scavenging activity. The neuroprotective and antioxidative effects of D138 were further evaluated in vivo using bilateral common carotid artery occlusion (BCCAO) mouse model and streptozotocin (STZ) diabetic mouse model. Our results indicated that treatment of D138 significantly ameliorated the hippocampal neuronal damage and the oxidative stress levels in these animal models. Moreover, D138 also reversed the behavioral deficiencies induced by BCCAO or STZ, as assessed by Y-maze test and fear conditioning test. In conclusion, all these findings support that D138 exerts free radical scavenging and neuroprotective activities and has the potentials to be a potent therapeutic candidate for brain oxidative damage induced by cerebral ischemia or diabetes mellitus.

Involvement of TRPV1 and AQP2 in hypertonic stress by xylitol in odontoblast cells.

PubMed

Tokuda, M; Fujisawa, M; Miyashita, K; Kawakami, Y; Morimoto-Yamashita, Y; Torii, M

2015-02-01

To examine the responses of mouse odontoblast-lineage cell line (OLC) cultures to xylitol-induced hypertonic stress. OLCs were treated with xylitol, sucrose, sorbitol, mannitol, arabinose and lyxose. Cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay. The expression of transient receptor potential vanilloids (TRPV) 1, 3 and 4 was detected using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. The expression of aquaporin (AQP) 2 was detected using immunofluorescence and Western blotting analysis. The expression of interleukin-6 (IL-6) under xylitol-induced hypertonic stress was assessed using an enzyme-linked immunosorbent assay (ELISA). Small interfering ribonucleic acid (siRNA) for AQP-2 was used to inhibition assay. Xylitol-induced hypertonic stress did not decrease OLC viability, unlike the other sugars tested. OLCs expressed TRPV1, 3 and 4 as well as AQP2. Xylitol inhibited lipopolysaccharide (LPS)-induced IL-6 expression after 3 h of hypertonic stress. TRPV1 mRNA expression was upregulated by xylitol. Costimulation with HgCl2 (AQP inhibitor) and Ruthenium red (TRPV1 inhibitor) decreased cell viability with xylitol stimulation. OLCs treated with siRNA against TRPV1 exhibited decreased cell viability with xylitol stimulation. OLCs have high-cell viability under xylitol-induced hypertonic stress, which may be associated with TRPV1 and AQP2 expressions.

[Determination of the healing effect of Piper aduncum (spiked pepper or matico) on human fibroblasts].

PubMed

Paco, Karen; Ponce-Soto, Luis Alberto; Lopez-Ilasaca, Marco; Aguilar, José L

2016-01-01

To evaluate the healing effect of a Piper aduncum ethanol-water extract on an adult human dermal fibroblast cell line (hDFa). After obtaining the extract via solid-liquid extraction, concentration, and lyophilization, extract proteins were purified using reverse phase high-performance liquid chromatography, identified using tandem mass spectrometry of tryptic peptides, and analyzed using MALDI-TOF-TOF on an ABSciex4800 mass spectrometer. Half maximum effective concentration values (EC50), half maximum inhibiting concentration (IC50), and percentages of cell proliferation were determined using tetrazolium salt assays. Cell migration was evaluated using a "scratch assay". Growth factor expression in cells was analyzed via quantitative real-time reverse transcription polymerase chain reaction. Against the hDFa cell line, the extract had an IC50 of 200 μg/mL and EC50 of 103.5 µg/mL. In the proliferation assay, protein K2 (obtained from the extract) exhibited increased proliferative activity relative to other treatments (1 µg/mL); this agent also exhibited increased activity (50 µg/mL) in the fibroblast migration assay.Furthermore, the relative expression of platelet-derived growth factor increased by 8.6-fold in the presence of K2 protein relative to the control. The hydroethanolic extract of Piper aduncum and its component proteins increased the proliferation and migration of hDFa and increased the expression of growth factors involved in the healing process.

Spathaspora passalidarum selected for resistance to AFEX hydrolysate shows decreased cell yield.

PubMed

Su, Yi-Kai; Willis, Laura B; Rehmann, Lars; Smith, David; Jeffries, Thomas W

2018-06-21

This study employed cell recycling, batch adaptation, cell mating and high throughput screening to select adapted Spathaspora passalidarum strains with improved fermentative ability.The most promising candidate, YK208-E11 (E11) showed a 3-fold increase in specific fermentation rate compared to the parental strain and an ethanol yield greater than 0.45 g/g substrate while co-utilizing cellobiose, glucose, and xylose. Further characterization showed that strain E11 also makes 40% less biomass compared to the parental strain when cultivated in rich media under aerobic conditions. A tetrazolium agar overlay assay in the presence of respiration inhibitors, including rotenone, antimycin A, KCN, and salicylhydroxamic acid elucidated the nature of the mutational events. Results indicated that E11 has a deficiency in its respiration system that could contribute to its low cell yield. Strain E11 was subjected to whole genome sequencing and a ∼11 kb deletion was identified; the open reading frames absent in strain E11 code for proteins with predicted functions in respiration, cell division and the actin cytoskeleton, and may contribute to the observed physiology of the adapted strain. Results of the tetrazolium overlay also suggest that cultivation on xylose affects the respiration capacity in the wild-type strain, which could account for its faster fermentation of xylose as compared to glucose. These results support our previous finding that S. passalidarum has highly unusual physiological responses to xylose under oxygen limitation.

Cytotoxicity Test Based on Human Cells Labeled with Fluorescent Proteins: Fluorimetry, Photography, and Scanning for High-Throughput Assay.

PubMed

Kalinina, Marina A; Skvortsov, Dmitry A; Rubtsova, Maria P; Komarova, Ekaterina S; Dontsova, Olga A

2018-06-01

High- and medium-throughput assays are now routine methods for drug screening and toxicology investigations on mammalian cells. However, a simple and cost-effective analysis of cytotoxicity that can be carried out with commonly used laboratory equipment is still required. The developed cytotoxicity assays are based on human cell lines stably expressing eGFP, tdTomato, mCherry, or Katushka2S fluorescent proteins. Red fluorescent proteins exhibit a higher signal-to-noise ratio, due to less interference by medium autofluorescence, in comparison to green fluorescent protein. Measurements have been performed on a fluorescence scanner, a plate fluorimeter, and a camera photodocumentation system. For a 96-well plate assay, the sensitivity per well and the measurement duration were 250 cells and 15 min for the scanner, 500 cells and 2 min for the plate fluorimeter, and 1000 cells and less than 1 min for the camera detection. These sensitivities are similar to commonly used MTT (tetrazolium dye) assays. The used scanner and the camera had not been previously applied for cytotoxicity evaluation. An image processing scheme for the high-resolution scanner is proposed that significantly diminishes the number of control wells, even for a library containing fluorescent substances. The suggested cytotoxicity assay has been verified by measurements of the cytotoxicity of several well-known cytotoxic drugs and further applied to test a set of novel bacteriotoxic compounds in a medium-throughput format. The fluorescent signal of living cells is detected without disturbing them and adding any reagents, thus allowing to investigate time-dependent cytotoxicity effects on the same sample of cells. A fast, simple and cost-effective assay is suggested for cytotoxicity evaluation based on mammalian cells expressing fluorescent proteins and commonly used laboratory equipment.

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

PubMed Central

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

2016-01-01

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

Potential protection of green tea polyphenols against 1800 MHz electromagnetic radiation-induced injury on rat cortical neurons.

PubMed

Liu, Mei-Li; Wen, Jian-Qiang; Fan, Yu-Bo

2011-10-01

Radiofrequency electromagnetic fields (EMF) are harmful to public health, but the certain anti-irradiation mechanism is not clear yet. The present study was performed to investigate the possible protective effects of green tea polyphenols against electromagnetic radiation-induced injury in the cultured rat cortical neurons. In this study, green tea polyphenols were used in the cultured cortical neurons exposed to 1800 MHz EMFs by the mobile phone. We found that the mobile phone irradiation for 24 h induced marked neuronal cell death in the MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazolium bromide) and TUNEL (TdT mediated biotin-dUTP nicked-end labeling) assay, and protective effects of green tea polyphenols on the injured cortical neurons were demonstrated by testing the content of Bcl-2 Assaciated X protein (Bax) in the immunoprecipitation assay and Western blot assay. In our study results, the mobile phone irradiation-induced increases in the content of active Bax were inhibited significantly by green tea polyphenols, while the contents of total Bax had no marked changes after the treatment of green tea polyphenols. Our results suggested a neuroprotective effect of green tea polyphenols against the mobile phone irradiation-induced injury on the cultured rat cortical neurons.

Screening of antiproliferative effect of aqueous extracts of plant foods consumed in México on the breast cancer cell line MCF-7.

PubMed

García-Solís, Pablo; Yahia, Elhadi M; Morales-Tlalpan, Verónica; Díaz-Muñoz, Mauricio

2009-01-01

We evaluated the antiproliferative effect of aqueous extracts of 14 plant foods consumed in Mexico on the breast cancer cell line MCF-7. The plant foods used were avocado, black sapote, guava, mango, prickly pear cactus stems (called nopal in Mexico, cooked and raw), papaya, pineapple, four different cultivars of prickly pear fruit, grapes and tomato. β-Carotene, total phenolics and gallic acid contents and the antioxidant capacity, measured by the ferric reducing/antioxidant power and the 2,2-diphenyl-1,1-picrylhydrazyl radical scavenging assays, were analyzed in each aqueous extract. Only the papaya extract had a significant antiproliferative effect measured with the methylthiazolydiphenyl-tetrazolium bromide assay. We did not notice a relationship between the total phenolic content and the antioxidant capacity with antiproliferative effect. It is suggested that each extract of plant food has a unique combination of the quantity and quality of phytochemicals that could determine its biological activity. Besides, papaya represents a very interesting fruit to explore its antineoplastic activities.

Assessment of bacterial endospore viability with fluorescent dyes.

PubMed

Laflamme, C; Lavigne, S; Ho, J; Duchaine, C

2004-01-01

To validate three fluorescence viability assays designed primarily for vegetative cells on pure Bacillus endospores. Purified fresh and gamma-irradiated Bacillus endospores (Bacillus cereus, B. coagulans and two strains of B. subtilis) were used. The viability assays were: 5-cyano-2,3-diotolyl tetrazolium chloride (CTC) to test respiratory activity and early germination, DiBAC4(3) and Live/Dead BacLight to measure membrane energization and permeabilization, respectively. Gamma irradiation treatment completely eliminated spore culturability and was used as negative control. The untreated spores showed respiratory activity after 1 h of incubation and this was characteristic of almost 100% of spores after 24 h. The membrane potential assessment gave no answer about spore viability. A lower proportion of untreated spores had permeabilized membrane compared with gamma-irradiated spores using Live/Dead BacLight (P < 0.02). It is possible to use CTC and Live/Dead BacLight to rapidly test endospore viability and evaluate the proportion of spores in a preparation that could not be recovered with plate count. This study shows that fluorescence tests could be applied to assess viability in potentially pathogenic Bacillus spore preparations within 1 h.

Immunocytogenetic effects of gonadotropin releasing hormone analogue: Triptorelin Pamoate (Decapeptyl) during in vitro fertilization treatment.

PubMed

Al-Qashi, S; Al-Qaoud, K M; Ja'fer, M; Khali, A M

2006-10-01

In this study, the immunocytogenetic effects of Decapeptyl (Triptorelin Pamoate) were assessed in the peripheral blood lymphocytes of females undergoing in vitro fertilization (IVF) treatment. Blood samples were taken from 34 females (23 treated and 11 controls), cultured and examined for sister chromatid exchanges (SCE) and cell replication index (CRI). The SCE frequency increased around ovulation time in the controls, and around the time of human chorionic gonadotropin administration in the IVF group. However, the SCE rate was significantly higher in the latter group. Furthermore, the white blood cells (WBC) count was significantly higher on the day of ovum pick up compared to the day preceding luteinizing hormone (LH) and follicle stimulating hormone (FSH) treatment. Similar observations were recorded with respect to phagocytic activity tested by nitroblue tetrazolium (NBT) assay. The nitric oxide production abilities of macrophages were not significantly changed in the LH, FSH-treated group relative to its control. Finally, the 50% complement hemolytic activity (CH50) assay results indicated that Decapeptyl lacks a significant potential to affect the complement system.

Evaluation of whole cigarette smoke induced oxidative stress in A549 and BEAS-2B cells.

PubMed

Zhang, Shimin; Li, Xiang; Xie, Fuwei; Liu, Kejian; Liu, Huimin; Xie, Jianping

2017-09-01

Cigarette smoke is a complex and oxidative aerosol. Previous researches on the hazards of cigarette smoke mainly focused on the adverse bioeffects induced by its condensates or gas vapor phase, which ignored the dynamic processes of smoking and the cigarette smoke aging. To overcome these disadvantages, we performed air-liquid interface exposure of whole smoke, which used native and unmodified smoke and ensured the exposure similar to physiological inhalation. Our results indicated that whole cigarette smoke induced lung epithelial cells (A549) and bronchial epithelial cells (BEAS-2B) damages in cytotoxicity assays (methyl thiazoly tetrazolium and neutral red uptake assays). In addition, A549 and BEAS-2B cells showed oxidative damages in whole smoke exposure, with concentration change of several biomarkers (reduced and oxidized glutathione, malondialdehyde, 4-hydroxyhydroxy-2-nonenal, extracellular superoxide dismutase, and 8-hydroxyl deoxyguanosine). These results indicate that whole smoke-induced oxidative stress occurs in two different kinds of cells at air-liquid interface. Copyright © 2017 Elsevier B.V. All rights reserved.

Cytotoxicity Evaluation and Magnetic Characteristics of Mechano-thermally Synthesized CuNi Nanoparticles for Hyperthermia

NASA Astrophysics Data System (ADS)

Amrollahi, P.; Ataie, A.; Nozari, A.; Seyedjafari, E.; Shafiee, A.

2015-03-01

CuNi alloys are very well known, both in academia and industry, based on their wide range of applications. In the present investigation, the previously synthesized Cu0.5Ni0.5 nanoparticles (NPs) by mechano-thermal method were studied more extensively. Phase composition and morphology of the samples were studied by employing x-ray diffraction (XRD), field-emission scanning electron microscopy (FESEM), and transmission electron microscopy (TEM) techniques. The Curie temperature ( T c) was determined by differential scanning calorimetry (DSC). In vitro cytotoxicity was studied through methyl-thiazolyl-tetrazolium (MTT) assay. XRD and FESEM results indicated the formation of single-phase Cu0.5Ni0.5. TEM micrographs showed that the mean particle size of powders is 20 nm. DSC results revealed that T c of mechano-thermally synthesized Cu0.5Ni0.5 is 44 °C. The MTT assay results confirmed the viability and proliferation of human bone marrow stem cells in contact with Cu0.5Ni0.5 NPs. In summary, the fabricated particles were demonstrated to have potential in low concentrations for cancer treatment applications.

Apoptotic effect of chalcone derivatives of 2-acetylthiophene in human breast cancer cells.

PubMed

Fogaça, Tatiana B; Martins, Rosiane M; Begnini, Karine R; Carapina, Caroline; Ritter, Marina; de Pereira, Claudio M P; Seixas, Fabiana K; Collares, Tiago

2017-02-01

A variety of chalcones have demonstrated cytotoxic activity toward several cancer cell lines. This study aimed to investigate the cytotoxicity of four chalcones derivatives of 2-acetylthiophene in human breast cancer cell lines. MCF-7 and MDA-MB-231 cells were treated with synthesized chalcones and the cytotoxicity was evaluated by tetrazolium dye (MTT), live/dead, and DAPI assays. Chalcones significantly decreased MCF-7 and MDA-MB-231 cells viability in vitro in a dose dependent manner. After 48h treatment, the IC 50 values ranging from 5.52 to 34.23μM. Chalcone 3c displayed the highest cytotoxic activity from all the tested compounds. Cytotoxic effects of compounds were confirmed in the live/dead assay. In addition, DAPI staining revealed that these compounds induce death by apoptosis. The data speculate that chalcone derivatives of 2-acetylthiophene may represent a source of therapeutic agents for human breast cancer. Copyright © 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Phagocytosis of PLGA Microparticles in Rat Peritoneal Exudate Cells: A Time-Dependent Study

NASA Astrophysics Data System (ADS)

Gomes, Anderson De Jesus; Nain Lunardi, Claure; Henrique Caetano, Flávio; Orive Lunardi, Laurelúcia; da Hora Machado, Antonio Eduardo

2006-07-01

With the purpose of enhancing the efficacy of microparticle-encapsulated therapeutic agents, in this study we evaluated the phagocytic ability of rat peritoneal exudate cells and the preferential location of poly(D,L-lactide-co-glycolic acid) (PLGA) microparticles inside these cells. The microparticles used were produced by a solvent evaporation method and were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Size distribution analysis using DLS and SEM showed that the particles were spherical, with diameters falling between 0.5 and 1.5 [mu]m. Results from cell adhesion by SEM assay, indicated that the PLGA microparticles are not toxic to cells and do not cause any distinct damage to them as confirmed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Among the large variety of cell populations found in the peritoneal exudates (neutrophils, eosinophils, monocytes, and macrophages), TEM showed that only the latter phagocytosed PLGA microparticles, in a time-dependent manner. The results obtained indicate that the microparticles studied show merits as possible carriers of drugs for intracellular delivery.

Candida albicans Biofilms Do Not Trigger Reactive Oxygen Species and Evade Neutrophil Killing

PubMed Central

Xie, Zhihong; Thompson, Angela; Sobue, Takanori; Kashleva, Helena; Xu, Hongbin; Vasilakos, John; Dongari-Bagtzoglou, Anna

2012-01-01

Neutrophils are found within Candida albicans biofilms in vivo and could play a crucial role in clearing the pathogen from biofilms forming on catheters and mucosal surfaces. Our goal was to compare the antimicrobial activity of neutrophils against developing and mature C. albicans biofilms and identify biofilm-specific properties mediating resistance to immune cells. Antibiofilm activity was measured with the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)2H-tetrazolium-5-carboxanilide assay and a molecular Candida viability assay. Reactive oxygen species generation was assessed by measuring fluorescence of 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester in preloaded neutrophils. We found that mature biofilms were resistant to leukocytic killing and did not trigger reactive oxygen species, even though neutrophils retained their viability and functional activation potential. Beta-glucans found in the extracellular matrix negatively affected antibiofilm activities. We conclude that these polymers act as a decoy mechanism to prevent neutrophil activation and that this represents an important innate immune evasion mechanism of C. albicans biofilms. PMID:23033146

Molecular docking study, synthesis and biological evaluation of Mannich bases as Hsp90 inhibitors.

PubMed

Gupta, Sayan Dutta; Bommaka, Manish Kumar; Mazaira, Gisela I; Galigniana, Mario D; Subrahmanyam, Chavali Venkata Satya; Gowrishankar, Naryanasamy Lachmana; Raghavendra, Nulgumnalli Manjunathaiah

2015-09-01

The ubiquitously expressed heat shock protein 90 is an encouraging target for the development of novel anticancer agents. In a program directed towards uncovering novel chemical scaffolds against Hsp90, we performed molecular docking studies using Tripos-Sybyl drug designing software by including the required conserved water molecules. The results of the docking studies predicted Mannich bases derived from 2,4-dihydroxy acetophenone/5-chloro 2,4-dihydroxy acetophenone as potential Hsp90 inhibitors. Subsequently, a few of them were synthesized (1-6) and characterized by IR, (1)H NMR, (13)C NMR and mass spectral analysis. The synthesized Mannich compounds were evaluated for their potential to suppress Hsp90 ATPase activity by the colorimetric Malachite green assay. Subsequently, the molecules were screened for their antiproilferative effect against PC3 pancreatic carcinoma cells by adopting the 3-(4,5-dimethythiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay method. The activity profile of the identified derivatives correlated well with their docking results. Copyright © 2015 Elsevier B.V. All rights reserved.

Growth inhibition of malignant melanoma by intermediate frequency alternating electric fields, and the underlying mechanisms.

PubMed

Chen, H; Liu, R; Liu, J; Tang, J

2012-01-01

This study investigated the antitumour effects of intermediate frequency alternating electric fields (IF-AEF) in a murine melanoma cell line (B16F10) and a mouse tumour model. IF-AEF was applied at 100 kHz. Proliferation of B16F10 cells in vitro was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. IF-AEF was applied in vivo to mice bearing B16F10 tumours. Terminal deoxy nucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay for apoptosis, and immunohistochemical detection of CD34 and vascular endothelial growth factor (VEGF), were performed. IF-AEF inhibited the proliferation of B16F10 cells in an electrical intensity and time-dependent manner. Treatment with IF-AEF for 7 days significantly inhibited the growth of tumours compared with untreated controls. IF-AEF induced apoptosis in vivo and reduced CD34-positive cell numbers; CD34 is a special marker of microvessel density. IF-AEF reduced microvessel density related to tumour growth and may serve as a therapeutic strategy for cancer treatment.

Kinetic study of an enzymic cycling system coupled to an enzymic step: determination of alkaline phosphatase activity.

PubMed Central

Valero, E; Varón, R; García-Carmona, F

1995-01-01

A kinetic study is made of a system consisting of a specific enzymic cycling assay coupled to an enzymic reaction. A kinetic analysis of this system is presented, and the accumulation of chromophore involved in the cycle is seen to be parabolic, i.e. the rate of the reaction increases continuously with constant acceleration. The system is illustrated by the measurement of alkaline phosphatase activity using beta-NADP+ as substrate. The enzymes alcohol dehydrogenase and diaphorase are used to cycle beta-NAD+ in the presence of ethanol and p-Iodonitrotetrazolium Violet. During each turn of the cycle, one molecule of the tetrazolium salt is reduced to an intensely coloured formazan. A simple procedure for evaluating the kinetic parameters involved in the system and for optimizing this cycling assay is described. The method is applicable to the measurement of any enzyme, and its amplification capacity as well as the simplicity of determining kinetic parameters enable it to be employed in enzyme immunoassays to increase the magnitude of the measured response. PMID:7619054

Preliminary in vitro evaluation of the anti-proliferative activity of guanylhydrazone derivatives.

PubMed

França, Paulo H B; Da Silva-Júnior, Edeildo F; Aquino, Pedro G V; Santana, Antônio E G; Ferro, Jamylle N S; De Oliveira Barreto, Emiliano; Do Ó Pessoa, Cláudia; Meira, Assuero Silva; De Aquino, Thiago M; Alexandre-Moreira, Magna S; Schmitt, Martine; De Araújo-Júnior, João X

2016-03-01

Guanylhydrazones have shown promising antitumor activity in preclinical tumor models in several studies. In this study, we aimed at evaluating the cytotoxic effect of a series of synthetic guanylhydrazones. Different human tumor cell lines, by including HCT-8 (colon carcinoma), MDA-MB-435 (melanoma) and SF-295 (glioblastoma) were continuous exposed to guanylhydrazone derivatives for 72 hours and growth inhibition of tumor cell lines and macrophages J774 was measured using tetrazolium salt (MTT) assay. Compounds 7, 11, 16 and 17 showed strong cytotoxic activity with IC50 values lower than 10 μmol L(-1) against four tumor cell lines. Among them, 7 was less toxic to non-tumor cells. Finally, obtained data suggest that guanylhydrazones may be regarded as potential lead compounds for the design of novel anticancer agents.

Combination of Fluorescence In Situ Hybridization with Staining Techniques for Cell Viability and Accumulation of PHA and polyP in Microorganisms in Complex Microbial Systems

NASA Astrophysics Data System (ADS)

Nielsen, Jeppe Lund; Kragelund, Caroline; Nielsen, Per Halkjær

Fluorescence in situ hybridization (FISH) can be combined with a number of staining techniques to reveal the relationships between the microorganisms and their function in complex microbial systems with a single-cell resolution. In this chapter, we have focused on staining methods for intracellular storage compounds (polyhydroxyalkanoates, polyphosphate) and a measure for cell viability, reduction of the tetrazolium-based redox stain CTC. These protocols are optimized for the study of microorganisms in waste-water treatment (activated sludge and biofilms), but they may also be used with minor modifications in many other ecosystems.

Computational Chemistry Toolkit for Energetic Materials Design

DTIC Science & Technology

2006-11-01

industry are aggressively engaged in efforts to develop multiscale modeling and simulation methodologies to model and analyze complex phenomena across...energetic materials design. It is hoped that this toolkit will evolve into a collection of well-integrated multiscale modeling methodologies...Experimenta Theoreticala This Work 1-5-Diamino-4- methyl- tetrazolium nitrate 8.4 41.7 47.5 1-5-Diamino-4- methyl- tetrazolium azide 138.1 161.6

Development of the NBT assay as a marker of sperm oxidative stress.

PubMed

Tunc, Ozlem; Thompson, Jeremy; Tremellen, Kelton

2010-02-01

Oxidative stress is a well-established cause of male infertility, with reactive oxygen species (ROS) causing infertility principally by impairing sperm motility and DNA integrity. Currently, most clinics do not test their infertile patients for the presence of oxidative stress because the available tests are expensive or difficult to perform. As antioxidant therapy may improve sperm DNA integrity and pregnancy outcomes, it has become apparent that there is an unmet clinical need for an inexpensive and easy-to-perform assay to identify sperm oxidative stress. The aim of this study was to develop a standardized protocol for performing a photometric nitro blue tetrazolium (NBT) assay for the measurement of seminal ROS production via production of coloured formazan, whilst correlating these results with impaired sperm function (motility and DNA integrity). Semen samples from 21 fertile and 36 male aetiology infertile men were assessed for ROS production (NBT assay), sperm DNA integrity (TUNEL), apoptosis (Annexin V) and sperm motility. Infertile men's semen contained on average fourfold higher levels of ROS than fertile men. The production of ROS by sperm was positively correlated with sperm DNA fragmentation and apoptosis, whilst being negatively correlated with sperm motility. Receiver-operating characteristic plot analysis established a cut-off point of 24 microg formazan/10(7) sperm having a sensitivity of 91.7% and a specificity of 81% for determining the fertility status of an individual. This study has been successful in establishing a standardized protocol for performing a photometric seminal NBT assay that has significant clinical utility in identifying men with impaired fertility because of oxidative stress.

Monitoring the Genotoxic and Cytotoxic Potential and the Presence of Pesticides and Hydrocarbons in Water of the Sinos River Basin, Southern Brazil.

PubMed

Bianchi, Eloisa; Lessing, Gustavo; Brina, Karisa Roxo; Angeli, Larissa; Andriguetti, Natália Bordin; Peruzzo, Jaqueline Regina Soares; do Nascimento, Carlos Augusto; Spilki, Fernando Rosado; Ziulkoski, Ana Luiza; da Silva, Luciano Basso

2017-04-01

The Sinos River is one of the most polluted rivers in Brazil. The purpose of this work was to monitor the presence of some pesticides and hydrocarbons as well as the genotoxic and cytotoxic potential on HEp-2 cells from water samples collected at seven sites in the Sinos River Basin (SRB), southern Brazil. Nine samples were taken from the three main rivers in the SRB and used as a solution to dilute the HEp-2 cell culture medium after microfiltration. Twenty-four pesticides and 19 hydrocarbons were measured. Cytotoxicity was assessed by methyl thiazolyl tetrazolium (MTT) and neutral red (NR) assays, in which cells were exposed to different concentrations of the water samples for 24 h. Genotoxicity of the microfiltrated raw water samples was assessed by comet assay after 6 and 24 h of exposure. Among the chemicals analyzed, only the 2,4-D, dichloromethane, tetrachloroethene, chloroform, bromodichloromethane, styrene, and toluene were detected, but they were all lower than the limit established by Brazilian regulations. Twenty samples from a total of 60 had a cytotoxic effect in the MTT assay and 30 in the NR assay. The comet assay indicated the presence of genotoxic substances in the water at the seven locations monitored. Temporal and spatial variation was observed in the cytotoxicity and genotoxicity assays. Results indicated that the water in all stretches of the SRB is contaminated and it can cause harmful effects to humans and to the aquatic biota. This HEp-2 cell-line approach can be an additional tool for environmental monitoring.

Andrographolide Sensitizes Ras-Transformed Cells to Radiation in vitro and in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hung, Shih-Kai; Department of Radiation Oncology, Buddhist Dalin Tzu Chi General Hospital, Chiayi, Taiwan; Tzu Chi University School of Medicine, Hualian, Taiwan

2010-07-15

Purpose: Increasing the sensitivity of tumor cells to radiation is a major goal of radiotherapy. The present study investigated the radiosensitizing effects of andrographolide and examined the molecular mechanisms of andrographolide-mediated radiosensitization. Methods and Materials: An H-ras-transformed rat kidney epithelial (RK3E) cell line was used to measure the radiosensitizing effects of andrographolide in clonogenic assays, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide assays, and a xenograft tumor growth model. The mechanism of andrographolide-sensitized cell death was analyzed using annexin V staining, caspase 3 activity assays, and terminal transferase uridyl nick end labeling assays. The roles of nuclear factor kappa B (NF-{kappa}B) and Akt inmore » andrographolide-mediated sensitization were examined using reporter assays, electrophoretic mobility shift assays, and Western blotting. Results: Concurrent andrographolide treatment (10 {mu}M, 3 h) sensitized Ras-transformed cells to radiation in vitro (sensitizer enhancement ratio, 1.73). Andrographolide plus radiation (one dose of 300 mg/kg peritumor andrographolide and one dose of 6 Gy radiation) resulted in significant tumor growth delay (27 {+-} 2.5 days) compared with radiation alone (22 {+-} 1.5 days; p <.05). Radiation induced apoptotic markers (e.g., caspase-3, membrane reversion, DNA fragmentation), and andrographolide treatment did not promote radiation-induced apoptosis. However, the protein level of activated Akt was significantly reduced by andrographolide. NF-{kappa}B activity was elevated in irradiated Ras-transformed cells, and andrographolide treatment significantly reduced radiation-induced NF-{kappa}B activity. Conclusion: Andrographolide sensitized Ras-transformed cells to radiation both in vitro and in vivo. Andrographolide-mediated radiosensitization was associated with downregulation of Akt and NF-{kappa}B activity. These observations indicate that andrographolide is a novel radiosensitizing agent with potential application in cancer radiotherapy.« less

Immunomodulatory activity of methanolic extracts of fruits and bark of Ficus glomerata Roxb. in mice and on human neutrophils.

PubMed

Heroor, Sanjeev; Beknal, Arun Kumar; Mahurkar, Nitin

2013-01-01

To evaluate the immunomodulatory activity of methanolic extracts of fruit and bark of Ficus glomerata Roxb. on cyclophosphamide-induced myelosuppression in mice and the phagocytic effect on human neutrophils. Methanolic extracts of fruits and bark of Ficus glomerata Roxb. at two dose levels of 250 and 500 mg/kg p.o. were administered for 13 days to albino mice and cyclophosphamide (30 mg/kg i.p.) was administered on 11th, 12th, and 13th days, 1 hour after the administration of the respective treatment. On 14th day blood was collected and the hematological parameters were evaluated. The two extracts in the concentration range 100, 50, 25, 12 and 6.25 μg were also tested for phagocytic effect on human neutrophils using the in vitro models-nitroblue tetrazolium (NBT) dye test, phagocytosis of Candida albicans, and chemotaxis assay. Methanolic extracts of fruit and bark of Ficus glomerata Roxb. showed significant counteracting effect (P < 0.01) to cyclophosphamide-induced reduction in total WBC, differential leucocyte count, platelet counts, RBC counts, and hemoglobin levels. The extracts of the plant in the concentration range 100, 50, 25, 12, and 6.25 μg also showed significant (P < 0.01) phagocytic effect on human neutrophils in the parameters studied. Methanolic extracts of fruits and bark of Ficus glomerata Roxb. exhibited immunomodulatory property in both in vivo and in vitro models.

Neuroprotective action and free radical scavenging activity of Guttiferone-A, a naturally occurring prenylated benzophenone.

PubMed

Nuñez-Figueredo, Y; García-Pupo, L; Ramírez-Sánchez, J; Alcántara-Isaac, Y; Cuesta-Rubio, O; Hernández, R D; Naal, Z; Curti, C; Pardo-Andreu, G L

2012-12-01

Reactive oxygen species (ROS) are important mediators in a number of neurodegenerative diseases and molecules capable of scavenging ROS may be a feasible strategy for protecting neuronal cells. We previously demonstrated a powerful iron-chelating action of Guttiferone-A (GA), a naturally occurring polyphenol, on oxidative stress injuries initiated by iron overload. Here we addressed the neuroprotective potential of GA in hydrogen peroxide and glutamate-induced injury on rat's primary culture of cortical neurons and PC12 cells, respectively, and antioxidant properties concerning scavenging and anti-lipoperoxidative activities in cell-free models. The decrease in cell viability induced by each of the toxins, assessed by [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) assay, was significantly attenuated by GA. In addition, GA was found to be a potent antioxidant, as shown by (i) inhibition of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical reduction (EC50=20.0 μM), (ii) prevention against chemically or electrochemically generated superoxide radicals, (iii) inhibition of spontaneous brain lipid peroxidation and (iv) interference with the Fenton reaction. These results indicate that GA exerts neuroprotective effects against H2O2 or glutamate toxicity and its antioxidant activity, demonstrated in vitro, could be at least partly involved. They also suggest a promising potential for GA as a therapeutic agent against neurodegenerative diseases involving ROS and oxidative damage. © Georg Thieme Verlag KG Stuttgart · New York.

Cytotoxicity of different extracts of arial parts of Ziziphus spina-christi on Hela and MDA-MB-468 tumor cells

PubMed Central

Jafarian, Abbas; Zolfaghari, Behzad; Shirani, Kobra

2014-01-01

Background: It has been shown that plants from the family Rhamnaceae possess anticancer activity. In this study, we sought to determine if Ziziphus spina-christi, a species from this family, has cytotoxic effect on cancer cell lines. Materials and Methods: Using maceration method, different extracts of leaves of Z. spina-christi were prepared. Hexane, chloroform, chloroform-methanol (9:1), methanol-water (7:1) methanol, butanol and water were used for extraction, after preliminary phytochemical analyses were done. The cytotoxic activity of the extracts against Hela and MDA-MB-468 tumor cells was evaluated by MTT assay. Briefly, cells were seeded in microplates and different concentrations of extracts were added. After incubation of cells for 72 h, their viability was evaluated by addition of tetrazolium salt solution. After 3 h medium was aspirated, dimethyl sulfoxide was added and absorbance was determined at 540 nm with an ELISA plate reader. Extracts were considered cytotoxic when more than 50% reduction on cell survival was observed. Results: Hexane, chloroform, chloroform-methanol, butanol, methanol-water and aqueous extracts of Z. spina-christi significantly and concentration-dependently reduced viability of Hela and MAD-MB-468 cells. In the both cell lines, chloroform-methanol extract of Z. spina-christi was more potent than the other extracts. Results: From the finding of this study it can be concluded that Z. spina-christi is a good candidate for further study for new cytotoxic agents. PMID:24627846

Some Strychnos spinosa (Loganiaceae) leaf extracts and fractions have good antimicrobial activities and low cytotoxicities.

PubMed

Isa, Adamu Imam; Awouafack, Maurice Ducret; Dzoyem, Jean Paul; Aliyu, Mohammed; Magaji, Rabiu AbduSsalam; Ayo, Joseph Olusegun; Eloff, Jacobus Nicolaas

2014-11-27

Strychnos spinosa Lam. is a deciduous tree used in traditional medicine to treat infectious diseases. This study is designed to determine the antimicrobial, antioxidant and cytotoxic activities of extracts and fractions from leaves of S. spinosa. Extracts were obtained by maceration with acetone, methanol and dichloromethane/methanol (1/1) while fractions were prepared by liquid-liquid fractionation of the acetone extract. A broth serial microdilution method with tetrazolium violet as growth indicator was used to determine the minimum inhibitory concentration (MIC) against fungi, Gram-positive and Gram-negative bacteria. The antioxidant activity was determined using free-radical-scavenging assays, and the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide reduction assay was used to determine cytotoxicity. Four extracts and five fractions had good to weak antimicrobial activity with MICs ranging from 0.04 to >1.25 mg/ml against both fungi and bacteria. The chloroform and ethyl acetate fractions had an MIC of 0.08 mg/ml against Aspergillus fumigatus. The n-butanol fraction had an MIC of 0.04 mg/ml against Cryptococcus neoformans. The hexane and chloroform fractions had an MIC of 0.08 mg/ml against Staphylococcus aureus. The antioxidant activities were much lower than that of the positive controls. Except for the alkaloid extract, all the extracts and fractions had free-radical-scavenging activity (IC50 ranging from 33.66 to 314.30 μg/ml). The cytotoxicity on Vero cells was reasonable to low with LC50 values ranging between 30.56 and 689.39 μg/ml. The acetone extract and the chloroform fraction had the highest antibacterial activity. By solvent-solvent fractionation it was possible to increase the activity against A. fumigatus and to decrease the cytotoxicity leading to a potentially useful product to protect animals against aspergillosis. Our results therefore support the use of S. spinosa leaves in traditional medicine to treat infectious diseases.

Antimicrobial Efficacy of Salvadora persica Extracts on a Monospecies Biofilm on Orthodontic Brackets In Vitro.

PubMed

Halawany, Hassan S; Abraham, Nimmi B; Siddiqui, Yunus M; Balto, Hanan A; Jacob, Vimal

2016-01-01

The oral cavity is a rich ecosystem with a plethora of microorganisms, and different components of fixed orthodontic appliances may contribute to a shift in the balance of oral ecology. The purpose of this study was to investigate the antimicrobial potential of hexane and ethanol extracts of Salvadora persica on a monospecies biofilm model established on orthodontic brackets in vitro. Streptococcus mutans biofilm was formed on mini diamond orthodontic brackets following three days of anaerobic incubation at 37˚C. The bacterial cell viability of this biofilm was measured after their exposure to saline, hexane extract of S. persica, ethanol extract of S. persica and 0.2% chlorhexidine using 3-(4, 5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) assay. On half of the brackets, the colony forming units (CFU) were counted. Both experiments were performed in triplicate. The absorbance values obtained from the MTS reduction assay after exposure to the different test agents showed a decline in the bacterial cell viability of the S. mutans biofilm as follows: chlorhexidine (+)0.05). The CFU counts of S. mutans obtained from chlorhexidine exposure were lower than from hexane and ethanol extracts. S. persica extracts were found to have antimicrobial effects on S. mutans biofilm established in vitro on orthodontic brackets suggestive of its potential use as an oral antimicrobial agent for orthodontic patients.

[Effects of basic fibroblast growth factor and vascular endothelial growth factor on the proliferation, migration and adhesion of human periodontal ligament stem cells in vitro].

PubMed

Zhang, Rong; Zhang, Mian; Li, Cheng-hua; Wang, Peng-cheng; Chen, Fang; Wang, Qin-tao

2013-05-01

To evaluate the effects of basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF) on the proliferation, migration, and adhesion of human periodontal ligament stem cells (PDLSC) in vitro. Human PDLSC were cultured in vitro using tissue culture method.The cells were cultured and incubated with various concentrations of FGF-2 and VEGF [A:α-MEM with 2% fetal bovine serum (FBS) (control 1); B:A supplemented with 20 µg/L FGF-2; C:A supplemented with 10 µg/L VEGF; D:A supplemented with 20 µg/L FGF-2 and 10 µg/L VEGF; E:α-MEM with 10% FBS (control 2); F:E supplemented with 20 µg/L FGF-2; G:E supplemented with 10 µg/L VEGF; H:E supplemented with 20 µg/L FGF-2 and 10 µg/L VEGF]. Soluble tetrazolium salts assay was used to evaluate the proliferative capacity on the 1st, 3rd, 5th and 7th d. Then the groups were changed according to result of the proliferation assay (control:α-MEM with 2% FBS; FGF-2 group:control supplemented with 20 µg/L FGF-2; VEGF:control supplemented with 10 µg/L VEGF; Combination group:control supplemented with 20 µg/L FGF-2 and 10 µg/L VEGF). The cell cycle, migration and adhesion capacities were evaluated using flow cytometer, soluble tetrazolium salts assay, cell adhesion assay and scratch wound-healing motility assay. In 2% volume fraction serum containing medium, FGF-2 and VEGF did not stimulate the cell proliferation. However, in 10% serum condition, in groups treated with FGF-2 for 3,5 or 7 d, the A value was (1.22 ± 0.17, 2.15 ± 0.19, 2.72 ± 0.11) respectively, which were significantly higher than that in the control group (0.76 ± 0.16, 1.25 ± 0.06, 1.64 ± 0.09) (P < 0.01) while lower than that in the group treated with FGF-2 and VEGF in combination on the 5 th and 7 th d (2.46 ± 0.17, 3.18 ± 0.27) ( P < 0.05). The A value in the VEGF group on the 5 th and 7 th d is higher than the control group while lower than the FGF-2 group (1.66 ± 0.05, 2.13 ± 0.13) (P < 0.05). Flow cytometer showed that the proliferation index in VEGF group [(34.3 ± 2.0)% ] were significantly lower than those in FGF-2 [(46.8 ± 3.2)%] group and (FGF-2+ VEGF) group [(45.0 ± 4.0)%] but higher than in the control group [(14.5 ± 1.7)%] (P < 0.01). The cell migration assay indicated that the group stimulated with FGF-2 showed no migration promoted effect. Cell adhesion assay showed that the ratio of the adhesive cells number to the original cells number is greater in the FGF-2 group (79 ± 4) than in the VEGF group (62 ± 4) (P < 0.05). Light microscope identified a better cellular morphology on the adhesive surface in the group with FGF-2 than groups without FGF-2. Both FGF-2 and VEGF could simulate the proliferation of PDLSC in a dose dependent manner, and showed an synergistic effect. FGF-2 was more effective to promote the adhesive capacity of PDLSC compared with VEGF. VEGF could facilitate the migration of PDLSC to the wound side.

Radioprotective effects of delphinidin on normal human lung cells against proton beam exposure

PubMed Central

Kim, Hyun Mi; Kim, Suk Hee

2018-01-01

BACKGROUND/OBJECTIVES Exposure of the normal lung tissue around the cancerous tumor during radiotherapy causes serious side effects such as pneumonitis and pulmonary fibrosis. Radioprotectors used during cancer radiotherapy could protect the patient from side effects induced by radiation injury of the normal tissue. Delphinidin has strong antioxidant properties, and it works as the driving force of a radioprotective effect by scavenging radiation-induced reactive oxygen species (ROS). However, no studies have been conducted on the radioprotective effect of delphinidin against high linear energy transfer radiation. Therefore, this study was undertaken to evaluate the radioprotective effects of delphinidin on human lung cells against a proton beam. MATERIALS/METHODS Normal human lung cells (HEL 299 cells) were used for in vitro experiments. The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay assessed the cytotoxicity of delphinidin and cell viability. The expression of radiation induced cellular ROS was measured by the 2′-7′-dicholordihydrofluorescein diacetate assay. Superoxide dismutase activity assay and catalase activity assay were used for evaluating the activity of corresponding enzymes. In addition, radioprotective effects on DNA damage-induced cellular apoptosis were evaluated by Western blot assay. RESULTS Experimental analysis, including cell survival assay, MTT assay, and Western blot assay, revealed the radioprotective effects of delphinidin. These include restoring the activities of antioxidant enzymes of damaged cells, increase in the levels of pro-survival protein, and decrease of pro-apoptosis proteins. The results from different experiments were compatible with each to provide a substantial conclusion. CONCLUSION Low concentration (2.5 µM/mL) of delphinidin administration prior to radiation exposure was radioprotective against a low dose of proton beam exposure. Hence, delphinidin is a promising shielding agent against radiation, protecting the normal tissues around a cancerous tumor, which are unintentionally exposed to low doses of radiation during proton therapy. PMID:29399295

The anthraquinones rubiadin and its 1-methyl ether isolated from Heterophyllaea pustulata reduces Candida tropicalis biofilms formation.

PubMed

Marioni, Juliana; da Silva, María Angel; Cabrera, José Luis; Montoya, Susana C Núñez; Paraje, María Gabriela

2016-11-15

Candida tropicalis is increasingly becoming among the most commonly isolated pathogens causing fungal infections with an important biofilm-forming capacity. This study addresses the antifungal effect of rubiadin (AQ1) and rubiadin 1-methyl ether (AQ2), two photosensitizing anthraquinones (AQs) isolated from Heterophyllaea pustulata, against C. tropicalis biofilms, by studying the cellular stress and antioxidant response in two experimental conditions: darkness and irradiation. The combination with Amphotericin B (AmB) was assayed to evaluate the synergic effect. Biofilms of clinical isolates and reference strain of Candida tropicalis were treated with AQs (AQ1 or AQ2) and/or AmB, and the biofilms depletion was studied by crystal violet and confocal scanning laser microscopy (CSLM). The oxidant metabolites production and the response of antioxidant defense system were also evaluated under dark and irradiation conditions, being the light a trigger for photo-activation of the AQs. The Reactive Oxygen Species (ROS) were detected by the reduction of Nitro Blue Tetrazolium test, and Reactive Nitrogen Intermediates (RNI) by the Griess assay. ROS accumulation was also detected inside biofilms by using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) probe, which was visualized by CSLM. Superoxide dismutase (SOD) activity and the total antioxidant capacity of biofilms were measured by spectrophotometric methods. The minimun inhibitory concentration for sessile cells (SMIC) was determined for each AQs and AmB. The fractional inhibitory concentration index (FICI) was calculated for the combinations of each AQ with AmB by the checkerboard microdilution method. Biofilm reduction of both strains was more effective with AQ1 than with AQ2. The antifungal effect was mediated by an oxidative and nitrosative stress under irradiation, with a significant accumulation of endogenous ROS detected by CSLM and an increase in the SOD activity. Thus, the prooxidant-antioxidant balance was altered especially by AQ1. The best synergic combination with AmB was also obtained with AQ1 (80.5%) (FICI=0.74). Under irradiation, the oxidative stress was the predominant effect, altering the prooxidant-antioxidant balance, which may be the cause of the irreversible cell injury in the biofilm. Our results showed synergism of these natural AQs with AmB. Therefore, the photosensitizing AQ1 could be an alternative for the Candida infections treatment, which deserves further investigation. Copyright © 2016 Elsevier GmbH. All rights reserved.

Permeation of superoxide anion through the bilayer of vesicles of a synthetic amphiphile.

PubMed

Gomes, L F; Cuccovia, I M; Chaimovich, H; Barbieri, D H; Politi, M J

1993-10-10

Large unilamellar vesicles, prepared with dioctadecyldimethylammonium chloride, entrap nitroblue tetrazolium. Addition of solid KO2, or production of superoxide anion by riboflavin photolysis, to nitroblue tetrazolium-containing dioctadecyldimethylammonium vesicles results in the formation of monoformazan above the phase-transition temperature of the bilayer. Below the phase-transition temperature the yield of monoformazan is negligible. These results demonstrate that superoxide anion permeates vesicles above the phase-transition temperature of the bilayer.

Combination of Osthole and Cisplatin Against Rhabdomyosarcoma TE671 Cells Yielded Additive Pharmacologic Interaction by Means of Isobolographic Analysis.

PubMed

Jarząb, Agata; Łuszczki, Jarogniew; Guz, Małgorzata; Skalicka-Woźniak, Krystyna; Hałasa, Marta; Smok-Kalwat, Jolanta; Polberg, Krzysztof; Stepulak, Andrzej

2018-01-01

Osthole is a simple coumarin that has been found to have anticancer, anti-inflammatory, antiviral, anticoagulant, anticonvulsant and antiallergic activities. The aim of this study was to analyze the combined anti-proliferative effect of cisplatin (CDDP) and osthole on a rhabdomyosarcoma cell line, and assess the pharmacology of drug-drug interaction between these drugs using isobolographic analysis. The anticancer actions of osthole in combination with CDDP were evaluated using the tetrazolium dye-based MTT cell proliferation assay. Osthole and CDDP applied together augmented their anti-cancer activities and yielded an additive type of pharmacologic interaction by means of isobolographic analysis. Combined therapy using osthole and cisplatin could be suggested as a potential chemotherapy regimen against rhabdomyosarcoma. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Cytochemical and Cytofluorometric Evidence for Guard Cell Photosystems 1

PubMed Central

Vaughn, Kevin C.; Outlaw, William H.

1983-01-01

Evidence for photosynthetic linear electron transport in guard cells was obtained with two sensitive methods of high spacial resolution. Light-dependent diaminobenzidine oxidation (an indicator of PSI) and DCMU-sensitive, light-dependent thiocarbamyl nitroblue tetrazolium reduction (an indicator of PSII) were observed in guard cell plastids of Hordeum vulgare L. cv Himalaya using electron microscopic cytochemical procedures. DCMU-sensitive Chl a fluorescence induction (an indicator of PSII) was detected in individual guard cell pairs of Vicia faba L. cv Longpod using an ultramicrofluorometer. At least for these species, we conclude these results are proof for the presence of PSII in guard cell chloroplasts, which until now has been somewhat controversial. Images Fig. 2 Fig. 1 PMID:16662840

ZnO Nanoparticles Treatment Induces Apoptosis by Increasing Intracellular ROS Levels in LTEP-a-2 Cells.

PubMed

Wang, Caixia; Hu, Xiaoke; Gao, Yan; Ji, Yinglu

2015-01-01

Owing to the wide use of novel nanoparticles (NPs) such as zinc oxide (ZnO) in all aspects of life, toxicological research on ZnO NPs is receiving increasing attention in these days. In this study, the toxicity of ZnO NPs in a human pulmonary adenocarcinoma cell line LTEP-a-2 was tested in vitro. Log-phase cells were exposed to different levels of ZnO NPs for hours, followed by colorimetric cell viability assay using tetrazolium salt and cell survival rate assay using trypan blue dye. Cell morphological changes were observed by Giemsa staining and light microscopy. Apoptosis was detected by using fluorescence microscopy and caspase-3 activity assay. Both intracellular reactive oxygen species (ROS) and reduced glutathione (GSH) were examined by a microplate-reader method. Results showed that ZnO NPs (≥ 0.01 μg/mL) significantly inhibited proliferation (P < 0.05) and induced substantial apoptosis in LTEP-a-2 cells after 4 h of exposure. The intracellular ROS level rose up to 30-40% corresponding to significant depletion (approximately 70-80%) in GSH content in LTEP-a-2 cells (P < 0.05), suggesting that ZnO NPs induced apoptosis mainly through increased ROS production. This study elucidates the toxicological mechanism of ZnO NPs in human pulmonary adenocarcinoma cells and provides reference data for application of nanomaterials in the environment.

In Vitro Study of a Liposomal Curcumin Formulation (Lipocurc™): Toxicity and Biological Activity in Synovial Fibroblasts and Macrophages.

PubMed

Kloesch, Burkhard; Gober, Lukas; Loebsch, Silvia; Vcelar, Brigitta; Helson, Lawrence; Steiner, Guenter

2016-01-01

The polyphenol curcumin is produced in the rhizome of Curcuma longa and exhibits potent anti-inflammatory, antioxidant, and chemopreventive activities. Due to the fact that curcumin is poorly soluble in water, many delivery systems have been developed to improve its solubility and bioavailability achieving optimum therapeutic application. In this study, we evaluated the biological effects of a liposomal curcumin formulation (Lipocurc™) on human synovial fibroblasts (SW982) and mouse macrophages (RAW264). Cellular uptake of liposomes was studied using calcein-loaded liposomes. Effects of Lipocurc™ on cell viability and proliferation were determined with Celltox green cytotoxicity assay and 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay, respectively. To induce cytokine/chemokine expression, the cells were stimulated with interleukin (IL)1β or lipopolysaccharide (LPS). The release of IL6, IL8, and tumor necrosis factor-alpha (TNFα) was quantified by enzyme-linked immunosorbent assay (ELISA). Data showed that the liposomal curcumin formulation Lipocurc™ was significantly less toxic to synovial fibroblasts and macrophages compared to non-encapsulated, free curcumin. Furthermore, Lipocurc™ effectively reduced pro-inflammatory cytokine/chemokine expression in synovial fibroblasts as well as in macrophages without affecting cell viability, suggesting that this curcumin nanoformulation might be a promising tool for the treatment of inflammatory diseases. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

The Design and Development of Potent Small Molecules as Anticancer Agents Targeting EGFR TK and Tubulin Polymerization

PubMed Central

Ihmaid, Saleh; Ahmed, Hany E. A.; Zayed, Mohamed F.

2018-01-01

Some novel anthranilate diamides derivatives 4a–e, 6a–c and 9a–d were designed and synthesized to be evaluated for their in vitro anticancer activity. Structures of all newly synthesized compounds were confirmed by infra-red (IR), high-resolution mass (HR-MS) spectra, 1H nuclear magnetic resonance (NMR) and 13C nuclear magnetic resonance (NMR) analyses. Cytotoxic screening was performed according to (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium (MTT) assay method using erlotinib as a reference drug against two different types of breast cancer cells. The molecular docking study was performed for representative compounds against two targets, epidermal growth factor receptor (EGFR) and tubulin in colchicine binding site to assess their binding affinities in order to rationalize their anticancer activity in a qualitative way. The data obtained from the molecular modeling was correlated with that obtained from the biological screening. These data showed considerable anticancer activity for these newly synthesized compounds. Biological data for most of the anthranilate diamide showed excellent activity with nanomolar or sub nanomolar half maximal inhibitory concentration (IC50) values against tumor cells. EGFR tyrosine kinase (TK) inhibition assay, tubulin inhibition assay and apoptosis analysis were performed for selected compounds to get more details about their mechanism of action. Extensive structure activity relationship (SAR) analyses were also carried out. PMID:29385728

Cytotoxic and genotoxic studies of essential oil from Rosa damascene Mill., Kashan, Iran.

PubMed

Shokrzadeh, Mohammad; Habibi, Emran; Modanloo, Mona

2017-08-01

Aim Rosa damascene Mill. belongs to the family of Roseaceae and its essential oil is produced in large amounts in Iran. The wide application of rose oil has raised questions about potential adverse health effects. We have investigated cytotoxic activity and genotoxic effects of Rosa oil from Kashan, Iran. Methods The cytotoxic effect and IC50 of the essential oil on the cell lines was studied followed by MTT assay. In this assay mitochondrial oxidoreductase enzymes with reducing the tetrazolium dye MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) reflect the number of viable cells. Genotoxic effect of the oil was evaluated by micronucleus assay by evaluating produced micronuclei due to cytogenetic damage in binucleated lymphocytes. Results The results showed that essential oil significantly had cytotoxic and genotoxic effects at doses over 10µg/mL (p<0.05). Also, essential oil of Rose showed lower IC50 in cancer cell line (A549) in comparison with the normal cell line (NIH3T3). Conclusion Cytotoxic and genotoxic properties of essential oil of Rose in Kashan, Iran, are safe at a dose of 10µg/mL. Also, a good cytotoxic effect was shown and could be introduced as an anticancer compound. Further studies are needed with regard to anti-cancer effects of Rose essential oil. Copyright© by the Medical Assotiation of Zenica-Doboj Canton.

Assessment of Cytotoxic Activity of Rosemary (Rosmarinus officinalis L.), Turmeric (Curcuma longa L.), and Ginger (Zingiber officinale R.) Essential Oils in Cervical Cancer Cells (HeLa).

PubMed

Santos, P A S R; Avanço, G B; Nerilo, S B; Marcelino, R I A; Janeiro, V; Valadares, M C; Machinski, Miguel

2016-01-01

The objective of this study was to evaluate the cytotoxic activity of rosemary (REO, Rosmarinus officinalis L.), turmeric (CEO, Curcuma longa L.), and ginger (GEO, Zingiber officinale R.) essential oils in HeLa cells. Cytotoxicity tests were performed in vitro , using tetrazolium (MTT) and neutral red assays for evaluation of antiproliferative activity by different mechanisms, trypan blue assay to assess cell viability and evaluation of cell morphology for Giemsa to observe the cell damage, and Annexin V to evaluate cell death by apoptosis. CEO and GEO exhibited potent cytotoxic activity against HeLa cells. IC 50 obtained was 36.6  μ g/mL for CEO and 129.9  μ g/mL for GEO. The morphology of HeLa cells showed condensation of chromatin, loss of cell membrane integrity with protrusions (blebs), and cell content leakage for cells treated with CEO and GEO, from the lowest concentrations studied, 32.81  μ g/mL of CEO and 32.12  μ g/mL of GEO. The Annexin V assay revealed a profile of cell death by apoptosis for both CEO and GEO. The results indicate cytotoxic activity in vitro for CEO and GEO, suggesting potential use as anticancer agents for cervical cancer cells.

Assessment of Cytotoxic Activity of Rosemary (Rosmarinus officinalis L.), Turmeric (Curcuma longa L.), and Ginger (Zingiber officinale R.) Essential Oils in Cervical Cancer Cells (HeLa)

PubMed Central

Santos, P. A. S. R.; Avanço, G. B.; Nerilo, S. B.; Marcelino, R. I. A.; Janeiro, V.; Valadares, M. C.

2016-01-01

The objective of this study was to evaluate the cytotoxic activity of rosemary (REO, Rosmarinus officinalis L.), turmeric (CEO, Curcuma longa L.), and ginger (GEO, Zingiber officinale R.) essential oils in HeLa cells. Cytotoxicity tests were performed in vitro, using tetrazolium (MTT) and neutral red assays for evaluation of antiproliferative activity by different mechanisms, trypan blue assay to assess cell viability and evaluation of cell morphology for Giemsa to observe the cell damage, and Annexin V to evaluate cell death by apoptosis. CEO and GEO exhibited potent cytotoxic activity against HeLa cells. IC50 obtained was 36.6 μg/mL for CEO and 129.9 μg/mL for GEO. The morphology of HeLa cells showed condensation of chromatin, loss of cell membrane integrity with protrusions (blebs), and cell content leakage for cells treated with CEO and GEO, from the lowest concentrations studied, 32.81 μg/mL of CEO and 32.12 μg/mL of GEO. The Annexin V assay revealed a profile of cell death by apoptosis for both CEO and GEO. The results indicate cytotoxic activity in vitro for CEO and GEO, suggesting potential use as anticancer agents for cervical cancer cells. PMID:28042599

Effects of ascorbate on leucocytes: Part II. Effects of ascorbic acid and calcium and sodium ascorbate on neutrophil phagocytosis and post-phagocytic metabolic activity.

PubMed

Anderson, R

1979-09-01

The effects of ascorbic acid and calcium and sodium ascorbate at a concentration range of 10(-6)M - 10(-1)M on polymorphonuclear leucocyte (PMN) phagocytosis of Candida albicans and post-phagocytic nitroblue tetrazolium (NBT) reduction, hexose monophosphate shunt (HMS) activity and myeloperoxidase-mediated iodination of ingested protein were investigated. Phagocytosis of C. albicans was unaffected by ascorbate concentrations of 10(-6)M - 10(-2)M; however, progressive inhibition was observed at concentrations of 10(-2)M upwards. Enhancement of resting and stimulated HMS activity and NBT reduction was evident at ascorbate concentrations of 10(-5) M - 10(-2)M. The stimulations of HMS activity and NBT reduction was independent of myeloperoxidase iodination of ingested protein and this latter function was strongly inhibited by ascorbate. Concentrations of ascorbic acid and calcium and sodium ascorbate which caused inhibition of phagocytosis and HMS activity were the same as those which mediated stimulation of cell motility, indicating that independent cellular mechanisms may govern motility and phagocytosis.

Evaluation of antioxidant and neuroprotective effect of Hippophae rhamnoides (L.) on oxidative stress induced cytotoxicity in human neural cell line IMR32

PubMed Central

Shivapriya, S.; Ilango, K.; Dubey, G.P.

2015-01-01

Aim and objective Hippophae rhamnoides is an edible, nutrient rich plant found in the northern regions of India. It belongs to the family Elaeagnaceae and is well known for its traditional pharmacological activities. The present study was aimed to investigate the antioxidant and neuroprotective activities of H. rhamnoides. Methodology The hydroalcoholic extract of H. rhamnoides was evaluated for free radical scavenging activity using DPPH, hydroxyl radical scavenging and ferric thiocyanate assays. In vitro neuroprotective activity was assessed on human neuroblastoma cell line-IMR32 against hydrogen peroxide (H2O2) induced cytotoxicity. The neuroprotective effect was determined by measuring the cell viability through tetrazolium dye MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reducing assay and propidium iodide (PI) staining. Also the intracellular reactive oxygen species (ROS) activity was assessed using dichloro-dihydro-fluorescein diacetate (DCFDA) assay by flowcytometer. Results The results of the study demonstrated that H. rhamnoides extract possesses potential free radical scavenging activity. The IC50 value for DPPH and OH radical scavenging assay was 70.92 μg/ml and 0.463 mg/ml, also the extract was also found to have considerable level of lipid peroxidation activity. The neuroprotective effect of H. rhamnoides was confirmed by its cell viability enhancing capacity against hydrogen peroxide induced cell cytotoxicity. The extract acted on IMR32 cells in a dose dependent manner as observed through PI and MTT assays. The percentage intracellular ROS activity was reduced by 60–70% in treated cells compared to H2O2 control. Conclusion Thus the outcome of the study suggests that H. rhamnoides acts as a neuroprotectant against oxidative stress induced neurodegeneration. PMID:26288571

Baicalin Down-Regulates IL-1β-Stimulated Extracellular Matrix Production in Nasal Fibroblasts

PubMed Central

Shin, Jae-Min; Kang, Ju-Hyung; Lee, Seoung-Ae; Park, Il-Ho; Lee, Heung-Man

2016-01-01

Purpose Baicalin, a Chinese herbal medicine, has anti-fibrotic and anti-inflammatory effects. The aims of present study were to investigate the effects of baicalin on the myofibroblast differentiation, extracellular matrix production, migration, and collagen contraction of interleukin (IL)-1β-stimulated nasal fibroblasts and to determine the molecular mechanism of baicalin in nasal fibroblasts. Methods Nasal fibroblasts were isolated from the inferior turbinate of patients. Baicalin was used to treat IL-1β-stimulated nasal fibroblasts. To evaluate cytotoxicity, a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay was used. The expression levels of α-smooth muscle actin (SMA), fibronectin, phospho-mitogen-activated protein kinase (p-MAPK), p-Akt, p-p50, p-p65, and p-IκBα were measured by western blotting, reverse transcription-polymerase chain reaction (RT—PCR),or immunofluorescence staining. Fibroblast migration was analyzed with scratch assays and transwell migration assays. Total collagen was evaluated with the Sircol collagen assay. Contractile activity was measured with a collagen gel contraction assay. Results Baicalin (0–50 μM) had no significant cytotoxic effects in nasal fibroblasts. The expression of α–SMA and fibronectin were significantly down-regulated in baicalin-treated nasal fibroblasts. Migration, collagen production, and contraction of IL-1β-stimulated nasal fibroblasts were significantly inhibited by baicalin treatment. Baicalin also significantly down-regulated p-MAPK, p-Akt, p-p50, p-p65, and p-IκBα in IL-1β-stimulated nasal fibroblasts. Conclusions We showed that baicalin down-regulated myofibroblast differentiation, extracellular matrix production, migration, and collagen contraction via the MAPK and Akt/ NF-κB pathways in IL-1β-stimulated nasal fibroblasts. PMID:28002421

Migration Rate Inhibition of Breast Cancer Cells Treated by Caffeic Acid and Caffeic Acid Phenethyl Ester: An In Vitro Comparison Study.

PubMed

Kabała-Dzik, Agata; Rzepecka-Stojko, Anna; Kubina, Robert; Jastrzębska-Stojko, Żaneta; Stojko, Rafał; Wojtyczka, Robert Dariusz; Stojko, Jerzy

2017-10-19

One of the deadliest cancers among women is a breast cancer. Research has shown that two natural substances occurring in propolis, caffeic acid (CA) and caffeic acid phenethyl ester (CAPE), have significant anticancer effects. The purpose of our in vitro study was to compare cytotoxic activity and migration rate inhibition using CA and CAPE (doses of 50 and 100 µm) against triple-negative, MDA-MB-231 breast adenocarcinoma line cells, drawn from Caucasian women. Viability was measured by XTT-NR-SRB assay (Tetrazolium hydroxide-Neutral Red-Sulforhodamine B) for 24 h and 48 h periods. Cell migration for wound healing assay was taken for 0 h, 8 h, 16 h, and 24 h periods. CAPE displayed more than two times higher cytotoxicity against MDA-MB-231 cells. IC 50 values for the XTT assay were as follows: CA for 24 h and 48 h were 150.94 µM and 108.42 µM, respectively, while CAPE was 68.82 µM for 24 h and 55.79 µM for 48 h. For the NR assay: CA was 135.85 µM at 24 h and 103.23 µM at 48 h, while CAPE was 64.04 µM at 24 h and 53.25 µM at 48 h. For the SRB assay: CA at 24 h was 139.80 µM and at 48 h 103.98 µM, while CAPE was 66.86 µM at 24 h and 47.73 µM at 48 h. Both agents suspended the migration rate; however, CAPE displayed better activity. Notably, for the 100 µM CAPE dose, motility of the tested breast carcinoma cells was halted.

Histone Deacetylase Inhibitors Enhance Cytotoxicity Towards Breast Tumors While Preserving the Wound-Healing Function of Adipose-Derived Stem Cells.

PubMed

Koko, Kiavash R; Chang, Shaohua; Hagaman, Ashleigh L; Fromer, Marc W; Nolan, Ryan S; Gaughan, John P; Zhang, Ping; Carpenter, Jeffrey P; Brown, Spencer A; Matthews, Martha; Bird, Dorothy

2017-06-01

Paclitaxel improves the oncologic response of breast cancer resections; however, it may negatively affect the wound-healing potential of human adipose-derived stem cells (hASCs) for fat grafting and reconstructive surgery. Histone deacetylase inhibitors (HDACis) modify the epigenetic regulation of gene expression and stabilize microtubules similarly to paclitaxel, thus, creating a synergistic mechanism of cell cycle arrest. We aim to combine these drugs to enhance cytotoxicity towards breast cancer cells, while preserving the wound-healing function of hASCs for downstream reconstructive applications. Triple negative breast cancer cells (MBA-MB-231) and hASCs (institutional review board-approved clinical isolates) were treated with a standard therapeutic dose of paclitaxel (1.0 μM) or with low-dose paclitaxel (0.1 μM) combined with the HDACi suberoylanilide hydroxamic acid or trichostatin A. Cell viability, gene expression, apoptosis, and wound-healing/migration were measured via methylthiazol tetrazolium assay, quantitative real-time polymerase chain reaction, annexin V assay, and fibroblast scratch assay, respectively. Combined HDACi and low-dose paclitaxel therapy maintained cytotoxicity towards breast cancer cells and preserved adipose-derived stem cell viability. Histone deacetylase inhibitor demonstrated selective anti-inflammatory effects on adipose-derived stem cell gene expression and decreased expression of the proapoptotic gene FAS. Furthermore, HDACi therapy did not increase relative apoptosis within hASCs. A scratch assay demonstrated enhanced wound healing among injured fibroblasts indirectly co-cultured with HDACi-treated hASCs. Combining HDACi with low-dose paclitaxel improved cytotoxicity towards breast cancer cells and preserved hASC viability. Furthermore, enhanced wound healing was observed by improved migration in a fibroblast scratch assay. These results suggest that the addition of HDACi to taxane chemotherapy regimens may improve oncologic results and wound-healing outcomes after reconstructive surgery.

Reduction of Norwalk Virus, Poliovirus 1, and Bacteriophage MS2 by Ozone Disinfection of Water

PubMed Central

Shin, Gwy-Am; Sobsey, Mark D.

2003-01-01

Norwalk virus and other human caliciviruses (noroviruses) are major agents of gastroenteritis, and water is a major route of their transmission. In an effort to control Norwalk virus in drinking water, Norwalk virus reduction by bench-scale ozone disinfection was determined using quantitative reverse transcription (RT)-PCR for virus assays. Two other enteric viruses, poliovirus 1 and coliphage MS2, were included for comparison, and their reductions were assayed by infectivity assays as well as by RT-PCR. Virus reductions by ozone were determined using a dose of 0.37 mg of ozone/liter at pH 7 and 5°C for up to 5 min. Based on two RT-PCR assays, the reductions of Norwalk virus were >3 log10 within a contact time of 10 s, and these were similar to the reductions of the other two viruses determined by the same assay methods. Also, the virus reductions detected by RT-PCR assays were similar to those detected by infectivity assays, indicating that the RT-PCR assay is a reliable surrogate assay for both culturable and nonculturable viruses disinfected with ozone. Overall, the results of this study indicate that Norwalk virus as well as other enteric viruses can be reduced rapidly and extensively by ozone disinfection and that RT-PCR is a useful surrogate assay for both culturable and nonculturable viruses disinfected with ozone. PMID:12839770

Reduction of Norwalk virus, poliovirus 1, and bacteriophage MS2 by ozone disinfection of water.

PubMed

Shin, Gwy-Am; Sobsey, Mark D

2003-07-01

Norwalk virus and other human caliciviruses (noroviruses) are major agents of gastroenteritis, and water is a major route of their transmission. In an effort to control Norwalk virus in drinking water, Norwalk virus reduction by bench-scale ozone disinfection was determined using quantitative reverse transcription (RT)-PCR for virus assays. Two other enteric viruses, poliovirus 1 and coliphage MS2, were included for comparison, and their reductions were assayed by infectivity assays as well as by RT-PCR. Virus reductions by ozone were determined using a dose of 0.37 mg of ozone/liter at pH 7 and 5 degrees C for up to 5 min. Based on two RT-PCR assays, the reductions of Norwalk virus were >3 log(10) within a contact time of 10 s, and these were similar to the reductions of the other two viruses determined by the same assay methods. Also, the virus reductions detected by RT-PCR assays were similar to those detected by infectivity assays, indicating that the RT-PCR assay is a reliable surrogate assay for both culturable and nonculturable viruses disinfected with ozone. Overall, the results of this study indicate that Norwalk virus as well as other enteric viruses can be reduced rapidly and extensively by ozone disinfection and that RT-PCR is a useful surrogate assay for both culturable and nonculturable viruses disinfected with ozone.

Gemcitabine inhibits proliferation and induces apoptosis in human pancreatic cancer PANC-1 cells.

PubMed

Yong-Xian, Gui; Xiao-Huan, Li; Fan, Zhang; Guo-Fang, Tian

2016-10-01

The aim of the study is to investigate the underlying molecular mechanisms by which gemcitabine (gem) inhibits proliferation and induces apoptosis in human pancreatic cancer PANC-1 cells in vitro. After PANC-1 cells had been treated by indicated concentration (0, 5, and 25 mg/L) of gem for 48 h, cell proliferation was evaluated by 3'-(4, 5 dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay; cell morphology was observed by transmission electron microscopy; Expression of c-IAP2 and Bcl-2 proteins was analyzed by Western blot; the activity of caspase-3 and -9 was detected by spectrophotometry. Gem significantly inhibited cell proliferation and could induce apoptosis of human pancreatic cancer PANC-1 cells, with a dose-dependent manner. Western blot analysis showed that gem significantly reduced c-IAP2 and Bcl-2 proteins expression level (P < 0.05). Spectrophotometric assay showed that gem significantly increased caspase-3 and -9 activity in PANC-1 cells. Gem could induce apoptosis of human pancreatic cancer PANC-1 cells, probably through downregulating c-IAP2 and Bcl-2 expression levels, and at the same time activating caspase-3 and -9.

Comparison between effects of free curcumin and curcumin loaded NIPAAm-MAA nanoparticles on telomerase and PinX1 gene expression in lung cancer cells.

PubMed

Badrzadeh, Fariba; Akbarzadeh, Abolfazl; Zarghami, Nosratollah; Yamchi, Mohammad Rahmati; Zeighamian, Vahide; Tabatabae, Fateme Sadate; Taheri, Morteza; Kafil, Hossein Samadi

2014-01-01

Herbal compounds such as curcumin which decrease telomerase and gene expression have been considered as beneficial tools for lung cancer treatment. In this article, we compared the effects of pure curcumin and curcumin-loaded NIPAAm-MAA nanoparticles on telomerase and PinX1 gene expression in a lung cancer cell line. A tetrazolium-based assay was used for determination of cytotoxic effects of curcumin on the Calu-6 lung cancer cell line and telomerase and pinX1 gene expression was measured with real-time PCR. MTT assay showed that Curcumin-loaded NIPAAm-MAA inhibited the growth of the Calu-6 lung cancer cell line in a time and dose-dependent manner. Our q-PCR results showed that the expression of telomerase gene was effectively reduced as the concentration of curcumin-loaded NIPAAm-MAA increased while expression of the PinX1 gene became elevated. The results showed that curcumin- loaded- NIPAAm-MAA exerted cytotoxic effects on the Calu-6 cell line through down-regulation of telomerase and stimulation of pinX1 gene expression. NIPPAm-MAA could be good carrier for such kinds of hydrophobic agent.

Effect of 940 nm low-level laser therapy on osteogenesis in vitro

NASA Astrophysics Data System (ADS)

Jawad, Mohammed Mahmood; Husein, Adam; Azlina, Ahmad; Alam, Mohammad Khursheed; Hassan, Rozita; Shaari, Rumaizi

2013-12-01

Bone regeneration is essential in medical treatment, such as in surgical bone healing and orthodontics. The aim of this study is to examine the effect of different powers of 940 nm diode low-level laser treatment (LLLT) on osteoblast cells during their proliferation and differentiation stages. A human fetal osteoblast cell line was cultured and treated with LLLT. The cells were divided into experimental groups according to the power delivered and periods of exposure per day for each laser power. The (3-(4,5-dimethylthiazol-2yl)-2,5 diphenyl tetrazolium bromide) (MTT) assay was used to determine cell proliferation. Both alkaline phosphatase and osteocalcin activity assays were assessed for cell differentiation. All treatment groups showed a significant increase in cell proliferation and differentiation compared to the control group. Regarding the exposure time, the subgroups treated with the LLLT for 6 min showed higher proliferation and differentiation rates for the powers delivered, the 300-mW LLLT group significantly increased the amount of cell proliferation. By contrast, the 100 and 200 mW groups showed significantly greater amounts of cell differentiation. These results suggest that the use of LLLT may play an important role in stimulating osteoblast cells for improved bone formation.

Synthesis, characterization and biological evaluation of ruthenium flavanol complexes against breast cancer

NASA Astrophysics Data System (ADS)

Singh, Ashok Kumar; Saxena, Gunjan; Sahabjada; Arshad, M.

2017-06-01

Four Ru(II) DMSO complexes (M1R-M4R) having substituted flavones viz. 3-Hydroxy-2-(4-methoxyphenyl)-4H-chromen-4-one (HL1), 3-Hydroxy-2-(4-nitrophenyl)-4H-chromen-4-one (HL2), 3-Hydroxy-2-(4-dimethylaminophenyl)-4H-chromen-4-one (HL3) and 3-Hydroxy-2-(4-chlorophenyl)-4H-chromen-4-one (HL4) were synthesized and characterized by elemental analysis, IR, UV-Vis, 1H NMR spectroscopies and ESI-MS. The molecular structures of the complexes were investigated by integrated spectroscopic and computational techniques (DFT). Both ligands as well as their complexes were screened for anticancer activities against breast cancer cell lines MCF-7. Cytotoxicity was assayed by MTT [3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay. All ligands and their complexes exhibited significant cytotoxic potential of 5-40 μM concentration at incubation period of 24 h. The cell cytotoxicity increased significantly in a concentration-dependent manner. In this series of compounds, HL2 (IC50 17.2 μM) and its complex M2R (IC50 16 μM) induced the highest cytotoxicity.

Some South African Rubiaceae Tree Leaf Extracts Have Antimycobacterial Activity Against Pathogenic and Non-pathogenic Mycobacterium Species.

PubMed

Aro, Abimbola O; Dzoyem, Jean P; Hlokwe, Tiny M; Madoroba, Evelyn; Eloff, Jacobus N; McGaw, Lyndy J

2015-07-01

Tuberculosis (TB) caused by Mycobacterium tuberculosis remains an ongoing threat to human health. Many plant species contain antimycobacterial compounds, which may serve as template molecules for new anti-TB drugs. The Rubiaceae family is the largest family of trees in southern Africa, and preliminary evidence revealed antimycobacterial activity in several species of the genus, motivating further studies. Leaf extracts of 15 tree species from the Rubiaceae family were screened for antimycobacterial activity against pathogenic M. tuberculosis and non-pathogenic Mycobacterium smegmatis, Mycobacterium aurum and Mycobacterium bovis BCG (Bacillus Calmette-Guérin) using a twofold serial microdilution assay. Cytotoxicity was determined using a tetrazolium-based colorimetric assay against C3A liver cells and Vero kidney cells. Minimum inhibitory concentration values as low as 0.04 mg/mL against M. smegmatis and M. tuberculosis were recorded. Activity against M. aurum was the best predictor of activity against pathogenic M. tuberculosis (correlation coefficient = 0.9). Bioautography indicated at least 40 different antimycobacterial compounds in the extracts. Cytotoxicity of the extracts varied, and Oxyanthus speciosus had the most promising selectivity index values. Copyright © 2015 John Wiley & Sons, Ltd.

Molecular size and origin do not influence the harmful side effects of hydroxyethyl starch on human proximal tubule cells (HK-2) in vitro.

PubMed

Bruno, Raphael R; Neuhaus, Winfried; Roewer, Norbert; Wunder, Christian; Schick, Martin A

2014-09-01

Recently, clinical trials revealed renal impairment induced by hydroxyethyl starch (HES) in septic patients. In prior studies, we managed to demonstrate that HES accumulated in renal proximal tubule cells (PTCs). The related pathomechanism has not yet been discovered. To validate our hypothesis that the HES molecule itself is harmful, regardless of its molecule size or origin, we conducted a comprehensive study to elucidate the influences of different HES preparations on PTC viability in vitro. Cell viability of human PTC was measured with a cytotoxicity assay, quantifying the reduction of tetrazolium salt to colored formazan. Experiments were performed by assessing the influence of different carrier solutions of HES (balanced, nonbalanced, culture medium), different average molecular weights (70, 130, 200 kDa), different origins (potato or corn derived), and various durations of incubation (2-21 hours). Furthermore, HES 130/0.4 was fractionated by ultrafiltration, and the impact on cell viability of average single-size fractions with <3, 3 to 10, 10 to 30, 30 to 50, 50 to 100, and >100 kDa was investigated. We also tested the possible synergistic effects of inflammation induced by tumor necrosis factor-α. All tested HES solutions, regardless of origin or carrier matrix, decreased cell viability in an equivalent, dose-dependent manner. Coincubation with tumor necrosis factor-α did not reduce HES-induced reduction of cell viability. Minor differences were detected comparing 70, 130, and 200 kDa preparations. Analysis of fractionated HES revealed that each fraction decreased cell viability. Even small HES molecules (10-30 kDa) were significantly deleterious. For the first time, we were able to show that only the total mass of HES molecules applied is responsible for the harmful impact on renal PTC in vitro. Neither molecular size nor their origin showed any relevance.

Kaempferol acts through mitogen-activated protein kinases and protein kinase B/AKT to elicit protection in a model of neuroinflammation in BV2 microglial cells

PubMed Central

Park, SE; Sapkota, K; Kim, S; Kim, H; Kim, SJ

2011-01-01

BACKGROUND AND PURPOSE Kaempferol, a dietary flavonoid and phyto-oestrogen, is known to have anti-inflammatory properties. Microglial activation has been implicated in various neurodegenerative diseases. Anti-inflammatory effects of kaempferol and the underlying mechanisms were investigated by using LPS-stimulated microglial BV2 cells. EXPERIMENTAL APPROACH Cell viability was measured using MTT and neutral red assays. elisa, Western blot, immunocytochemistry and electrophoretic mobility-shift assay were used to analyse NO, PGE2, TNF-α and IL-1β production, inducible NOS (iNOS), COX-2 expression and the involvement of signalling pathways such as toll-like receptor-4 (TLR4), MAPK cascades, PKB (AKT) and NF-κB. Accumulation of reaction oxygen species (ROS) was measured by nitroblue tetrazolium and 2′7′-dichlorofluorescein diacetate assay. Matrix metalloproteinase activity was investigated by zymography and immunoblot assay. Phagocytotic activity was assessed by use of latex beads. KEY RESULTS Kaempferol significantly attenuated LPS-induced NO, PGE2, TNF-α, IL-1β and ROS production and phagocytosis in a concentration-dependent manner. Kaempferol suppressed the expression of iNOS, COX-2, MMP-3 and blocked the TLR4 activation. Moreover, kaempferol inhibited LPS-induced NF-κB activation and p38 MAPK, JNK and AKT phosphorylation. CONCLUSION AND IMPLICATIONS Kaempferol was able to reduce LPS-induced inflammatory mediators through the down-regulation of TLR4, NF-κB, p38 MAPK, JNK and AKT suggesting that kaempferol has therapeutic potential for the treatment of neuroinflammatory diseases. PMID:21449918

Glucose Oxidase-Mediated Polymerization as a Platform for Dual-Mode Signal Amplification and Biodetection

PubMed Central

Berron, Brad J; Johnson, Leah M; Ba, Xiao; McCall, Joshua D; Alvey, Nicholas J; Anseth, Kristi S; Bowman, Christopher N

2011-01-01

We report the first use of a polymerization-based ELISA substrate solution employing enzymatically mediated radical polymerization as a dual-mode amplification strategy. Enzymes are selectively coupled to surfaces to generate radicals that subsequently lead to polymerization-based amplification (PBA) and biodetection. Sensitivity and amplification of the polymerization-based detection system were optimized in a microwell strip format using a biotinylated microwell surface with a glucose oxidase (GOx)–avidin conjugate. The immobilized GOx is used to initiate polymerization, enabling the detection of the biorecognition event visually or through the use of a plate reader. Assay response is compared to that of an enzymatic substrate utilizing nitroblue tetrazolium in a simplified assay using biotinylated wells. The polymerization substrate exhibits equivalent sensitivity (2 µg/mL of GOx-avidin) and over three times greater signal amplification than this traditional enzymatic substrate since each radical that is enzymatically generated leads to a large number of polymerization events. Enzyme-mediated polymerization proceeds in an ambient atmosphere without the need for external energy sources, which is an improvement upon previous PBA platforms. Substrate formulations are highly sensitive to both glucose and iron concentrations at the lowest enzyme concentrations. Increases in amplification time correspond to higher assay sensitivities with no increase in non-specific signal. Finally, the polymerization substrate generated a signal to noise ratio of 14 at the detection limit (156 ng/mL) in an assay of transforming growth factor-beta. Biotechnol. Bioeng. 2011; 108:1521–1528. © 2011 Wiley Periodicals, Inc. PMID:21337335

Andrographolide radiosensitizes human esophageal cancer cell line ECA109 to radiation in vitro.

PubMed

Wang, Z-M; Kang, Y-H; Yang, X; Wang, J-F; Zhang, Q; Yang, B-X; Zhao, K-L; Xu, L-P; Yang, L-P; Ma, J-X; Huang, G-H; Cai, J; Sun, X-C

2016-01-01

To explore the radiosensitivity of andrographolide on esophageal cancer cell line ECA109. The inhibition effects of andrographolide were measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. Clonogenic survival assay was used to evaluate the effects of andrographolide on the radiosensitivity of esophageal cancer cells. Immunofluorescence was employed to examine Bax expression. The changes in cell cycle distribution and apoptosis were assayed using flow cytometry. The expression of NF-κb/Cleaved-Caspase3/Bax/Bcl-2 was measured using Western blot analysis. DNA damage was detected via γ-H2AX foci counting. With a clear dose and time effects, andrographolide was found to inhibit the proliferation of esophageal cell line ECA109. The results of the clonogenic survival assay show that andrographolide could markedly enhance radiosensitivity (P < 0.05) with a sensitizing enhancement ratio of 1.28. Andrographolide caused a dose-dependent increase in Cleaved-Caspase3/Bax protein expression and a decrease in Bcl-2/NF-κb expression. Apoptosis in andrographolide-treated ECA-109 increased significantly compared with the apoptosis in the simple drug and radiation combined with drug groups (P < 0.001; P < 0.05). Moreover, compared with the independent radiation group, the andrographolide combined with radiation group increased the number of DNA double chain breaks. Andrographolide can increase the radiosensitivity of esophageal cell line ECA109. This result may be associated with the decrease in the NF-κb level and the induced apoptosis of esophageal cancer cells. © 2014 International Society for Diseases of the Esophagus.

The prosurvival role of autophagy in Resveratrol-induced cytotoxicity in human U251 glioma cells

PubMed Central

2009-01-01

Background Previous study reported that resveratrol has anti-tumor activity. In this study, we investigated the involvement of autophagy in the resveratrol-induced apoptotic death of human U251 glioma cells. Methods The growth inhibition of U251 cells induced by resveratrol was assessed with methyl thiazolyl tetrazolium (MTT). The activation of autophagy and proapoptotic effect were characterized by monodansylcadaverine labeling and Hoechst stain, respectively. Mitochondrialtransmembrane potential (ΔΨm) was measured as a function of drug treatment using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). The role of autophagy and apoptosis in the resveratrol-induced death of U251 cells was assessed using autophagic and caspase inhibitors. Immunofluorescence, flow cytometry, and Western blot analysis were used to study the apoptotic and autophagic mechanisms. Results Methyl thiazolyl tetrazolium (MTT) assays indicated that resveratrol decreased the viability of U251 cells in a dose- and time-dependent manner. Flow cytometry analysis indicated that resveratrol increased cell population at sub-G1 phase, an index of apoptosis. Furthermore, resveratrol-induced cell death was associated with a collapse of the mitochondrial membrane potential. The pan-caspase inhibitor Z-VAD-fmk suppressed resveratrol-induced U251 cell death. Resveratrol stimulated autophagy was evidenced by punctuate monodansylcadaverine(MDC) staining and microtubule-associated protein light chain 3 (LC3) immunoreactivty. Resveratrol also increased protein levels of beclin 1 and membrane form LC3 (LC3-II). Autophagy inhibitors 3-methylademine (3-MA) and bafilomycin A1 sensitized the cytotoxicity of resveratrol. Conclusion Together, these findings indicate that resveratrol induces autophagy in human U251 glioma cells and autophagy suppressed resveratrol-induced apoptosis. This study thus suggests that autophagy inhibitors can increase the cytotoxicity of resveratrol to glioma cells. PMID:19566920

Chemical characterization and cytotoxic, genotoxic, and mutagenic properties of Baccharis trinervis (Lam, Persoon) from Colombia and Brazil.

PubMed

Jaramillo-García, Victoria; Trindade, Cristiano; Lima, Elisiane; Guecheva, Temenouga N; Villela, Izabel; Martinez-Lopez, Wilner; Corrêa, Dione S; Ferraz, Alexandre de B F; Moura, Sidnei; Sosa, Milton Quintana; Da Silva, Juliana; Henriques, João Antônio Pegas

2018-03-01

Baccharis trinervis (Lam, Persoon) leaves are used in the traditional medicine for the treatment of high fevers, edema, inflammation, sores and muscle cramps, snakebites and as antiseptic. To investigate the cytotoxic, genotoxic, and mutagenic effects of extracts and fractions of B. trinervis from Brazil and Colombia in Chinese Hamster Ovary (CHO) cells, and to examine the mutagenic activity in Salmonella typhimurium. Aqueous extracts (AE) of aerial parts of B. trinervis from Brazil (B) and Colombia (C) were fractioned in ethyl acetate fraction (EAF), butanol extract (BF), and aqueous residue fraction (ARF). Qualitative chemical screening and determination of total flavonoid content were made. Identification of chemical constituents was performed by High Performance Liquid Chromatography (HPLC) and High Resolution Mass Spectrometry (HRMS). For the in vitro tests, CHO cells were treated for 3h with extracts and fractions. The cytotoxic activity was evaluated by clonal survival and 3-(4.5-dimethylthiazole-2-yl)-2.5-biphenyl tetrazolium bromide reduction assay (MTT). Genotoxic and mutagenic effects were evaluated by the alkaline comet assay and Cytokinesis-blockage micronucleus test (CBMN), respectively. Additionally, Salmonella/microsome assay was carried out to determinate the mutagenic effects in EAF from Brazil and Colombia. Phytochemical analyses indicated the presence of saponins and flavonoids. AE and EAF were the samples with the highest quantity of total flavonoids. HPLC showed the presence of luteolin only in AEC, and caffeic acid, ellagic acid, rosmarinic acid, and rutin were identified in AEB and AEC (AEC>AEB). The HRMS in positive mode of EAFB and EAFC showed presence of two carboxylic acids, coumarin, and two terpenoids. In addition, were identified one terpenoid and two carboxylic acids in AE, BF and ARF of B. trinervis from both countries in negative mode. Dose-dependent cytotoxic effects were observed in CHO cells treated with B. trinervis extracts and fractions by using clonal survival and MTT at concentrations higher than 0.05mg/mL. All the extracts and fractions induced DNA strand breaks in CHO cells with dose-dependent response, mostly EAFB and EAFC. The EAF from Brazil and Colombia showed mutagenic effect at 0.5mg/mL, while the other fractions did not show a significant difference in relation to the control. No mutagenic effects were found in EAF from both countries by the Salmonella/microsome assay. Cytotoxic and genotoxic effects were demonstrated in all extracts and fractions used, although only EAF showed mutagenic effects by CBMN, but not by Salmonella/microsome assay. Our results suggest that flavonoids, phenylpropanoids, coumarins, and diterpenes may be responsible for the cytotoxic, genotoxic and mutagenic effects observed. Copyright © 2017 Elsevier B.V. All rights reserved.

Influence of the Testa on Seed Dormancy, Germination, and Longevity in Arabidopsis1

PubMed Central

Debeaujon, Isabelle; Léon-Kloosterziel, Karen M.; Koornneef, Maarten

2000-01-01

The testa of higher plant seeds protects the embryo against adverse environmental conditions. Its role is assumed mainly by controlling germination through dormancy imposition and by limiting the detrimental activity of physical and biological agents during seed storage. To analyze the function of the testa in the model plant Arabidopsis, we compared mutants affected in testa pigmentation and/or structure for dormancy, germination, and storability. The seeds of most mutants exhibited reduced dormancy. Moreover, unlike wild-type testas, mutant testas were permeable to tetrazolium salts. These altered dormancy and tetrazolium uptake properties were related to defects in the pigmentation of the endothelium and its neighboring crushed parenchymatic layers, as determined by vanillin staining and microscopic observations. Structural aberrations such as missing layers or a modified epidermal layer in specific mutants also affected dormancy levels and permeability to tetrazolium. Both structural and pigmentation mutants deteriorated faster than the wild types during natural aging at room temperature, with structural mutants being the most strongly affected. PMID:10677433

Protective effect of Edaravone against hypoxia-induced cytotoxicity in osteoblasts MC3T3-E1 cells.

PubMed

Cao, Bo; Chai, Chunxiang; Zhao, Sishun

2015-12-01

Edaravone is a newly developed clinical medicine for the treatment of acute cerebral infarction. Reduced blood supply to bones (hypoxia) has been involved in the pathological development of osteoporosis. In this study, we investigated the effect of Edaravone and its latent mechanism on hypoxia-induced cell toxicity in MC3T3-E1 cells. Cell viability was determined by the 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Intracellular reactive oxygen species (ROS) and nitric oxide (NO) were determined by the fluorescence dyes 2',7'-dichlorofluorescein diacetate (DCFH-DA) and 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA), respectively. mRNA and proteins were determined by real-time polymerase chain reaction and Western blot analysis, respectively. Edaravone significantly restored the hypoxia-induced reduction of MC3T3-E1 cell viability and inhibited lactate dehydrogenase release. In addition, we found that Edaravone inhibits the generation of ROS and NO. Hoechst staining results indicated that the nuclear condensation characteristic of apoptosis was increased in MC3T3-E1 cells after hypoxia exposure, which was significantly suppressed by Edaravone treatment. Mechanistically, we found that Edaravone markedly reduced the expression of cleaved caspase-3 and blunted the release of cytochrome c. These findings strongly suggested that Edaravone suppresses hypoxia-induced cytotoxicity in MC3T3-E1 cells. The pleiotropic effects of Edaravone on hypoxia exposure in osteoblasts suggest potential antiosteoporosis mechanisms of Edaravone. © 2015 International Union of Biochemistry and Molecular Biology.

Comparative assessment of antibiotic susceptibility of coagulase-negative staphylococci in biofilm versus planktonic culture as assessed by bacterial enumeration or rapid XTT colorimetry.

PubMed

Cerca, Nuno; Martins, Silvia; Cerca, Filipe; Jefferson, Kimberly K; Pier, Gerald B; Oliveira, Rosário; Azeredo, Joana

2005-08-01

To quantitatively compare the antibiotic susceptibility of biofilms formed by the coagulase-negative staphylococci (CoNS) Staphylococcus epidermidis and Staphylococcus haemolyticus with the susceptibility of planktonic cultures. Several CoNS strains were grown planktonically or as biofilms to determine the effect of the mode of growth on the level of susceptibility to antibiotics with different mechanisms of action. The utility of a new, rapid colorimetric method that is based on the reduction of a tetrazolium salt (XTT) to measure cell viability was tested by comparison with standard bacterial enumeration techniques. A 6 h kinetic study was performed using dicloxacillin, cefazolin, vancomycin, tetracycline and rifampicin at the peak serum concentration of each antibiotic. In planktonic cells, inhibitors of cell wall synthesis were highly effective over a 3 h period. Biofilms were much less susceptible than planktonic cultures to all antibiotics tested, particularly inhibitors of cell wall synthesis. The susceptibility to inhibitors of protein and RNA synthesis was affected by the biofilm phenotype to a lesser degree. Standard bacterial enumeration techniques and the XTT method produced equivalent results both in biofilms and planktonic assays. This study provides a more accurate comparison between the antibiotic susceptibilities of planktonic versus biofilm populations, because the cell densities in the two populations were similar and because we measured the concentration required to inhibit bacterial metabolism rather than to eradicate the entire bacterial population. While the biofilm phenotype is highly resistant to antibiotics that target cell wall synthesis, it is fairly susceptible to antibiotics that target RNA and protein synthesis.

Evaluation of the activity of new cationic carbosilane dendrimers on trophozoites and cysts of Acanthamoeba polyphaga.

PubMed

Heredero-Bermejo, Irene; Copa-Patiño, Jose Luis; Soliveri, Juan; Fuentes-Paniagua, Elena; de la Mata, Francisco Javier; Gomez, Rafael; Perez-Serrano, Jorge

2015-02-01

Dendrimers are repetitively branched molecules with a broad spectrum of applications, mainly for their antimicrobial properties and as nanocarriers for other molecules. Recently, our research group have synthesized and studied their activity against Acanthamoeba sp., causative agent of a severe ocular disease in humans: Acanthamoeba keratitis. New cationic carbosilane dendrimers were tested against the protozoa forms at different concentrations and for different incubation times. Trophozoite viability was determined by manual counting and cyst viability by observing excystment in microplates with fresh culture medium. Cytotoxicity was checked on HeLa cells using the microculture tetrazolium assay. Alterations were observed by optical microscopy and by flow cytometry staining with propidium iodide. Six out of the 18 dendrimers tested were non-cytotoxic and effective against the trophozoite form, having one of them (dendrimer 14 with an IC50 of 2.4 + 0.1 mg/L) a similar activity to chlorhexidine digluconate (IC50 1.7 + 0.1 mg/L). This dendrimer has a polyphenoxo core and a sulphur atom close to the six -NH3+ terminal groups. On the other hand, only two dendrimers showed some effect against cysts (dendrimers 14 and 17). However, their minimum cysticidal concentrations were cytotoxic and less effective than the control drug. The alterations on the amoeba morphology produced by the treatment with dendrimers were size reduction, increased complexity, loss of acanthopodia and cell membrane disruption. In conclusion, these results suggest that some dendrimers may be studied in animal models to test their effect and that new dendrimers with similar features should be synthesized.

The effects of diazinon and cypermethrin on the differentiation of neuronal and glial cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Flaskos, J.; Harris, W.; Sachana, M.

2007-03-15

Diazinon and cypermethrin are pesticides extensively used in sheep dipping. Diazinon is a known anti-cholinesterase, but there is limited information regarding its molecular mechanism of action. This paper describes the effects of diazinon and cypermethrin at a morphological and molecular level on differentiating mouse N2a neuroblastoma and rat C6 glioma cell lines. Concentrations up to 10 {mu}M of both compounds and their mixture had no effect on the viability of either cell line, as determined by methyl blue tetrazolium reduction and total protein assays. Microscopic analysis revealed that 1 {mu}M and 10 {mu}M diazinon but not cypermethrin inhibited the outgrowthmore » of axon-like processes in N2a cells after a 24-h exposure but neither compound affected process outgrowth by differentiating C6 cells at these concentrations. Under these conditions, 10 {mu}M diazinon inhibited AChE slightly compared to the control after a 4-h exposure but not after 24 h. Western blotting analysis showed that morphological changes were associated with reduced cross-reactivity with antibodies that recognize the neurofilament heavy chain (NFH), microtubule associated protein MAP 1B and HSP-70 compared to control cell extracts, whereas reactivity with anti-{alpha}-tubulin antibodies was unchanged. Aggregation of NFH was observed in cell bodies of diazinon-treated N2a cells, as determined by indirect immunofluorescence staining. These data demonstrate that diazinon specifically targets neurite outgrowth in neuronal cells and that this effect is associated with disruption of axonal cytoskeleton proteins, whereas cypermethrin has no effect on the same parameters.« less

Inhibition of human polymorphonuclear leukocyte function by components of human colostrum and mature milk.

PubMed

Pickering, L K; Cleary, T G; Caprioli, R M

1983-04-01

To compare the effect of human colostrum (days 1 to 3 postpartum) and mature milk (days 170 +/- 24 postpartum) on the function of polymorphonuclear leukocytes (PMNL), Ficoll-Hypaque-separated PMNL from the blood of 60 healthy volunteers were incubated with whole colostrum, colostral lipid, and colostral aqueous phase from 30 mothers, or with mature whole milk and its separated components from 30 mothers, and tested for resting and zymosan-stimulated oxidative metabolism, functional activity, and the presence of Fc receptors. Stimulated oxygen consumption, quantitative nitroblue tetrazolium dye reduction, [1-(14)C]glucose utilization, and Fc receptors were significantly (P < 0.05 to P < 0.001) less in PMNL exposed to whole human colostrum or colostral lipid than in non-lipid-exposed cells or cells exposed to the aqueous phase of colostrum. In contrast, PMNL exposed to whole mature milk or to its lipid or aqueous phase caused no significant decrease in any of these parameters when compared to nonexposed cells. In assays of phagocytosis, colostral PMNL or blood PMNL exposed to colostral lipid had a significant (P < 0.001) decrease in their ability to ingest [methyl-(3)H]thymidine-labeled Staphylococcus aureus when compared to non-lipid-exposed PMNL. Blood PMNL exposed to lipid from mature milk had no decrease in ability to ingest S. aureus. Analysis of total lipid and total and individual fatty acid content revealed a uniform increase in all components in mature milk when compared to colostrum. Lipid or lipid-soluble material present in human colostrum but not mature milk causes inhibition of phagocytosis and respiratory burst-related activities of PMNL.

Bioactivity of calcium phosphate bioceramic coating fabricated by laser cladding

NASA Astrophysics Data System (ADS)

Zhu, Yizhi; Liu, Qibin; Xu, Peng; Li, Long; Jiang, Haibing; Bai, Yang

2016-05-01

There were always strong expectations for suitable biomaterials used for bone regeneration. In this study, to improve the biocompatiblity of titanium alloy, calcium phosphate bioceramic coating was obtained by laser cladding technology. The microstructure, phases, bioactivity, cell differentiation, morphology and resorption lacunae were investigated by optical microscope (OM), x-ray diffraction (XRD), methyl thiazolyl tetrazolium (MTT) assay, tartrate-resistant acid phosphatase (TRAP) staining and scanning electronic microscope (SEM), respectively. The results show that bioceramic coating consists of three layers, which are a substrate, an alloyed layer and a ceramic layer. Bioactive phases of β-tricalcium phosphate (β-TCP) and hydroxyapatite (HA) were found in ceramic coating. Osteoclast precursors have excellent proliferation on the bioceramic surface. The bioceramics coating could be digested by osteoclasts, which led to the resorption lacunae formed on its surface. It revealed that the gradient bioceramic coating has an excellent bioactivity.

Physicochemical and biological properties of oxovanadium(IV), cobalt(II) and nickel(II) complexes with oxydiacetate anions.

PubMed

Wyrzykowski, Dariusz; Kloska, Anna; Pranczk, Joanna; Szczepańska, Aneta; Tesmar, Aleksandra; Jacewicz, Dagmara; Pilarski, Bogusław; Chmurzyński, Lech

2015-03-01

The potentiometric and conductometric titration methods have been used to characterize the stability of series of VO(IV)-, Co(II)- and Ni(II)-oxydiacetato complexes in DMSO-water solutions containing 0-50 % (v/v) DMSO. The influence of DMSO as a co-solvent on the stability of the complexes as well as the oxydiacetic acid was evaluated. Furthermore, the reactivity of the complexes towards superoxide free radicals was assessed by employing the nitro blue tetrazolium (NBT) assay. The biological properties of the complexes were investigated in relation to their cytoprotective activity against the oxidative damage generated exogenously by using hydrogen peroxide in the Human Dermal Fibroblasts adult (HDFa) cell line as well as to their antimicrobial activity against the bacteria (Bacillus subtilis, Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis). The relationship between physicochemical and biological properties of the complexes was discussed.

AKT Axis, miR-21, and RECK Play Pivotal Roles in Dihydroartemisinin Killing Malignant Glioma Cells

PubMed Central

Shao, Ying-Ying; Zhang, Tao-Lan; Wu, Lan-Xiang; Zou, He-Cun; Li, Shuang; Huang, Jin; Zhou, Hong-Hao

2017-01-01

Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, is known to play important roles in inhibiting proliferation rate, inducing apoptosis, as well as hindering the metastasis and invasion of glioma cells, but the underlying mechanisms are still unclear so far. In this study, methyl thiazolyl tetrazolium (MTT), colony-forming, wound healing, invasion, and apoptosis assays were performed to investigate the effect of DHA on malignant glioma cells. Results showed that DHA induced apoptosis of malignant glioma cells through Protein Kinase B (AKT) axis, induced death of malignant glioma cells by downregulating miR-21, and inhibited the invasion of malignant glioma cells corresponding with up-regulation of the reversion-inducing-cysteine-rich protein with kazal motifs (RECK). These results revealed that AKT axis, miR-21, and RECK play pivotal roles in DHA killing malignant glioma cells, suggesting that DHA is a potential agent for treating glioma. PMID:28208619

Antioxidant properties of Aller-7, a novel polyherbal formulation for allergic rhinitis.

PubMed

D'Souza, P; Amit, A; Saxena, V S; Bagchi, D; Bagchi, M; Stohs, S J

2004-01-01

Allergic rhinitis, a frequently occurring immunological disorder affecting men, women and children worldwide, is a state of hypersensitivity that occurs when the body overreacts to a substance such as pollen, mold, mites or dust. Allergic rhinitis exerts inflammatory response and irritation of the nasal mucosal membranes leading to sneezing; stuffy/runny nose; nasal congestion; and itchy, watery and swollen eyes. A novel, safe polyherbal formulation (Aller-7/NR-A2) has been developed for the treatment of allergic rhinitis using a unique combination of extracts from seven medicinal plants including Phyllanthus emblica, Terminalia chebula, Terminalia bellerica, Albizia lebbeck, Piper nigrum, Zingiber officinale and Piper longum. In this study, the antioxidant efficacy of Aller-7 was investigated by various assays including hydroxyl radical scavenging assay, superoxide anion scavenging assay, 1,1-diphenyl-2-picryl hydrazyl (DPPH) and 2,2-azinobis-ethyl-benzothiozoline-sulphonic acid diammonium salt (ABTS) radical scavenging assays. The protective effect of Aller-7 on free radical-induced lysis of red blood cells and inhibition of nitric oxide release by Aller-7 in lipopolysaccharide-stimulated murine macrophages were determined. Aller-7 exhibited concentration-dependent scavenging activities toward biochemically generated hydroxyl radicals (IC50 741.73 microg/ml); superoxide anion (IC50 24.65 microg/ml by phenazine methosulfate-nicotinamide adenine dinucleotide [PMS-NADH] assay and IC50 4.27 microg/ml by riboflavin/nitroblue tetrazolium [NBT] light assay), nitric oxide (IC50 16.34 microg/ml); 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical (IC50 5.62 microg/ml); and 2,2-azinobis-ethyl-benzothiozoline-sulphonic acid diammonium salt (ABTS) radical (IC50 7.35 microg/ml). Aller-7 inhibited free radical-induced hemolysis in the concentration range of 20-80 microg/ml. Aller-7 also significantly inhibited nitric oxide release from lipopolysaccharide-stimulated murine macrophages. These results demonstrate that Aller-7 is a potent scavenger of free radicals and that it may serve.

Circular Noncoding RNA HIPK3 Mediates Retinal Vascular Dysfunction in Diabetes Mellitus.

PubMed

Shan, Kun; Liu, Chang; Liu, Bai-Hui; Chen, Xue; Dong, Rui; Liu, Xin; Zhang, Yang-Yang; Liu, Ban; Zhang, Shu-Jie; Wang, Jia-Jian; Zhang, Sheng-Hai; Wu, Ji-Hong; Zhao, Chen; Yan, Biao

2017-10-24

The vascular complications of diabetes mellitus are the major causes of morbidity and mortality among people with diabetes. Circular RNAs are a class of endogenous noncoding RNAs that regulate gene expression in eukaryotes. In this study, we investigated the role of circular RNA in retinal vascular dysfunction induced by diabetes mellitus. Quantitative polymerase chain reactions, Sanger sequencing, and Northern blots were conducted to detect circular HIPK3 (circHIPK3) expression pattern on diabetes mellitus-related stresses. MTT (3-[4,5-dimethythiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assays, EdU (5-ethynyl-2'-deoxyuridine) incorporation assays, Transwell migration assays, and Matrigel assays were conducted to detect the role of circHIPK3 in retinal endothelial cell function in vitro. Retinal trypsin digestion, vascular permeability assays, and ELISA assays were conducted to detect the role of circHIPK3 in retinal vascular dysfunction in vivo. Bioinformatics analysis, luciferase activity assays, RNA pull-down assays, and in vitro studies were conducted to reveal the mechanism of circHIPK3-mediated retinal vascular dysfunction. circHIPK3 expression was significantly upregulated in diabetic retinas and retinal endothelial cells following stressors related to diabetes mellitus. circHIPK3 silencing or overexpressing circHIPK3 changed retinal endothelial cell viability, proliferation, migration, and tube formation in vitro. circHIPK3 silencing in vivo alleviated retinal vascular dysfunction, as shown by decreased retinal acellular capillaries, vascular leakage, and inflammation. circHIPK3 acted as an endogenous miR-30a-3p sponge to sequester and inhibit miR-30a-3p activity, which led to increased vascular endothelial growth factor-C, FZD4, and WNT2 expression. Ectopic expression of miR-30a-3p mimicked the effect of circHIPK3 silencing on vascular endothelial phenotypes in vivo and in vitro. The circular RNA circHIPK3 plays a role in diabetic retinopathy by blocking miR-30a function, leading to increased endothelial proliferation and vascular dysfunction. These data suggest that circular RNA is a potential target to control diabetic proliferative retinopathy. © 2017 American Heart Association, Inc.

6-mercaptopurine transport in human lymphocytes: Correlation with drug-induced cytotoxicity

PubMed Central

CONKLIN, Laurie S.; CUFFARI, Carmen; OKAZAKI, Toshihiko; MIAO, Yinglei; SAATIAN, Bahman; CHEN, Tian-E.; TSE, Ming; BRANT, Steven R.; LI, Xuhang

2013-01-01

OBJECTIVE 6-mercaptopurine (6-MP) is efficacious in the treatment of inflammatory bowel disease (IBD). However, about one-third of patients respond poorly to therapy. This study aimed to characterize the inherent differences in 6-MP transport that may contribute to the differences in treatment responses. METHODS Intracellular 6-MP accumulation was assayed in Epstein–Barr virus (EBV)-transformed lymphocytes from IBD patients, using 14C-radiolabeled 6-MP. Cell proliferation was determined by methyl thiazolyl tetrazolium (MTT) assay. Apoptosis was assayed based on the activation of caspase 3. The expressions of 15 potential 6-MP transporters were evaluated by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS Intracellular 6-MP accumulation, varying significantly among patients, was carrier-dependent and partially sodium-dependent. 6-MP cytotoxicity was, at least in part, due to apoptosis and correlated with intracellular drug accumulation. The efflux transporters did not appear to contribute to the variability of intracellular drug accumulation between patients, since none correlated with drug accumulation or cyto-toxicity. Rather, differential expression of five influx/uptake transporters might be a key contributor to the difference in the accumulation of and susceptibility to the drug. CONCLUSIONS The heterogeneity of the drug transporters may be the reason for the therapeutic sensitivity of 6-MP in IBD patients. As the 6-MP uptake is a carrier-mediated and partially sodium-dependent process, future studies are necessary to evaluate the role of the putative transporters and their correlation with drug sensitivity in patients. PMID:22257476

Effects of canonical NF-κB signaling pathway on the proliferation and odonto/osteogenic differentiation of human stem cells from apical papilla.

PubMed

Li, Junjun; Yan, Ming; Wang, Zilu; Jing, Shuanglin; Li, Yao; Liu, Genxia; Yu, Jinhua; Fan, Zhipeng

2014-01-01

NF-κB signaling pathway plays a complicated role in the biological functions of mesenchymal stem cells. However, the effects of NF-κB pathway on the odonto/osteogenic differentiation of stem cells from apical papilla (SCAPs) remain unclear. The present study was designed to evaluate the effects of canonical NF-κB pathway on the osteo/odontogenic capacity of SCAPs in vitro. Western blot results demonstrated that NF-κB pathway in SCAPs was successfully activated by TNF-α or blocked by BMS-345541. NF-κB pathway-activated SCAPs presented a higher proliferation activity compared with control groups, as indicated by dimethyl-thiazol-diphenyl tetrazolium bromide assay (MTT) and flow cytometry assay (FCM). Wound scratch assay revealed that NF-κB pathway-activated SCAPs presented an improved migration capacity, enhanced alkaline phosphatase (ALP) activity, and upregulated mineralization capacity of SCAPs, as compared with control groups. Meanwhile, the odonto/osteogenic markers (ALP/ALP, RUNX2/RUNX2, OSX/OSX, OCN/OCN, OPN/OPN, BSP/BSP, DSPP/DSP, and DMP-1/DMP-1) in NF-κB pathway-activated SCAPs were also significantly upregulated as compared with control groups at both protein and mRNA levels. However, NF-κB pathway-inhibited SCAPs exhibited a lower proliferation/migration capacity, and decreased odonto/osteogenic ability in comparison with control groups. Our findings suggest that classical NF-κB pathway plays a paramount role in the proliferation and committed differentiation of SCAPs.

Calcium Hydroxide-induced Proliferation, Migration, Osteogenic Differentiation, and Mineralization via the Mitogen-activated Protein Kinase Pathway in Human Dental Pulp Stem Cells.

PubMed

Chen, Luoping; Zheng, Lisha; Jiang, Jingyi; Gui, Jinpeng; Zhang, Lingyu; Huang, Yan; Chen, Xiaofang; Ji, Jing; Fan, Yubo

2016-09-01

Calcium hydroxide has been extensively used as the gold standard for direct pulp capping in clinical dentistry. It induces proliferation, migration, and mineralization in dental pulp stem cells (DPSCs), but the underlying mechanisms are still unclear. The aim of this study was to investigate the role of the mitogen-activated protein (MAP) kinase pathway in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Human DPSCs between passages 3 and 6 were used. DPSCs were preincubated with inhibitors of MAP kinases and cultured with calcium hydroxide. The phosphorylated MAP kinases were detected by Western blot analysis. Cell viability was analyzed via the methylthiazol tetrazolium assay. Cell migration was estimated using the wound healing assay. Alkaline phosphatase (ALP) expression was analyzed using the ALP staining assay. Mineralization was studied by alizarin red staining analysis. Calcium hydroxide significantly promoted the phosphorylation of the c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase. The inhibition of JNK and p38 signaling abolished calcium hydroxide-induced proliferation of DPSCs. The inhibition of JNK, p38, and extracellular signal-regulated kinase signaling suppressed the migration, ALP expression, and mineralization of DPSCs. Our study showed that the MAP kinase pathway was involved in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Cytotoxic and apoptotic effects of chalcone derivatives of 2-acetyl thiophene on human colon adenocarcinoma cells.

PubMed

de Vasconcelos, Alana; Campos, Vinicius Farias; Nedel, Fernanda; Seixas, Fabiana Kömmling; Dellagostin, Odir A; Smith, Kevin R; de Pereira, Cláudio Martin Pereira; Stefanello, Francieli Moro; Collares, Tiago; Barschak, Alethéa Gatto

2013-06-01

Recent studies report that chalcones exhibit cytotoxicity to human cancer cell lines. Typically, the form of cell death induced by these compounds is apoptosis. In the context of the discovery of new anticancer agents and in light of the antitumour potential of several chalcone derivatives, in the present study, we synthesized and tested the cytotoxicity of six chalcone derivatives on human colon adenocarcinoma cells. Six derivatives of 3-phenyl-1-(thiophen-2-yl) prop-2-en-1-one were prepared and characterized on the basis of their (1) H and (13) C NMR spectra. HT-29 cells were treated with synthesized chalcones on two concentrations by three different incubation times. Cells were evaluated by cell morphology, Tetrazolium dye (MTT) colorimetric assay, live/dead, flow cytometry (annexin V) and gene expression analyses to determine the cytotoxic way. Chalcones 3-(4-bromophenyl)-1-(thiophen-2-yl)prop-2-en-1-one (C06) and 3-(2-nitrophenyl)-1-(thiophen-2-yl)prop-2-en-1-one (C09) demonstrated higher cytotoxicity than other chalcones as shown by cell morphology, live/dead and MTT assays. In addition, C06 induced apoptosis on flow cytometry annexin V assay. These data were confirmed by a decreased expression of anti-apoptotic genes and increased pro-apoptotic genes. Our findings indicate in summary that the cytotoxic activity of chalcone C06 on colorectal carcinoma cells occurs by apoptosis. Copyright © 2012 John Wiley & Sons, Ltd.

Comparison of methods for determining the effectiveness of antibacterial functionalized textiles.

PubMed

Haase, Hajo; Jordan, Lisa; Keitel, Laura; Keil, Claudia; Mahltig, Boris

2017-01-01

Antimicrobial functionalization of textiles is important for various applications, such as protection of textile materials from decomposition, generation of more effective wound dressings, and the prevention of infections or malodors resulting from bacterial growth. In order to test the efficacy of new products, their antibacterial activity needs to be evaluated. At present, several different procedures are being used for this purpose, hindering comparisons among different studies. The present paper compares five of these assays using a sample panel of different textiles functionalized with copper (Cu) and silver (Ag) as antibacterial agents, and discusses the suitability of these methods for different analytical requirements. Bacterial viability was determined by measuring the optical density at 600 nm, a colorimetric assay based on MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide) conversion, an agar diffusion assay, and colony formation, either after culturing in media containing textile samples, or after recovery from textiles soaked with bacterial suspension. All experiments were performed with a Gram-negative (Escherichia coli) and a Gram-positive (Staphylococcus warneri) model organism. In general, the results yielded by the different methods were of good comparability. To identify the most suitable test system for the particular type of antibacterial coating, several factors need to be taken into account, such as choosing appropriate endpoints for analyzing passive or active antibacterial effects, selection of relevant microorganisms, correcting for potential interference by leaching of colored textile coatings, required hands on time, and the necessary sensitivity.

Comparison of methods for determining the effectiveness of antibacterial functionalized textiles

PubMed Central

Jordan, Lisa; Keitel, Laura; Keil, Claudia; Mahltig, Boris

2017-01-01

Antimicrobial functionalization of textiles is important for various applications, such as protection of textile materials from decomposition, generation of more effective wound dressings, and the prevention of infections or malodors resulting from bacterial growth. In order to test the efficacy of new products, their antibacterial activity needs to be evaluated. At present, several different procedures are being used for this purpose, hindering comparisons among different studies. The present paper compares five of these assays using a sample panel of different textiles functionalized with copper (Cu) and silver (Ag) as antibacterial agents, and discusses the suitability of these methods for different analytical requirements. Bacterial viability was determined by measuring the optical density at 600 nm, a colorimetric assay based on MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide) conversion, an agar diffusion assay, and colony formation, either after culturing in media containing textile samples, or after recovery from textiles soaked with bacterial suspension. All experiments were performed with a Gram-negative (Escherichia coli) and a Gram-positive (Staphylococcus warneri) model organism. In general, the results yielded by the different methods were of good comparability. To identify the most suitable test system for the particular type of antibacterial coating, several factors need to be taken into account, such as choosing appropriate endpoints for analyzing passive or active antibacterial effects, selection of relevant microorganisms, correcting for potential interference by leaching of colored textile coatings, required hands on time, and the necessary sensitivity. PMID:29161306

The effect of resveratrol on protecting corneal epithelial cells from cytotoxicity caused by moxifloxacin and benzalkonium chloride.

PubMed

Tsai, Tzu-Yun; Chen, Ta-Ching; Wang, I-Jong; Yeh, Chao-Yuan; Su, Ming-Jai; Chen, Ruey-Hua; Tsai, Tzu-Hsun; Hu, Fung-Rong

2015-02-10

Moxifloxacin (MOX), a fourth generation fluoroquinolone (FQ), has a wide antibacterial spectrum, but may show cytotoxicity characterized by high productions of reactive oxygen species (ROS). This study investigated the protective role of a common antioxidant agent, resveratrol (trans-3,5,4'-trihydroxystilbene), against the cytotoxicity caused by MOX. Experiments were performed with a human corneal epithelial cell line (HCECs; ATCC-CRL-11515). Another commonly used FQ, levofloxacin (LEV), and the most commonly used preservatives, benzalkonium chloride (BAC), were also used for comparison with MOX. Cell viability and morphologic changes after treatment were evaluated with trypan blue exclusion assay, propidium iodine/annexin V-FITC staining, and flow cytometry. Chemiluminescence immunoassay was used for ROS quantification. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, wound healing assay, and intracellular detections of oxidative stress were performed to evaluate the effects of resveratrol. The MOX group, similar to the BAC group, showed significant cell shrinkage and death compared with the LEV group. High ROS production in HCECs of MOX group was observed both by chemiluminescence immunoassay and intracellular images. Within the observations of MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay, live cell images, and wound healing process in vitro, the cytotoxic effects of the MOX and BAC groups were opposed by resveratrol. Human corneal epithelial cells pretreated with resveratrol demonstrated better cell viability and healing rate in the early stage. The protective effects of antioxidant agents indicate that MOX, similar to BAC, causes oxidative stress-related cell damage. The results also inspired us to think about a "supplementary regimen" to increase safety and decrease the adverse effect in the treatment of corneal infections. Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.

The inhibitory effects of capillarisin on cell proliferation and invasion of prostate carcinoma cells.

PubMed

Tsui, Ke-Hung; Chang, Ying-Ling; Yang, Pei-Shan; Hou, Chen-Pang; Lin, Yu-Hsiang; Lin, Bing-Wei; Feng, Tsui-Hsia; Juang, Horng-Heng

2018-04-01

Capillarisin (Cap), an active component of Artemisia capillaris root extracts, is characterized by its anti-inflammatory, anti-oxidant and anti-cancer properties. Nevertheless, the functions of Cap in prostate cancer have not been fully explored. We evaluated the potential actions of Cap on the cell proliferation, migration and invasion of prostate carcinoma cells. Cell proliferation and cell cycle distribution were measured by water-soluble tetrazolium-1 and flow cytometry assays. The expression of cyclins, p21, p27, survivin, matrix metallopeptidase (MMP2 and MMP9) were assessed by immunoblotting assays. Effects of Cap on invasion and migration were determined by wound closure and matrigel transmigration assays. The constitutive and interlukin-6 (IL-6)-inducible STAT3 activation of prostate carcinoma cells were determined by immunoblotting and reporter assays. Capillarisin inhibited androgen-independent DU145 and androgen-dependent LNCaP cell growth through the induction of cell cycle arrest at the G0/G1 phase by upregulating p21 and p27 while downregulating expression of cyclin D1, cyclin A and cyclin B. Cap decreased protein expression of survivin, MMP-2, and MMP-9 and therefore blocked the migration and invasion of DU145 cells. Cap suppressed constitutive and IL-6-inducible STAT3 activation in DU145 and LNCaP cells. Our data indicate that Cap blocked cell growth by modulation of p21, p27 and cyclins. The inhibitory effects of Cap on survivin, MMP-2, MMP-9 and STAT3 activation may account for the suppression of invasion in prostate carcinoma cells. Our data suggest that Cap might be a therapeutic agent in treating advanced prostate cancer with constitutive STAT3 or IL-6-inducible STAT3 activation. © 2017 John Wiley & Sons Ltd.

Pirfenidone inhibits transforming growth factor β1-induced extracellular matrix production in nasal polyp-derived fibroblasts.

PubMed

Shin, Jae-Min; Park, Joo-Hoo; Park, Il-Ho; Lee, Heung-Man

2015-01-01

Pirfenidone has been shown to have antifibrotic and anti-inflammatory effects in the lungs. The purpose of this study was to evaluate the inhibitory effects of pirfenidone on transforming growth factor (TGF)-β1-induced myofibroblast differentiation and extracellular matrix accumulation. We also determined the molecular mechanisms of pirfenidone in nasal polyp-derived fibroblasts (NPDF). NPDFs were isolated from nasal polyps from eight patients who had chronic rhinosinusitis with nasal polyp. Pirfenidone was used to treat TGF-β1-induced NPDFs. Cytotoxicity was evaluated by using a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay. Fibroblast migration was evaluated with scratch assays. Expression levels of α-smooth muscle actin (SMA), fibronectin, and phosphorylated Smad2/3 were determined by Western blot and/or reverse transcription-polymerase chain reaction and immunofluorescent staining. Total collagen production was analyzed with the Sircol collagen assay and contractile activity was measured by a collagen gel contraction assay. Pirfenidone (0-2 mg/mL) has no significant cytotoxic effects in TGF-β1-induced NPDFs. Migration of NPDFs was significantly inhibited by pirfenidone treatment. The expression levels of α-SMA and fibronectin were significantly reduced in pirfenidone-treated NPDFs. Collagen contraction and production were also significantly decreased by pirfenidone treatment. Finally, pirfenidone significantly inhibited phosphorylation of the Smad2/3 pathway in TGF-β1-induced NPDFs. Pirfenidone has an inhibitory effect on TGF-β1-induced migration, myofibroblast differentiation (α-SMA), extracellular matrix accumulation, and collagen contraction by blocking the phosphorylation of Smad2/3 pathways in NPDFs. Thus, pirfenidone may inhibit TGF-β1-induced extracellular matrix by regulating Smad2/3.

Nano-sized calcium phosphate particles for periodontal gene therapy.

PubMed

Elangovan, Satheesh; Jain, Shardool; Tsai, Pei-Chin; Margolis, Henry C; Amiji, Mansoor

2013-01-01

Growth factors such as platelet-derived growth factor (PDGF) have significantly enhanced periodontal therapy outcomes with a high degree of variability, mostly due to the lack of continual supply for a required period of time. One method to overcome this barrier is gene therapy. The aim of this in vitro study is to evaluate PDGF-B gene delivery in fibroblasts using nano-sized calcium phosphate particles (NCaPP) as vectors. NCaPP incorporating green fluorescent protein (NCaPP-GFP) and PDGF-B (NCaPP-PDGF-B) plasmids were synthesized using an established precipitation system and characterized using transmission electron microscopy and 1.2% agarose gel electrophoresis. Biocompatibility and transfection of the nanoplexes in fibroblasts were evaluated using cytotoxicity assay and florescence microscopy, respectively. Polymerase chain reaction and enzyme-linked immunosorbent assay were performed to evaluate PDGF-B transfection after different time points of treatments, and the functionality of PDGF-B transfection was evaluated using the cell proliferation assay. Synthesized NCaPP nanoplexes incorporating the genes of GFP and PDGF-B were spherical in shape and measured about 30 to 50 nm in diameter. Gel electrophoresis confirmed DNA incorporation and stability within the nanoplexes, and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium reagent assay demonstrated their biocompatibility in fibroblasts. In vitro transfection studies revealed a higher and longer lasting transfection after NCaPP-PDGF-B treatment, which lasted up to 96 hours. Significantly enhanced fibroblast proliferation observed in NCaPP-PDGF-B-treated cells confirmed the functionality of these nanoplexes. NCaPP demonstrated higher levels of biocompatibility and efficiently transfected PDGF plasmids into fibroblasts under described in vitro conditions.

Arsenic induces functional re-expression of estrogen receptor α by demethylation of DNA in estrogen receptor-negative human breast cancer.

PubMed

Du, Juan; Zhou, Nannan; Liu, Hongxia; Jiang, Fei; Wang, Yubang; Hu, Chunyan; Qi, Hong; Zhong, Caiyun; Wang, Xinru; Li, Zhong

2012-01-01

Estrogen receptor α (ERα) is a marker predictive for response of breast cancers to endocrine therapy. About 30% of breast cancers, however, are hormone- independent because of lack of ERα expression. New strategies are needed for re-expression of ERα and sensitization of ER-negative breast cancer cells to selective ER modulators. The present report shows that arsenic trioxide induces reactivated ERα, providing a target for therapy with ER antagonists. Exposure of ER-negative breast cancer cells to arsenic trioxide leads to re-expression of ERα mRNA and functional ERα protein in in vitro and in vivo. Luciferase reporter gene assays and 3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays show that, upon exposure to arsenic trioxide, formerly unresponsive, ER-negative MDA-MB-231 breast cancer cells become responsive to ER antagonists, 4-hydroxytamoxifen and ICI 182,780. Furthermore, methylation- specific PCR and bisulfite-sequencing PCR assays show that arsenic trioxide induces partial demethylation of the ERα promoter. A methyl donor, S-adenosylmethionine (SAM), reduces the degree of arsenic trioxide-induced re-expression of ERα and demethylation. Moreover, Western blot and ChIP assays show that arsenic trioxide represses expression of DNMT1 and DNMT3a along with partial dissociation of DNMT1 from the ERα promoter. Thus, arsenic trioxide exhibits a previously undefined function which induces re-expression ERα in ER-negative breast cancer cells through demethylation of the ERα promoter. These findings could provide important information regarding the application of therapeutic agents targeting epigenetic changes in breast cancers and potential implication of arsenic trioxide as a new drug for the treatment of ER-negative human breast cancer.

Cytotoxicity of β-D-glucose/sucrose-coated silver nanoparticles depends on cell type, nanoparticles concentration and time of incubation

NASA Astrophysics Data System (ADS)

Vergallo, Cristian; Panzarini, Elisa; Carata, Elisabetta; Ahmadi, Meysam; Mariano, Stefania; Tenuzzo, Bernardetta Anna; Dini, Luciana

2016-06-01

The use of silver NanoParticles (AgNPs) in several consumer commercialized products, like food contact materials, medical devices and cosmetics has increased significantly, owing to their antibacterial and antifungal properties. Even though the NPs are widely diffused, due to the great variety in size, coating or shape, controversial data on their possible detrimental health effects still exist. Herein, by performing an easy and fast green method synthesis, we used β-D-glucose/sucrose to stabilize AgNPs and avoid the release of cytotoxic soluble silver ions Ag+ in the culture medium. The cytotoxic effects of these β-D-Glucose/Sucrose-Coated AgNPs (AgNPs-GS) was assessed on two cell culture models, which are human liver HepG2 and human Peripheral Blood Lymphocytes (PBLs) cells. AgNPs-GS, as determined by Transmission Electron Microscopy (TEM) analyses, had an average diameter of 30±5 nm, a spherical shape and were well-dispersed in the freshly-prepared solution. In addition, they were found spectrophotometrically stable throughout the experiment. Cytotoxicity, determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, was evaluated by using two AgNPs-GS amounts, indicated as highest (10×103 of NPs/cell) and lowest (2×103 NPs/cell) concentration for 6, 12 and 24 h. The highest concentration of AgNPs-GS was significantly cytotoxic for both HepG2 and PBLs cells at all times, when compared with the negative control; conversely, the lowest amount of AgNPs-GS was toxic only for HepG2 cells. A significant increase of Reactive Oxygen Species (ROS) levels, determined by Nitro Blue Tetrazolium (NBT) reduction assay, was observed only in PBLs after treatment with NPs, by reaching maximum levels after the incubation with the lowest amount of NPs for 24 h. Significant morphological changes, depending on NPs/cell amount, characteristic of cell toxicity, like shape, cytoplasm, and nucleus alterations, were observed in lymphocytes and HepG2 cells exposed to AgNPs. The results indicate that HepG2 cells are more responsive to AgNPs treatment than PBLs. It is generally believed that cellular oxidative stress induces toxicity of NPs; however, in this study we did not detect any AgNPs-induced oxidative stress in HepG2, thus suggesting an alternative mechanism of toxicity in this cell line. In the whole, our findings suggest that AgNPs-GS-induced toxicity strictly depends on cell type, NPs amount and time of treatment.

In Vitro Effects of Serotonin, Melatonin, and Other Related Indole Compounds on Amyloid-β Kinetics and Neuroprotection.

PubMed

Hornedo-Ortega, Ruth; Da Costa, Grégory; Cerezo, Ana B; Troncoso, Ana M; Richard, Tristan; Garcia-Parrilla, M Carmen

2018-02-01

Amyloid-β peptide is the main component of senile plaques in Alzheimer's disease. The inhibition of amyloid-β peptide assembly, the destabilization of amyloid-β peptide aggregates, and the decrease of its cytotoxicity for the prevention of neuronal death are considered neuroprotective effects. In this work, the protective effects against amyloid-β peptide aggregation and cytotoxicity of eight indolic compounds are evaluated: tryptophan, tryptamine, serotonin, tryptophol, N-acetylserotonin, 3-indoleacetic acid, tryptophan ethyl ester, and melatonin. Thioflavin T spectroscopic assay, transmission electron microscopy, western blotting, circular dichroism, NMR, cell viability (thiazolyl blue tetrazolium bromide assay), quantitative PCR, and heme oxygenase activity are used. Serotonin is the most effective compound for inhibiting amyloid-β peptide aggregation. Almost all the indolic compounds tested prevent amyloid-β peptide-induced and increase cell viability, being between 9 and 25%. Melatonin and serotonin are the most active. Moreover, serotonin increased the expression of SIRT-1 and 2, heat shock protein 70, and heme oxygenase activity, this being a possible mechanism underlying the observed neuroprotective effect. Melatonin and other related indolic compounds, mainly serotonin, show an inhibitory and destabilizing effect on amyloid-β peptide fibril formation and they possess neuroprotective properties related to the vitagenes system. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Emodin Inhibits Migration and Invasion of Human Endometrial Stromal Cells by Facilitating the Mesenchymal-Epithelial Transition Through Targeting ILK.

PubMed

Zheng, Qiaomei; Xu, Ying; Lu, Jingjing; Zhao, Jing; Wei, Xuan; Liu, Peishu

2016-11-01

To determine whether emodin facilitates the mesenchymal-epithelial transition (MET) of endometrial stromal cells (ESCs) as well as to explore the mechanism through which emodin favored the MET of ESCs. Cell viability was tested by methyl thiazolyl tetrazolium assay. Cell migration and invasion abilities were detected by transwell assays. Levels of integrin-linked kinase (ILK) and epithelial-mesenchymal transition (EMT)-related proteins were detected by Western blot. Upregulated ILK and increased abilities of migration and invasion were confirmed in the eutopic and ectopic ESCs (EuSCs and EcSCs), especially in the EcSCs. After treated with emodin, the expression of ILK was statistically downregulated in EcSCs, resulting in the MET and decreased migration and invasion abilities of EcSCs. Additionally, silencing of the ILK gene in EcSCs also achieved the above-mentioned effects, which were strengthened by emodin. Furthermore, exogenous expression of ILK in control ESCs (CSCs) resulted in the EMT and increased abilities of migration and invasion of CSCs, which can be abrogated by emodin. Besides, exogenous expression of ILK also abrogated the effects of emodin on CSCs. Emodin inhibits the migration and invasion abilities of human ESCs by facilitating the MET through targeting ILK. © The Author(s) 2016.

BH3 mimetics inhibit growth of chondrosarcoma--a novel targeted-therapy for candidate models.

PubMed

Morii, Takeshi; Ohtsuka, Kouki; Ohnishi, Hiroaki; Mochizuki, Kazuo; Yoshiyama, Akira; Aoyagi, Takayuki; Hornicek, Francis J; Ichimura, Shoichi

2014-11-01

Chondrosarcoma is refractory to conventional chemotherapy. BH-3 mimetics ABT-737 and ABT-263 are synthetic small-molecule inhibitors of anti-apoptotic proteins B-cell lymphoma-2 (Bcl2) and Bcl-xL, which play a critical role in survival of chondrosarcoma cells. Chondrosarcoma cell lines SW-1353 and CS-1 were used as the disease model. We used immunoblotting to assess the expression of target molecules Bcl2 and Bcl-xL, and the apoptotic inducers Bcl2-associated X (Bax) and Bcl2-antagonist/killer (Bak). In vitro growth inhibition by BH-3 mimetics was confirmed by photomicroscopic cell counting and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. Apoptotic induction was confirmed by Enzyme-Linked ImmunoSorbent Assay (ELISA). In vivo growth inhibition was assessed in a non-obese diabetic/severe combined immunodeficient (NOD/SCID) mouse model. Expression of the target and effector molecules was confirmed in chondrosarcoma cell lines. BH3 mimetics significantly inhibited cell growth and induced apoptosis in vitro. Administration of ABT-263 inhibited chondrosarcoma growth and improved survival in a mouse model. BH3 mimetics represent a novel treatment modality for chondrosarcoma. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Effects of drinking desalinated seawater on cell viability and proliferation.

PubMed

Macarrão, Camila Longhi; Bachi, André Luis Lacerda; Mariano, Mario; Abel, Lucia Jamli

2017-06-01

Desalination of seawater is becoming an important means to address the increasing scarcity of freshwater resources in the world. Seawater has been used as drinking water in the health, food, and medical fields and various beneficial effects have been suggested, although not confirmed. Given the presence of 63 minerals and trace elements in drinking desalinated seawater (63 DSW), we evaluated their effects on the behavior of tumorigenic and nontumorigenic cells through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and annexin-V-fluorescein isothiocyanate/propidium iodide staining. Our results showed that cell viability and proliferation in the presence of 63 DSW were significantly greater than in mineral water and in the presence of fetal bovine serum in a dose-dependent manner. Furthermore, 63 DSW showed no toxic effect on murine embryonic fibroblast (NIH-3T3) and murine melanoma (B16-F10) cells. In another assay, we also showed that pre-treatment of non-adherent THP-1 cells with 63 DSW reduces apoptosis incidence, suggesting a protective effect against cell death. We conclude that cell viability and proliferation were improved by the mineral components of 63 DSW and this effect can guide further studies on health effects associated with DSW consumption.

Silver nanoparticles synthesized with Rumex hymenosepalus extracts: effective broad-spectrum microbicidal agents and cytotoxicity study.

PubMed

Rodríguez-León, Ericka; Íñiguez-Palomares, Ramón A; Navarro, Rosa Elena; Rodríguez-Beas, César; Larios-Rodríguez, Eduardo; Alvarez-Cirerol, Francisco J; Íñiguez-Palomares, Claudia; Ramírez-Saldaña, Maricela; Hernández Martínez, Javier; Martínez-Higuera, Aarón; Galván-Moroyoqui, José Manuel; Martínez-Soto, Juan Manuel

2017-08-21

We synthesized silver nanoparticles using Rumex hymenosepalus root extract (Rh). Nanoparticles were subjected to a purification process and final product is a composite of Rh and silver nanoparticles (AgNPsC). Transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS) were used to perform a microstructure study. Additionally, two fractions (RhA and RhB) were obtained from the original extract by filtration with tetrahydrofuran (THF); both fractions were analyzed using UV-Vis spectroscopy, Fourier transform infrared spectroscopy (FT-IR), and 2,2-diphenyl-1-picrylhydrazyl (DPPH); total polyphenol content was also determined. Separate inhibition tests for AgNPsC and RhA and RhB were applied to Gram-positive bacteria, Gram-negative bacteria, and yeast (Candida albicans) using the well diffusion method. Extract fractions were found to have inhibitory effects only over Gram-positive bacteria, and silver nanoparticles showed inhibitory effects over all the evaluated microorganisms. Cytotoxicity was evaluated using the tetrazolium dye (MTT) assay in mononuclear peripheral blood cells. In addition, we assessment AgNPsC in THP-1 monocyte cell line, using the cell viability estimation by trypan blue dye exclusion test (TB) and Live/Dead (LD) cell viability assays by confocal microscopy.

A comparison of in vitro cytotoxicity assays in medical device regulatory studies.

PubMed

Liu, Xuemei; Rodeheaver, Denise P; White, Jeffrey C; Wright, Ann M; Walker, Lisa M; Zhang, Fan; Shannon, Stephen

2018-06-06

Medical device biocompatibility testing is used to evaluate the risk of adverse effects on tissues from exposure to leachates/extracts. A battery of tests is typically recommended in accordance with regulatory standards to determine if the device is biocompatible. In vitro cytotoxicity, a key element of the standards, is a required endpoint for all types of medical devices. Each validated cytotoxicity method has different methodology and acceptance criteria that could influence the selection of a specific test. In addition, some guidances are more specific than others as to the recommended test methods. For example, the International Organization for Standardization (ISO 1 ) cites preference for quantitative methods (e.g., tetrazolium (MTT/XTT), neutral red (NR), or colony formation assays (CFA)) over qualitative methods (e.g., elution, agar overlay/diffusion, or direct), while a recent ISO standard for contact lens/lens care solutions specifically requires a qualitative direct test. Qualitative methods are described in United States Pharmacopeia (USP) while quantitative CFAs are listed in Japan guidance. The aim of this review is to compare the methodologies such as test article preparation, test conditions, and criteria for six cytotoxicity methods recommended in regulatory standards in order to inform decisions on which method(s) to select during the medical device safety evaluation. Copyright © 2018. Published by Elsevier Inc.

Maslinic acid, a natural triterpenoid compound from Olea europaea, protects cortical neurons against oxygen-glucose deprivation-induced injury.

PubMed

Qian, Yisong; Guan, Teng; Tang, Xuzhen; Huang, Longfei; Huang, Menghao; Li, Yunman; Sun, Hongbin

2011-11-16

Maslinic acid is a triterpenoid compound present in plants of Olea europaea. This compound has been reported to have potent antioxidant, anti-cancer, anti-HIV and anti-inflammatory activities. In this study, we investigated the neuroprotective effect of maslinic acid and its mechanism of action. With presence or absence of maslinic acid, cortical neurons were subjected to 1h of oxygen-glucose deprivation and 24h of reoxygenation. Cell injury was determined by lactate dehydrogenase (LDH) measurement and 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay. Neuronal apoptosis was evaluated by flow cytometry assay, caspase-3 expression/activity, caspase-9 activity and Bcl-2/Bax ratio. Nitric Oxide (NO) production and inducible nitric oxide synthase (iNOS) expression were also detected. Results showed that maslinic acid dose-dependently ameliorated neuron injury and apoptosis. Maslinic acid treatment normalized the caspase expression/activation and increased the Bcl-2/Bax ratio. In addition, maslinic acid inhibited oxygen-glucose deprivation-induced NO production and iNOS expression. These results indicated that maslinic acid has beneficial effects on hypoxic neurons by suppressing iNOS activation, which may, in turn, provide neuroprotection. Copyright © 2011 Elsevier B.V. All rights reserved.

Anticancer Activity of a Hexapeptide from Skate (Raja porosa) Cartilage Protein Hydrolysate in HeLa Cells

PubMed Central

Pan, Xin; Zhao, Yu-Qin; Hu, Fa-Yuan; Chi, Chang-Feng; Wang, Bin

2016-01-01

In this study, the hexapeptide Phe-Ile-Met-Gly-Pro-Tyr (FIMGPY), which has a molecular weight of 726.9 Da, was separated from skate (Raja porosa) cartilage protein hydrolysate using ultrafiltration and chromatographic methods, and its anticancer activity was evaluated in HeLa cells. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay indicated that FIMGPY exhibited high, dose-dependent anti-proliferation activities in HeLa cells with an IC50 of 4.81 mg/mL. Acridine orange/ethidium bromide (AO/EB) fluorescence staining and flow cytometry methods confirmed that FIMGPY could inhibit HeLa cell proliferation by inducing apoptosis. Western blot assay revealed that the Bax/Bcl-2 ratio and relative intensity of caspase-3 in HeLa cells treated with 7-mg/mL FIMGPY were 2.63 and 1.83, respectively, significantly higher than those of the blank control (p < 0.01). Thus, FIMGPY could induce apoptosis by upregulating the Bax/Bcl-2 ratio and caspase-3 activation. Using a DNA ladder method further confirmed that the anti-proliferation activity of FIMGPY was attributable to its role in inducing apoptosis. These results suggest that FIMGPY from skate cartilage protein hydrolysate may have applications as functional foods and nutraceuticals for the treatment and prevention of cancer. PMID:27537897

In Vitro and In Vivo Antimalarial Evaluations of Myrtle Extract, a Plant Traditionally Used for Treatment of Parasitic Disorders

PubMed Central

Naghibi, Farzaneh; Esmaeili, Somayeh; Abdullah, Noor Rain; Nateghpour, Mehdi; Taghvai, Mahdieh; Kamkar, Siamak; Mosaddegh, Mahmoud

2013-01-01

Based on the collected ethnobotanical data from the Traditional Medicine and Materia Medica Research Center (TMRC), Iran, Myrtus communis L. (myrtle) was selected for the assessment of in vitro and in vivo antimalarial and cytotoxic activities. Methanolic extract of myrtle was prepared from the aerial parts and assessed for antiplasmodial activity, using the parasite lactate dehydrogenase (pLDH) assay against chloroquine-resistant (K1) and chloroquine-sensitive (3D7) strains of Plasmodium falciparum. The 4-day suppressive test was employed to determine the parasitemia suppression of the myrtle extract against P. berghei   in vivo. The IC50 values of myrtle extract were 35.44 µg/ml against K1 and 0.87 µg/ml against 3D7. Myrtle extract showed a significant suppression of parasitaemia (84.8 ± 1.1% at 10 mg/kg/day) in mice infected with P. berghei after 4 days of treatment. Cytotoxic activity was carried out against mammalian cell lines using methyl thiazol tetrazolium (MTT) assay. No cytotoxic effect on mammalian cell lines up to 100 µg/mL was shown. The results support the traditional use of myrtle in malaria. Phytochemical investigation and understanding the mechanism of action would be in our upcoming project. PMID:24455686

Isolation and biological activities of decanal, linalool, valencene, and octanal from sweet orange oil.

PubMed

Liu, Kehai; Chen, Qiulin; Liu, Yanjun; Zhou, Xiaoyan; Wang, Xichang

2012-11-01

Product 1 (82.25% valencene), product 2 (73.36% decanal), product 3 (78.12% octanal), and product 4 (90.61% linalool) were isolated from sweet orange oil by combined usage of molecular distillation and column chromatography. The antioxidant activity of sweet orange oil and these products was investigated using 2,2-diphenyl-1-picrylhydrazyl and reducing power assays. In this test, product 1 (82.25% valencene), product 2 (73.36% decanal), and product 4 (90.61% linalool) had antioxidant activity, but lower than sweet orange oil. The antimicrobial activity was investigated in order to evaluate their efficacy against 5 microorganisms. The results showed that sweet orange oil, product 2 (73.36% decanal), product 3 (78.12% octanal), and product 4 (90.61% linalool) had inhibitory and bactericidal effect on the test microorganisms (except Penicillium citrinum). Valencene did not show any inhibitory effect. Saccharomyces cerivisiae was more susceptible, especially to the crude sweet orange oil (minimal inhibitory concentration 6.25 μL/mL). The cytotoxicity was evaluated on Hela cells using the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. All test samples showed significant cytotoxicity on the cell lines with IC(50) values much less than 20 μg/mL. © 2012 Institute of Food Technologists®

Investigation of photobiomodulation potentiality by 635 and 809 nm lasers on human osteoblasts.

PubMed

Bölükbaşı Ateş, Gamze; Ak Can, Ayşe; Gülsoy, Murat

2017-04-01

Photobiomodulation (PBM) describes light-induced photochemical reactions achieved by the application of red or near infrared lasers/LED light with low energy densities. This noninvasive and painless method has been used in some clinical areas but controversial outcomes demand a skeptical look for its promising and potential effects. In this detailed in vitro study, the osteoblast cells were irradiated with 635 and 809 nm diode lasers at energy densities of 0.5, 1, and 2 J/cm 2 . Cell viability, proliferation, bone formation, and osteoblast differentiation were evaluated by methylthiazole tetrazolium (MTT) assay, Alamar Blue assay, acridine orange/propidium iodide staining, alkaline phosphatase (ALP) activity, Alizarin red staining, and reverse-transcription polymerase chain reaction (RT-PCR) to test the expression of collagen type I, ALPL, and osteocalcin. The results indicate that studied energy doses have a transient effect (48 h after laser irradiation) on the osteoblast viability and proliferation. Similarly, laser irradiation did not appear to have any effect on ALP activity. These results were confirmed by RT-PCR analysis of osteoblast markers. This study suggests that several irradiation parameters and variations in the methods should be clearly established in the laboratory before laser treatment becomes a postulated application for bone tissue regeneration in clinical level.

Response of UMR 106 cells exposed to titanium oxide and aluminum oxide nanoparticles.

PubMed

Di Virgilio, Ana L; Reigosa, Miguel; de Mele, Monica Fernández Lorenzo

2010-01-01

The cytotoxicity potential of TiO(2) and Al(2)O(3) nanoparticles (NP) in UMR 106 cells was studied by evaluating the lysosomal activity with neutral red uptake assay (NR), and the mitochondrial activity with tetrazolium MTT test. Different NP concentrations (10-300 microg/mL range) were used. A significant (p < 0.001) increase in the absorbance (stronger for TiO(2) NP) was detected in both NR and MTT assays after 24-h exposure to the NP. However, the total cell proteins and the cell proliferation rate demonstrated (p < 0.05) that the cell viability decreased after 96 h exposure to NP. The formation of NP-containing vesicles within the cells was observed by transmission electronic microscopy. Such event could explain the high cellular activity detected during the early stages of exposure not related to the increase in cell viability. Results showed that the effects of NP on cell lines are dependent on the chemical composition of the particles, their concentration, exposure time, and the type of treated cell. It can be concluded that the presence of TiO(2) and Al(2)O(3) NP in the cell surroundings can lead to cytotoxic effects. In the case of osteoblast cells, such events may induce osseointegration failures in orthopedic and dental implants that release NP.

Effects of Angiotensin Inhibitor Valsartan on the Expression of the Angiotensin II 1 Receptor, Matrix Metalloproteinases -2 and -9 in Human Bladder Cancer Cell Lines.

PubMed

Yang, Delin; Huo, Qian; Luan, Ting; Wang, Jiansong; Tang, Zhaoran; Wang, Haifeng

2016-08-01

In order to investigate how valsartan-the angiotensin II 1 receptor (AT1R) antagonist-affects the expressions of AT1R antigen, matrix metalloproteinases (MMPs) -2 and -9 in carcinoma of urinary bladder (CUB) cell lines with different invasive abilities. Three cell lines, EJ-M3, EJ, and BIU-87, with different invasive abilities were cultured and treated with valsartan. Cell proliferation states were determined by the methyl thiazolyl tetrazolium (MTT) method. The expressions at protein level and gene level were determined by Western blot and real-time fluorescence reverse transcription polymerase chain reaction (RT-PCR), respectively. The invasive abilities and migratory abilities of the three cell lines were determined by Transwell in vitro cell invasion assay and wound healing assay, respectively. MTT results show that valsartan can inhibit the proliferation of CUB cells, and the inhibition effect is enhanced with the increase of concentration. AngII promotes the MMP2 and MMP9 expressions (both protein and gene levels) in CUB cells through AT1R, but their expressions can be effectively inhibited by valsartan, the AngII inhibitor. AngII inhibitor may become a novel drug that can inhibit CUB metastasis and prolong the survival of CUB patients.

Anticancer Activity of a Hexapeptide from Skate (Raja porosa) Cartilage Protein Hydrolysate in HeLa Cells.

PubMed

Pan, Xin; Zhao, Yu-Qin; Hu, Fa-Yuan; Chi, Chang-Feng; Wang, Bin

2016-08-16

In this study, the hexapeptide Phe-Ile-Met-Gly-Pro-Tyr (FIMGPY), which has a molecular weight of 726.9 Da, was separated from skate (Raja porosa) cartilage protein hydrolysate using ultrafiltration and chromatographic methods, and its anticancer activity was evaluated in HeLa cells. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay indicated that FIMGPY exhibited high, dose-dependent anti-proliferation activities in HeLa cells with an IC50 of 4.81 mg/mL. Acridine orange/ethidium bromide (AO/EB) fluorescence staining and flow cytometry methods confirmed that FIMGPY could inhibit HeLa cell proliferation by inducing apoptosis. Western blot assay revealed that the Bax/Bcl-2 ratio and relative intensity of caspase-3 in HeLa cells treated with 7-mg/mL FIMGPY were 2.63 and 1.83, respectively, significantly higher than those of the blank control (p < 0.01). Thus, FIMGPY could induce apoptosis by upregulating the Bax/Bcl-2 ratio and caspase-3 activation. Using a DNA ladder method further confirmed that the anti-proliferation activity of FIMGPY was attributable to its role in inducing apoptosis. These results suggest that FIMGPY from skate cartilage protein hydrolysate may have applications as functional foods and nutraceuticals for the treatment and prevention of cancer.

Exploring Bioactive Properties of Marine Cyanobacteria Isolated from the Portuguese Coast: High Potential as a Source of Anticancer Compounds

PubMed Central

Costa, Margarida; Garcia, Mónica; Costa-Rodrigues, João; Costa, Maria Sofia; Ribeiro, Maria João; Fernandes, Maria Helena; Barros, Piedade; Barreiro, Aldo; Vasconcelos, Vitor; Martins, Rosário

2013-01-01

The oceans remain a major source of natural compounds with potential in pharmacology. In particular, during the last few decades, marine cyanobacteria have been in focus as producers of interesting bioactive compounds, especially for the treatment of cancer. In this study, the anticancer potential of extracts from twenty eight marine cyanobacteria strains, belonging to the underexplored picoplanktonic genera, Cyanobium, Synechocystis and Synechococcus, and the filamentous genera, Nodosilinea, Leptolyngbya, Pseudanabaena and Romeria, were assessed in eight human tumor cell lines. First, a crude extract was obtained by dichloromethane:methanol extraction, and from it, three fractions were separated in a Si column chromatography. The crude extract and fractions were tested in eight human cancer cell lines for cell viability/toxicity, accessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and lactic dehydrogenase release (LDH) assays. Eight point nine percent of the strains revealed strong cytotoxicity; 17.8% showed moderate cytotoxicity, and 14.3% assays showed low toxicity. The results obtained revealed that the studied genera of marine cyanobacteria are a promising source of novel compounds with potential anticancer activity and highlight the interest in also exploring the smaller filamentous and picoplanktonic genera of cyanobacteria. PMID:24384871

Immunization with a novel chimeric peptide representing B and T cell epitopes from HER2 extracellular domain (HER2 ECD) for breast cancer.

PubMed

Mahdavi, Manijeh; Keyhanfar, Mehrnaz; Jafarian, Abbas; Mohabatkar, Hassan; Rabbani, Mohammad

2014-12-01

Because of direct stimulating immune system against disease, vaccination or active immunotherapy is preferable compared to passive immunotherapy. For this purpose, a newly designed chimeric peptide containing epitopes for both B and T cells from HER2 ECD subdomain III was proposed. To evaluate the effects of the active immunization, a discontinuous B cell epitope peptide was selected based on average antigenicity by bioinformatics analysis. The selected peptide was collinearly synthesized as a chimera with a T helper epitope from the protein sequence of measles virus fusion (208-302) using the GPSL linker. Three mice were immunized with the chimeric peptide. Reactive antibodies with HER2 protein in ELISA and immunofluorescence assays with no cross-reactivity were generated. The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay indicated that the anti-peptide sera had inhibitory effects on proliferation of SK-BR-3 cells. Hence, the newly designed, discontinuous chimeric peptide representing B and T cell epitopes from subdomain III of HER2-ECD can form the basis for future vaccines design, where these data can be applied for monoclonal antibody production targeting the distinct epitope of HER2 receptor compared to the two broadly used anti-HER2 monoclonal antibodies, Herceptin and pertuzumab.

Validation of an automated colony counting system for group A Streptococcus.

PubMed

Frost, H R; Tsoi, S K; Baker, C A; Laho, D; Sanderson-Smith, M L; Steer, A C; Smeesters, P R

2016-02-08

The practice of counting bacterial colony forming units on agar plates has long been used as a method to estimate the concentration of live bacteria in culture. However, due to the laborious and potentially error prone nature of this measurement technique, an alternative method is desirable. Recent technologic advancements have facilitated the development of automated colony counting systems, which reduce errors introduced during the manual counting process and recording of information. An additional benefit is the significant reduction in time taken to analyse colony counting data. Whilst automated counting procedures have been validated for a number of microorganisms, the process has not been successful for all bacteria due to the requirement for a relatively high contrast between bacterial colonies and growth medium. The purpose of this study was to validate an automated counting system for use with group A Streptococcus (GAS). Twenty-one different GAS strains, representative of major emm-types, were selected for assessment. In order to introduce the required contrast for automated counting, 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) dye was added to Todd-Hewitt broth with yeast extract (THY) agar. Growth on THY agar with TTC was compared with growth on blood agar and THY agar to ensure the dye was not detrimental to bacterial growth. Automated colony counts using a ProtoCOL 3 instrument were compared with manual counting to confirm accuracy over the stages of the growth cycle (latent, mid-log and stationary phases) and in a number of different assays. The average percentage differences between plating and counting methods were analysed using the Bland-Altman method. A percentage difference of ±10 % was determined as the cut-off for a critical difference between plating and counting methods. All strains measured had an average difference of less than 10 % when plated on THY agar with TTC. This consistency was also observed over all phases of the growth cycle and when plated in blood following bactericidal assays. Agreement between these methods suggest the use of an automated colony counting technique for GAS will significantly reduce time spent counting bacteria to enable a more efficient and accurate measurement of bacteria concentration in culture.

Effects of benzalkonium chloride on growth and survival of Chang conjunctival cells.

PubMed

De Saint Jean, M; Brignole, F; Bringuier, A F; Bauchet, A; Feldmann, G; Baudouin, C

1999-03-01

The aim of this study was to investigate the action of benzalkonium chloride (BAC), used as a preservative in most ophthalmic topical solutions, on epithelial conjunctival cells in vitro. A continuous human conjunctival cell line (Wong-Kilbourne derivative of Chang conjunctiva) was exposed to BAC solutions at various concentrations (0.1%-0.0001%) during a period of 10 minutes. Cells were examined before treatment and 3, 24, 48, and 72 hours later, after reexposure to normal cell culture conditions. Cell number and viability were assessed with crystal violet and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide colorimetric assays. The expression of the apoptotic marker Apo 2.7, nuclear antigen p53, membrane proteins Fas and Fas ligand, and DNA content was studied by flow cytometry. Morphologic aspects of cell nuclei were analyzed on slides with a nucleic acid-specific dye, 4',6'-diamidino-2-phenylindole dihydrochloride. Cytoskeleton was labeled with a monoclonal anti-pancytokeratin antibody. In addition, apoptosis was measured by DNA electrophoresis assays in agarose gel. Cell exposure to 0.1% and 0.05% BAC induced cell lysis immediately after treatment. All cells (100%) treated with 0.01% BAC died in a delayed manner within 24 hours, with most of the characteristics of apoptosis (chromatin condensation and DNA fragmentation, reduction in cell volume, expression of the apoptotic marker Apo 2.7, and apoptotic changes in DNA content). Aliquots of 0.005%, 0.001%, 0.0005%, and 0.0001% BAC induced growth arrest and apoptotic cell death in a dose-dependent manner between 24 and 72 hours after treatment. The expressions of Fas and p53 did not vary after BAC treatment. Fas ligand was always negative. These results suggest that BAC induces cell growth arrest and death at a concentration as low as 0.0001%. The mode of BAC-induced cell death is dose-dependent. Cells die by necrosis after BAC treatment at high concentrations and by apoptosis if low concentrations of BAC are applied. This new aspect of in vitro toxicity of BAC could in part explain some ocular surface disorders observed in patients undergoing long-term topical treatments with preservative-containing drugs.

Role and Mechanism of Microglial Activation in Iron-Induced Selective and Progressive Dopaminergic Neurodegeneration

PubMed Central

Yan, Zhao-fen; Gao, Jun-hua; Sun, Li; Huang, Xi-yan; Liu, Zhuo; Yu, Shu-yang; Cao, Chen-Jie; Zuo, Li-jun; Chen, Ze-Jie; Hu, Yang; Wang, Fang; Hong, Jau-shyong; Wang, Xiao-min

2016-01-01

Parkinson’s disease (PD) patients have excessive iron depositions in substantia nigra (SN). Neuroinflammation characterized by microglial activation is pivotal for dopaminergic neurodegeneration in PD. However, the role and mechanism of microglial activation in iron-induced dopaminergic neurodegeneration in SN remain unclear yet. This study aimed to investigate the role and mechanism of microglial β-nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) activation in iron-induced selective and progressive dopaminergic neurodegeneration. Multiple primary midbrain cultures from rat, NOX2+/+ and NOX2−/− mice were used. Dopaminergic neurons, total neurons, and microglia were visualized by immunostainings. Cell viability was measured by MTT assay. Superoxide (O2·−) and intracellular reactive oxygen species (iROS) were determined by measuring SOD-inhibitable reduction of tetrazolium salt WST-1 and DCFH-DA assay. mRNA and protein were detected by real-time PCR and Western blot. Iron induces selective and progressive dopaminergic neurotoxicity in rat neuron–microglia–astroglia cultures and microglial activation potentiates the neurotoxicity. Activated microglia produce a magnitude of O2·− and iROS, and display morphological alteration. NOX2 inhibitor diphenylene iodonium protects against iron-elicited dopaminergic neurotoxicity through decreasing microglial O2·− generation, and NOX2−/− mice are resistant to the neurotoxicity by reducing microglial O2·− production, indicating that iron-elicited dopaminergic neurotoxicity is dependent of NOX2, a O2·−-generating enzyme. NOX2 activation is indicated by the increased mRNA and protein levels of subunits P47 and gp91. Molecules relevant to NOX2 activation include PKC-σ, P38, ERK1/2, JNK, and NF-ΚBP65 as their mRNA and protein levels are enhanced by NOX2 activation. Iron causes selective and progressive dopaminergic neurodegeneration, and microglial NOX2 activation potentiates the neurotoxicity. PKC-σ, P38, ERK1/2, JNK, and NF-ΚBP65 are the potential molecules relevant to microglial NOX2 activation. PMID:24277523

A new microtitre plate screening method for evaluating the viability of aerobic respiring bacteria in high surface biofilms.

PubMed

Pérez, L M; Alvarez, B L; Codony, F; Fittipaldi, M; Adrados, B; Peñuela, G; Morató, J

2010-09-01

It is difficult to determine the effects of bactericidal compounds against bacteria in a biofilm because classical procedures for determining cell viability require several working days, multiple complicated steps and are frequently only applicable to cells in suspension. We attempt to develop a compact, inexpensive and versatile system to measure directly the extent of biofilm formation from water systems and to determine the viability of respiring bacteria in high surface biofilms. It has been reported that the reduction of tetrazolium sodium salts, such as XTT (sodium 3,3'-[1-[(phenylamino)carbonyl]-3,4-tetrazolium]Bis(4-methoxy)-6-nitro)benzene sulfonic acid hydrate), during active bacterial metabolism can be incorporated into a colorimetric method for quantifying cell viability. XTT is reduced to a soluble formazan compound during bacterial aerobic metabolism such that the amount of formazan generated is proportional to the bacterial biomass. We show here, for the first time, that this colorimetric approach can be used to determine the metabolic activity of adherent aerobic bacteria in a biofilm as a measure of cell viability. This technique has been used to estimate viability and proliferation of bacteria in suspension, but this is the first application to microbial communities in a real undisturbed biofilm. This simple new system can be used to evaluate the complex biofilm community without separating the bacteria from their support. Thus, the results obtained by this practice may be more representative of the circumstances in a natural system, opening the possibility to multiple potential applications.

Evaluation of colorimetric assays for analyzing reductively methylated proteins: Biases and mechanistic insights.

PubMed

Brady, Pamlea N; Macnaughtan, Megan A

2015-12-15

Colorimetric protein assays, such as the Coomassie blue G-250 dye-binding (Bradford) and bicinchoninic acid (BCA) assays, are commonly used to quantify protein concentration. The accuracy of these assays depends on the amino acid composition. Because of the extensive use of reductive methylation in the study of proteins and the importance of biological methylation, it is necessary to evaluate the impact of lysyl methylation on the Bradford and BCA assays. Unmodified and reductively methylated proteins were analyzed using the absorbance at 280 nm to standardize the concentrations. Using model compounds, we demonstrate that the dimethylation of lysyl ε-amines does not affect the proteins' molar extinction coefficients at 280 nm. For the Bradford assay, the responses (absorbance per unit concentration) of the unmodified and reductively methylated proteins were similar, with a slight decrease in the response upon methylation. For the BCA assay, the responses of the reductively methylated proteins were consistently higher, overestimating the concentrations of the methylated proteins. The enhanced color formation in the BCA assay may be due to the lower acid dissociation constants of the lysyl ε-dimethylamines compared with the unmodified ε-amine, favoring Cu(II) binding in biuret-like complexes. The implications for the analysis of biologically methylated samples are discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

Evaluation of Colorimetric Assays for Analyzing Reductively Methylated Proteins: Biases and Mechanistic Insights

PubMed Central

Brady, Pamlea N.; Macnaughtan, Megan A.

2015-01-01

Colorimetric protein assays, such as the Coomassie blue G-250 dye-binding (Bradford) and bicinchoninic acid (BCA) assays, are commonly used to quantify protein concentration. The accuracy of these assays depends on the amino acid composition. Because of the extensive use of reductive methylation in the study of proteins and the importance of biological methylation, it is necessary to evaluate the impact of lysyl methylation on the Bradford and BCA assays. Unmodified and reductively methylated proteins were analyzed using the absorbance at 280 nm to standardize the concentrations. Using model compounds, we demonstrate that the dimethylation of lysyl ε-amines does not affect the proteins’ molar extinction coefficients at 280 nm. For the Bradford assay, the response (absorbance per unit concentration) of the unmodified and reductively methylated proteins were similar with a slight decrease in the response upon methylation. For the BCA assay, the responses of the reductively methylated proteins were consistently higher, overestimating the concentrations of the methylated proteins. The enhanced color-formation in the BCA assay may be due to the lower acid dissociation constants of the lysyl ε-dimethylamines, compared to the unmodified ε-amine, favoring Cu(II) binding in biuret-like complexes. The implications for the analysis of biologically methylated samples are discussed. PMID:26342307

Evaluation of the antifungal effect of EDTA, a metal chelator agent, on Candida albicans biofilm.

PubMed

Casalinuovo, I A; Sorge, R; Bonelli, G; Di Francesco, P

2017-03-01

Candida albicans biofilm is frequently found on artificial surfaces and the infections related to biofilm are difficult to eliminate, as they require the removal of artificial devices and treatment with antifungal drugs. Nowadays, fungal growth in biofilms is difficult to eradicate with conventional antifungal drugs such as fluconazole. Among chelating agents, disodium salt-Ethylene Diamine Tetraacetic Acid (EDTA) is known to have antifungal activity. In this study, we examined the in vitro activity of the EDTA and the antifungal drug fluconazole against C. albicans mature biofilm. C. albicans ATCC 20191, fluconazole-susceptible strain, was grown at an inoculum starter of 1 x 106 cells/ml for 72 h in 24-well microtiter plates and was further treated for 24 h with EDTA and/or fluconazole. Antifungal activities in biofilms were expressed as reduction in optical density (OD) determined by a 2,3-bis (2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide (XTT) colorimetric assay and compared to untreated biofilms. Colorimetric readings revealed that EDTA alone (at 25 and 2.5 mM) significantly reduced fungal metabolic activity in preformed biofilms. Also, EDTA combined with fluconazole significantly reduced the growth of biofilm when compared to biofilm treated with fluconazole alone (at 25 and 2.5 µg/ml). Our data suggest that the employment of EDTA or other chemicals destabilizers of the biofilm matrix, in combination with antifungal drugs, could lead to the development of new strategies for the management of infections associated to Candida biofilm. Another relevant result of our study suggests that the initial cell concentration, probably through mechanisms of quorum sensing, affects the cellular viability during the process of biofilm formation.

In Vitro Interactions between Aspirin and Amphotericin B against Planktonic Cells and Biofilm Cells of Candida albicans and C. parapsilosis

PubMed Central

Zhou, Yabin; Wang, Ganggang; Li, Yutang; Liu, Yang; Song, Yu; Zheng, Wenshuai; Zhang, Ning; Hu, Xiaoyan; Yan, Shikun

2012-01-01

The increase in drug resistance and invasion caused by biofilm formation brings enormous challenges to the management of Candida infection. Aspirin's antibiofilm activity in vitro was discovered recently. The spectrophotometric method and the XTT {2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide} reduction assay used for data generation make it possible to evaluate fungal biofilm growth accurately. The combined use of the most commonly used methods, the fractional inhibitory concentration index (FICI) and a newly developed method, the ΔE model, which uses the concentration-effect relationship over the whole concentration range instead of using the MIC index alone, makes the interpretation of results more reliable. As an attractive tool for studying the pharmacodynamics of antimicrobial agents, time-kill curves can provide detailed information about antimicrobial efficacy as a function of both time and concentration. In the present study, in vitro interactions between aspirin (acetylsalicylic acid [ASA]) and amphotericin B (AMB) against planktonic cells and biofilm cells of Candida albicans and C. parapsilosis were evaluated by the checkerboard microdilution method and the time-kill test. Synergistic and indifferent effects were found for the combination of ASA and AMB against planktonic cells, while strong synergy was found against biofilm cells analyzed by FICI. The ΔE model gave more consistent results with FICI. The positive interactions in concentration were also confirmed by the time-kill test. Moreover, this approach also revealed the pharmacodynamics changes of ASA and synergistic action on time. Our findings suggest a potential clinical use for combination therapy with ASA and AMB to augment activity against biofilm-associated infections. PMID:22391539

Poly(lactic-co-glycolic acid)(PLGA)/TiO2 nanotube bioactive composite as a novel scaffold for bone tissue engineering: In vitro and in vivo studies.

PubMed

Eslami, Hossein; Azimi Lisar, Hamidreza; Jafarzadeh Kashi, Tahereh Sadat; Tahriri, Mohammadreza; Ansari, Mojtaba; Rafiei, Tohid; Bastami, Farshid; Shahin-Shamsabadi, Alireza; Mashhadi Abbas, Fatemeh; Tayebi, Lobat

2018-05-01

The aim of this study was to synthesize and characterize novel three-dimensional porous scaffolds made of poly (lactic-co-glycolic acid)/TiO 2 nanotube (TNT) composite microspheres for bone tissue engineering applications. The incorporation of TNT greatly increases mechanical properties of PLGA/TNT microsphere-sintered scaffold. The experimental results exhibit that the PLGA/0.5 wt% TNT scaffold sintered at 100 °C for 3 h showed the best mechanical properties and a proper pore structure for tissue engineering. Biodegradation test ascertained that the weight of both PLGA and PLGA/PLGA/0.5 wt% TiO 2 nanotube composites slightly reduced during the first 4 weeks following immersion in SBF solution. Moreover, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and alkaline phosphatase activity (ALP activity) results represent increased cell viability for PLGA/0.5%TNT composite scaffold in comparison to the control group. In vivo studies show the amount of bone formation for PLGA/TNT was approximately twice of pure PLGA. Vivid histologic images of the newly generated bone on the implants further supported our test results. Eventually, a mathematical model showed that both PLGA and PLGA/TNT scaffolds' mechanical properties follow an exponential trend with time as their degradation occurs. By a three-dimensional finite element model, a more monotonous distribution of stress was present in the scaffold due to the presence of TNT with a reduction in maximum stress on bone. Copyright © 2018 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Comparative assessment of antibiotic susceptibility of coagulase-negative staphylococci in biofilm versus planktonic culture as assessed by bacterial enumeration or rapid XTT colorimetry

PubMed Central

Cerca, Nuno; Martins, Silvia; Cerca, Filipe; Jefferson, Kimberly K.; Pier, Gerald B.; Oliveira, Rosário; Azeredo, Joana

2005-01-01

Objectives To quantitatively compare the antibiotic susceptibility of biofilms formed by the coagulase-negative staphylococci (CoNS) Staphylococcus epidermidis and Staphylococcus haemolyticus with the susceptibility of planktonic cultures. Methods Several CoNS strains were grown planktonically or as biofilms to determine the effect of the mode of growth on the level of susceptibility to antibiotics with different mechanisms of action. The utility of a new, rapid colorimetric method that is based on the reduction of a tetrazolium salt (XTT) to measure cell viability was tested by comparison with standard bacterial enumeration techniques. A 6 h kinetic study was performed using dicloxacillin, cefazolin, vancomycin, tetracycline and rifampicin at the peak serum concentration of each antibiotic. Results In planktonic cells, inhibitors of cell wall synthesis were highly effective over a 3 h period. Biofilms were much less susceptible than planktonic cultures to all antibiotics tested, particularly inhibitors of cell wall synthesis. The susceptibility to inhibitors of protein and RNA synthesis was affected by the biofilm phenotype to a lesser degree. Standard bacterial enumeration techniques and the XTT method produced equivalent results both in biofilms and planktonic assays. Conclusions This study provides a more accurate comparison between the antibiotic susceptibilities of planktonic versus biofilm populations, because the cell densities in the two populations were similar and because we measured the concentration required to inhibit bacterial metabolism rather than to eradicate the entire bacterial population. While the biofilm phenotype is highly resistant to antibiotics that target cell wall synthesis, it is fairly susceptible to antibiotics that target RNA and protein synthesis. PMID:15980094

Glutaraldehyde cross-linking of amniotic membranes affects their nanofibrous structures and limbal epithelial cell culture characteristics.

PubMed

Lai, Jui-Yang; Ma, David Hui-Kang

2013-01-01

Given that the cells can sense nanometer dimensions, the chemical cross-linking-mediated alteration in fibrillar structure of collagenous tissue scaffolds is critical to determining their cell culture performances. This article explores, for the first time, the effect of nanofibrous structure of glutaraldehyde (GTA) cross-linked amniotic membrane (AM) on limbal epithelial cell (LEC) cultivation. Results of ninhydrin assays demonstrated that the amount of new cross-links formed between the collagen chains is significantly increased with increasing the cross-linking time from 1 to 24 hours. By transmission electron microscopy, the AM treated with GTA for a longer duration exhibited a greater extent of molecular aggregation, thereby leading to a considerable increase in nanofiber diameter and resistance against collagenase degradation. In vitro biocompatibility studies showed that the samples cross-linked with GTA for 24 hours are not well-tolerated by the human corneal epithelial cell cultures. When the treatment duration is less than 6 hours, the biological tissues cross-linked with GTA for a longer time may cause slight reductions in 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, and anti-inflammatory activities. Nevertheless, significant collagen molecular aggregation also enhances the stemness gene expression, indicating a high ability of these AM matrices to preserve the progenitors of LECs in vitro. It is concluded that GTA cross-linking of collagenous tissue materials may affect their nanofibrous structures and corneal epithelial stem cell culture characteristics. The AM treated with GTA for 6 hours holds promise for use as a niche for the expansion and transplantation of limbal epithelial progenitor cells.

In Vitro Interactions between Tacrolimus and Azoles against Candida albicans Determined by Different Methods▿

PubMed Central

Sun, Shujuan; Li, Yan; Guo, Qiongjie; Shi, Changwen; Yu, Jinlong; Ma, Lin

2008-01-01

Combination therapy could be of use for the treatment of fungal infections, especially those caused by drug-resistant fungi. However, the methods and approaches used for data generation and result interpretation need further optimizing. The fractional inhibitory concentration index (FICI) is the most commonly used method, but it has several drawbacks in characterizing antifungal drug interaction. Alternatively, some new methods can be used such as the ΔE model (difference between the predicted and measured fungal growth percentages) and the response surface approach, which uses the concentration-effect relationship over the whole concentration range instead of just the MIC. In the present study, in vitro interactions between tacrolimus (FK506) and three azoles—fluconazole (FLC), itraconazole (ITR), and voriconazole (VRC)-against Candida albicans were evaluated by the checkerboard microdilution method and time-killing test. The intensity of the interactions was determined by visual reading and the spectrophotometric method in a checkerboard assay, and the nature of the interactions was assessed by nonparametric models of FICI and ΔE. Colony counting and colorimetric viable detection methods (2,3-bis {2-methoxy-4-nitro-5-[(sulfenylamino) carbonyl]-2H-tetrazolium hydroxide} [XTT] reduction test) were used for evaluating the combination antifungal effects over time. Synergistic and indifferent effects were found for the combination of FK506 and azoles against azole-sensitive strains, while strong synergy was found against azole-resistant strains analyzed by FICI. The ΔE model gave more consistent results with FICI. The positive interactions were also confirmed by the time-killing test. Our findings suggest a potential role for combination therapy with calcineurin pathway inhibitors and azoles to augment activity against resistant C. albicans. PMID:18056277

Once the Light Touch to the Brain: Cytotoxic Effects of Low-Dose Gamma-Ray, Laser Light, and Visible Light on Rat Neuronal Cell Culture

PubMed Central

Cakir, Murteza; Colak, Abdullah; Calikoglu, Cagatay; Taspinar, Numan; Sagsoz, Mustafa Erdem; Kadioglu, Hakan Hadi; Hacimuftuoglu, Ahmet; Seven, Sabriye

2016-01-01

Objective: We aimed to evaluate the effects of gamma-ray, laser light, and visible light, which neurons are commonly exposed to during treatment of various cranial diseases, on the viability of neurons. Materials and Methods: Neuronal cell culture was prepared from the frontal cortex of 9 newborn rats. Cultured cells were irradiated with gamma-ray for 1–10 min by 152Eu, 241Am, and 132Ba isotopes, visible light for 1–160 min, and laser light for 0.2–2 seconds. The MTT tetrazolium reduction assay was used to assess the number of viable cells in the neuronal cell cultures. Wavelength dispersive X-ray fluorescence spectrometer was used to determine Na, K, and Ca levels in cellular fluid obtained from neuronal cell culture plaques. Results: Under low-dose radiation with 152Eu, 241Am, and 132Ba isotopes, cell viability insignificantly decreased with time (p>0.05). On the other hand, exposure to visible light produced statistically significant decrease in cell viability at both short- (1–10 min) and long-term (20–160 min). Cell viability did not change with 2 seconds of laser exposure. Na, K, and Ca levels significantly decreased with gamma-ray and visible light. The level of oxidative stress markers significantly changed with gamma-ray. Conclusion: In conclusion, while low dose gamma-ray has slight to moderate apoptotic effect in neuronal cell cultures by oxidative stress, long-term visible light induces remarkable apoptosis and cell death. Laser light has no significant effect on neurons. Further genetic studies are needed to clarify the chronic effect of visible light on neuronal development and functions. PMID:27551168

In vitro enzyme-mimic activity and in vivo therapeutic potential of HSJ-0017, a novel Mn porphyrin-based antioxidant enzyme mimic.

PubMed

Li, Bao-qiu; Dong, Xin; Li, Na; Gao, Ji-you; Yuan, Qiang; Fang, Shi-hong; Gong, Xian-chang; Wang, Shu-juan; Wang, Feng-shan

2014-10-01

Manganese (III) 5, 10, 15, 20-tetrakis [3-(2-(2-methoxy)-ethoxy) ethoxy] phenyl porphyrin chloride, designated HSJ-0017, is a novel antioxidant enzyme mimic. The aim of the present study was to investigate the enzyme-mimic activity and the therapeutic potential of HSJ-0017 in free radical-related diseases. Superoxide dismutase (SOD) mimic activity was measured by the nitroblue tetrazolium chloride monohydrate reduction assay. Catalase (CAT) mimic activity was measured based on the decomposition of hydrogen peroxide. The antitumor, radioprotective and chemoprotective effects of HSJ-0017 were evaluated in H22 or S180 tumor-bearing Kunming mice. The anti-inflammatory and hepatoprotective effects were, respectively, evaluated in histamine-induced edema model and CCl4-induced hepatic damage model in Wistar rats. HSJ-0017 over a concentration range of 0.001-10 µmol/L significantly inhibited the generation of superoxide anion. Significant hydrogen peroxide scavenging activity was observed when the concentration of HSJ-0017 was higher than 0.01 µmol/L. HSJ-0017 at a dose of 3.0 mg/kg exhibited significant antitumor effect on S180 tumor xenografts, whereas no significant antitumor effect was observed in H22 tumor xenografts. HSJ-0017 at a dose of 3.0 mg/kg enhanced the antitumor effects of radiotherapy and chemotherapy, and reduced their toxicity. However, HSJ-0017 counteracted the antitumor effects of radiotherapy when administered simultaneously with radiotherapy. HSJ-0017 showed significant anti-inflammatory and hepatoprotective effects. Our results demonstrate that HSJ-0017 exhibits antioxidant, antitumor, anti-inflammatory, radioprotective, chemoprotective, and hepatoprotective effects. It is a potent dual SOD/CAT mimic. © 2014 by the Society for Experimental Biology and Medicine.

In Vitro and In Vivo Activities of Pterostilbene against Candida albicans Biofilms

PubMed Central

Li, De-Dong; Zhao, Lan-Xue; Mylonakis, Eleftherios; Hu, Gan-Hai; Zou, Yong; Huang, Tong-Kun; Yan, Lan

2014-01-01

Pterostilbene (PTE) is a stilbene-derived phytoalexin that originates from several natural plant sources. In this study, we evaluated the activity of PTE against Candida albicans biofilms and explored the underlying mechanisms. In 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assays, biofilm biomass measurement, confocal laser scanning microscopy, and scanning electron microscopy, we found that ≤16 μg/ml PTE had a significant effect against C. albicans biofilms in vitro, while it had no fungicidal effect on planktonic C. albicans cells, which suggested a unique antibiofilm effect of PTE. Then we found that PTE could inhibit biofilm formation and destroy the maintenance of mature biofilms. At 4 μg/ml, PTE decreased cellular surface hydrophobicity (CSH) and suppressed hyphal formation. Gene expression microarrays and real-time reverse transcription-PCR showed that exposure of C. albicans to 16 μg/ml PTE altered the expression of genes that function in morphological transition, ergosterol biosynthesis, oxidoreductase activity, and cell surface and protein unfolding processes (heat shock proteins). Filamentation-related genes, especially those regulated by the Ras/cyclic AMP (cAMP) pathway, including ECE1, ALS3, HWP1, HGC1, and RAS1 itself, were downregulated upon PTE treatment, indicating that the antibiofilm effect of PTE was related to the Ras/cAMP pathway. Then, we found that the addition of exogenous cAMP reverted the PTE-induced filamentous growth defect. Finally, with a rat central venous catheter infection model, we confirmed the in vivo activity of PTE against C. albicans biofilms. Collectively, PTE had strong activities against C. albicans biofilms both in vitro and in vivo, and these activities were associated with the Ras/cAMP pathway. PMID:24514088

Umbelliprenin is cytotoxic against QU-DB large cell lung cancer cell line but anti-proliferative against A549 adenocarcinoma cells

PubMed Central

2012-01-01

Background Umbelliprenin is a natural compound, belonging to the class of sesquiterpene coumarins. Recently, umbelliprenin has attracted the researchers' attention for its antitumor activities against skin tumors. Its effect on lung cancer is largely unknown. The aim of our study was to investigate the effects of this natural compound, which is expected to have low adverse effects, on lung cancer. Methods The QU-DB large cell and A549 adenocarcinoma lung cancer cell lines were treated with umbelliprenin. IC50 values were estimated using methyl thiazolely diphenyl-tetrazolium bromide (MTT) assay, in which a decrease in MTT reduction can occur as a result of cell death or cell proliferation inhibition. To quantify the rate of cell death at IC50 values, flow cytometry using Annexin V-FITC (for apoptotic cells), and propidium iodide (for necrotic cells) dyes were employed. Results Data from three independent MTT experiments in triplicate revealed that IC50 values for QU-DB and A549 were 47 ± 5.3 μM and 52 ± 1.97 μM, respectively. Annexin V/PI staining demonstrated that umbelliprenin treatment at IC50 induced 50% cell death in QU-DB cells, but produced no significant death in A549 cells until increasing the umbelliprenin concentration to IC80. The pattern of cell death was predominantly apoptosis in both cell lines. When peripheral blood mononuclear cells were treated with 50 μM and less concentrations of umbelliprenin, no suppressive effect was observed. Conclusions We found cytotoxic/anti-proliferative effects of umbelliprenin against two different types of lung cancer cell lines. PMID:23351548

Glutaraldehyde cross-linking of amniotic membranes affects their nanofibrous structures and limbal epithelial cell culture characteristics

PubMed Central

Lai, Jui-Yang; Ma, David Hui-Kang

2013-01-01

Given that the cells can sense nanometer dimensions, the chemical cross-linking-mediated alteration in fibrillar structure of collagenous tissue scaffolds is critical to determining their cell culture performances. This article explores, for the first time, the effect of nanofibrous structure of glutaraldehyde (GTA) cross-linked amniotic membrane (AM) on limbal epithelial cell (LEC) cultivation. Results of ninhydrin assays demonstrated that the amount of new cross-links formed between the collagen chains is significantly increased with increasing the cross-linking time from 1 to 24 hours. By transmission electron microscopy, the AM treated with GTA for a longer duration exhibited a greater extent of molecular aggregation, thereby leading to a considerable increase in nanofiber diameter and resistance against collagenase degradation. In vitro biocompatibility studies showed that the samples cross-linked with GTA for 24 hours are not well-tolerated by the human corneal epithelial cell cultures. When the treatment duration is less than 6 hours, the biological tissues cross-linked with GTA for a longer time may cause slight reductions in 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, and anti-inflammatory activities. Nevertheless, significant collagen molecular aggregation also enhances the stemness gene expression, indicating a high ability of these AM matrices to preserve the progenitors of LECs in vitro. It is concluded that GTA cross-linking of collagenous tissue materials may affect their nanofibrous structures and corneal epithelial stem cell culture characteristics. The AM treated with GTA for 6 hours holds promise for use as a niche for the expansion and transplantation of limbal epithelial progenitor cells. PMID:24204144

Biodegradable mixed MPEG-SS-2SA/TPGS micelles for triggered intracellular release of paclitaxel and reversing multidrug resistance

PubMed Central

Dong, Kai; Yan, Yan; Wang, Pengchong; Shi, Xianpeng; Zhang, Lu; Wang, Ke; Xing, Jianfeng; Dong, Yalin

2016-01-01

In this study, a type of multifunctional mixed micelles were prepared by a novel biodegradable amphiphilic polymer (MPEG-SS-2SA) and a multidrug resistance (MDR) reversal agent (d-α-tocopheryl polyethylene glycol succinate, TPGS). The mixed micelles could achieve rapid intracellular drug release and reversal of MDR. First, the amphiphilic polymer, MPEG-SS-2SA, was synthesized through disulfide bonds between poly (ethylene glycol) monomethyl ether (MPEG) and stearic acid (SA). The structure of the obtained polymer was similar to poly (ethylene glycol)-phosphatidylethanolamine (PEG-PE). Then the mixed micelles, MPEG-SS-2SA/TPGS, were prepared by MPEG-SS-2SA and TPGS through the thin film hydration method and loaded paclitaxel (PTX) as the model drug. The in vitro release study revealed that the mixed micelles could rapidly release PTX within 24 h under a reductive environment because of the breaking of disulfide bonds. In cell experiments, the mixed micelles significantly inhibited the activity of mitochondrial respiratory complex II, also reduced the mitochondrial membrane potential, and the content of adenosine triphosphate, thus effectively inhibiting the efflux of PTX from cells. Moreover, in the confocal laser scanning microscopy, cellular uptake and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assays, the MPEG-SS-2SA/TPGS micelles achieved faster release and more uptake of PTX in Michigan Cancer Foundation-7/PTX cells and showed better antitumor effects as compared with the insensitive control. In conclusion, the biodegradable mixed micelles, MPEG-SS-2SA/TPGS, could be potential vehicles for delivering hydrophobic chemotherapeutic drugs in MDR cancer therapy. PMID:27785018

In vitro antibacterial and cytotoxicity assessments of an orthodontic bonding agent containing benzalkonium chloride.

PubMed

Saito, Kayo; Hayakawa, Tohru; Kawabata, Rihito; Meguro, Daijiro; Kasai, Kazutaka

2009-03-01

To assess the antibacterial activity and cytotoxicity of an orthodontic bonding material containing an antibacterial agent. Superbond C&B (4-methacryloxyethyl trimellitate anhydride/methyl methacrylate-tri-n-butyl borane [4-META/MMA-TBB]) resin was mixed with benzalkonium chloride (BAC) to obtain final BAC concentrations of 0.25%, 0.75%, 1.25%, 1.75%, 2.5%, and 5.0% (wt/ wt). Antibacterial activity against Streptococcus mutans and Streptococcus sobrinus was evaluated by soaking the BAC-resin in distilled water at 37 degrees C for periods of 30, 90, and 180 days. Antibacterial activity of the BAC-resin was measured by the disk diffusion method, and the inhibition zone around each sample was measured and recorded. For evaluation of cytotoxicity, BAC-resin samples were put into cell culture inserts placed above human gingival cells and were incubated at 37 degrees C for 1, 3, and 6 days. Cytotoxicity was assessed with a tetrazolium bromide reduction assay. The antibacterial activity of BAC-incorporated resin samples decreased significantly after immersion in water for 180 days, regardless of BAC concentration. The antibacterial activity of nonimmersed resin containing 0.25% or 1.75% BAC was comparable with that of 5.0% BAC-resin immersed for 180 days. In cytotoxicity tests, most cells died when exposed to resins containing 1.75%, 2.5%, and 5% BAC. No difference was observed between resins containing 0.25% and 0.75% BAC at 1, 3, and 6 days of culture. The addition of BAC to 4-META/MMA-TBB resin confers an antibacterial effect even after immersion in water, and 4-META/MMA-TBB resin containing 0.25% to 0.75% BAC has no significant cytotoxic effect.

A high-throughput seed germination assay for root parasitic plants

PubMed Central

2013-01-01

Background Some root-parasitic plants belonging to the Orobanche, Phelipanche or Striga genus represent one of the most destructive and intractable weed problems to agricultural production in both developed and developing countries. Compared with most of the other weeds, parasitic weeds are difficult to control by conventional methods because of their life style. The main difficulties that currently limit the development of successful control methods are the ability of the parasite to produce a tremendous number of tiny seeds that may remain viable in the soil for more than 15 years. Seed germination requires induction by stimulants present in root exudates of host plants. Researches performed on these minute seeds are until now tedious and time-consuming because germination rate is usually evaluated in Petri-dish by counting germinated seeds under a binocular microscope. Results We developed an easy and fast method for germination rate determination based on a standardized 96-well plate test coupled with spectrophotometric reading of tetrazolium salt (MTT) reduction. We adapted the Mosmann’s protocol for cell cultures to germinating seeds and determined the conditions of seed stimulation and germination, MTT staining and formazan salt solubilization required to obtain a linear relationship between absorbance and germination rate. Dose–response analyses were presented as applications of interest for assessing half maximal effective or inhibitory concentrations of germination stimulants (strigolactones) or inhibitors (ABA), respectively, using four parameter logistic curves. Conclusion The developed MTT system is simple and accurate. It yields reproducible results for germination bioassays of parasitic plant seeds. This method is adapted to high-throughput screenings of allelochemicals (stimulants, inhibitors) or biological extracts on parasitic plant seed germination, and strengthens the investigations of distinctive features of parasitic plant germination. PMID:23915294

Automated image-based assay for evaluation of HIV neutralization and cell-to-cell fusion inhibition.

PubMed

Sheik-Khalil, Enas; Bray, Mark-Anthony; Özkaya Şahin, Gülsen; Scarlatti, Gabriella; Jansson, Marianne; Carpenter, Anne E; Fenyö, Eva Maria

2014-08-30

Standardized techniques to detect HIV-neutralizing antibody responses are of great importance in the search for an HIV vaccine. Here, we present a high-throughput, high-content automated plaque reduction (APR) assay based on automated microscopy and image analysis that allows evaluation of neutralization and inhibition of cell-cell fusion within the same assay. Neutralization of virus particles is measured as a reduction in the number of fluorescent plaques, and inhibition of cell-cell fusion as a reduction in plaque area. We found neutralization strength to be a significant factor in the ability of virus to form syncytia. Further, we introduce the inhibitory concentration of plaque area reduction (ICpar) as an additional measure of antiviral activity, i.e. fusion inhibition. We present an automated image based high-throughput, high-content HIV plaque reduction assay. This allows, for the first time, simultaneous evaluation of neutralization and inhibition of cell-cell fusion within the same assay, by quantifying the reduction in number of plaques and mean plaque area, respectively. Inhibition of cell-to-cell fusion requires higher quantities of inhibitory reagent than inhibition of virus neutralization.

On methods for the detection of reactive oxygen species generation by human spermatozoa: analysis of the cellular responses to catechol oestrogen, lipid aldehyde, menadione and arachidonic acid.

PubMed

Aitken, R J; Smith, T B; Lord, T; Kuczera, L; Koppers, A J; Naumovski, N; Connaughton, H; Baker, M A; De Iuliis, G N

2013-03-01

Oxidative stress is known to have a major impact on human sperm function and, as a result, there is a need to develop sensitive methods for measuring reactive oxygen species (ROS) generation by these cells. A variety of techniques have been developed for this purpose including chemiluminescence (luminol and lucigenin), flow cytometry (MitoSOX Red, dihydroethidium, 4,5-diaminofluorescein diacetate and 2',7'-dichlorodihydrofluorescein diacetate) and spectrophotometry (nitroblue tetrazolium). The relative sensitivity of these assays and their comparative ability to detect ROS generated in different subcellular compartments of human spermatozoa, have not previously been investigated. To address this issue, we have compared the performance of these assays when ROS generation was triggered with a variety of reagents including 2-hydroxyestradiol, menadione, 4-hydroxynonenal and arachidonic acid. The results revealed that menadione predominantly induced release of ROS into the extracellular space where these metabolites could be readily detected by luminol-peroxidase and, to a lesser extent, 2',7'-dichlorodihydrofluorescein. However, such sensitivity to extracellular ROS meant that these assays were particularly vulnerable to interference by leucocytes. The remaining reagents predominantly elicited ROS generation by the sperm mitochondria and could be optimally detected by MitoSOX Red and DHE. Examination of spontaneous ROS generation by defective human spermatozoa revealed that MitoSOX Red was the most effective indicator of oxidative stress, thereby emphasizing the general importance of mitochondrial dysregulation in the aetiology of defective sperm function. © 2013 American Society of Andrology and European Academy of Andrology.

Effect of doxycycline on epithelial-mesenchymal transition via the p38/Smad pathway in respiratory epithelial cells.

PubMed

Shin, Jae-Min; Kang, Ju-Hyung; Lee, Seoung-Ae; Park, Il-Ho; Lee, Heung-Man

2017-03-01

Doxycycline has antibacterial and anti-inflammatory effects, and it also suppresses collagen biosynthesis. This study aimed to confirm the effects and mechanism of doxycycline on transforming growth factor (TGF) beta 1 induced epithelial-mesenchymal transition and cell migration in A549 and primary nasal epithelial cells. A 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay and phalloidin-fluorescein isothiocyanate staining were used to evaluate cytotoxicity and cellular morphologic changes. Western blot and immunofluorescence staining were used to determine the expression levels of E-cadherin, vimentin, alpha-smooth muscle actin, fibronectin, phosphorylated Smad2/3, and mitogen-activated protein kinases. Scratch and transwell migration assays were used to assess cellular migration ability. Doxycycline (0-10 μg/mL) had no significant cytotoxic effects in A549 and primary nasal epithelial cells. Increased expression of mesenchymal markers, including vimentin, alpha-smooth muscle actin, and fibronectin in TGF beta 1 induced A549 cells were downregulated by doxycycline treatment. In contrast, E-cadherin expression was upregulated in TGF beta 1 induced A549 cells. An in vitro cell migration assay showed that doxycycline also inhibited the ability of TGF beta 1 induced migration. Doxycycline treatment suppressed the activation of Smad2/3 and p38, whereas its inhibitory effects were similar to each element-specific inhibitor in A549 and primary nasal epithelial cells. Doxycycline inhibited TGF beta 1 induced epithelial-to-mesenchymal transition and migration by targeting Smad2/3 and p38 signal pathways in respiratory epithelial cells.

Bismuth subsalicylate nanoparticles with anaerobic antibacterial activity for dental applications

NASA Astrophysics Data System (ADS)

Vega-Jiménez, A. L.; Almaguer-Flores, A.; Flores-Castañeda, M.; Camps, E.; Uribe-Ramírez, M.; Aztatzi-Aguilar, O. G.; De Vizcaya-Ruiz, A.

2017-10-01

In recent years, nanomaterials have been used in the medical-dental field as new alternative antimicrobial agents. Bismuth subsalicylate (BSS) has been used as an antimicrobial agent, but the effect of BSS in the form of nanoparticles (BSS-nano) as a potential antimicrobial agent has not been tested, in specific against bacteria responsible for periodontal disease. The aim of this study was to evaluate the antibacterial effect of BSS-nano against oral anaerobic bacteria and to assess the safety of BSS-nano by evaluating their cytotoxicity in human gingival fibroblast (HGF-1) cells. BSS-nano were synthesized by laser ablation and were previously physico-chemically characterized using in vitro assays. The antibacterial activity was measured using the tetrazolium-based XTT assay, and cytotoxicity was determined using lactate dehydrogenase (LDH) and MTS assays in HGF-1 cells. Transmission electron microscopy of HGF-1 exposed to BSS-nano was also performed. BSS-nano was shown to have a primary size of 4-22 nm and a polygonal shape. Among the tested bacterial strains, those with a greater sensitivity to BSS-nano (highest concentration of 21.7 μg ml-1) were A. actinomycetemcomitans, C. gingivalis, and P. gingivalis. BSS-nano at a concentration of 60 μg ml-1 showed low cytotoxicity (6%) in HFG-1 cells and was mainly localized intracellularly in acidic vesicles. Our results indicate that the concentration of BSS-nano used as an effective antibacterial agent does not induce cytotoxicity in mammalian cells; thus, BSS-nano can be applied as an antibacterial agent in dental materials or antiseptic solutions.

Effects of Canonical NF-κB Signaling Pathway on the Proliferation and Odonto/Osteogenic Differentiation of Human Stem Cells from Apical Papilla

PubMed Central

Li, Junjun; Yan, Ming; Wang, Zilu; Jing, Shuanglin; Li, Yao; Liu, Genxia; Yu, Jinhua; Fan, Zhipeng

2014-01-01

Background Information. NF-κB signaling pathway plays a complicated role in the biological functions of mesenchymal stem cells. However, the effects of NF-κB pathway on the odonto/osteogenic differentiation of stem cells from apical papilla (SCAPs) remain unclear. The present study was designed to evaluate the effects of canonical NF-κB pathway on the osteo/odontogenic capacity of SCAPs in vitro. Results. Western blot results demonstrated that NF-κB pathway in SCAPs was successfully activated by TNF-α or blocked by BMS-345541. NF-κB pathway-activated SCAPs presented a higher proliferation activity compared with control groups, as indicated by dimethyl-thiazol-diphenyl tetrazolium bromide assay (MTT) and flow cytometry assay (FCM). Wound scratch assay revealed that NF-κB pathway-activated SCAPs presented an improved migration capacity, enhanced alkaline phosphatase (ALP) activity, and upregulated mineralization capacity of SCAPs, as compared with control groups. Meanwhile, the odonto/osteogenic markers (ALP/ALP, RUNX2/RUNX2, OSX/OSX, OCN/OCN, OPN/OPN, BSP/BSP, DSPP/DSP, and DMP-1/DMP-1) in NF-κB pathway-activated SCAPs were also significantly upregulated as compared with control groups at both protein and mRNA levels. However, NF-κB pathway-inhibited SCAPs exhibited a lower proliferation/migration capacity, and decreased odonto/osteogenic ability in comparison with control groups. Conclusion. Our findings suggest that classical NF-κB pathway plays a paramount role in the proliferation and committed differentiation of SCAPs. PMID:24864235

Green synthesis of silver nanoparticles from aqueous leaf extract of Pomegranate (Punica granatum) and their anticancer activity on human cervical cancer cells

NASA Astrophysics Data System (ADS)

Sarkar, Sonia; Kotteeswaran, Venkatesan

2018-06-01

Plants contain different important phytochemicals that can be used as a potential treatment for various ailments including cancer. The green synthesis of silver nanoparticles from the extract of different plant parts has gained a wide range of engrossment among the researchers due to its unique optical and structural property. The aim of this study is green synthesis of silver nanoparticles from the aqueous leaf extract of pomegranate (Punica granatum) and to investigate its anticancer activity on human cervical cancer cells (HeLa). The synthesis of silver nanoparticle was depicted by the colour change from golden yellowish to dark brownish, UV-visible spectral analysis gave a characteristic surface plasmon absorption peak at . Further morphological characterization was done by Zeta potential where the size analysis was depicted to be 46.1 nm and zeta potential as . Fourier transform infrared spectroscopy (FTIR) inferred 3 intense sharp peaks at , , , confirmed the presence of flavonoids and polyphenols. The scanning electron microscopy (SEM) analysis with energy diffraction spectroscopy (EDS) confirmed the presence of silver nanoparticles with size ranged from to . X-ray diffraction (XRD) confirmed the crystallographic nature of silver. The cell proliferation activity of nanoparticles was tested by 3, ‑4, 5 dimethylthiazol-2,5 diphenyl tetrazolium bromide (MTT) assay where the inhibitory concentration () was found at inhibiting of HeLa cell line. The anticancer activity of nanoparticles was determined by lactate dehydrogenase (LDH) assay where showed of cytotoxicity. Furthermore, the anticancer property of nanoparticles was confirmed by the DNA fragmentation assay.

Friction Stir Processing of Stainless Steel for Ascertaining Its Superlative Performance in Bioimplant Applications.

PubMed

Perumal, G; Ayyagari, A; Chakrabarti, A; Kannan, D; Pati, S; Grewal, H S; Mukherjee, S; Singh, S; Arora, H S

2017-10-25

Substrate-cell interactions for a bioimplant are driven by substrate's surface characteristics. In addition, the performance of an implant and resistance to degradation are primarily governed by its surface properties. A bioimplant typically degrades by wear and corrosion in the physiological environment, resulting in metallosis. Surface engineering strategies for limiting degradation of implants and enhancing their performance may reduce or eliminate the need for implant removal surgeries and the associated cost. In the current study, we tailored the surface properties of stainless steel using submerged friction stir processing (FSP), a severe plastic deformation technique. FSP resulted in significant microstructural refinement from 22 μm grain size for the as-received alloy to 0.8 μm grain size for the processed sample with increase in hardness by nearly 1.5 times. The wear and corrosion behavior of the processed alloy was evaluated in simulated body fluid. The processed sample demonstrated remarkable improvement in both wear and corrosion resistance, which is explained by surface strengthening and formation of a highly stable passive layer. The methylthiazol tetrazolium assay demonstrated that the processed sample is better in supporting cell attachment, proliferation with minimal toxicity, and hemolysis. The athrombogenic characteristic of the as-received and processed samples was evaluated by fibrinogen adsorption and platelet adhesion via the enzyme-linked immunosorbent assay and lactate dehydrogenase assay, respectively. The processed sample showed less platelet and fibrinogen adhesion compared with the as-received alloy, signifying its high thromboresistance. The current study suggests friction stir processing to be a versatile toolbox for enhancing the performance and reliability of currently used bioimplant materials.

Pretreatment of Ferulic Acid Protects Human Dermal Fibroblasts against Ultraviolet A Irradiation

PubMed Central

Hahn, Hyung Jin; Kim, Ki Bbeum; Bae, Seunghee; Choi, Byung Gon; An, Sungkwan

2016-01-01

Background Approximately 90%~99% of ultraviolet A (UVA) ray reaches the Earth's surface. The deeply penetrating UVA rays induce the formation of reactive oxygen species (ROS), which results in oxidative stress such as photoproducts, senescence, and cell death. Thus, UVA is considered a primary factor that promotes skin aging. Objective Researchers investigated whether pretreatment with ferulic acid protects human dermal fibroblasts (HDFs) against UVA-induced cell damages. Methods HDF proliferation was analyzed using the water-soluble tetrazolium salt assay. Cell cycle distribution and intracellular ROS levels were assessed by flow cytometric analysis. Senescence was evaluated using a senescence-associated β-galactosidase assay, while Gadd45α promoter activity was analyzed through a luciferase assay. The expression levels of superoxide dismutase 1 (SOD1), catalase (CAT), xeroderma pigmentosum complementation group A and C, matrix metalloproteinase 1 and 3, as well as p21 and p16 were measured using quantitative real-time polymerase chain reaction. Results Inhibition of proliferation and cell cycle arrest were detected in cells that were irradiated with UVA only. Pretreatment with ferulic acid significantly increased the proliferation and cell cycle progression in HDFs. Moreover, ferulic acid pretreatment produced antioxidant effects such as reduced DCF intensity, and affected SOD1 and CAT mRNA expression. These effects were also demonstrated in the analysis of cell senescence, promoter activity, expression of senescent markers, and DNA repair. Conclusion These results demonstrate that ferulic acid exerts protective effects on UVA-induced cell damages via anti-oxidant and stress-inducible cellular mechanisms in HDFs. PMID:27904274

Cytotoxic and radioprotective effects of Podophyllum hexandrum.

PubMed

Shukla, Sandeep Kumar; Chaudhary, Pankaj; Prem Kumar, Indracanti; Afrin, Farhat; Puri, Satish Chandra; Qazi, Ghulam Nabi; Sharma, Rakesh Kumar

2006-07-01

Podophyllum hexandrum, a herb thriving in Himalayas has already been reported to exhibit antitumor and radioprotective properties. Present study was undertaken to unravel the possible mechanism responsible for the cytotoxic and radioprotective properties of REC-2001, a fraction isolated from the rhizome of P. hexandrum using murine peritoneal macrophages and plasmid DNA as model systems. Cell death, levels of intracellular reactive oxygen species (ROS) and apoptosis were studied employing trypan blue exclusion assay, dichlorofluorescein diacetate and DNA fragmentation assay, respectively. Superoxide anions, hydroxyl radicals and DNA damage were estimated following nitroblue tetrazolium, 2-deoxyribose degradation and plasmid DNA relaxation assays, respectively. Pre-irradiation administration of REC-2001 to peritoneal macrophages in the concentration range of 25-200μg/ml significantly reduced radiation induced ROS generation, DNA damage, apoptosis and cell killing in comparison to radiation control group indicating radioprotective potential. Studies with plasmid DNA indicated the ability of REC-2001 to inhibit 20Gy induced single and double strand breaks further supporting the antioxidative potential. However, REC-2001 in a dose-dependent fashion induced cell death, ROS and DNA fragmentation indicating the cytotoxic nature. REC-2001, in presence of 100μM copper sulfate, generated significant amount of hydroxyl radicals and superoxide anions indicating ability to act as a pro-oxidant in presence of metal ions. The superoxide anion generation was found to be sensitive to metal chelators like EDTA and deferoxamine mesylate (DFR). These results suggest that the ability of REC-2001 to act as a pro-oxidant in presence of metal ions and antioxidant in presence of free radicals might be responsible for cytotoxic and radioprotective properties.

In vitro effects of preserved and unpreserved anti-allergic drugs on human corneal epithelial cells.

PubMed

Guzman-Aranguez, Ana; Calvo, Patricia; Ropero, Inés; Pintor, Jesús

2014-11-01

Treatment with topical eye drops for long-standing ocular diseases like allergy can induce detrimental side effects. The purpose of this study was to investigate in vitro cytotoxicity of commercially preserved and unpreserved anti-allergic eye drops on the viability and barrier function of monolayer and stratified human corneal-limbal epithelial cells. Cells were treated with unpreserved ketotifen solution, benzalkonium chloride (BAC)-containing anti-allergic drugs (ketotifen, olopatadine, levocabastine) as well as BAC alone. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to determine cell viability. Effects of compounds on barrier function were analyzed measuring transepithelial electrical resistance (TEER) to determine paracellular permeability and rose bengal assays to evaluate transcellular barrier formation. The BAC-preserved anti-allergic formulations and BAC alone significantly reduced cell viability, monolayer cultures being more sensitive to damage by these solutions. Unpreserved ketotifen induced the least diminution in cell viability. The extent of decrease of cell viability was clearly dependent of BAC presence, but it was also affected by the different types of drugs when the concentration of BAC was low and the short time of exposure. Treatment with BAC-containing anti-allergic drugs and BAC alone resulted in increased paracellular permeability and loss of transcellular barrier function as indicated by TEER measurement and rose bengal assays. The presence of the preservative BAC in anti-allergic eye drop formulations contributes importantly to the cytotoxic effects induced by these compounds. Stratified cell cultures seem to be a more relevant model for toxicity evaluation induced on the ocular surface epithelia than monolayer cultures.

6-Gingerol Mediates its Anti Tumor Activities in Human Oral and Cervical Cancer Cell Lines through Apoptosis and Cell Cycle Arrest.

PubMed

Kapoor, Vaishali; Aggarwal, Sadhna; Das, Satya N

2016-04-01

6-Gingerol, a potent nutraceutical, has been shown to have antitumor activity in different tumors, although its mechanism of action is not well understood. In this study, we evaluated antitumor activities of 6-gingerol on human oral (SCC4, KB) and cervical cancer (HeLa) cell lines with or without wortmannin, rapamycin, and cisplatin. Tumor cell proliferation was observed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium, inner salt assay, cell cycle analysis by propidium iodide labeling and flow cytometry, apoptosis by Annexin-V binding assay, and caspase activity by chemiluminescence assay. 6-Gingerol showed dose-dependent cytotoxicity in all three cell lines. Combinations of 6-gingerol with wortmannin and cisplatin showed additive effects, while with rapamycin, it showed 50% cytotoxicity that was equivalent to IC50 of 6-gingerol alone. Treatment with 6-gingerol resulted in G2-phase arrest in KB and HeLa cells and S-phase arrest in SCC4 cells. 6-Gingerol, wortmannin, and rapamycin treatment showed almost two-fold higher expression of caspase 3 in all cell lines. The results imply that 6-gingerol either alone or in combination with PI-3 K inhibitor and cisplatin may provide better therapeutic effects in oral and cervical carcinoma. Thus, 6-gingerol appears to be a safe and potent chemotherapeutic/chemopreventive compound acting through cell cycle arrest and induction of apoptosis in human oral and cervical tumor cells. Copyright © 2016 John Wiley & Sons, Ltd.

Combination of NRP1-mediated iRGD with 5-fluorouracil suppresses proliferation, migration and invasion of gastric cancer cells.

PubMed

Zhang, Li; Xing, Yanfeng; Gao, Qi; Sun, Xuejun; Zhang, Di; Cao, Gang

2017-09-01

Gastric cancer is one of the most of common cancers in the world. 5-Fluorouracil (5-FU) has been identified as one of the standard first-line chemotherapy drugs for locally advanced or metastatic gastric cancer. However, poor tumor penetration, bad selectivity and toxic side effects are the major limitations for the application of chemotherapy drugs in anticancer therapy. Recently, plenty of studies demonstrate that the novel tumor-homing peptide iRGD could promote the tumor-penetrating capability of chemotherapy drugs in multiple cancers, and neuropilin-1 (NRP1) protein is the critical mediator for iRGD. Here,we found that NRP1 protein expression was significantly up-regulated in gastric cancer tissues and cell lines by Immunohistochemistry and Western blot. And elevated NRP1 was notably associated with tumor differentiation (P=0.021), tumor size (P=0.004), tumor stage(P=0.028), lymph node metastasis(P=0.032), TNM tumor stage (P=0.006) and poorer prognosis. Functionally, the data of Methyl thiazolyl tetrazolium (MTT) assay, Colony formation assay and Transwell assay revealed that NRP1 could facilitate gastric cancer cells proliferation, migration and invasion. Furthermore, iRGD could strengthen the chemotherapy effect of 5-FU on gastric cancer cells through NRP1. Taken together, NPR1 might be a promising tumor target for gastric cancer, and combination of iRGD with 5-FU may be a novel and valuable approach to improving the prognosis of gastric cancer patients. Copyright © 2017. Published by Elsevier Masson SAS.

Synthesis of {111} Facet-Exposed MgO with Surface Oxygen Vacancies for Reactive Oxygen Species Generation in the Dark.

PubMed

Hao, Ying-Juan; Liu, Bing; Tian, Li-Gang; Li, Fa-Tang; Ren, Jie; Liu, Shao-Jia; Liu, Ying; Zhao, Jun; Wang, Xiao-Jing

2017-04-12

Seeking a simple and moderate route to generate reactive oxygen species (ROS) for antibiosis is of great interest and challenge. This work demonstrates that molecule transition and electron rearrangement processes can directly occur only through chemisorption interaction between the adsorbed O 2 and high-energy {111} facet-exposed MgO with abundant surface oxygen vacancies (SOVs), hence producing singlet oxygen and superoxide anion radicals without light irradiation. These ROS were confirmed by electron paramagnetic resonance, in situ Raman, and scavenger experiments. Furthermore, heat plays a crucial role for the electron transfer process to accelerate the formation of ·O 2 - , which is verified by temperature kinetic experiments of nitro blue tetrazolium reduction in the dark. Therefore, the presence of oxygen vacancy can be considered as an intensification of the activation process. The designed MgO is acquired in one step via constructing a reduction atmosphere during the combustion reaction process, which has an ability similar to that of noble metal Pd to activate molecular oxygen and can be used as an effective bacteriocide in the dark.

Species Differentiation of Group D Streptococci

PubMed Central

Papavassiliou, J.

1962-01-01

Three hundred and fourteen strains of group D streptococci were studied by means of a number of tests. The majority of the strains were identified as Streptococcus faecalis (83 strains), Streptococcus faecium (131 strains), or Streptococcus bovis (32 strains). Several strains (47 or nearly 15%) either shared characteristics of two species or were completely atypical. S. faecalis and S. bovis were more easily identified than S. faecium, which is not sharply defined from the other species and could be subdivided into several fermentative types on the basis of fermentation of arabinose, mannitol, sorbitol, glycerol, and sucrose. The value of some characteristics in species identification is discussed. Growth in the presence of potassium tellurite 1:2,500 and in the presence of 6.5% NaCl and fermentation of arabinose, glycerol, and raffinose are very important tests for the identification of the three species. The reduction of tetrazolium salts, the reduction of litmus milk, and the fermentation of sorbitol may serve as complementary tests for the same purpose. For the differentiation of these three species the “pattern of reactions” is more important than single tests. PMID:14483707

Neuroprotective compounds of Tilia amurensis

PubMed Central

Lee, Bohyung; Weon, Jin Bae; Eom, Min Rye; Jung, Youn Sik; Ma, Choong Je

2015-01-01

Background: Tilia amurensis (Tiliacese) has been used for anti-tumor and anti-inflammatory in Korea, China, and Japan. Objective: In this study, we isolated five compounds from T. amurensis and determined whether protected neuronal cells against glutamate-induced oxidative stress in HT22 cells. Materials and Methods: Compounds were isolated using chromatographic techniques including silica gel, Sephadex LH-20 open column and high performance liquid chromatography analysis, and evaluated neuroprotective effect in HT22 cells by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Results: β-D-fructofuranosyl α-D-glucopyranoside (1), (-)-epicatechin (2), nudiposide (3), lyoniside (4), and scopoletin (5) were isolated by bioactivity-guided fractionation from the ethyl acetate fraction of T. amurensis. Among them, (-)-epicatechin, nudiposide, lyoniside, and scopoletin had significant neuroprotective activities against glutamate-injured neurotoxicity in HT22 cells. Conclusion: These results demonstrated that compound two, three, four, and five have a pronounced protective effect against glutamate-induced neurotoxicity in HT22 cells. PMID:26664019

Antibacterial properties and cytocompatibility of tantalum oxide coatings with different silver content

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Heng-Li; Chang, Yin-Yu, E-mail: yinyu@mail2000.com.tw; Chen, Hung-Jui

Tantalum (Ta) oxides and their coatings have been proved to increase their applications in the biomedical fields by improving osseointegration and wear resistance. In this study, Ta oxide coatings containing different proportions of Ag are deposited on SS304 materials. A twin-gun magnetron sputtering system is used to deposit the tantalum oxide-Ag coating. In this study, Staphylococcus aureus, which exhibits physiological commensalism on the human skin, nares, and mucosal and oral areas, is chosen as the model for in vitro antibacterial analyses via a fluorescence staining method using Syto9. The cytocompatibility and adhesive morphology of human skin fibroblast cells (CCD-966SK) onmore » the coatings are also determined by using the microculture tetrazolium assay. This study shows that Ta{sub 2}O{sub 5} and Ta{sub 2}O{sub 5}-Ag coatings with 12.5 at. % of Ag exhibit improved antibacterial effects against S. aureus and have good skin fibroblast cell cellular biocompatibility.« less

Corrosion assessment and enhanced biocompatibility analysis of biodegradable magnesium-based alloys

NASA Astrophysics Data System (ADS)

Pompa, Luis Enrique

Magnesium alloys have raised immense interest to many researchers because of its evolution as a new third generation material. Due to their biocompatibility, density, and mechanical properties, magnesium alloys are frequently reported as prospective biodegradable implant materials. Moreover, magnesium based alloys experience a natural phenomena to biodegrade in aqueous solutions due to its corrosive activity, which is excellent for orthopedic and cardiovascular applications. However, major concerns with such alloys are fast and non-uniform corrosion degradation. Controlling the degradation rate in the physiological environment determines the success of an implant. In this investigation, three grades of magnesium alloys: AZ31B, AZ91E and ZK60A were studied for their corrosion resistance and biocompatibility. Scanning electron microscopy, energy dispersive spectroscopy, atomic force microscopy and contact angle meter are used to study surface morphology, chemistry, roughness and wettability, respectively. Additionally, the cytotoxicity of the leached metal ions was evaluated by a tetrazolium based bio-assay, MTS.

Antitumor effects evaluation of a novel porphyrin derivative in photodynamic therapy.

PubMed

Li, Jian-Wei; Wu, Zhong-Ming; Magetic, Davor; Zhang, Li-Jun; Chen, Zhi-Long

2015-12-01

In this paper, the antitumor activity of a novel porphyrin-based photosensitizer 5,10,15,20-tetrakis[(5-diethylamino)pentyl] porphyrin (TDPP) was reported in vitro and in vivo. The photophysical and cellular properties of TDPP were investigated. The singlet oxygen generation quantum yield of TDPP was detected; it showed a high singlet oxygen quantum yield of 0.52. The intracellular distribution of photosensitizer was detected with laser scanning confocal microscopy. The efficiency of TDPP-photodynamic therapy (PDT) in vitro was analyzed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and in situ trypan blue exclusion test. Treated with a 630-nm laser, TDPP can kill cultured human esophageal cancer cell line (Eca-109) cells and reduce the growth of Eca-109 xenograft tumors significantly in BABL/c nude mice. And histopathological study was also used to confirm the antitumor effect. It has the perspective to be developed as a new antitumor drug in photodynamic therapy and deserves further investigation.

A porphyrin-based metal–organic framework as a pH-responsive drug carrier

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Wenxin; Hu, Quan; Jiang, Ke

A low cytotoxic porphyrin-based metal–organic framework (MOF) PCN-221, which exhibited high PC12 cell viability via 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyl tetrazolium (MTT) assay, was selected as an oral drug carrier. Methotrexate (MTX) was chosen as the model drug molecule which was absorbed into inner pores and channels of MOFs by diffusion. PCN-221 showed high drug loading and sustained release behavior under physiological environment without “burst effect”. The controlled pH-responsive release of drugs by PCN-221 revealed its promising application in oral drug delivery. - Graphical abstract: The porous crystals PCN-221 with pore openings (MOF) PCN-221 was prepared exhibiting low cytotoxicity. PCN-221 showed high drug Methotrexatemore » loading and controlled pH-responsive release of Methotrexate. - Highlights: • A porphyrin-based metal–organic framework (MOF) PCN-221 was prepared showing low cytotoxicity. • PCN-221 showed high drug Methotrexate loading. • PCN-221 showed controlled pH-responsive release of Methotrexate.« less

Synthesis and in vitro antiproliferative activity of 2-methyl-3-(2-piperazin-1-yl-ethyl)-pyrido[1,2-a]pyrimidin-4-one derivatives against human cancer cell lines.

PubMed

Mallesha, Lingappa; Mohana, Kikkeri N; Veeresh, Bantal; Alvala, Ravi; Mallika, Alvala

2012-01-01

A series of new 2-methyl-3-(2-piperazin-1-yl-ethyl)-pyrido[1,2-a]pyrimidin-4-one derivatives 6a-j were synthesized by a nucleophilic substitution reaction of 2-methyl-3-(2-piperazin-1-ylethyl)-pyrido[1,2-a]pyrimidin-4-one with various sulfonyl chlorides. The compounds were characterized by different spectral studies. All the compounds were evaluated for their antiproliferative effect using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay method against four human cancer cell lines (K562, Colo-205, MDA-MB 231, IMR-32) for the time period of 24 h. Among the series, compounds 6d, 6e and 6i showed good activity on all cell lines except K562, whereas the other compounds in the series exhibited moderate activity. Compound 6d could be a potential anticancer agent and therefore deserves further research.

Bupivacaine induces apoptosis via ROS in the Schwann cell line.

PubMed

Park, C J; Park, S A; Yoon, T G; Lee, S J; Yum, K W; Kim, H J

2005-09-01

Local anesthetics have been generally accepted as being safe. However, recent clinical trials and basic studies have provided strong evidence for the neurotoxicity of local anesthetics, especially through apoptosis. We hypothesized that local anesthetics cause neural complications through Schwann cell apoptosis. Among local anesthetics tested on the Schwann cell line, RT4-D6P2T, bupivacaine significantly induced cell death, measured by the methyl tetrazolium (MTT) assay, in a dose- (LD50 = 476 microM) and time-dependent manner. The bupivacaine-induced generation of reactive oxygen species (ROS), which was initiated within 5 hrs and preceded the activation of caspase-3 and poly ADP-ribose polymerase (PARP) degradation, was suggested to trigger apoptosis, exhibited by Hoechst 33258 nuclear staining and DNA fragmentation. Furthermore, concomitant block of ROS by anti-oxidants significantly inhibited bupivacaine-induced apoptosis. Among the local anesthetics for peripheral neural blocks, bupivacaine induced apoptosis in the Schwann cell line, which may be associated with ROS production.

Antimicrobial effect of sophoraflavanone G isolated from Sophora flavescens against mutans streptococci.

PubMed

Kim, Chun Sung; Park, Soon-Nang; Ahn, Sug-Joon; Seo, Young-Woo; Lee, Young-Ju; Lim, Yun Kyong; Freire, Marcelo Oliveira; Cho, Eugene; Kook, Joong-Ki

2013-02-01

In this study, the antibacterial properties of sophoraflavanone G isolated from the methanol extract of Sophora flavescens were tested against 16 strains of mutans streptococci to screen and determine the optimal concentration of anti-caries natural extract. The antimicrobial activity was evaluated by measuring minimum bactericidal concentration (MBC). The cell viability of normal human gingival fibroblast (NHGF) cells was tested using the methyl thiazolyl tetrazolium assay after exposure to sophoraflavanone G. The data showed that sophoraflavanone G had a remarkable antimicrobial effect on the bacteria tested with an MBC ranging from 0.5 μg/ml to 4 μg/ml. Sophoraflavanone G had no cytotoxic effect on NHGF cells at concentrations where it produced an antimicrobial effect. These findings demonstrate that sophoraflavanone G has strong antimicrobial activity against mutans streptococci and could be useful in the development of novel oral hygiene products, such as a gargle solution or dentifrice. Copyright © 2012 Elsevier Ltd. All rights reserved.

Chemical composition of the essential oil from basil (Ocimum basilicum Linn.) and its in vitro cytotoxicity against HeLa and HEp-2 human cancer cell lines and NIH 3T3 mouse embryonic fibroblasts.

PubMed

Kathirvel, Poonkodi; Ravi, Subban

2012-01-01

This study examines the chemical composition and in vitro anticancer activity of the essential oil from Ocimum basilicum Linn. (Lamiaceae), cultivated in the Western Ghats of South India. The chemical compositions of basil fresh leaves were identified by GC-MS: 11 components were identified. The major constituents were found to be methyl cinnamate (70.1%), linalool (17.5%), β-elemene (2.6%) and camphor (1.52%). The results revealed that this plant may belong to the methyl cinnamate and linalool chemotype. A methyl thiazol tetrazolium assay was used for in vitro cytotoxicity screening against the human cervical cancer cell line (HeLa), human laryngeal epithelial carcinoma cell line (HEp-2) and NIH 3T3 mouse embryonic fibroblasts. The IC(50) values obtained were 90.5 and 96.3 µg mL(-1), respectively, and the results revealed that basil oil has potent cytotoxicity.

Anti-collagenase, anti-elastase and anti-oxidant activities of extracts from 21 plants

PubMed Central

Thring, Tamsyn SA; Hili, Pauline; Naughton, Declan P

2009-01-01

Background Owing to their roles in tissue remodelling in health and disease, several studies have reported investigations on plant extracts as inhibitors of proteinases and as anti-oxidants. Methods The anti-ageing and anti-oxidant properties of 23 plant extracts (from 21 plant species) were assessed as anti-elastase and anti-collagenase activities and in selected anti-oxidant assays along with phenolic content. Results Anti-elastase activities were observed for nine of the extracts with inhibitory activity in the following order: white tea (~89%), cleavers (~58%), burdock root (~51%), bladderwrack (~50%), anise and angelica (~32%). Anti-collagenase activities were exhibited by sixteen plants of which the highest activity was seen in white tea (~87%), green tea (~47%), rose tincture (~41%), and lavender (~31%). Nine plant extracts had activities against both elastase (E) and collagenase (C) and were ranked in the order of white tea (E:89%, C:87%) > bladderwrack (E:50%, C:25%) > cleavers (E:58%, C:7%) > rose tincture (E:22%, C:41%) > green tea (E:10%: C:47%) > rose aqueous (E: 24%, C:26%) > angelica (E:32%, C:17%) > anise (E:32%, C:6%) > pomegranate (E:15%, C:11%). Total phenolic content varied between 0.05 and 0.26 mg gallic acid equivalents (GAE)/mL with the exception of white tea (0.77 mg GAE/mL). For anti-oxidant assessment, the Trolox equivalent anti-oxidant capacity (TEAC) assay revealed activity for all extracts. White tea had the highest activity equivalent to ~21 μM Trolox for a 6.25 μg aliquot. In addition, seven extracts exhibited activities = 10 μM Trolox with witch hazel (6.25 μg = 13 μM Trolox) and rose aqueous (6.25 μg = 10 μM Trolox) showing very high activities at low concentrations. A high activity for white tea was also found in the superoxide dismutase (SOD) assay in which it exhibited ~88% inhibition of reduction of nitroblue tetrazolium. High activities were also observed for green tea (86.41%), rose tincture (82.77%), witch hazel (82.05%) and rose aqueous (73.86%). Conclusion From a panel of twenty three plant extracts, some one dozen exhibit high or satisfactory anti-collagenase or anti-elastase activities, with nine having inhibitory activity against both enzymes. These included white tea which was found to have very high phenolic content, along with high TEAC and SOD activities. PMID:19653897

Cytotoxic effect of magnetic iron oxide nanoparticles synthesized via seaweed aqueous extract.

PubMed

Namvar, Farideh; Rahman, Heshu Sulaiman; Mohamad, Rosfarizan; Baharara, Javad; Mahdavi, Mahnaz; Amini, Elaheh; Chartrand, Max Stanley; Yeap, Swee Keong

2014-01-01

Magnetic iron oxide nanoparticles (Fe3O4 MNPs) are among the most useful metal nanoparticles for multiple applications across a broad spectrum in the biomedical field, including the diagnosis and treatment of cancer. In previous work, we synthesized and characterized Fe3O4 MNPs using a simple, rapid, safe, efficient, one-step green method involving reduction of ferric chloride solution using brown seaweed (Sargassum muticum) aqueous extract containing hydroxyl, carboxyl, and amino functional groups mainly relevant to polysaccharides, which acts as a potential stabilizer and metal reductant agent. The aim of this study was to evaluate the in vitro cytotoxic activity and cellular effects of these Fe3O4 MNPs. Their in vitro anticancer activity was demonstrated in human cell lines for leukemia (Jurkat cells), breast cancer (MCF-7 cells), cervical cancer (HeLa cells), and liver cancer (HepG2 cells). The cancer cells were treated with different concentrations of Fe3O4 MNPs, and an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was used to test for cytotoxicity, resulting in an inhibitory concentration 50 (IC50) value of 23.83±1.1 μg/mL (HepG2), 18.75±2.1 μg/mL (MCF-7), 12.5±1.7 μg/mL (HeLa), and 6.4±2.3 μg/mL (Jurkat) 72 hours after treatment. Therefore, Jurkat cells were selected for further investigation. The representative dot plots from flow cytometric analysis of apoptosis showed that the percentages of cells in early apoptosis and late apoptosis were increased. Cell cycle analysis showed a significant increase in accumulation of Fe3O4 MNP-treated cells at sub-G1 phase, confirming induction of apoptosis by Fe3O4 MNPs. The Fe3O4 MNPs also activated caspase-3 and caspase-9 in a time-response fashion. The nature of the biosynthesis and therapeutic potential of Fe3O4 MNPs could pave the way for further research on the green synthesis of therapeutic agents, particularly in nanomedicine, to assist in the treatment of cancer.

Cytotoxic effect of magnetic iron oxide nanoparticles synthesized via seaweed aqueous extract

PubMed Central

Namvar, Farideh; Rahman, Heshu Sulaiman; Mohamad, Rosfarizan; Baharara, Javad; Mahdavi, Mahnaz; Amini, Elaheh; Chartrand, Max Stanley; Yeap, Swee Keong

2014-01-01

Magnetic iron oxide nanoparticles (Fe3O4 MNPs) are among the most useful metal nanoparticles for multiple applications across a broad spectrum in the biomedical field, including the diagnosis and treatment of cancer. In previous work, we synthesized and characterized Fe3O4 MNPs using a simple, rapid, safe, efficient, one-step green method involving reduction of ferric chloride solution using brown seaweed (Sargassum muticum) aqueous extract containing hydroxyl, carboxyl, and amino functional groups mainly relevant to polysaccharides, which acts as a potential stabilizer and metal reductant agent. The aim of this study was to evaluate the in vitro cytotoxic activity and cellular effects of these Fe3O4 MNPs. Their in vitro anticancer activity was demonstrated in human cell lines for leukemia (Jurkat cells), breast cancer (MCF-7 cells), cervical cancer (HeLa cells), and liver cancer (HepG2 cells). The cancer cells were treated with different concentrations of Fe3O4 MNPs, and an MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was used to test for cytotoxicity, resulting in an inhibitory concentration 50 (IC50) value of 23.83±1.1 μg/mL (HepG2), 18.75±2.1 μg/mL (MCF-7), 12.5±1.7 μg/mL (HeLa), and 6.4±2.3 μg/mL (Jurkat) 72 hours after treatment. Therefore, Jurkat cells were selected for further investigation. The representative dot plots from flow cytometric analysis of apoptosis showed that the percentages of cells in early apoptosis and late apoptosis were increased. Cell cycle analysis showed a significant increase in accumulation of Fe3O4 MNP-treated cells at sub-G1 phase, confirming induction of apoptosis by Fe3O4 MNPs. The Fe3O4 MNPs also activated caspase-3 and caspase-9 in a time-response fashion. The nature of the biosynthesis and therapeutic potential of Fe3O4 MNPs could pave the way for further research on the green synthesis of therapeutic agents, particularly in nanomedicine, to assist in the treatment of cancer. PMID:24899805

Cytotoxicity assessments of Portulaca oleracea and Petroselinum sativum seed extracts on human hepatocellular carcinoma cells (HepG2).

PubMed

Farshori, Nida Nayyar; Al-Sheddi, Ebtesam Saad; Al-Oqail, Mai Mohammad; Musarrat, Javed; Al-Khedhairy, Abdulaziz Ali; Siddiqui, Maqsood Ahmed

2014-01-01

The Pharmacological potential, such as antioxidant, anti-inflammatory, and antibacterial activities of Portulaca oleracea (PO) and Petroselinum sativum (PS) extracts are well known. However, the preventive properties against hepatocellular carcinoma cells have not been explored so far. Therefore, the present investigation was designed to study the anticancer activity of seed extracts of PO and PS on the human hepatocellular carcinoma cells (HepG2). The HepG2 cells were exposed with 5-500 μg/ml of PO and PS for 24 h. After the exposure, cell viability by 3-(4,5-dimethylthiazol-2yl)-2,5-biphenyl tetrazolium bromide (MTT) assay, neutral red uptake (NRU) assay, and cellular morphology by phase contrast inverted microscope were studied. The results showed that PO and PS extracts significantly reduced the cell viability of HepG2 in a concentration dependent manner. The cell viability was recorded to be 67%, 31%, 21%, and 17% at 50, 100, 250, and 500 μg/ml of PO, respectively by MTT assay and 91%, 62%, 27%, and 18% at 50, 100, 250, and 500 μg/ml of PO, respectively by NRU assay. PS exposed HepG2 cells with 100 μg/ml and higher concentrations were also found to be cytotoxic. The decrease in the cell viability at 100, 250, and 500 μg/ml of PS was recorded as 70%, 33%, and 15% by MTT assay and 63%, 29%, and 17%, respectively by NRU assay. Results also showed that PO and PS exposed cells reduced the normal morphology and adhesion capacity of HepG2 cells. HepG2 cells exposed with 50 μg/ml and higher concentrations of PO and PS lost their typical morphology, become smaller in size, and appeared in rounded bodies. Our results demonstrated preliminary screening of anticancer activity of Portulaca oleracea and Petroselinum sativum extracts against HepG2 cells, which can be further used for the development of a potential therapeutic anticancer agent.

Radiation sensitivity of Merkel cell carcinoma cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leonard, J.H.; Ramsay, J.R.; Birrell, G.W.

1995-07-30

Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT)more » assay was used for these lines, to estimate cell growth after {gamma} irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to {gamma} irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution. 25 refs., 3 figs., 1 tab.« less

Magnolol pretreatment attenuates heat stress-induced IEC-6 cell injury.

PubMed

Mei, Chen; He, Sha-Sha; Yin, Peng; Xu, Lei; Shi, Ya-Ran; Yu, Xiao-Hong; Lyu, An; Liu, Feng-Hua; Jiang, Lin-Shu

2016-06-01

Heat stress (HS) is an important environmental stressor that adversely influences livestock during the summer. The aim of this study was to investigate whether magnolol protects against HS-induced intestinal epithelial cell injury. An intestinal epithelial cell line (IEC-6) was subjected to HS at 42 °C, with and without magnolol pretreatment. Cell injury was detected by monitoring lactate dehydrogenase (LDH) release. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay was used to assess cell proliferation and viability, including identifying effective concentrations of magnolol. Flow cytometry confirmed G1-phase cell-cycle arrest and its alleviation by magnolol. Active DNA synthesis was measured by incorporation of nucleic acid 5-ethynyl-2'-deoxyuridine (EdU). G1-phase cell-cycle-related gene expression was assessed by real-time reverse transcription polymerase chain reaction (RT-PCR) and levels of G1-phase-related proteins by Western blotting. HS induced IEC-6 cell injury and decreased cell viability, as demonstrated by data from LDH and MTS assays, respectively. Based on a number of criteria, IEC-6 cells subjected to HS were arrested in the G1 phase of the cell cycle. Magnolol pretreatment decreased HS-induced cell injury through relief of this cell-cycle arrest. Magnolol pretreatment attenuates HS-induced injury in IEC-6 cells. Magnolol is potentially promising as a protective strategy for HS in livestock.

Scavenging capacity of strawberry tree (Arbutus unedo L.) leaves on free radicals.

PubMed

Oliveira, Ivo; Coelho, Valentim; Baltasar, Raquel; Pereira, José Alberto; Baptista, Paula

2009-07-01

Despite strawberry tree (Arbutus unedo L.) leaves had a long use in traditional medicine due to its antiseptic, diuretic, astringent and depurative properties, the potential of their antioxidant activity are still lacking. Our study goals to assess the antioxidant and free radical scavenging potential of water, ethanol, methanol and diethyl ether extracts of A. unedo leaves. Total phenols content was achieved spectrophotometrically using Folin-Ciocalteau reagent with gallic acid as standard. Antioxidant activity was evaluated using three different methods: reducing power of iron (III)/ferricyanide complex assay, scavenging effect on DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals and scavenging effect on superoxide radicals by using the PMS-NADH-nitroblue tetrazolium system. Ethanol extracts of A. unedo leaves were the highest in reducing power (IC(50) 232.7 microg/mL) and DPPH scavenging effect (IC(50) 63.2 microg/mL) followed by water extracts (with IC(50) of 287.7 and 73.7 microg/mL, respectively); whereas diethyl ether extracts were the lowest. In the scavenging on superoxide radical assay, methanol extracts obtained the best results (IC(50) 6.9 microg/mL). For all the methods tested the antioxidant activity was concentration dependent. In accordance with antioxidant activity, highest total phenols content were found in ethanol, followed by water, methanol and diethyl ether extract. The results indicated that A. unedo leaves are a potential source of natural antioxidants.

Corrosive effects of fluoride on titanium: investigation by X-ray photoelectron spectroscopy, atomic force microscopy, and human epithelial cell culturing.

PubMed

Stájer, Anette; Ungvári, Krisztina; Pelsoczi, István K; Polyánka, Hilda; Oszkó, Albert; Mihalik, Erzsébet; Rakonczay, Zoltán; Radnai, Márta; Kemény, Lajos; Fazekas, András; Turzó, Kinga

2008-11-01

High fluoride (F(-)) concentrations and acidic pH impair the corrosion resistance of titanium (Ti). Effects of F(-)-containing caries-preventive prophylactic rinses, and gels on Ti were investigated by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Human epithelial cell attachment and proliferation were investigated by dimethylthiazol-diphenyl tetrazolium bromide (MTT) and protein content assays. Aqueous 1% NaF solution (3800 ppm F(-), pH 4.5) or high (12,500 ppm) F(-) content gel (pH 4.8) strongly corroded the surface and modified its composition. XPS revealed formation of a strongly bound F(-)-containing complex (Na(2)TiF(6)). AFM indicated an increase in roughness (R(a)) of the surfaces: 10-fold for the NaF solution and smaller for the gel or a mouthwash (250 ppm F(-), pH 4.4). MTT revealed that cell attachment was significantly increased by the gel, but was not disturbed by either the mouthwash or the NaF. Cell proliferation determined by MTT decreased significantly only for the NaF-treated samples; protein content assay experiments showed no such effect. This study indicates that epithelial cell culturing results can depend on the method used, and the adverse effects of a high F(-) concentration and low pH should be considered when prophylactic gels are applied by patients with Ti implants or other dental devices.

Biocompatibility and antibacterial activity of nitrogen-doped titanium dioxide nanoparticles for use in dental resin formulations

PubMed Central

Zane, Andrew; Zuo, Ranfang; Villamena, Frederick A; Rockenbauer, Antal; Digeorge Foushee, Ann Marie; Flores, Kristin; Dutta, Prabir K; Nagy, Amber

2016-01-01

The addition of antibacterial functionality to dental resins presents an opportunity to extend their useful lifetime by reducing secondary caries caused by bacterial recolonization. In this study, the potential efficacy of nitrogen-doped titanium dioxide nanoparticles for this purpose was determined. Nitrogen doping was carried out to extend the ultraviolet absorbance into longer wavelength blue light for increased biocompatibility. Titanium dioxide nanoparticles (approximately 20–30 nm) were synthesized with and without nitrogen doping using a sol–gel method. Ultraviolet–Visible spectroscopy indicated a band of trap states, with increasing blue light absorbance as the concentration of the nitrogen dopant increased. Electron paramagnetic resonance measurements indicated the formation of superoxide and hydroxyl radicals upon particle exposure to visible light and oxygen. The particles were significantly toxic to Escherichia coli in a dose-dependent manner after a 1-hour exposure to a blue light source (480 nm). Intracellular reactive oxygen species assay demonstrated that the particles caused a stress response in human gingival epithelial cells when exposed to 1 hour of blue light, though this did not result in detectable release of cytokines. No decrease in cell viability was observed by water-soluble tetrazolium dye assay. The results show that nitrogen-doped titanium dioxide nanoparticles have antibacterial activity when exposed to blue light, and are biocompatible at these concentrations. PMID:27980404

Biocompatibility and antibacterial activity of nitrogen-doped titanium dioxide nanoparticles for use in dental resin formulations.

PubMed

Zane, Andrew; Zuo, Ranfang; Villamena, Frederick A; Rockenbauer, Antal; Digeorge Foushee, Ann Marie; Flores, Kristin; Dutta, Prabir K; Nagy, Amber

The addition of antibacterial functionality to dental resins presents an opportunity to extend their useful lifetime by reducing secondary caries caused by bacterial recolonization. In this study, the potential efficacy of nitrogen-doped titanium dioxide nanoparticles for this purpose was determined. Nitrogen doping was carried out to extend the ultraviolet absorbance into longer wavelength blue light for increased biocompatibility. Titanium dioxide nanoparticles (approximately 20-30 nm) were synthesized with and without nitrogen doping using a sol-gel method. Ultraviolet-Visible spectroscopy indicated a band of trap states, with increasing blue light absorbance as the concentration of the nitrogen dopant increased. Electron paramagnetic resonance measurements indicated the formation of superoxide and hydroxyl radicals upon particle exposure to visible light and oxygen. The particles were significantly toxic to Escherichia coli in a dose-dependent manner after a 1-hour exposure to a blue light source (480 nm). Intracellular reactive oxygen species assay demonstrated that the particles caused a stress response in human gingival epithelial cells when exposed to 1 hour of blue light, though this did not result in detectable release of cytokines. No decrease in cell viability was observed by water-soluble tetrazolium dye assay. The results show that nitrogen-doped titanium dioxide nanoparticles have antibacterial activity when exposed to blue light, and are biocompatible at these concentrations.

Inhibition of Bacteria Associated with Wound Infection by Biocompatible Green Synthesized Gold Nanoparticles from South African Plant Extracts

PubMed Central

Elbagory, Abdulrahman M.; Meyer, Mervin; Cupido, Christopher N.

2017-01-01

Unlike conventional physical and chemical methods, the biogenic synthesis of gold nanoparticles (GNPs) is considered a green and non-toxic approach to produce biocompatible GNPs that can be utilized in various biomedical applications. This can be achieved by using plant-derived phytochemicals to reduce gold salt into GNPs. Several green synthesized GNPs have been shown to have antibacterial effects, which can be applied in wound dressings to prevent wound infections. Therefore, the aim of this study is to synthesize biogenic GNPs from the South African Galenia africana and Hypoxis hemerocallidea plants extracts and evaluate their antibacterial activity, using the Alamar blue assay, against bacterial strains that are known to cause wound infections. Additionally, we investigated the toxicity of the biogenic GNPs to non-cancerous human fibroblast cells (KMST-6) using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. In this paper, spherical GNPs, with particle sizes ranging from 9 to 27 nm, were synthesized and fully characterized. The GNPs from H. hemerocallidea exhibited antibacterial activity against all the tested bacterial strains, whereas GNPs produced from G. africana only exhibited antibacterial activity against Pseudomonas aeruginosa. The GNPs did not show any significant toxicity towards KMST-6 cells, which may suggest that these nanoparticles can be safely applied in wound dressings. PMID:29186826

Cytotoxicity and cytokine expression induced by silorane and methacrylate-based composite resins.

PubMed

Longo, Daniele Lucca; Paula-Silva, Francisco Wanderley Garcia; Faccioli, Lucia Helena; Gatón-Hernández, Patrícia Maria; Queiroz, Alexandra Mussolino de; Silva, Léa Assed Bezerra da

2016-01-01

The aim of this study was to evaluate cytotoxicity and cytokine production induced by light-cured or non-light-cured methacrylate-based and silorane composite resins in RAW 264.7 macrophages. Cells were stimulated with the extracts from light-cured or non-light-cured composite resins. After incubation for 24 h, cytotoxicity was assessed with the lactate dehydrogenase (LDH) and methyl thiazolyl tetrazolium (MTT) assays, and total protein was quantified using the Lowry method. TNF-α detection was examined with an enzyme-linked immunosorbent assay (ELISA) conducted with cell supernatants after cell stimulation for 6, 12, and 24 h. Data were analyzed using one-way analysis of variance (ANOVA) and Tukey's post hoc test (α=0.05). KaloreTM and FiltekTM Silorane were cytotoxic with or without light curing (p<0.05) after 24 h of incubation. KaloreTM stimulated the early production of TNF-α in comparison with control (p<0.05), whereas FiltekTM Silorane did not affect TNF-α levels after 6 and 12 h (p>0.05). However, after 24 h FiltekTM Silorane inhibited the production of TNF-α (p<0.05). KaloreTM and FiltekTM Silorane were cytotoxic regardless of light curing. The extract obtained from KaloreTM after 15 days of incubation stimulated the production of TNF-α, unlike that obtained from FiltekTM Silorane.

Effect of brief exposure to mitomycin C on cultured human nasal mucosa fibroblasts.

PubMed

Hu, D; Sires, B S; Tong, D C; Royack, G A; Oda, D

2000-03-01

To observe the effect of mitomycin C (MMC) on cultured human nasal mucosa fibroblasts. Cultured human nasal mucosa fibroblasts were exposed to MMC (0.1-0.4 mg/ml) for 1 to 5 minutes. The viability of the fibroblasts was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay; DNA fragmentation (apoptosis) by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL); apoptotic percentage by flow cytometry; and morphology by light microscopy. A portion of the fibroblasts survived the mitomycin treatment and showed evidence of regrowth within 2 to 3 days. These cells reached confluence in 5 to 7 days. The inhibition rates by MTT assay of 0.4 mg/ml MMC for 5-minute exposures was 31.3%. Dose-response effect was noted with the lower concentrations and shorter exposure times where the inhibition rates were lower (but not significantly so). DNA fragmentation was observed in fibroblasts 24 hours after MMC exposure (0.4 mg/ml) for 5 minutes compared with normal control. The apoptotic rate of fibroblasts treated by 0.4 mg/ml MMC was significantly higher than the control (p < 0.05). Short MMC exposure times have a variable cytotoxic effect and inhibit proliferation of cultured nasal mucosa fibroblasts. MMC also can induce apoptosis with a 5-minute exposure time. Therefore, it is possible that MMC has a complex effect in dacryocystorhinostomy.

Effect of Cinnamomum osmophloeum Kanehira Leaf Aqueous Extract on Dermal Papilla Cell Proliferation and Hair Growth

PubMed Central

Wen, Tung-Chou; Li, Yuan-Sheng; Rajamani, Karthyayani; Harn, Horng-Jyh; Lin, Shinn-Zong; Chiou, Tzyy-Wen

2018-01-01

In this study, we explored the effect of the water extract of Cinnamomum osmophloeum Kanehira (COK) leaves on hair growth by in vitro and in vivo assays. Using an in vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, it was found that the proliferation of rat vibrissae and human hair dermal papilla cells (hDPCs) was significantly enhanced by the COK leaf extract treatment. As determined by quantitative real-time polymerase chain reaction (RT-PCR), the messenger RNA (mRNA) levels of some hair growth–related factors including vascular endothelial growth factor, keratinocyte growth factor (KGF), and transforming growth factor-β2 were found to be higher in the cultured hDPCs exposed to COK leaf extract than those in the untreated control group. In the hair-depilated C57BL/6 mouse model, the stimulation of hair growth was demonstrated in the group of COK leaf extract treatment. Both photographical and histological observations revealed the promotion of the anagen phase in the hair growth cycle by the COK leaf extract in the C57BL/6 mice. Finally, the ultra performance liquid chromatography (UPLC) showed that the COK extract contained mostly cinnamic aldehyde and a small amount of cinnamic acid. The results suggest that the COK leaf extract may find use for the treatment of hair loss. PMID:29637818

Vitamin K1 enhances sorafenib-induced growth inhibition and apoptosis of human malignant glioma cells by blocking the Raf/MEK/ERK pathway

PubMed Central

2012-01-01

Background The combined effects of anticancer drugs with nutritional factors against tumor cells have been reported previously. This study characterized the efficacy and possible mechanisms of the combination of sorafenib and vitamin K1 (VK1) on glioma cell lines. Methods We examined the effects of sorafenib, VK1 or their combination on the proliferation and apoptosis of human malignant glioma cell lines (BT325 and U251) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, flow cytometry and 4′,6-diamidino-2-phenylindole (DAPI) assay. The signaling pathway changes were detected by western blotting. Results Sorafenib, as a single agent, showed antitumor activity in a dose-dependent manner in glioma cells, but the effects were more pronounced when used in combination with VK1 treatment. Sorafenib in combination with VK1 treatment produced marked potentiation of growth inhibition and apoptosis, and reduced expression of phospho-mitogen-activated protein kinase kinase (MEK) and phospho-extracellular signal-regulated kinase (ERK). Furthermore, the expression levels of antiapoptotic proteins Bcl-2 and Mcl-1 were significantly reduced. Conclusions Our findings indicated that VK1 enhanced the cytotoxicity effect of sorafenib through inhibiting the Raf/MEK/ERK signaling pathway in glioma cells, and suggested that sorafenib in combination with VK1 maybe a new therapeutic option for patients with gliomas. PMID:22520038

Human fibroblast-derived extracellular matrix constructs for bone tissue engineering applications.

PubMed

Tour, Gregory; Wendel, Mikael; Tcacencu, Ion

2013-10-01

We exploited the biomimetic approach to generate constructs composed of synthetic biphasic calcium phosphate ceramic and extracellular matrix (SBC-ECM) derived from adult human dermal fibroblasts in complete xeno-free culture conditions. The construct morphology and composition were assessed by scanning electron microscopy, histology, immunohistochemistry, Western blot, glycosaminoglycan, and hydroxyproline assays. Residual DNA quantification, endotoxin testing, and local inflammatory response after implantation in a rat critical-sized calvarial defect were used to access the construct biocompatibility. Moreover, in vitro interaction of human mesenchymal stem cells (hMSCs) with the constructs was studied. The bone marrow- and adipose tissue-derived mesenchymal stem cells were characterized by flow cytometry and tested for osteogenic differentiation capacity prior seeding onto SBC-ECM, followed by alkaline phosphatase, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and real-time quantitative polymerase chain reaction to assess the osteogenic differentiation of hMSCs after seeding onto the constructs at different time intervals. The SBC-ECM constructs enhanced osteogenic differentiation of hMSCs in vitro and exhibited excellent handling properties and high biocompatibility in vivo. Our results highlight the ability to generate in vitro fibroblast-derived ECM constructs in complete xeno-free conditions as a step toward clinical translation, and the potential use of SBC-ECM in craniofacial bone tissue engineering applications. Copyright © 2013 Wiley Periodicals, Inc., a Wiley Company.

Nicotine promotes proliferation and collagen synthesis of chondrocytes isolated from normal human and osteoarthritis patients.

PubMed

Ying, Xiaozhou; Cheng, Shaowen; Shen, Yue; Cheng, Xiaojie; An Rompis, Ferdinand; Wang, Wei; Lin, Zhongqin; Chen, Qingyu; Zhang, Wei; Kou, Dongquan; Peng, Lei; Tian, Xin Qiao; Lu, Chuan Zhu

2012-01-01

The aims of the study were to show the direct effect of nicotine with different concentrations (0, 25, 50, and 100 ng/ml) on chondrocytes isolated from normal human and osteoarthritis patients, respectively. Microscopic observation was performed during the culture with an inverted microscope. Methyl thiazolyl tetrazolium (MTT) assay method was adopted to observe the influence of nicotine on the proliferation of chondrocytes, and real-time PCR and ELISA were used to assay the mRNA and protein expression of type II collagen and aggrecan, respectively. We discovered that the OA chondrocytes were similar to fibroblasts in shape and grow slower than normal chondrocytes. The proliferation of the two kinds of chondrocytes was increased in a concentration-dependent manner and in a time-dependent manner (P<0.05). Also, we found that the mRNA level of type II collagen were upregulated under 25-100 ng/ml nicotine doses both in the two kinds of chondrocytes compared with control. The expression of protein levels of type II collagen were synthesized in line with the increase in mRNA. No effect was observed on aggrecan synthesis with any nicotine dose. We concluded that nicotine has the same effect on both chondrocytes, obtained either from osteoarthritis patients or from normal human, and the positive effect of smoking in OA may relate to the alteration in metabolism of chondrocytes.

[Effect of Shexiang Baoxin Pills on isoprenaline-induced myocardial cell hypertrophy and Cx43 expression].

PubMed

Tang, Fen; Jiang, Zhentao; Tan, Wenting; Long, Junrong; Liu, Shengquan; Chu, Chun

2017-08-28

To observe the effects of Shexiang Baoxin Pill (SBP) on isoprenaline (Iso)-induced changes in myocardial cell volume, shape, and connexin 43 (Cx43) expression.
 Methods: H9C2 myocardial cells were randomly divided into a control group, a Iso group and a Iso+SBP group. After 72 h of culture, the average surface area of H9C2 cells was measured under phase contrast microscope. Bicinchoninic acid (BCA) protein assay was carried out to determine the concentration of proteins. The survival rate of myocardial cells was measured by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, and the Cx43 expression was detected by Western blot.
 Results: The mean surface area and Cx43 concentration in Iso-treated myocardial cells were increased under the phase contrast microscope (P<0.05). Compared with the Iso group, the mean surface area was decreased, and the Cx43 concentration was reduced in the Iso+SBP group (both P<0.05). Compared with the control group, the Cx43 expression was obviously down-regulated in the H9C2 cells of the Iso group (P<0.05); while compared with the Iso group, the Cx43 expression was obviously up-regulated in the Iso+SBP group (P<0.05).
 Conclusion: Shexiang Baoxin Pills can prevent Iso-induced myocardial hypertrophy and down-regulate Cx43 expression.

Chrysin reduces proliferation and induces apoptosis in the human prostate cancer cell line pc-3.

PubMed

Samarghandian, Saeed; Afshari, Jalil Tavakkol; Davoodi, Saeideh

2011-01-01

Honey is a common household product with many medicinal uses described in traditional medicine. Only recently has its antioxidant properties and preventive effects against disease been highlighted. Chrysin is a natural flavone commonly found in honey that has been shown to be an antioxidant agent. In this study, we investigated the antiproliferative and apoptotic effects of honey and chrysin on cultured human prostate cancer cells. Cells were cultured in RPMI medium and treated with different concentrations of honey and chrysin for three consecutive days. Cell viability was quantitated by the 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The percentage of apoptotic cells was determined by flow cytometry using Annexin V-fluorescein isothiocyanate. The MTT assay revealed that both compounds had an antiproliferative effect on PC-3 cells in a dose- and time-dependent manner. The IC50 values for honey and chrysin against PC-3 cells were 2.5% and 24.5% after 48 h and 1.8% and 8.5% after 72 h, respectively. Chrysin induced apoptosis in PC-3 cells, as determined by flow cytometry. Our results suggest that honey has anti-proliferative effects on prostate cancer cells and the effects are mainly due to chrysin. Therefore, chrysin may be a potential compound for both cancer prevention and treatment. Further in vivo investigation is needed to support the use of chrysin in cancer therapy.

Comparative study on toxicity of methylmercury chloride and methylmercury hydroxide to the human neuroblastoma cell line SH-SY5Y.

PubMed

Patnaik, Rajashree; Padhy, Rabindra N

2018-05-11

Toxicities of methylmercury chloride (CH 3 HgCl) and methylmercury hydroxide (CH 3 HgOH) to cultured neuroblastoma cell line SH-SY5Y in vitro are evaluated. This is the comparative study between two methylmercury compounds to find out the extent of toxicity of these compounds are toxic to SH-SY5Y cell line. Both cytotoxicity and genotoxicity experiments were carried out to find out the more toxic compound. For cytotoxicity study, four staining assay methods independently with trypan blue (TB), acridine orange/ethidium bromide (AO/EB), 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT), and neutral red (NR) were used and the comet assay method was done for genotoxicity study. The obtained toxicity data were used for probit analysis. In cytotoxicity, CH 3 HgCl had minimum inhibitory concentration (MIC) value in each assay method as 3 mg/L invariably; LC 25 values were in the range 7.41 to 10.23 mg/L, and LC 50 values were 14.79 to 15.48 mg/L; while LC 75 values were 20.89 to 26.91 mg/L. Moreover, LC 100 value was 30 mg/L, known from comet assay experiments for CH 3 HgCl. Similarly for CH 3 HgOH, the MIC value in each assay method was invariably 3 mg/L, the LC 25 values were in the range 12.58 to 16.59 mg/L, and LC 50 values were 19.49 to 23.44 mg/L; LC 75 values were 27.54 to 30.90 mg/L and LC 100 value was 42 mg/L in each assay done for cytotoxicity and genotoxicity studies. Computed DNA fragmentation indices in comet assays were 98.6 ± 0.57 30 mg/L with CH 3 HgCl and 76 ± 5.29 30 mg/L with CH 3 HgOH. This study clearly indicated that methylmercury chloride is more toxic than methylmercury hydroxide to SH-SY5Y cell line. Toxicity of Hg had been quantified with in vitro cultured human neuroblastoma cell line; since it has neurotoxic effects, its neural evaluation has implications in environmental health issues.

Safety assessment of nanoparamagnetic contrast agents with different coatings for molecular MRI

NASA Astrophysics Data System (ADS)

Azizian, Gholamreza; Riyahi-Alam, Nader; Haghgoo, Soheila; Saffari, Mojtaba; Zohdiaghdam, Reza; Gorji, Ensieh

2013-04-01

Despite the wide application of gadolinium as a contrast agent for magnetic resonance imaging (MRI), there is a serious lack of information on its toxicity. Gadolinium and gadolinium oxide (Gd-oxide) are used as contrast agents for magnetic resonance imaging (MRI). There are methods for reducing toxicity of these materials, such as core nanoparticles coating or conjugating. Therefore, for toxicity evaluation, we compared the viability of commercial contrast agents in MRI (Gd-DTPA) and three nanoparticles with the same core Gd2O3 and small particulate gadolinium oxide or SPGO (< 40 nm) but different coatings of diethyleneglycol (DEG) as Gd2O3-DEG and methoxy polyethylene glycol-silane (mPEG-silane: 550 and 2000 Dalton) as SPGO-mPEG-silane550 and SPGO-mPEG-silane2000, respectively, in the SK-MEL3 cell line, by light microscopy, MTT assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, and the LDH assay detecting lactate dehydrogenase activity. The viability values were not statistically different between the three nanoparticles and Gd-DTPA. The MTT and LDH assay results showed that Gd2O3-DEG nanoparticles were more toxic than Gd-DTPA and other nanoparticles. Also, SPGO-mPEG-silane2000 was more biocompatible than other nanoparticles. The obtained results did not show any significant increase in cytotoxicity of the nanoparticles and Gd-DTPA, neither dose-dependent nor time-dependent. Therefore, DEG and PEG, due to their considerable properties and irregular sizes (different molecular weights), were selected as the useful surface covering materials of nanomagnetic particles that could reveal noticeable relaxivity and biocompatibility characteristics.

Mobile phone radiation alters proliferation of hepatocarcinoma cells.

PubMed

Ozgur, Elcin; Guler, Goknur; Kismali, Gorkem; Seyhan, Nesrin

2014-11-01

This study investigated the effects of intermittent exposure (15 min on, 15 min off for 1, 2, 3, or 4 h, at a specific absorption rate of 2 W/kg) to enhanced data rates for global system for mobile communication evolution-modulated radiofrequency radiation (RFR) at 900- and 1,800-MHz frequencies on the viability of the Hepatocarcinoma cells (Hep G2). Hep G2 cell proliferation was measured by a colorimetric assay based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenases in viable cells. Cell injury was evaluated by analyzing the levels of lactate dehydrogenase (LDH) and glucose released from lysed cells into the culture medium. Morphological observation of the nuclei was carried out by 4',6-diamidino-2-phenylindole (DAPI) staining using fluorescence microscopy. In addition, TUNEL assay was performed to confirm apoptotic cell death. It was observed that cell viability, correlated with the LDH and glucose levels, changed according to the frequency and duration of RFR exposure. Four-hour exposure produced more pronounced effects than the other exposure durations. 1,800-MHz RFR had a larger impact on cell viability and Hep G2 injury than the RFR at 900 MHz. Morphological observations also supported the biochemical results indicating that most of the cells showed irregular nuclei pattern determined by using the DAPI staining, as well as TUNEL assay which shows DNA damage especially in the cells after 4 h of exposure to 1,800-MHz RFR. Our results indicate that the applications of 900- and 1,800-MHz (2 W/kg) RFR cause to decrease in the proliferation of the Hep G2 cells after 4 h of exposure. Further studies will be conducted on other frequency bands of RFR and longer duration of exposure.

Serenoa repens extracts promote hair regeneration and repair of hair loss mouse models by activating TGF-β and mitochondrial signaling pathway.

PubMed

Zhu, H-L; Gao, Y-H; Yang, J-Q; Li, J-B; Gao, J

2018-06-01

Plenty of plant extracts have been used for treating hair loss. This study aims to investigate the effects of liposterolic extracts of Serenoa repens (LSESr) on hair cell growth and regeneration of hair, and clarify the associated mechanisms. Human keratinocyte cells (HACAT) were cultured, incubated with dihydrotestosterone (DHT) and treated with LSESr. Cell viability was examined by using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H- tetrazolium bromide (MTT) assay. Hair loss C57BL/6 mouse model was established by inducing with DHT. Hair growth, density, and thickness were evaluated. Back skin samples were collected and stained with hematoxylin and eosin (HE) assay. B-cell lymphoma-2 (Bcl-2), Bcl-2 associated protein X (Bax), cleaved caspase 3 and transforming growth factor β2 (TGF-β2) were examined using Western blot assay. LSESr treatment significantly increased HACAT cell viabilities compared to DHT-only treated cells (p<0.05). LSESr treatment post injection of DHT significantly converted skin color from pink to gray and increased hair density, weight and thickness compared to DHT-only treated mice (p<0.05). LSESr treatment significantly triggered follicle growth and decreased inflammatory response. LSESr treatment significantly decreased TGF-β2 and cleaved caspase 3 expression of hair loss mouse models compared to that of DHT treated mice (p<0.05). LSESr treatment significantly enhanced Bcl-2 expression and reduced Bax expression compared to that of DHT treated mice (p<0.05). Meanwhile, effects of LSESr were substantial even achieving to the potential of finasteride. LSESr promoted the hair regeneration and repair of hair loss mouse models by activating TGF-β signaling and mitochondrial signaling pathway.

A Vitex agnus-castus extract inhibits cell growth and induces apoptosis in prostate epithelial cell lines.

PubMed

Weisskopf, M; Schaffner, W; Jundt, G; Sulser, T; Wyler, S; Tullberg-Reinert, H

2005-10-01

Extracts of Vitex agnus-castus fruits (VACF) are described to have beneficial effects on disorders related to hyperprolactinemia (cycle disorders, premenstrual syndrome). A VACF extract has recently been shown to exhibit antitumor activities in different human cancer cell lines. In the present study, we explored the antiproliferative effects of a VACF extract with a particular focus on apoptosis-inducing and potential cytotoxic effects. Three different human prostate epithelial cell lines (BPH-1, LNCaP, PC-3) representing different disease stages and androgen responsiveness were chosen. The action of VACF on cell viability was assessed using the WST-8-tetrazolium assay. Cell proliferation in cells receiving VACF alone or in combination with a pan-caspase inhibitor (Z-VAD-fmk) was quantified using a Crystal Violet assay. Flow cytometric cell cycle analysis and measurement of DNA fragmentation using an ELISA method were used for studying the induction of apoptosis. Lactate dehydrogenase (LDH) activity was determined as a marker of cytotoxicity. The extract inhibited proliferation of all three cell lines in a concentration-dependent manner with IC (50) values below 10 microg/mL after treatment for 48 h. Cell cycle analysis and DNA fragmentation assays suggest that part of the cells were undergoing apoptosis. The VACF-induced decrease in cell number was partially inhibited by Z-VAD-fmk, indicating a caspase-dependent apoptotic cell death. However, the concentration-dependent LDH activity of VACF treated cells indicated cytotoxic effects as well. These data suggest that VACF contains components that inhibit proliferation and induce apoptosis in human prostate epithelial cell lines. The extract may be useful for the prevention and/or treatment not only of benign prostatic hyperplasia but also of human prostate cancer.

Arsenic trioxide inhibits Ewing's sarcoma cell invasiveness by targeting p38(MAPK) and c-Jun N-terminal kinase.

PubMed

Zhang, Shuai; Guo, Wei; Ren, Ting-Ting; Lu, Xin-Chang; Tang, Guo-Qing; Zhao, Fu-Long

2012-01-01

Ewing's sarcoma is the second most frequent primary malignant bone tumor, mainly affecting children and young adults. The notorious metastatic capability of this tumor aggravates patient mortality and remains a problem to be overcome. We investigated the effect of arsenic trioxide (As₂O₃) on the metastasis capability of Ewing's sarcoma cells. We performed 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide assays to choose appropriate concentrations of As₂O₃ for the experiments. Migration, invasion, and adhesion assays were performed to assess the effect of As₂O₃ on the metastasis of Ewing's sarcoma. Immunofluorescent staining was used to observe cytoskeleton reorganization in Ewing's sarcoma cells treated with As₂O₃. Changes in matrix metalloproteinase-9 expression and the mitogen-activated protein kinase (MAPK) pathway were investigated using western blot. Inhibitors of p38(MAPK) (sb202190) and c-Jun NH₂-terminal kinase (JNK, sp600125) were used in invasion assays to determine the effect of p38(MAPK) and JNK. We found that As₂O₃ may markedly inhibit the migration and invasion capacity of Ewing's sarcoma cells with structural rearrangements of the actin cytoskeleton. The expressions of matrix metalloproteinase-9, phosphor-p38(MAPK), and phosphor-JNK were suppressed by As₂O₃ treatment in a dose-dependent manner. The inhibitors of p38(MAPK) (sb202190) and JNK (sp600125) enhanced the inhibition induced by As₂O₃, which was counteracted by anisomycin, an activating agent of p38(MAPK) and JNK. Taken together, our results demonstrate that As₂O₃ can inhibit the metastasis capability of RD-ES and A-673 cells and may have new therapeutic value for Ewing's sarcoma.

Evaluation of Anti-Metastatic Potential of the Combination of Fisetin with Paclitaxel on A549 Non-Small Cell Lung Cancer Cells.

PubMed

Klimaszewska-Wiśniewska, Anna; Hałas-Wiśniewska, Marta; Grzanka, Alina; Grzanka, Dariusz

2018-02-27

The identification and development of new agents with a therapeutic potential as well as novel drug combinations are gaining the attention of scientists and clinicians as a plausible approach to improve therapeutic regimens for chemoresistant tumors. We have recently reported that the flavonoid fisetin (FIS), at physiologically attainable concentrations, acts synergistically with clinically achievable doses of paclitaxel (PTX) to produce growth inhibitory and pro-death effects on A549 human non-small cell lung cancer (NSCLC) cells. To further investigate a potential therapeutic efficacy of the combination of fisetin with paclitaxel, we decided to assess its impact on metastatic capability of A549 cells as well as its toxicity toward normal human lung fibroblast. Cell viability, cell migration, and invasion were measured by thiazolyl blue tetrazolium bromide (MTT) assay, wound healing assay, and Transwell chamber assay, respectively. The expression of metastasis-related genes was assessed with quantitative reverse transcriptase real-time polymerase chain reaction (qRT-PCR). Actin and vimentin filaments were examined under the fluorescence microscope. The combination of FIS and PTX significantly reduced cancer cell migration and invasion, at least partially, through a marked rearrangement of actin and vimentin cytoskeleton and the modulation of metastasis-related genes. Most of these effects of the combination treatment were significantly greater than those of individual agents. Paclitaxel alone was even more toxic to normal cells than the combination of this drug with the flavonoid, suggesting that FIS may provide some protection against PTX-mediated cytotoxicity. The combination of FIS and PTX is expected to have a synergistic anticancer efficacy and a significant potential for the treatment of NSCLC, however, further in vitro and in vivo studies are required to confirm this preliminary evidence.

The XTT Cell Proliferation Assay Applied to Cell Layers Embedded in Three-Dimensional Matrix

PubMed Central

Huyck, Lynn; Ampe, Christophe

2012-01-01

Abstract Cell proliferation, a main target in cancer therapy, is influenced by the surrounding three-dimensional (3D) extracellular matrix (ECM). In vitro drug screening is, thus, optimally performed under conditions in which cells are grown (embedded or trapped) in dense 3D matrices, as these most closely mimic the adhesive and mechanical properties of natural ECM. Measuring cell proliferation under these conditions is, however, technically more challenging compared with two-dimensional (2D) culture and other “3D culture conditions,” such as growth on top of a matrix (pseudo-3D) or in spongy scaffolds with large pore sizes. Consequently, such measurements are only slowly applied on a wider scale. To advance this, we report on the equal quality (dynamic range, background, linearity) of measuring the proliferation of cell layers embedded in dense 3D matrices (collagen, Matrigel) compared with cells in 2D culture using the easy (one-step) and in 2D well-validated, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT)-assay. The comparison stresses the differences in proliferation kinetics and drug sensitivity of matrix-embedded cells versus 2D culture. Using the specific cell-layer-embedded 3D matrix setup, quantitative measurements of cell proliferation and cell invasion are shown to be possible in similar assay conditions, and cytostatic, cytotoxic, and anti-invasive drug effects can thus be reliably determined and compared in physiologically relevant settings. This approach in the 3D matrix holds promise for improving early-stage, high-throughput drug screening, targeting either highly invasive or highly proliferative subpopulations of cancers or both. PMID:22574651

Doxycycline inhibits TGF-β1-induced extracellular matrix production in nasal polyp-derived fibroblasts.

PubMed

Shin, Jae-Min; Park, Joo-Hoo; Park, Il-Ho; Lee, Heung-Man

2016-03-01

Doxycycline has been shown to have antibacterial and anti-inflammatory effects and suppresses collagen biosynthesis. The purpose of this study was to evaluate the effects of doxycycline on transforming growth factor (TGF)-β1-induced myofibroblast differentiation and extracellular matrix production in nasal polyp-derived fibroblasts (NPDFs). We also determined the molecular mechanisms of action for doxycycline. NPDFs were isolated from nasal polyps from 8 patients. Doxycycline was used to pretreat TGF-β1-induced NPDFs. Cytotoxicity was evaluated using a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay. Expression levels of α-smooth muscle actin (SMA) and fibronectin were measured using Western blot, reverse-transcription polymerase chain reaction, and immunofluorescence staining. Total collagen production was analyzed with the Sircol collagen assay, while mitogen-activated protein kinase (MAPK) and NF-κB activation were determined using Western blot analysis. Luciferase assay was used to evaluate the transcriptional activity of NF-κB. Although doxycycline (0 to 40 μg/mL) had no significant cytotoxic effects in TGF-β1-induced NPDFs, it significantly reduced the expression levels of α-SMA, fibronectin, and collagen in TGF-β1-induced NPDFs in a dose-dependent manner. Doxycycline also inhibited the TGF-β1-induced activation of p38, c-Jun NH2 -terminal kinase (JNK), and NF-κB, and its inhibitory effects were similar to those of the specific inhibitors for each. Doxycycline has an inhibitory effect on TGF-β1-induced myofibroblast differentiation and extracellular matrix production via the p38 and JNK/NF-κB signal pathways in NPDFs. © 2015 ARS-AAOA, LLC.

Effect of doxycycline on transforming growth factor-beta-1-induced matrix metalloproteinase 2 expression, migration, and collagen contraction in nasal polyp-derived fibroblasts.

PubMed

Shin, Jae-Min; Park, Joo-Hoo; Kang, Byungjin; Lee, Seoung-Ae; Park, Il-Ho; Lee, Heung-Man

2016-11-01

It is well known that doxycycline has antibacterial and anti-inflammatory effects. In this study, we aimed to investigate the effects of doxycycline on the transforming growth factor (TGF) beta 1-induced matrix metalloproteinase (MMP) 2 expression, migration, and collagen contraction, and to determine its molecular mechanism on nasal polyp-derived fibroblasts (NPDF). NPDFs were isolated from the nasal polyps of six patients. Doxycycline was used to pretreat TGF-beta-1-induced NPDFs and ex vivo organ cultures of nasal polyps. Cytotoxicity was evaluated by using a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay. Smad2/3 is one of the major transcription factors of TGF-beta signaling. The expression levels of MMP2 and Smad2/3 were measured by using Western blotting, reverse transcription-polymerase chain reaction, and immunofluorescence staining. The enzymic activity of MMP2 was analyzed by using gelatin zymography. Fibroblast migration was evaluated by using transwell migration assays. Contractile activity was measured by a collagen gel contraction assay. The expression level of MMP2 in nasal polyp tissues increased in comparison with inferior turbinate tissues. TGF-beta-1-induced NPDFs were not affected by doxycycline (0-40 μg/mL). The expression levels of MMP2 and activation of Smad2/3 in TGF-beta-1-induced NPDFs and in organ cultures of nasal polyps were significantly downregulated with doxycycline pretreatment. Doxycycline also reduced TGF-beta-1-induced fibroblast migration and collagen contraction in NPDFs. Doxycycline inhibited TGF-beta-1-induced MMP2 expression, migration, and collagen contraction via the Smad2/3 signal pathways in NPDFs.

6-Mercaptopurine transport in human lymphocytes: correlation with drug-induced cytotoxicity.

PubMed

Conklin, Laurie S; Cuffari, Carmen; Okazaki, Toshihiko; Miao, Yinglei; Saatian, Bahman; Chen, Tian-E; Tse, Ming; Brant, Steven R; Li, Xuhang

2012-02-01

  6-mercaptopurine (6-MP) is efficacious in the treatment of inflammatory bowel disease (IBD). However, about one-third of patients respond poorly to therapy. This study aimed to characterize the inherent differences in 6-MP transport that may cotribute to the differences in treatment responses.   Intracellular 6-MP accumulation was assayed in Epstein-Barr virus (EBV)-transformed lymphocytes from IBD patients, using (14) C-radiolabeled 6-MP. Cell proliferation was determined by methyl thiazolyl tetrazolium (MTT) assay. Apoptosis was assayed based on the activation of caspase 3. The expressions of 15 potential 6-MP transporters were evaluated by reverse transcription-polymerase chain reaction (RT-PCR).   Intracellular 6-MP accumulation, varying significantly among patients, was carrier-dependent and partially sodium-dependent. 6-MP cytotoxicity was, at least in part, due to apoptosis and correlated with intracellular drug accumulation. The efflux transporters did not appear to contribute to the variability of intracellular drug accumulation between patients, since none correlated with drug accumulation or cytotoxicity. Rather, differential expression of five influx/uptake transporters might be a key contributor to the difference in the accumulation of and susceptibility to the drug.   The heterogeneity of the drug transporters may be the reason for the therapeutic sensitivity of 6-MP in IBD patients. As the 6-MP uptake is a carrier-mediated and partially sodium-dependent process, future studies are necessary to evaluate the role of the putative transporters and their correlation with drug sensitivity in patients. © 2012 The Authors. Journal of Digestive Diseases © 2012 Chinese Medical Association Shanghai Branch, Chinese Society of Gastroenterology, Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine and Blackwell Publishing Asia Pty Ltd.

Comprehensive Analysis of Temporal Alterations in Cellular Proteome of Bacillus subtilis under Curcumin Treatment

PubMed Central

Reddy, Panga Jaipal; Sinha, Sneha; Ray, Sandipan; Sathe, Gajanan J.; Chatterjee, Aditi; Prasad, T. S. Keshava; Dhali, Snigdha; Srikanth, Rapole; Panda, Dulal; Srivastava, Sanjeeva

2015-01-01

Curcumin is a natural dietary compound with antimicrobial activity against various gram positive and negative bacteria. This study aims to investigate the proteome level alterations in Bacillus subtilis due to curcumin treatment and identification of its molecular/cellular targets to understand the mechanism of action. We have performed a comprehensive proteomic analysis of B. subtilis AH75 strain at different time intervals of curcumin treatment (20, 60 and 120 min after the drug exposure, three replicates) to compare the protein expression profiles using two complementary quantitative proteomic techniques, 2D-DIGE and iTRAQ. To the best of our knowledge, this is the first comprehensive longitudinal investigation describing the effect of curcumin treatment on B. subtilis proteome. The proteomics analysis revealed several interesting targets such UDP-N-acetylglucosamine 1-carboxyvinyltransferase 1, putative septation protein SpoVG and ATP-dependent Clp protease proteolytic subunit. Further, in silico pathway analysis using DAVID and KOBAS has revealed modulation of pathways related to the fatty acid metabolism and cell wall synthesis, which are crucial for cell viability. Our findings revealed that curcumin treatment lead to inhibition of the cell wall and fatty acid synthesis in addition to differential expression of many crucial proteins involved in modulation of bacterial metabolism. Findings obtained from proteomics analysis were further validated using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) assay for respiratory activity, resazurin assay for metabolic activity and membrane integrity assay by potassium and inorganic phosphate leakage measurement. The gene expression analysis of selected cell wall biosynthesis enzymes has strengthened the proteomics findings and indicated the major effect of curcumin on cell division. PMID:25874956

17beta-estradiol promotes the odonto/osteogenic differentiation of stem cells from apical papilla via mitogen-activated protein kinase pathway.

PubMed

Li, Yao; Yan, Ming; Wang, Zilu; Zheng, Yangyu; Li, Junjun; Ma, Shu; Liu, Genxia; Yu, Jinhua

2014-11-17

Estrogen plays an important role in the osteogenic differentiation of mesenchymal stem cells, while stem cells from apical papilla (SCAP) can contribute to the formation of dentin/bone-like tissues. To date, the effects of estrogen on the differentiation of SCAP remain unclear. SCAP was isolated and treated with 10⁻⁷ M 17beta-estradiol (E2). The odonto/osteogenic potency and the involvement of mitogen-activated protein kinase (MAPK) signaling pathway were subsequently investigated by using methyl-thiazolyl-tetrazolium (MTT) assay, and other methods. MTT and flow cytometry results demonstrated that E2 treatment had no effect on the proliferation of SCAP in vitro, while alkaline phosphatase (ALP) assay and alizarin red staining showed that E2 can significantly promote ALP activity and mineralization ability in SCAP. Real-time reverse transcription polymerase chain reaction (RT-PCR) and western blot assay revealed that the odonto/osteogenic markers (ALP, DMP1/DMP1, DSPP/DSP, RUNX2/RUNX2, OSX/OSX and OCN/OCN) were significantly upregulated in E2-treated SCAP. In addition, the expression of phosphor-p38 and phosphor-JNK in these stem cells was enhanced by E2 treatment, as was the expression of the nuclear downstream transcription factors including phosphor-Sp1, phosphor-Elk-1, phosphor-c-Jun and phosphor-c-Fos, indicating the activation of MAPK signaling pathway during the odonto/osteogenic differentiation of E2-treated SCAP. Conversely, the differentiation of E2-treated SCAP was inhibited in the presence of MAPK specific inhibitors. The ondonto/osteogenic differentiation of SCAP is enhanced by 10⁻⁷ M 17beta-estradiol via the activation of MAPK signaling pathway.

Comprehensive analysis of temporal alterations in cellular proteome of Bacillus subtilis under curcumin treatment.

PubMed

Reddy, Panga Jaipal; Sinha, Sneha; Ray, Sandipan; Sathe, Gajanan J; Chatterjee, Aditi; Prasad, T S Keshava; Dhali, Snigdha; Srikanth, Rapole; Panda, Dulal; Srivastava, Sanjeeva

2015-01-01

Curcumin is a natural dietary compound with antimicrobial activity against various gram positive and negative bacteria. This study aims to investigate the proteome level alterations in Bacillus subtilis due to curcumin treatment and identification of its molecular/cellular targets to understand the mechanism of action. We have performed a comprehensive proteomic analysis of B. subtilis AH75 strain at different time intervals of curcumin treatment (20, 60 and 120 min after the drug exposure, three replicates) to compare the protein expression profiles using two complementary quantitative proteomic techniques, 2D-DIGE and iTRAQ. To the best of our knowledge, this is the first comprehensive longitudinal investigation describing the effect of curcumin treatment on B. subtilis proteome. The proteomics analysis revealed several interesting targets such UDP-N-acetylglucosamine 1-carboxyvinyltransferase 1, putative septation protein SpoVG and ATP-dependent Clp protease proteolytic subunit. Further, in silico pathway analysis using DAVID and KOBAS has revealed modulation of pathways related to the fatty acid metabolism and cell wall synthesis, which are crucial for cell viability. Our findings revealed that curcumin treatment lead to inhibition of the cell wall and fatty acid synthesis in addition to differential expression of many crucial proteins involved in modulation of bacterial metabolism. Findings obtained from proteomics analysis were further validated using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) assay for respiratory activity, resazurin assay for metabolic activity and membrane integrity assay by potassium and inorganic phosphate leakage measurement. The gene expression analysis of selected cell wall biosynthesis enzymes has strengthened the proteomics findings and indicated the major effect of curcumin on cell division.

Isothiocyanates Reduce Mercury Accumulation via an Nrf2-Dependent Mechanism during Exposure of Mice to Methylmercury

PubMed Central

Toyama, Takashi; Shinkai, Yasuhiro; Yasutake, Akira; Uchida, Koji; Yamamoto, Masayuki

2011-01-01

Background: Methylmercury (MeHg) exhibits neurotoxicity through accumulation in the brain. The transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) plays an important role in reducing the cellular accumulation of MeHg. Objectives: We investigated the protective effect of isothiocyanates, which are known to activate Nrf2, on the accumulation of mercury after exposure to MeHg in vitro and in vivo. Methods: We used primary mouse hepatocytes in in vitro experiments and mice as an in vivo model. We used Western blotting, luciferase assays, atomic absorption spectrometry assays, and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assays, and we identified toxicity in mice based on hind-limb flaccidity and mortality. Results: The isothiocyanates 6-methylsulfinylhexyl isothiocyanate (6-HITC) and sulforaphane (SFN) activated Nrf2 and up-regulated downstream proteins associated with MeHg excretion, such as glutamate-cysteine ligase, glutathione S-transferase, and multidrug resistance–associated protein, in primary mouse hepatocytes. Under these conditions, intracellular glutathione levels increased in wild-type but not Nrf2-deficient primary mouse hepatocytes. Pretreatment with 6-HITC and SFN before MeHg exposure suppressed cellular accumulation of mercury and cytotoxicity in wild-type but not Nrf2-deficient primary mouse hepatocytes. In comparison, in vivo administration of MeHg to Nrf2-deficient mice resulted in increased sensitivity to mercury concomitant with an increase in mercury accumulation in the brain and liver. Injection of SFN before administration of MeHg resulted in a decrease in mercury accumulation in the brain and liver of wild-type, but not Nrf2-deficient, mice. Conclusions: Through activation of Nrf2, 6-HITC and SFN can suppress mercury accumulation and intoxication caused by MeHg intake. PMID:21382770

Anti-tumor activity of the ATR inhibitor AZD6738 in HER2 positive breast cancer cells.

PubMed

Kim, Hee-Jun; Min, Ahrum; Im, Seock-Ah; Jang, Hyemin; Lee, Kyung Hun; Lau, Alan; Lee, Miso; Kim, Seongyeong; Yang, Yaewon; Kim, Jungeun; Kim, Tae Yong; Oh, Do-Youn; Brown, Jeffrey; O'Connor, Mark J; Bang, Yung-Jue

2017-01-01

Ataxia telangiectasia and Rad3-related (ATR) proteins are sensors of DNA damage, which induces homologous recombination (HR)-dependent repair. ATR is a master regulator of DNA damage repair (DDR), signaling to control DNA replication, DNA repair and apoptosis. Therefore, the ATR pathway might be an attractive target for developing new drugs. This study was designed to investigate the antitumor effects of the ATR inhibitor, AZD6738 and its underlying mechanism in human breast cancer cells. Growth inhibitory effects of AZD6738 against human breast cancer cell lines were studied using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (methyl thiazolyl tetrazolium, MTT) assay. Cell cycle analysis, Western blotting, immunofluorescence and comet assays were also performed to elucidate underlying mechanisms of AZD6738 action. Anti-proliferative and DDR inhibitory effects of AZD6738 were demonstrated in human breast cancer cell lines. Among 13 cell lines, the IC 50 values of nine cell lines were less than 1 μmol/L using MTT assay. Two cell lines, SK-BR-3 and BT-474, were chosen for further evaluation focused on human epidermal growth factor receptor 2 (HER2)-positive breast cancer cells. Sensitive SK-BR-3 but not the less sensitive BT-474 breast cancer cells showed increased level of apoptosis and S phase arrest and reduced expression levels of phosphorylated check-point kinase 1 (CHK1) and other repair markers. Decreased functional CHK1 expression induced DNA damage accumulation due to HR inactivation. AZD6738 showed synergistic activity with cisplatin. Understanding the antitumor activity and mechanisms of AZD6738 in HER2-positive breast cancer cells creates the possibility for future clinical trials targeting DDR in HER2-positive breast cancer treatment. © 2016 UICC.

In vitro antimicrobial activity of natural toxins and animal venoms tested against Burkholderia pseudomallei

PubMed Central

Perumal Samy, R; Pachiappan, A; Gopalakrishnakone, P; Thwin, Maung M; Hian, Yap E; Chow, Vincent TK; Bow, Ho; Weng, Joseph T

2006-01-01

Background Burkholderia pseudomallei are the causative agent of melioidosis. Increasing resistance of the disease to antibiotics is a severe problem in treatment regime and has led to intensification of the search for new drugs. Antimicrobial peptides are the most ubiquitous in nature as part of the innate immune system and host defense mechanism. Methods Here, we investigated a group of venoms (snakes, scorpions and honey bee venoms) for antimicrobial properties against two strains of Gram-negative bacteria Burkholderia pseudomallei by using disc-diffusion assay for in vitro susceptibility testing. The antibacterial activities of the venoms were compared with that of the isolated L-amino acid oxidase (LAAO) and phospholipase A2 (PLA2s) enzymes. MICs were determined using broth dilution method. Bacterial growth was assessed by measurement of optical density at the lowest dilutions (MIC 0.25 mg/ml). The cell viability was measured using tetrazolium salts (XTT) based cytotoxic assay. Results The studied venoms showed high antimicrobial activity. The venoms of C. adamanteus, Daboia russelli russelli, A. halys, P. australis, B. candidus and P. guttata were equally as effective as Chloramphenicol and Ceftazidime (30 μg/disc). Among those tested, phospholipase A2 enzymes (crotoxin B and daboiatoxin) showed the most potent antibacterial activity against Gram-negative (TES) bacteria. Naturally occurring venom peptides and phospholipase A2 proved to possess highly potent antimicrobial activity against Burkholderia pseudomallei. The XTT-assay results showed that the cell survival decreased with increasing concentrations (0.05–10 mg/mL) of Crotalus adamanteus venom, with no effect on the cell viability evident at 0.5 mg/mL. Conclusion This antibacterial profile of snake venoms reported herein will be useful in the search for potential antibacterial agents against drug resistant microorganisms like B. pseudomallei. PMID:16784542

Acupuncture inhibits the decrease in brain catecholamine contents and the impairment of passive avoidance task in ovariectomized mice.

PubMed

Toriizuka, K; Okumura, M; Iijima, K; Haruyama, K; Cyong, J C

1999-01-01

The effects of acupuncture on the disorders elicited by abnormalities of endocrine system were investigated in ovariectomized mice. Female mice (strain; C57BL/6) were ovariectomized (OVX) and acupuncture points, Shenshu ([Japanese pictograph see text] : BL23) on both side of the back were continuously stimulated by subcutaneous needles for 20 days. After completion of experimental sessions, animals were sacrificed and specific brain regions were assayed for catecholamine contents by high performance liquid chromatography with electro chemical detector (ECD-HPLC). The mitogenic activities of splenic lymphocytes were measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTS) assay and alkaline phosphatase (ALP) assay. Furthermore, the effects of needle stimulation on learning and memory ability were studied by the step-through type passive avoidance test. Norepinephrine and dopamine contents in the frontoparietal cerebral cortex, ventral hippocampus and olfactory bulb were decreased in the OVX group, and both MTS activity and ALP activity were decreased 20 days after ovariectomy. The mean latent period was also shortened in the passive avoidance test in the OVX group. However, applying needle stimulation increased norepinephrine and dopamine contents in the brain regions, and enhanced mitogenic activities of splenic lymphocytes. The stimulation also improved memory-related behavior. It was concluded from this study that after mice were stimulated by subcutaneous needle insertion, overall changes were observed in central nervous system (including retention of memory) and immune functions. The study suggests that acupuncture improves the memory loss and decrease of immune responses accompanying aging and/or menopause, and the that it may have an important role in medical care for the elderly.

Mitomycin C induces apoptosis in cultured corneal fibroblasts derived from type II granular corneal dystrophy corneas.

PubMed

Kim, Tae-im; Choi, Seung-il; Lee, Hyung Keun; Cho, Young Jae; Kim, Eung Kweon

2008-06-30

The present study investigated the effect of mitomycin C (MMC) on cell viability, apoptosis, and transforming growth factor beta-induced protein (TGFBIp) expression in cultured normal corneal fibroblasts and heterozygote or homozygote granular corneal dystrophy type II (GCD II) corneal fibroblasts. Keratocytes were obtained from normal cornea or from heterozygote or homozygote GCD II patients after lamellar or penetrating keratoplasty. To measure cell viability, corneal fibroblasts were incubated with 0.02% MMC for 3 h, 6 h, and 24 h or with 0%, 0.01%, 0.02%, and 0.04% MMC for 24 h and then tested using lactate dehydrogenase (LDH) and 3-[4,5-demethylthiazol-2,5-diphenyl-2H-tetrazolium bromide] (MTT) assays. To measure apoptosis, cells were analyzed by FACS analysis and annexin V staining. Bcl-xL, Bax, and TGFBI mRNA expression was measured using reverse transcription polymerase chain reaction (RT-PCR) assays. Cellular and media levels of TGFBIp protein were measured by immunoblotting. MTT and LDH assays showed that MMC reduced cell viability in all three cell types in a dose-dependent and time-dependent manner (p<0.05). FACS analysis and annexin V staining showed that MMC caused apoptosis with GCD II homozygote cells being most affected. RT-PCR analysis showed that MMC decreased Bcl-xL mRNA expression and increased Bax mRNA expression in all cell types. RT-PCR and immunoblotting analysis showed that MMC reduced TGFBI mRNA levels and cellular and media TGFBIp protein levels in all cell types. MMC induced apoptosis, and the effects of MMC were greatest in GCD II homozygote cells. MMC also reduced the production of TGFBIp in all three types of corneal fibroblasts. These findings may explain the additional therapeutic effect of MMC in GCD II patients.

The effect of growth hormone on fibroblast proliferation and keratinocyte migration.

PubMed

Lee, Sang Woo; Kim, Suk Hwa; Kim, Ji Youn; Lee, Yoonho

2010-04-01

The beneficial effects of growth hormones (GHs) on wound healing have been reported. Although the mechanism of how GH promotes wound healing is unclear, there are reports showing that the principal factor lies in the GH-stimulated production of IGF-1 in topical wounds. In this study, a human primary cell model was devised to examine how the topical application of GHs affects fibroblast proliferation and keratinocyte migration, which play fundamental roles in wound healing. The fibroblasts were cultured in media with different concentrations of GH. The amount of fibroblast proliferation was assessed using a tetrazolium-based colourimetric assay (MTT assay). The amount of newly formed IGF-I mRNA was measured by reverse transcription and polymerase chain reaction (RT-PCR). Keratinocyte migration was compared using a migration assay. Fibroblast proliferation was significantly higher in the experimental group than in the control group (the absorbance of 2.5IU L(-1) GH applied group: 0.3954+/-0.056, control group: 0.2943+/-0.0554, P<0.05), and the promotion of IGF-I formation by fibroblasts was observed. There was more keratinocyte migration in the experimental group than in the control group (the remaining gap in the 2.5IU L(-1) GH applied group after keratinocyte migration: 46.57+/-2.22% of the primary gap, control group: 75.14+/-3.44%, P<0.05). GH enhances the local formation of IGF-1, which activates fibroblast proliferation and keratinocyte migration. These results highlight the potential of the topical application of GHs in the treatment of wounds. Copyright 2009 British Association of Plastic, Reconstructive and Aesthetic Surgeons. All rights reserved.

Bioactivity and biocompatibility of hydroxyapatite-based bioceramic coatings on zirconium by plasma electrolytic oxidation.

PubMed

Aktuğ, Salim Levent; Durdu, Salih; Yalçın, Emine; Çavuşoğlu, Kültigin; Usta, Metin

2017-02-01

In the present work, hydroxyapatite (HAP)-based plasma electrolytic oxide (PEO) coatings were produced on zirconium at different current densities in a solution containing calcium acetate and β-calcium glycerophosphate by a single step. The phase structure, surface morphology, functional groups, thickness and roughness of the coatings were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), eddy current method and surface profilometer, respectively. The phases of cubic-zirconia, calcium zirconate and HAP were detected by XRD. The amount of HAP and calcium zirconate increased with increasing current density. The surface of the coatings was very porous and rough. Moreover, bioactivity and biocompatibility of the coatings were analyzed in vitro immersion simulated body fluid (SBF) and MTT (3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium bromide) assay, hemolysis assay and bacterial formation. The apatite-forming ability of the coatings was evaluated after immersion in SBF up to 28days. After immersion, the bioactivity of HAP-based coatings on zirconium was greater than the ones of uncoated zirconium and zirconium oxide-based surface. The bioactivity of PEO surface on zirconium was significantly improved under SBF conditions. The bacterial adhesion of the coatings decreased with increasing current density. The bacterial adhesion of the coating produced at 0.370A/cm 2 was minimum compared to uncoated zirconium coated at 0.260 and 0.292A/cm 2 . The hemocompatibility of HAP-based surfaces was improved by PEO. The cell attachment and proliferation of the PEO coatings were better than the one of uncoated zirconium according to MTT assay results. Copyright © 2016 Elsevier B.V. All rights reserved.

Dynamic perfusion bioreactor system for 3D culture of rat bone marrow mesenchymal stem cells on nanohydroxyapatite/polyamide 66 scaffold in vitro.

PubMed

Qian, Xu; Yuan, Fang; Zhimin, Zhu; Anchun, Mo

2013-08-01

The aim of the study was to investigate the biocompatibility and osteogenic effectiveness of the porous nanohydroxyapatite/polyamide 66 (n-HA/PA66) scaffold material that was cultured with the rat bone marrow mesenchymal stem cells (rBMSCs), under the static culture condition and the dynamic perfusion culture condition in vitro, and to investigate whether the 3D perfusion culture condition was better in provoking proliferation of rBMSCs than the 3D static culture condition. The Methyl thiazolyl tetrazolium (MTT) assay, alkaline phosphatase (ALP) activity assay, Osteocalcin (OCN) assay and scanning electron microscope (SEM) were used to observe the proliferation and differentiation of rBMSCs. The samples were respectively harvested at 1st, 3rd, 7th, 14th, and 21st days and effect comparisons were made between the two of the culture conditions. The results showed that values of MTT, ALP, and OCN were increased continuously and revealed a significant difference between the two culture conditions (p < 0.05). On the 14th day, SEM revealed calcified nodules 2-8 μm in diameter in the lamellar structure. Under the static culture condition, the pores were covered with the cells looking like a piece of blanket, but under the perfusion culture condition the cells were observed to have a 3D lamellar structure. In conclusion, the porous n-HA/PA66 scaffold material can be used as a good candidate material for the bone scaffold construction in the tissue engineering because of its excellent 3D structure, which can greatly improve the proliferation and differentiation of rBMSCs and make them proliferate and osteogenesis even better under the perfusion culture condition. Copyright © 2013 Wiley Periodicals, Inc.

In Vitro Effects of Preserved and Unpreserved Anti-Allergic Drugs on Human Corneal Epithelial Cells

PubMed Central

Calvo, Patricia; Ropero, Inés; Pintor, Jesús

2014-01-01

Abstract Purpose: Treatment with topical eye drops for long-standing ocular diseases like allergy can induce detrimental side effects. The purpose of this study was to investigate in vitro cytotoxicity of commercially preserved and unpreserved anti-allergic eye drops on the viability and barrier function of monolayer and stratified human corneal-limbal epithelial cells. Methods: Cells were treated with unpreserved ketotifen solution, benzalkonium chloride (BAC)-containing anti-allergic drugs (ketotifen, olopatadine, levocabastine) as well as BAC alone. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to determine cell viability. Effects of compounds on barrier function were analyzed measuring transepithelial electrical resistance (TEER) to determine paracellular permeability and rose bengal assays to evaluate transcellular barrier formation. Results: The BAC-preserved anti-allergic formulations and BAC alone significantly reduced cell viability, monolayer cultures being more sensitive to damage by these solutions. Unpreserved ketotifen induced the least diminution in cell viability. The extent of decrease of cell viability was clearly dependent of BAC presence, but it was also affected by the different types of drugs when the concentration of BAC was low and the short time of exposure. Treatment with BAC-containing anti-allergic drugs and BAC alone resulted in increased paracellular permeability and loss of transcellular barrier function as indicated by TEER measurement and rose bengal assays. Conclusions: The presence of the preservative BAC in anti-allergic eye drop formulations contributes importantly to the cytotoxic effects induced by these compounds. Stratified cell cultures seem to be a more relevant model for toxicity evaluation induced on the ocular surface epithelia than monolayer cultures. PMID:25100331

Honey dilution impact on in vitro wound healing: Normoxic and hypoxic condition.

PubMed

Chaudhary, Amrita; Bag, Swarnendu; Barui, Ananya; Banerjee, Provas; Chatterjee, Jyotirmoy

2015-01-01

Honey is known as a popular healing agent against tropical infections and wounds. However, the effects of honey dilutions on keratinocyte (HaCaT) wound healing under hypoxic condition is still not explored. In this study, we examined whether honey dilution have wound healing potential under hypoxic stress. The antioxidant potential and healing efficacy of honey dilution on in vitro wound of human epidermal keratinocyte (HaCaT cells) under hypoxia (3% O2 ), and normoxia is explored by nitro blue tetrazolium assay. The cell survival % quantified by MTT assay to select four honey dilutions like 10, 1, 0.1, and 0.01 v/v% and the changes in cellular function was observed microscopically. Further, the cell proliferation, migration, cell-cell adhesion, and relevant gene expression were studied by flow cytometry, migration/scratch assay, immunocytochemistry, and reverse transcription-polymerase chain reaction, respectively. The expression pattern of cardinal molecular features viz. E-cadherin, cytoskeletal protein F-actin, p63, and hypoxia marker Hif 1α were examined. Honey dilution in 0.1% v/v combat wound healing limitations in vitro under normoxia and hypoxia (3%). Its wound healing potential was quantified by immunocytochemistry and real-time PCR for the associated molecular features that were responsible for cell proliferation and migration. Our data showed that honey dilution can be effective in hypoxic wound healing. Additionally, it reduced superoxide generation and supplied favorable bioambience for cell proliferation, migration, and differentiation during hypoxic wound healing. These findings may reveal the importance of honey as an alternative and cost effective therapeutic natural product for wound healing in hypoxic condition. © 2015 by the Wound Healing Society.

Anticancer activity of flavonol and flavan-3-ol rich extracts from Croton celtidifolius latex.

PubMed

Biscaro, Fernanda; Parisotto, Eduardo Benedetti; Zanette, Vanilde Citadini; Günther, Tania Mara Fischer; Ferreira, Eduardo Antonio; Gris, Eliana Fortes; Correia, João Francisco Gomes; Pich, Claus Tröger; Mattivi, Fulvio; Filho, Danilo Wilhelm; Pedrosa, Rozangela Curi

2013-06-01

Croton celtidifolius Baill (Euphorbiaceae) is a tree found in the Atlantic Forest in Southern Brazil, where it is commonly known as "Sangue-de-Dragão". Its red latex is used traditionally for treating ulcers, diabetes and cancer. To evaluate antitumor activities of Croton celtififolius latex in vitro and in vivo. Phytochemical analyses were conducted using HPLC-DAD-MS. Cytotoxic, nuclease and pro-apoptotic properties were determined using the tetrazolium salt assay (MTT), plasmid DNA damage assay and ethidium bromide (EB)/acridine orange methods, respectively, and antitumor activity was determined in the Ehrlich ascites carcinoma (EAC) mouse model. Phytochemical studies indicated a high phenol content of flavonols (45.67 ± 0.24 and 18.01 ± 0.23 mg/mL of myricetin and quercetin, respectively) and flavan-3-ols (114.12 ± 1.84 and 1527.41 ± 16.42 mg/L of epicatechin and epigallocatechin, respectively) in latex. These compounds reduced MCF-7 and EAC cell viability in the MTT assay (IC50 = 169.0 ± 1.8 and 187.0 ± 2.2 μg/mL, respectively). Latex compounds caused significant DNA fragmentation and increased the number of apoptotic cells (negative control (NC), 12%; latex, 41%) as indicated by differential staining in the EB/acridine orange assay. The in vivo latex treatment at 3.12 mg/kg/day reduced the body weight by 7.57 ± 2.04 g and increased median survival time to 17.5 days when compared to the NC group (13.0 days). In addition, the highest latex concentration inhibited tumor growth by 56%. These results agree with ethno-pharmacological reports showing cytotoxicity and antitumor activity of C. celtidifolius latex. The mechanism of antitumor action may be related to direct DNA fragmentation that reduces survival and induces apoptosis.

Influence of silicon doping of titanium nickelide near-surface layers on alloy cytocompatibility

NASA Astrophysics Data System (ADS)

Lotkov, A. I.; Matveev, A. L.; Artemyeva, L. V.; Meysner, S. N.; Matveeva, V. A.; Kudryashov, A. N.

2017-12-01

The cytocompatibility of titanium nickelide (TiNi) with near-surface layers doped with silicon ions was studied on mesenchymal stem cells of rat bone marrow cultivated in vitro. The cytotoxic effect of eluted components of material on the mesenchymal stem cells was determined using a RTCA iCELLigence cellular analyzer. The proliferative activity of mesenchymal stem cells cultivated in the presence or on the surfaces of titanium nickelide samples was estimated from the cell mitochondrial respiration rate in MTT tests using [2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium] tetrazolium salt. It is shown that ion plasma modification of near-surface layers of titanium nickelide with silicon improves the cytocompatibility of the alloy.

Microrespirometer chamber for determinations of viability in cell and organ cultures.

PubMed Central

Gabridge, M G

1976-01-01

The effects of chemical, physical, and infectious cytotoxic agents on primary and cultured cells were evaluated by measurements of oxygen uptake for various time periods. A newly developed respirometer used a Clark oxygen electrode in a 1.0-ml chamber, with provisions for constant mixing and for temperature control of both the sample and electrode chambers. The device was unique because the electrode and instrumentation were provided by a clinical blood-gas analyzer. Oxygen uptake by blank controls was negligible, whereas cells and tissue consumed oxygen at rates of approximately 1 to 5 mul/h in a dose- and temperature-dependent fashion. Cyanide, heat, and freeze-thaw lysis reduced the oxygen uptake to less than 0.6 mul/mg per h. Infection of trachea organ cultures with Mycoplasma pneumoniae significantly reduced relative ciliary activity, tetrazolium reduction capacity, and oxygen consumption in a coordinated fashion. Images PMID:985826

Resveratrol protects primary cortical neuron cultures from transient oxygen-glucose deprivation by inhibiting MMP-9.

PubMed

Gao, Dakuan; Huang, Tao; Jiang, Xiaofan; Hu, Shijie; Zhang, Lei; Fei, Zhou

2014-06-01

It was recently shown that resveratrol exerts neuroprotective effects against cerebral ischemia in mice. The aim of the present study was to further confirm these effects in in vitro primary cortical neuron cultures with transient oxygen-glucose deprivation (OGD), and to investigate whether these effects are due to the inhibition of matrix metalloproteinase-9 (MMP-9) and of cell apoptosis. Neuronal primary cultures of cerebral cortex were prepared from BALB/c mice embryos (13-15 days). Cells from 14- to 16-day cultures were subjected to OGD for 3 h, followed by 21 h of reoxygenation to simulate transient ischemia. Different doses of resveratrol were added into the culture medium during the simulation of transient ischemia. The effect of the extracellular signal-regulated kinase (ERK) inhibitor U0126 was studied by adding U0126 (5 µg/µl, 4 µl) into the culture medium during transient ischemia; as a control, we used treatment of cells with 50 µM of resveratrol. Cell viability was investigated using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) reduction assay. Cell apoptosis was assessed by flow cytometry. The effects of resveratrol on the expression of MMP-9 were analyzed by western blotting and reverse transcription-polymerase chain reaction (RT-PCR), while the levels of ERK, phosphorylated (p)-ERK, cleaved caspase-3, Bax and Bcl-2 were measured by western blotting. The results of the MTT assay showed that cell viability is significantly reduced by transient OGD. OGD induced cell apoptosis, the expression of Bax and the activation of caspase-3 and ERK, inhibited the expression of Bcl-2 and increased the expression of MMP-9, while these effects were reversed by treatment with resveratrol. The therapeutic efficacy of resveratrol was shown to be dose-dependent, with the most suitable dose range determined at 50-100 µM. Treatment with U0126 inhibited MMP-9 and Bax expression and caspase-3 activation, while it further promoted the expression of the anti-apoptotic molecule Bcl-2, suggesting that resveratrol inhibits MMP-9 expression and cell apoptosis by attenuating the activation of ERK1/2. In conclusion, OGD can induce apoptosis through canonical apoptotic signals and by regulating the expression of MMP-9; the anti-apoptotic activity of resveratrol and its inhibitory effect on MMP-9 expression contribute in the reduced activation of ERK.

Modified Polymeric Nanoparticles Exert In Vitro Antimicrobial Activity Against Oral Bacteria.

PubMed

Toledano-Osorio, Manuel; Babu, Jegdish P; Osorio, Raquel; Medina-Castillo, Antonio L; García-Godoy, Franklin; Toledano, Manuel

2018-06-14

Polymeric nanoparticles were modified to exert antimicrobial activity against oral bacteria. Nanoparticles were loaded with calcium, zinc and doxycycline. Ions and doxycycline release were measured by inductively coupled plasma optical emission spectrometer and high performance liquid chromatography. Porphyromonas gingivalis , Lactobacillus lactis , Streptoccocus mutans , gordonii and sobrinus were grown and the number of bacteria was determined by optical density. Nanoparticles were suspended in phosphate-buffered saline (PBS) at 10, 1 and 0.1 mg/mL and incubated with 1.0 mL of each bacterial suspension for 3, 12, and 24 h. The bacterial viability was assessed by determining their ability to cleave the tetrazolium salt to a formazan dye. Data were analyzed by ANOVA and Scheffe’s F ( p < 0.05). Doxycycline doping efficacy was 70%. A burst liberation effect was produced during the first 7 days. After 21 days, a sustained release above 6 µg/mL, was observed. Calcium and zinc liberation were about 1 and 0.02 µg/mL respectively. The most effective antibacterial material was found to be the Dox-Nanoparticles (60% to 99% reduction) followed by Ca-Nanoparticles or Zn-Nanoparticles (30% to 70% reduction) and finally the non-doped nanoparticles (7% to 35% reduction). P. gingivalis , S. mutans and L. lactis were the most susceptible bacteria, being S. gordonii and S. sobrinus the most resistant to the tested nanoparticles.

Schisandrin B protects PC12 cells by decreasing the expression of amyloid precursor protein and vacuolar protein sorting 35★

PubMed Central

Yan, Mingmin; Mao, Shanping; Dong, Huimin; Liu, Baohui; Zhang, Qian; Pan, Gaofeng; Fu, Zhiping

2012-01-01

PC12 cell injury was induced using 20 μM amyloid β-protein 25–35 to establish a model of Alzheimer's disease. The cells were then treated with 5, 10, and 25 μM Schisandrin B. Methylthiazolyldiphenyl-tetrazolium bromide assays and Hoechst 33342 staining results showed that with increasing Schisandrin B concentration, the survival rate of PC12 cells injured by amyloid β-protein 25–35 gradually increased and the rate of apoptosis gradually decreased. Reverse transcription-PCR, immunocytochemical staining and western blot results showed that with increasing Schisandrin B concentration, the mRNA and protein expression of vacuolar protein sorting 35 and amyloid precursor protein were gradually decreased. Vacuolar protein sorting 35 and amyloid precursor protein showed a consistent trend for change. These findings suggest that 5, 10, and 25 μM Schisandrin B antagonizes the cellular injury induced by amyloid β-protein 25–35 in a dose-dependent manner. This may be caused by decreasing the expression of vacuolar protein sorting 35 and amyloid precursor protein. PMID:25745458

Novel T-cell epitopes of ovalbumin in BALB/c mouse: Potential for peptide-immunotherapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Marie; Mine, Yoshinori

The identification of food allergen T-cell epitopes provides a platform for the development of novel immunotherapies. Despite extensive knowledge of the physicochemical properties of hen ovalbumin (OVA), a major egg allergen, the complete T-cell epitope map of OVA has surprisingly not been defined in the commonly used BALB/c mouse model. In this study, spleen cells obtained from OVA-sensitized mice were incubated in the presence of 12-mer overlapping synthetic peptides, constructed using the SPOTS synthesis method. Proliferative activity was assessed by 72-h in vitro assays with use of the tetrazolium salt WST-1 and led to identification of four mitogenic sequences, i.e.,more » A39R50, S147R158, K263E274, and A329E340. ELISA analyses of interferon (IFN)-{gamma} and interleukin (IL)-4 productions in cell culture supernatants upon stimulation with increasing concentrations of peptides confirmed their immunogenicity. Knowledge of the complete T-cell epitope map of OVA opens the way to a number of experimental investigations, including the exploration of peptide-based immunotherapy.« less

Activin A prevents neuron-like PC12 cell apoptosis after oxygen-glucose deprivation☆

PubMed Central

Xu, Guihua; He, Jinting; Guo, Hongliang; Mei, Chunli; Wang, Jiaoqi; Li, Zhongshu; Chen, Han; Mang, Jing; Yang, Hong; Xu, Zhongxin

2013-01-01

In this study, PC12 cells were induced to differentiate into neuron-like cells using nerve growth factor, and were subjected to oxygen-glucose deprivation. Cells were treated with 0, 10, 20, 30, 50, 100 ng/mL exogenous Activin A. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay and Hoechst 33324 staining showed that the survival percentage of PC12 cells significantly decreased and the rate of apoptosis significantly increased after oxygen-glucose deprivation. Exogenous Activin A significantly increased the survival percentage of PC12 cells in a dose-dependent manner. Reverse transcription-PCR results revealed a significant increase in Activin receptor IIA, Smad3 and Smad4 mRNA levels, which are key sites in the Activin A/Smads signaling pathway, in neuron-like cells subjected to oxygen-glucose deprivation, while mRNA expression of the apoptosis-regulation gene caspase-3 decreased. Our experimental findings indicate that exogenous Activin A plays an anti-apoptotic role and protects neurons by means of activating the Activin A/Smads signaling pathway. PMID:25206395

Effects on DNA repair in human lymphocytes exposed to the food dye tartrazine yellow.

PubMed

Soares, Bruno Moreira; Araújo, Taíssa Maíra Thomaz; Ramos, Jorge Amando Batista; Pinto, Laine Celestino; Khayat, Bruna Meireles; De Oliveira Bahia, Marcelo; Montenegro, Raquel Carvalho; Burbano, Rommel Mario Rodríguez; Khayat, André Salim

2015-03-01

Tartrazine is a food additive that belongs to a class of artificial dyes and contains an azo group. Studies about its genotoxic, cytotoxic and mutagenic effects are controversial and, in some cases, unsatisfactory. This work evaluated the potential in vitro cytotoxicity, genotoxicity and effects on DNA repair of human lymphocytes exposed to the dye. We assessed the cytotoxicity of tartrazine by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide test and the response of DNA repair through comet assay (alkaline version). We used different concentrations of the dye, ranging from 0.25-64.0 mM. The results demonstrated that tartrazine has no cytotoxic effects. However, this dye had a significant genotoxic effect at all concentrations tested. Although most of the damage was amenable to repair, some damage remained higher than positive control after 24 h of repair. These data demonstrate that tartrazine may be harmful to health and its prolonged use could trigger carcinogenesis. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

The effects of cetrorelix and triptorelin on the viability and steroidogenesis of cultured human granulosa luteinized cells.

PubMed

Metallinou, Chryssa; Köster, Frank; Diedrich, Klaus; Nikolettos, Nikos; Asimakopoulos, Byron

2012-01-01

We investigated the effects of the gonadotropin-releasing hormone (GnRH) agonist triptorelin as well the GnRH antagonist cetrorelix those of on the viability and steroidogenesis in human granulosa luteinized (hGL) cell cultures. The hGL cells were obtained from 34 women undergoing ovarian stimulation for IVF treatment. The cells were cultured for 48 h with or without 1 nM or 3 nM of cetrorelix or triptorelin in serum-free media. The cell viability was evaluated by the MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay. The concentrations of estradiol and progesterone in culture supernatants were measured by ELISA. Treatment with triptorelin slightly increased cell viability, whereas treatment with 3 nM cetrorelix led to a significant decrease. Estradiol concentrations were reduced with 3 nM triptorelin. Cultures treated with high-dose of either cetrorelix or triptorelin tended to secrete less progesterone than controls. Cetrorelix significantly reduces the viability of hGL cells. Triptorelin and cetrorelix may have minor effects on steroidogenesis. These results suggest that GnRH analogues may influence ovarian functions.

Synthesis and in vitro evaluation of a pH-sensitive PLA-PEG-folate based polymeric micelle for controlled delivery of docetaxel.

PubMed

Hami, Zahra; Amini, Mohsen; Ghazi-Khansari, Mahmoud; Rezayat, Seyed Mehdi; Gilani, Kambiz

2014-04-01

pH-responsive docetaxel-conjugated poly (lactic acid) (PLA)-polyethyleneglycol (PEG) micellar formulation was synthesized via acid labile hydrazone linkage. Levulinic acid (LEV) was used as a linker between docetaxel (DTX) and hydrazine. Targeted delivery of DTX was achieved by conjugation of folate to PEG segment. The DTX conjugated polymeric micelles were about 181 nm in diameter and their critical micelle concentration was 5.18 μg/ml. DTX was released from micelles in a pH-dependent manner. The results showed a significant difference in DTX release from polymeric micelles at pH 5.0 and pH 7.4. Cytotoxicity assays using methyl tetrazolium (MTT), neutral red (NR) and lactate dehydrogenase (LDH) demonstrated a decreased cytotoxic activity of the drug containing nanoconjugate compared with free DTX that appears to be contributed to the sustained release of drug from micelles. Based on these results, it is expected that this pH-responsive nanoconjugate is promising as a useful carrier for targeted delivery of anticancer agents. Copyright © 2014 Elsevier B.V. All rights reserved.

DNA methyltransferase mediates dose-dependent stimulation of neural stem cell proliferation by folate.

PubMed

Li, Wen; Yu, Min; Luo, Suhui; Liu, Huan; Gao, Yuxia; Wilson, John X; Huang, Guowei

2013-07-01

The proliferative response of neural stem cells (NSCs) to folate may play a critical role in the development, function and repair of the central nervous system. It is important to determine the dose-dependent effects of folate in NSC cultures that are potential sources of transplantable cells for therapies for neurodegenerative diseases. To determine the optimal concentration and mechanism of action of folate for stimulation of NSC proliferation in vitro, NSCs were exposed to folic acid or 5-methyltetrahydrofolate (5-MTHF) (0-200 μmol/L) for 24, 48 or 72 h. Immunocytochemistry and methyl thiazolyl tetrazolium assay showed that the optimal concentration of folic acid for NSC proliferation was 20-40 μmol/L. Stimulation of NSC proliferation by folic acid was associated with DNA methyltransferase (DNMT) activation and was attenuated by the DNMT inhibitor zebularine, which implies that folate dose-dependently stimulates NSC proliferation through a DNMT-dependent mechanism. Based on these new findings and previously published evidence, we have identified a mechanism by which folate stimulates NSC growth. Copyright © 2013 Elsevier Inc. All rights reserved.

In vitro assessment of the genotoxic and cytotoxic effects of boiled juice (tucupi) from Manihot esculenta Crantz roots.

PubMed

Cunha, L A; Mota, T C; Cardoso, P C S; Alcântara, D D F Á; Burbano, R M R; Guimarães, A C; Khayat, A S; Rocha, C A M; Bahia, M O

2016-10-05

The population of Pará (a state in Brazil) has a very characteristic food culture, as a majority of the carbohydrates consumed are obtained from cassava (Manihot esculenta Crantz) derivatives. Tucupi is the boiled juice of cassava roots that plays a major role in the culinary footprint of Pará. Before boiling, this juice is known as manipueira and contains linamarin, a toxic glycoside that can decompose to hydrogen cyanide. In this study, the cytotoxic and genotoxic effects of tucupi on cultured human lymphocytes were assessed using the comet assay and detection of apoptosis and necrosis by differential fluorescent staining with acridine orange-ethidium bromide. Tucupi concentrations (v/v) were determined using the methylthiazole tetrazolium biochemical test. Concentrations of tucupi that presented no genotoxic effects (2, 4, 8, and 16%) were used in our experiments. The results showed that under our study conditions, tucupi exerted no genotoxic effects; however, cytotoxic effects were observed with cell death mainly induced by necrosis. These effects may be related to the presence of hydrogen cyanide in the juice.

Decursin prevents cisplatin-induced apoptosis via the enhancement of antioxidant enzymes in human renal epithelial cells.

PubMed

Kim, Jeong Hwan; Jeong, Soo-Jin; Kwon, Hee-Young; Park, Sang Yoon; Lee, Hyo-Jung; Lee, Hyo-Jeong; Lieske, John Charles; Kim, Sung-Hoon

2010-01-01

Adverse effects, nephrotoxicity and hepatotoxicity, of anticancer drugs such as cisplatin have limited the usage for cancer therapy. Therefore, development or identification of supplement agents in anticancer drugs is attractive to reduce side effects and enhance antitumor activity. Here, we found that decursin isolated from Angelica gigas showed protective effects of cisplatin-induced damage in normal human primary renal epithelial cells (HRCs). We found that decursin significantly blocked cisplatin-induced cytotoxicity by 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay in HRCs. Further, we found that decursin inhibited sub-G1 and cell death by suppression of cleavage of caspase-3, -9 and poly(ADP-ribose) polymerase (PARP) induced by cisplatin treatment in HRCs. Importantly, decursin effectively restored the activities of Cu/Zn superoxide dismutase (SOD), catalase and glutathione peroxidase in cisplatin-treated HRCs. Taken together, our findings demonstrate that decurcin prevents cisplatin-induced cytotoxicity and apoptosis through the activation of antioxidant enzymes in HRCs and suggest further that combination of decursin might suppressed adverse effects of anticancer drugs in cancer patients.

[Protective effect of amlodipine on the cytotoxicity induced by contrast media in human kidney cells].

PubMed

Zhou, Xiao-rong; Duan, Shao-bin; Peng, You-ming; Liu, Fu-you; Ye, Yun; Liu, Rui-hong; Li, Gui-yuan

2007-10-01

To explore the protective effect of amlodipine on the cytotoxicity induced by contrast media (meglumine diatrizoate) in human kidney cells (HKC). An HKC line was used. The experiment was divided into 4 groups: a model group (diatrizoate 111g/L), a prevention group (diatrizoate 111g/L+amlodipine 10(-5)mol/L), an amlodipine control group (amlodipine 10(-5)mol/L), and a culture medium control group (simple none blood serum DMEM-F12 medium). Cytotoxicity induced by meglumine diatrizoate was analysed by methyl thiazolyl tetrazolium (MTT) test, lactate dehydrogenase (LDH) assay, Hochest33258 fluorescence stained cytospins, and flow cytometric DNA analysis. The protein expression of Bax was determined by Western blot, and caspase-3 activity was examined by fluorometric method. In the prevention group, the cell viability increased significantly (P<0.05), LDH levels decreased (P<0.05), and the apoptosis was lower than that of the model group (P<0.05) .Bax protein expression and caspase 3 activity decreased (P<0.05). Amlodipine can inhibit the HKC apoptosis and protect the renal tubule cell from injury induced by meglumine diatrizoate.

Cell Attachment and Proliferation of Human Adipose-Derived Stem Cells on PLGA/Chitosan Electrospun Nano-Biocomposite.

PubMed

Razavi, Shahnaz; Karbasi, Saeed; Morshed, Mohammad; Zarkesh Esfahani, Hamid; Golozar, Mohammad; Vaezifar, Sedigheh

2015-01-01

In this study, nano-biocomposite composed of poly (lactide-co-glycolide) (PLGA) and chitosan (CS) were electrospun through a single nozzle by dispersing the CS nano-powders in PLGA solution. The cellular behavior of human adipose derived stem cells (h-ADSCs) on random and aligned scaffolds was then evaluated. In this experimental study, the PLGA/CS scaffolds were prepared at the different ratios of 90/10, 80/20, and 70/30 (w/w) %. Morphology, cell adhesion and prolif- eration rate of h-ADSCs on the scaffolds were assessed using scanning electron microscope (SEM), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and trypan blue staining respectively. H-ADSCs seeded on the matrices indicated that the PLGA/CS composite matrix with aligned nanofibres and higher content of CS nano-powders gave significantly better performance than others in terms of cell adhesion and proliferation rate (P<0.05). We found that CS enhanced cell adhesion and proliferation rate, and aligned nanofibers guided cell growth along the longitudinal axis of the nanofibers, which would provide a beneficial approach for tissue engineering.

A Novel Photosensitizer 3¹,13¹-phenylhydrazine -Mppa (BPHM) and Its in Vitro Photodynamic Therapy against HeLa Cells.

PubMed

Li, Wenting; Tan, Guanghui; Cheng, Jianjun; Zhao, Lishuang; Wang, Zhiqiang; Jin, Yingxue

2016-04-29

Photodynamic therapy (PDT) has attracted widespread attention due to its potential in the treatment of various cancers. Porphyrinic pyropheophorbide-a (PPa) has been shown to be a potent photosensitizer in PDT experiments. In this paper, a C-3¹,13¹ bisphenylhydrazone modified methyl pyropheophorbide-a (BPHM) was designed and synthesized with the consideration that phenylhydrazone structure may extend absorption wavelength of methyl pyro-pheophorbide-a (Mppa), and make the photosensitizer potential in deep tumor treatment. The synthesis, spectral properties and in vitro photodynamic therapy (PDT) against human HeLa cervical cancer cell line was studied. Methyl thiazolyl tetrazolium (MTT) assay showed the title compound could achieve strong inhibition of cervical cancer cell viability under visible light (675 nm, 25 J/cm²). Cell uptake experiments were performed on HeLa cells. Morphological changes were examined and analyzed by fluorescent inverted microscope. In addition, the mechanism of the photochemical processes of PDT was investigated, which showed that the formation of singlet oxygen after treatment with PDT played a moderate important role.

Gum tragacanth stabilized green gold nanoparticles as cargos for Naringin loading: A morphological investigation through AFM.

PubMed

Rao, Komal; Imran, Muhammad; Jabri, Tooba; Ali, Imdad; Perveen, Samina; Shafiullah; Ahmed, Shakil; Shah, Muhammad Raza

2017-10-15

Gold nanoparticles (AuNPs) have attracted greater scientific interests for the construction of drugs loading cargos due to their biocompatibility, safety and facile surface modifications. This study deals with the fabrication of gum tragacanth (GT) green AuNPs as carrier for Naringin, a less water soluble therapeutic molecule. The optimized AuNPs were characterized through UV-vis spectroscopy, FT-IR and atomic force microscope (AFM). Naringin loaded nanoparticles were investigated for their bactericidal potentials using Tetrazolium Microplate assay. Morphological studies conducted via AFM revealed spherical shape for AuNPs with nano-range size and stabilized by GT multi-functional groups. The AuNPs acted as carrier for increased amount of Naringin. Upon loading in AuNPs, Naringin An increased in the bactericidal potentials of Naringin was observed after loading on AuNPs against various tested bacterial strains. This was further authenticated by the surface morphological analysis, showing enhanced membrane destabilizing effects of loaded Naringin. The results suggest that GT stabilized green AuNPs can act as effective delivery vehicles for enhancing bactericidal potentials of Naringin. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cellular compatibility of highly degradable bioactive ceramics for coating of metal implants.

PubMed

Radetzki, F; Wohlrab, D; Zeh, A; Delank, K S; Mendel, T; Berger, G; Syrowatka, F; Mayr, O; Bernstein, A

2011-01-01

Resorbable ceramics can promote the bony integration of implants. Their rate of degradation should ideally be synchronized with bone regeneration. This study examined the effect of rapidly resorbable calcium phosphate ceramics 602020, GB14, 305020 on adherence, proliferation and morphology of human bone-derived cells (HBDC) in comparison to β-TCP. The in vitro cytotoxicity was determined by the microculture tetrazolium (MTT) assay. HBDC were grown on the materials for 3, 7, 11, 15 and 19 days and counted. Cell morphology, cell attachment, cell spreading and the cytoskeletal organization of HBDC cultivated on the substrates were investigated using laser scanning microscopy and environmental scanning electron microscopy. All substrates supported sufficient cellular growth for 19 days and showed no cytotoxicity. On each material an identical cell colonisation of well communicating, polygonal, vital cells with strong focal contacts was verified. HBDC showed numerous well defined stress fibres which give proof of well spread and strongly anchored cells. Porous surfaces encouraged the attachment and spreading of HBDC. Further investigations regarding long term biomaterial/cell interactions in vitro and in vivo are required to confirm the utility of the new biomaterials.

Design, synthesis, evaluation, and molecular docking of ursolic acid derivatives containing a nitrogen heterocycle as anti-inflammatory agents.

PubMed

Wei, Zhi-Yu; Chi, Ke-Qiang; Wang, Ke-Si; Wu, Jie; Liu, Li-Ping; Piao, Hu-Ri

2018-06-01

Ursolic acid derivatives containing oxadiazole, triazolone, and piperazine moieties were synthesized in an attempt to develop potent anti-inflammatory agents. Structures of the synthesized compounds were elucidated by 1 H NMR, 13 C NMR, and HRMS. Most of the synthesized compounds showed pronounced anti-inflammatory effects at 100 mg/kg. In particular, compound 11b, which displayed the most potent anti-inflammatory activity of all of the compounds prepared, with 69.76% inhibition after intraperitoneal administration, was more potent than the reference drugs indomethacin and ibuprofen. The cytotoxicity of the compounds was also assessed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, and no compounds showed any appreciable cytotoxic activity (IC 50 >100 μmol/L). Furthermore, molecular docking studies of the synthesized compounds were performed to rationalize the obtained biological results. Overall, the results indicate that compound 11b could be a therapeutic candidate for the treatment of inflammation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Shape-dependent antibacterial effects of non-cytotoxic gold nanoparticles

PubMed Central

Penders, Jelle; Stolzoff, Michelle; Hickey, Daniel J; Andersson, Martin; Webster, Thomas J

2017-01-01

Gold nanoparticles (AuNPs) of various shapes (including spheres, stars and flowers), with similar dimensions, were synthesized and evaluated for their antibacterial effects toward Staphylococcus aureus, a bacterium responsible for numerous life-threatening infections worldwide. Optical growth curve measurements and Gompertz modeling showed significant AuNP shape- and concentration-dependent decreases in bacterial growth with increases in bacterial growth lag time. To evaluate prospective use in in vivo systems, the cytotoxicity of the same AuNPs was evaluated toward human dermal fibroblasts in vitro by 3-(4,5 dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) viability assays and confocal microscopy. No indication of any mammalian cell toxicity or morphological effects was found. Additionally, it was observed that the AuNPs were readily internalized in fibroblasts after 4 days of incubation. Most importantly, the results of the present study showed that gold nanoflowers in particular possessed the most promising non-cytotoxic mammalian cell behavior with the greatest shape-dependent antibacterial activity-promising properties for their future investigation in a wide range of anti-infection applications. PMID:28408817

Diethylentriaminepenta acetic acid glucose conjugates as a cell permeable iron chelator.

PubMed

Mosayebnia, Mona; Shafiee-Ardestani, Mehdi; Pasalar, Parvin; Mashayekhi, Mojgan; Amanlou, Massoud

2014-01-01

To find out whether DTPA-DG complex can enhance clearance of intracellular free iron. Diethylenetriaminepentaacetic acid-D-deoxy-glucosamine (DTPA-DG) was synthesized and examined for its activity as a cell-permeable iron chelator in human hepatocellular carcinoma (HEPG2) cell line exposed to high concentration of iron sulfate and compared with deferoxamine (DFO), a prototype iron chelator. The effect of DTPA-DG on cell viability was monitored using the 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide MTT assay as well. There was a significant increase of iron level after iron overload induction in HEPG2 cell culture. DTPA-DG presented a remarkable capacity to iron burden reducing with estimated 50% inhibitory concentration value of 65.77 nM. In fact, glycosyl moiety was gained access of DTPA to intracellular iron deposits through glucose transporter systems. DTPA-DG, more potent than DFO to sequester deposits of free iron with no profound toxic effect. The results suggest the potential of DTPA-DG in chelating iron and permitting its excretion from primary organ storage.

Modulation of Tumor Cell Metabolism by Laser Photochemotherapy with Cisplatin or Zoledronic Acid In Vitro.

PubMed

Heymann, Paul Günther Baptist; Henkenius, Katharina Sabine Elisabeth; Ziebart, Thomas; Braun, Andreas; Hirthammer, Klara; Halling, Frank; Neff, Andreas; Mandic, Robert

2018-03-01

Laser photochemotherapy is a new approach in cancer treatment using low-level laser therapy (LLLT) to enhance the effect of chemotherapy. In order to evaluate the effect of LLLT on tumor cells, HeLa cells were treated with cisplatin or zoledronic acid (ZA) followed by LLLT. Cell viability was evaluated with 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay. Oxidative phosphorylation and glycolysis were measured using extracellular flux analysis. Immunocytochemistry of heat-shock protein 70 (HSP70) and western blot analysis were performed. LLLT alone increased viability and was associated with lower oxidative phosphorylation but higher glycolysis rates. Cisplatin and ZA alone lowered cell viability, glycolysis and oxidative phosphorylation. This effect was significantly enhanced in conjunction with LLLT and was accompanied by reduced oxidative phosphorylation and collapse of glycolysis. Our observations indicate that LLLT may raise the cytotoxicity of cisplatin and ZA by modulating cellular metabolism, pointing to a possible application in cancer treatment. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Physicochemical characterization of solid dispersions of carbamazepine formulated by supercritical carbon dioxide and conventional solvent evaporation method.

PubMed

Sethia, Sundeep; Squillante, Emilio

2002-09-01

Solid dispersions of carbamazepine (CBZ) were formulated by supercritical fluid processing (SCP) and conventional solvent evaporation in polyethylene glycol (PEG) 8000 with either Gelucire 44/14 or vitamin E TPGS NF (d-alpha-tocopheryl PEG 1000 succinate). Formulations were evaluated by dissolution, scanning electron microscopy, powder X-ray diffraction, and differential scanning calorimetry, and excipient cytotoxicity in Caco-2 cells by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay. CBZ release was enhanced from supercritical fluid-treated CBZ and the CBZ/PEG 8000 (1:5), CBZ/PEG 8000/TPGS or Gelucire 44/14 (1:4:1) solid dispersions. The radically altered morphologies of SCP samples seen by scanning electron microscopy suggested polymorphic change that was confirmed by the X-ray diffraction and differential scanning calorimetry. Disappearance of the characteristic CBZ melting peak indicated that CBZ was dissolved inside the carrier system. Polymorphic change of CBZ during SCP led to faster dissolution. Therefore, SCP provides advantages over solid dispersions prepared by conventional processes. Copyright 2002 Wiley-Liss Inc.

Magnolol pretreatment attenuates heat stress-induced IEC-6 cell injury*

PubMed Central

Mei, Chen; He, Sha-sha; Yin, Peng; Xu, Lei; Shi, Ya-ran; Yu, Xiao-hong; Lyu, An; Liu, Feng-hua; Jiang, Lin-shu

2016-01-01

Objective: Heat stress (HS) is an important environmental stressor that adversely influences livestock during the summer. The aim of this study was to investigate whether magnolol protects against HS-induced intestinal epithelial cell injury. Materials and methods: An intestinal epithelial cell line (IEC-6) was subjected to HS at 42 °C, with and without magnolol pretreatment. Cell injury was detected by monitoring lactate dehydrogenase (LDH) release. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay was used to assess cell proliferation and viability, including identifying effective concentrations of magnolol. Flow cytometry confirmed G1-phase cell-cycle arrest and its alleviation by magnolol. Active DNA synthesis was measured by incorporation of nucleic acid 5-ethynyl-2'-deoxyuridine (EdU). G1-phase cell-cycle-related gene expression was assessed by real-time reverse transcription polymerase chain reaction (RT-PCR) and levels of G1-phase-related proteins by Western blotting. Results: HS induced IEC-6 cell injury and decreased cell viability, as demonstrated by data from LDH and MTS assays, respectively. Based on a number of criteria, IEC-6 cells subjected to HS were arrested in the G1 phase of the cell cycle. Magnolol pretreatment decreased HS-induced cell injury through relief of this cell-cycle arrest. Conclusions: Magnolol pretreatment attenuates HS-induced injury in IEC-6 cells. Magnolol is potentially promising as a protective strategy for HS in livestock. PMID:27256675

Upregulation of human DNA binding protein A (dbpA) in gastric cancer cells.

PubMed

Wang, Guo-rong; Zheng, Yan; Che, Xiang-ming; Wang, Xin-yang; Zhao, Jia-hui; Wu, Kai-jie; Zeng, Jin; Pan, Chen-en; He, Da-lin

2009-10-01

To determine the effect of human DNA binding protein (dbpA) on the biology of gastric cancer cells. DbpA expression was analyzed by Western blot analysis and immunofluorescence staining in gastric cancer tissues and cell lines. A dbpA-specific small interference (si) RNA was designed and synthesized. Suppressive effect of siRNA on dbpA expression was assessed by real-time RT-PCR. Transwell migration and colony formation assays were used to assess the inhibitory effects of dbpA siRNA on cell invasion and tumorigenesis in vitro. Drug-sensitivity was evaluated using a conventional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The expression of dbpA was upregulated in gastric cancer tissues and cell lines as compared to adjacent normal tissues or gastric epithelial cells. siRNA treatment successfully silenced dbpA expression. Silencing of dbpA increased expression of E-cadherin, decreased expression of adenomatous polyposis coli (APC), beta-catenin and cyclin D1, but had no effect on expression of NF-kappaB. Silencing of dbpA also suppressed cell invasion and colony formation of SGC7901 cells, and enhanced their chemosensitivity to 5-fluorouracil. DbpA plays an important role in the pathogenesis and development of gastric cancer, and the process involves E-cadherin, APC, beta-catenin and cyclin D1. Silencing of dbpA might be a novel therapeutic strategy for increasing chemosensitivity to 5-fluorouracil in gastric cancer.

Leishmanicidal activity of Yucatecan medicinal plants on Leishmania species responsible for cutaneous leishmaniasis.

PubMed

Getti, Giulia; Durgadoss, Priyanka; Domínguez-Carmona, Dafne; Martin-Quintal, Zhelmy; Peraza-Sánchez, Sergio; Peña-Rodriguez, Luis Manuel; Humber, David

2009-04-01

The leishmanicidal activity of 15 extracts and 4 pure metabolites obtained from Urechites andrieuxii, Colubrina greggii, Dorstenia contrajerva, and Tridax procumbens was evaluated using the newly developed MTS ({3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assay, optimized for promastigotes of Leishmania major, Leishmania tropica, and Leishmania aethiopica, as well as for L. aethiopica axenic amastigotes. The assay was then used for calculating the percentage of viable stationary phase parasites after a 24-hr treatment with each plant extract or pure metabolite. The 3 most active samples, 2 from C. greggii (NCG-5C and DCG-3A) and 1 from T. procumbens (TPZ-2A), showed LD50 values of 62.4, 7.2, and 18.5 microg/ml, respectively, on stationary promastigotes, and of 94.2, 27.1, and 95.2 microg/ml, on amastigotes of L. aethiopica. Moreover, TPZ-2A and DCG-3A significantly reduced the percentage of infected monocyte-derived macrophages (THP-I). The percentage of infected cells decreased from 69.9% +/- 2.5% to 20.8% +/- 2% when the cells were treated with the DCG-3A fraction and to 14.9% +/- 0.5% when treated with TPZ-2A, without significantly decreasing the number of human cells. These findings indicate the presence of potentially bioactive metabolites in the roots of C. greggii and in T. procumbens and reflect the importance of pursuing the bioassay-guided purification of these metabolites.

Liposome-based delivery of a boron-containing cholesteryl ester for high-LET particle-induced damage of prostate cancer cells: a boron neutron capture therapy study.

PubMed

Gifford, Ian; Vreeland, Wyatt; Grdanovska, Slavica; Burgett, Eric; Kalinich, John; Vergara, Vernieda; Wang, C-K Chris; Maimon, Eric; Poster, Dianne; Al-Sheikhly, Mohamad

2014-06-01

The efficacy of a boron-containing cholesteryl ester compound (BCH) as a boron neutron capture therapy (BNCT) agent for the targeted irradiation of PC-3 human prostate cancer cells was examined. Liposome-based delivery of BCH was quantified with inductively coupled plasma-mass spectrometry (ICP-MS) and high-performance liquid chromatography (HPLC). Cytotoxicity of the BCH-containing liposomes was evaluated with neutral red, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), and lactate dehydrogenase assays. Colony formation assays were utilized to evaluate the decrease in cell survival due to high-linear energy transfer (LET) particles resulting from (10)B thermal neutron capture. BCH delivery by means of encapsulation in a lipid bilayer resulted in a boron uptake of 35.2 ± 4.3 μg/10(9) cells, with minimal cytotoxic effects. PC-3 cells treated with BCH and exposed to a 9.4 × 10(11) n/cm(2) thermal neutron fluence yielded a 20-25% decrease in clonogenic capacity. The decreased survival is attributed to the generation of high-LET α particles and (7)Li nuclei that deposit energy in densely ionizing radiation tracks. Liposome-based delivery of BCH is capable of introducing sufficient boron to PC-3 cells for BNCT. High-LET α particles and (7)Li nuclei generated from (10)B thermal neutron capture significantly decrease colony formation ability in the targeted PC-3 cells.

Paroxetine Can Enhance Neurogenesis during Neurogenic Differentiation of Human Adipose-derived Stem Cells

PubMed Central

Jahromi, Maliheh; Razavi, Shahnaz; Amirpour, Nushin; Khosravizadeh, Zahra

2016-01-01

Background: Some antidepressant drugs can promote neuronal cell proliferation in vitro as well as hippocampal neurogenesis in human and animal models. Furthermore, adipose tissue is an available source of adult stem cells with the ability to differentiate in to multiple lineages. Therefore, human Adipose-Derived Stem Cells (hAD-SCs) may be a suitable source for regenerative medical applications. Since there is no evidence for the effect of Paroxetine as the most commonly prescribed antidepressant drug for neurogenic potential of hADSCs, an attempt was made to determine the effect of Paroxetine on proliferation and neural differentiation of hADSCs. Methods: ADSCs were isolated from human abdominal fat. These cells differentiated to neuron-like cells and were treated with Paroxetine. 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay and immunofluorescence technique were used for assessment of cell proliferation and neurogenic differentiation potential of induced cells, respectively. Results: MTT assay analysis showed that Paroxetine significantly increased the proliferation rate of induced hADSCs (p<0.05), while immunofluorescent staining indicated that Paroxetine treatment during neurogenic differentiation could enhance the mean percentage of Nestin and MAP2 (Microtubule-associated protein-2) positive cells but the mean percentage of GFAP (Glial acidic fibrillary protein) positive cells significantly decreased relative to control group (p<0.05). Conclusion: Our results provide evidence that Paroxetine can promote proliferation and differentiation rate during neurogenic differentiation of hADSCs. Moreover, Paroxetine can reduce gliogenesis of induced hADSCs during neurogenic differentiation. PMID:27920882

Immunomodulatory properties of titanium dioxide nanostructural materials.

PubMed

Latha, T Sree; Reddy, Madhava C; R Durbaka, Prasad V; Muthukonda, Shankar V; Lomada, Dakshayani

2017-01-01

Although titanium dioxide (TiO 2 ) nanostructural materials have been widely used in Biology and Medicine, very little is known about immunomodulation mechanism of these materials. Objectives of this study are to investigate in vitro immunomodulatory effects of TiO 2 . Immunosuppressant may lower immune responses and are helpful for the treatment of graft versus host diseases and autoimmune disorders. In this study, we used H 2 Ti 3 O 7 titanium dioxide nanotubes (TNT) nanotubes along with commercial TiO 2 nanoparticles (TNP) and TiO 2 fine particles (TFP). We investigated the in vitro immunomodulatory effects of TNP, TNT, and TFP using mixed lymphocyte reaction (MLR). Suppression was studied by 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Cytokine profile was measured by enzyme-linked immunosorbent assay (ELISA). The results from this study illustrated that the TiO 2 nanostructural materials strongly suppressed splenocytes proliferation in MLR. For TNP and TNT, at 50 μg/ml suppression of 20%-25% and 30%-35%, respectively, and for TFP at 100 μg/ml suppression was 25%-30% was observed. Suppression of splenocytes proliferation in the presence of TNP, TNT, and TFP demonstrated that these nanostructural materials probably block T-cell-mediated responses in vitro . Our ELISA results confirmed that significantly lower levels of Th1 type cytokines (interleukin-2, interferon-γ) in the 48 h MLR culture supernatants. Our data suggest that TiO 2 nanostructural materials suppress splenocytes proliferation by suppressing Th1 cytokines.

Protective effects of lactoferrin against intestinal mucosal damage induced by lipopolysaccharide in human intestinal Caco-2 cells.

PubMed

Hirotani, Yoshihiko; Ikeda, Kenji; Kato, Ryuji; Myotoku, Michiaki; Umeda, Takashi; Ijiri, Yoshio; Tanaka, Kazuhiko

2008-09-01

Indirect evidence suggests that lactoferrin (Lf), a major iron-binding protein in human milk, induces enterocyte growth and proliferation, depending on its concentration and affects the function and permeability of the intestinal mucosa. The bacterial endotoxin (lipopolysaccharide, LPS) is known to cause mucosal hyperpermeability in vivo. However, protective effects of Lf against LPS-mediated intestinal mucosal damage and barrier function in epithelial cells are not yet fully clarified. The aim of this study was to investigate whether Lf can reduce the cellular injury and alter epithelial hyperpermeability caused by LPS in human intestinal Caco-2 cells. When cell viability was measured by a WST-1 assay (tetrazolium salt-based assay), the protective effects against LPS-induced damage to Caco-2 cells were observed at doses of 800 and 1000 microg/ml Lf. The barrier function of Caco-2 monolayer tight junctions was assessed by measuring transepithelial electrical resistance (TEER) and permeability of FITC-labeled dextran 4000 (FD-4). The treatment of Caco-2 cells with Lf at doses of 400 and 1000 microg/ml significantly increased TEER as compared to treatment with LPS alone for 2 h (p<0.05). Further, at doses of 400 and 1000 microg/ml, Lf inhibited the enhancement of LPS-mediated permeability in Caco-2 cell monolayer. The results of this study suggest that Lf may have protective effects against LPS-mediated intestinal mucosal damage and impairment of barrier function in intestinal epithelial cells.

The endoplasmic reticulum stress inducer thapsigargin enhances the toxicity of ZnO nanoparticles to macrophages and macrophage-endothelial co-culture.

PubMed

Chen, Gui; Shen, Yuexin; Li, Xiyue; Jiang, Qin; Cheng, Shanshan; Gu, Yuxiu; Liu, Liangliang; Cao, Yi

2017-03-01

It was recently shown that exposure to ZnO nanoparticles (NPs) could induce endoplasmic reticulum (ER) stress both in vivo and in vitro, but the role of ER stress in ZnO NP induced toxicity remains unclear. Because macrophages are sensitive to ER stress, we hypothesized that stressing macrophages with ER stress inducer could enhance the toxicity of ZnO NPs. In this study, the effects of ER stress inducer thapsigargin (TG) on the toxicity of ZnO NPs to THP-1 macrophages were investigated. The results showed that TG enhanced ZnO NP induced cytotoxicity as revealed by water soluble tetrazolium-1 (WST-1) and neutral red uptake assays, but not lactate dehydrogenase (LDH) assay. ZnO NPs dose-dependently enhanced the accumulation of intracellular Zn ions without the induction of reactive oxygen species (ROS), and the presence of TG did not significantly affect these effects. In the co-culture, exposure of THP-1 macrophages in the upper chamber to ZnO NPs and TG significantly reduced the viability of human umbilical vein endothelial cells (HUVECs) in the lower chamber, but the release of tumor necrosis factor α (TNFα) was not induced. In summary, our data showed that stressing THP-1 macrophages with TG enhanced the cytotoxicity of ZnO NPs to macrophages and macrophage-endothelial co-cultures. Copyright © 2017 Elsevier B.V. All rights reserved.

Chrysin reduces proliferation and induces apoptosis in the human prostate cancer cell line pc-3

PubMed Central

Samarghandian, Saeed; Afshari, Jalil Tavakkol; Davoodi, Saeideh

2011-01-01

INTRODUCTION: Honey is a common household product with many medicinal uses described in traditional medicine. Only recently has its antioxidant properties and preventive effects against disease been highlighted. Chrysin is a natural flavone commonly found in honey that has been shown to be an antioxidant agent. In this study, we investigated the antiproliferative and apoptotic effects of honey and chrysin on cultured human prostate cancer cells. METHODS: Cells were cultured in RPMI medium and treated with different concentrations of honey and chrysin for three consecutive days. Cell viability was quantitated by the 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. The percentage of apoptotic cells was determined by flow cytometry using Annexin V-fluorescein isothiocyanate. RESULTS: The MTT assay revealed that both compounds had an antiproliferative effect on PC-3 cells in a dose- and time-dependent manner. The IC50 values for honey and chrysin against PC-3 cells were 2.5% and 24.5% after 48 h and 1.8% and 8.5% after 72 h, respectively. Chrysin induced apoptosis in PC-3 cells, as determined by flow cytometry. CONCLUSION: Our results suggest that honey has anti-proliferative effects on prostate cancer cells and the effects are mainly due to chrysin. Therefore, chrysin may be a potential compound for both cancer prevention and treatment. Further in vivo investigation is needed to support the use of chrysin in cancer therapy. PMID:21808878

[Viability and germination of Hechtia perotensis (Bromeliaceae) seed].

PubMed

Elizalde, Violeta; García, José Rodolfo; Peña-Valdivia, Cecilia Beatriz; Ybarra, Ma Carmen; Leyva, Otto Raúl; Trejo, Carlos

2017-03-01

Endemic populations of Hechtia perotensis have been described in Puebla and Veracruz, Mexico. Good quality seed collections can be used in conservation, research and ecological restoration. To evaluate seed quality of wild and endemic species, some compounds are used as effective promoters of germination, such as potassium nitrate (KNO3) and gibberellic acid (AG3), because they increase seed germination capacity and reduce latency. The triphenyl tetrazolium chloride (tetrazolium) test correlates seed viability because it is based on the activity of dehydrogenases in live tissues that catalyze mitochondrial respiration. The objective of this study was to obtain information on size and weight of capsules and seeds and seed germination and viability of H. perotensis, collected in Veracruz in the year 2012 and 2015. The hypotheses were 1) that seed germination and viability are independent of the year of collection, 2) that there is a tetrazolium concentration that can identify seed viability better than others, and 3) that pretreatment with KNO3 or AG3 improves seed germination. Seed germination was assessed using a completely randomized design with three treatments (control and the germination promoters 0.2 % KNO3 and 500 mg/L AG3), four treatments for the viability test (control, 0.2, 0.5 and 1.0 % of tetrazolium) and six replicates for each treatment. A total of one hundred seeds for germination experiments, and 25 seeds for the viability test were used. The results between and within years were analyzed with ANOVA and multiple comparison with the Tukey test. The proportion of non-germinated seeds was quantified along with the number of normal and abnormal seedlings, seeds with viable embryo, seeds without embryo, and seeds with low or no viability. On average, for the 2012 collected sample, 36 % had viable embryos, 7 % had low viability, 24 % were not viable and 33 % had no embryo. This result was significantly different from the 2015 sample, for which 87 % of seed showed viable embryos, 10 % had low viability, 0 % was not viable and 3 % had no embryo. Seed germination was also significantly different between years (22 and 92 %) Pregerminative treatments did not improve germination. Seed germination and viability of H. perotensis significantly varied between years of seed collection.

INT (2-(4-Iodophenyl)-3-(4-Nitrophenyl)-5-(Phenyl) Tetrazolium Chloride) Is Toxic to Prokaryote Cells Precluding Its Use with Whole Cells as a Proxy for In Vivo Respiration.

PubMed

Villegas-Mendoza, Josué; Cajal-Medrano, Ramón; Maske, Helmut

2015-11-01

Prokaryote respiration is expected to be responsible for more than half of the community respiration in the ocean, but the lack of a practical method to measure the rate of prokaryote respiration in the open ocean resulted in very few published data leaving the role of organotrophic prokaryotes open to debate. Oxygen consumption rates of oceanic prokaryotes measured with current methods may be biased due to pre-incubation size filtration and long incubation times both of which can change the physiological and taxonomic profile of the sample during the incubation period. In vivo INT reduction has been used in terrestrial samples to estimate respiration rates, and recently, the method was introduced and applied in aquatic ecology. We measured oxygen consumption rates and in vivo INT reduction to formazan in cultures of marine bacterioplankton communities, Vibrio harveyi and the eukaryote Isochrysis galbana. For prokaryotes, we observed a decrease in oxygen consumption rates with increasing INT concentrations between 0.05 and 1 mM. Time series after 0.5 mM INT addition to prokaryote samples showed a burst of in vivo INT reduction to formazan and a rapid decline of oxygen consumption rates to zero within less than an hour. Our data for non-axenic eukaryote cultures suggest poisoning of the eukaryote. Prokaryotes are clearly poisoned by INT on time scales of less than 1 h, invalidating the interpretation of in vivo INT reduction to formazan as a proxy for oxygen consumption rates.

A.D.A.M. test (Antibiofilm Dressing's Activity Measurement) - Simple method for evaluating anti-biofilm activity of drug-saturated dressings against wound pathogens.

PubMed

Junka, Adam F; Żywicka, Anna; Szymczyk, Patrycja; Dziadas, Mariusz; Bartoszewicz, Marzena; Fijałkowski, Karol

2017-12-01

In the present article, we propose a simple Antibiofilm Dressing's Activity Measurement (A.D.A.M.) test that allows to check in vitro a dressing's suitability against biofilm-related wound infections. To perform the test, three agar discs are covered with biofilm formed by the tested pathogen after which they are assembled one over another in the form of an agar plug and placed in the well of a 24-well plate. The top disc is covered with the analyzed dressing and the entire set is incubated for 24h. During this time, the investigated antimicrobial substance is released from the dressing and penetrates to subsequent biofilm-covered agar discs. Biofilm reduction is measured using 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) spectrometric assay and the results are compared to untreated control samples (agar plug covered with biofilm and without the dressing/or with a passive dressing placed on the top disc). Furthermore, in order to standardize the differences in penetrability of the drugs released from active dressings the results can be expressed as a dimensionless value referred to as the Penetrability Index. In summary, A.D.A.M. test is simple, cheap, can be performed practically in every clinical laboratory and takes no more time than routine microbiological diagnostics. Apart from measuring the released drug's activity, the A.D.A.M. test allows to assess drug penetrability (across three agar discs), reflecting real wound conditions, where microbes are frequently hidden under the necrotic tissue or cloth. In conclusion, the A.D.A.M. test produces a high volume of data that, when analyzed, can provide a researcher with a valuable hint concerning the applicability of active dressings against specific biofilm pathogens in a particular setting. Copyright © 2017 Elsevier B.V. All rights reserved.

Grape Seed Extract Dose-Responsively Decreases Disease Severity in a Rat Model of Mucositis; Concomitantly Enhancing Chemotherapeutic Effectiveness in Colon Cancer Cells

PubMed Central

Cheah, Ker Yeaw; Howarth, Gordon Stanley; Bastian, Susan Elaine Putnam

2014-01-01

Objective Mucositis is a serious disorder of the gastrointestinal tract that results from cancer chemotherapy. We investigated the effects of increasing grape seed extract doses on the severity of chemotherapy in a rat model and its coincident impact on chemotherapeutic effectiveness in colon cancer cells. Design Female Dark Agouti rats were gavaged with grape seed extract (400–1000 mg/kg) or water (day 3–11) and were injected intraperitoneally with 5-Fluorouracil (150 mg/kg) or saline (control) on day 9 to induce mucositis. Daily metabolic data were collected and rats were sacrificed on day 12. Intestinal tissues were collected for histological and myeloperoxidase analyses. Caco-2 cell viability was examined in response to grape seed extract in combination with 5-Fluorouracil by 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide) assay. Results Compared with 5-Fluorouracil controls, grape seed extract (400–1000 mg/kg) significantly decreased the histological damage score (P<0.05) in the jejunum. Grape seed extract (1000 mg/kg) increased jejunal crypt depth by 25% (P<0.05) in 5-Fluorouracil treated rats compared to 5-Fluorouracil controls, and attenuated the 5-Fluorouracil -induced reduction of mucosal thickness (25%, P<0.05). Grape seed extract (600 mg/kg) decreased myeloperoxidase activity by 55% (P<0.01) compared to 5-Fluorouracil controls. Grape seed extract was more effective at ameliorating 5-Fluorouracil induced intestinal injury, with effects most pronounced in the proximal jejunum. Grape seed extract (10–25 ug/mL) significantly enhanced the growth-inhibitory effects of 5-Fluorouracil by 26% (P<0.05) in Caco-2 cells and was more potent than 5-Fluorouracil at 50–100 µg/mL. Conclusion Grape seed extract may represent a new therapeutic option to decrease the symptoms of intestinal mucositis while concurrently impacting on the viability of colon cancer cells. PMID:24465501

The efficacy of Poly-β-Hydroxy Butyrate (PHB)/biosurfactant derived from Staphylococcus hominis against White Spot Syndrome Virus (WSSV) in Penaeus monodon.

PubMed

Monica, M; Priyanka, T; Akshaya, Murugesan; Rajeswari, V; Sivakumar, Lingappa; Somasundaram, S T; Shenbhagarathai, R

2017-12-01

White Spot Syndrome Virus (WSSV) is one of the most important causative agents of Penaeid shrimps diseases that incur heavy losses to the shrimp aquaculture. It has severe impact on the sustainability and the production of Penaeus monodon. Hence, the present study focussed on the investigation of Poly-β-hydroxybutyrate/biosurfactant as immunostimulants against WSSV infected shrimps. Infection of WSSV was periodically checked in all the experimental shrimps using PCR diagnostic kit. After ensuring all shrimps were free of viral infection, experiments were carried out to analyze the nonspecific immune responses (prophenol oxidase, nitro blue tetrazolium reduction assay and total haemocyte count) both in control and experimental group. Further, gills and muscles of Penaeus monodon were subjected to proteome analysis after treated it with PHB/biosurfactant independently in the concentration of 2% and 5% each. Increase in the level of haemocytes was observed in both PHB (26 ± 2 × 10⁴ cells)/biosurfactant (28 ± 2 × 10 4  cells) treated shrimps, when compared with control (17 ± 2 × 10⁴ cells). proPhenolOxidase (proPO) activity was also enhanced in treated groups compared to WSSV infected shrimps. Less production of superoxide anion was observed in control and treated groups. Differences in the protein expression was analyzed in muscle tissue of control, WSSV infected and PHB/biosurfactant treated shrimps. Our finding suggested that partial substitution of feed with 2% PHB and biosurfactant showed increased rate on the survival of WSSV infected P. monodon which might be due to either the over expression/down regulation of proteins that play a vital role in enhancing the immune system/the progression of the disease respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

Ceramide does not mediate the effect of tumour necrosis factor alpha on superoxide generation in human neutrophils.

PubMed Central

Yanaga, F; Watson, S P

1994-01-01

The effect of tumour necrosis factor alpha (TNF alpha) on superoxide generation in human neutrophils was investigated using the Nitro Blue Tetrazolium reduction assay. TNF alpha stimulated superoxide generation in a time- and concentration-dependent fashion. The maximally effective concentration of TNF alpha for superoxide generation was 10 nM and maximal response was obtained after 15-20 min. The monoclonal antibody (mAb), utr-1, which was raised against the 75 kDa receptor and behaves as an antagonist, had no effect on superoxide generation, but partially inhibited the response to TNF alpha. mAb htr-9, which was raised against the 55 kDa receptor and behaves as an agonist, mimicked the effect of TNF alpha, but with a lower maximal response. As it has been reported that ceramide might act as a second messenger to mediate many of the effects of TNF alpha, the effects of exogenous sphingomyelinase and the cell-permeable ceramide analogue, C2- ceramide, on production of superoxide anions, induction of priming in response to formylmethionyl-leucyl-phenylalanine, and cell-shape change were examined. Neither sphingomyelinase nor C2-ceramide mimicked the effect of TNF alpha. Ceramide is converted into ceramide 1-phosphate by ceramide kinase and we have measured levels of this metabolite to clarify the effect of TNF alpha on sphingomyelinase activity in neutrophils. Although exogenous sphingomyelinase increased the amount of ceramide 1-phosphate in a time-dependent manner, and C2-ceramide was rapidly converted into C2-ceramide phosphate, TNF alpha had no effect on the level of ceramide 1-phosphate. These results suggest that TNF alpha stimulates superoxide generation through both the 55 kDa and 75 kDa receptors, but that ceramide does not act as an intracellular mediator for TNF alpha in human neutrophils. Images Figure 4 PMID:8141790

Effects of matrine on the proliferation of HT29 human colon cancer cells and its antitumor mechanism

PubMed Central

CHANG, CHENG; LIU, SHAO-PING; FANG, CHUN-HUA; HE, REN-SHENG; WANG, ZHEN; ZHU, YOU-QING; JIANG, SHAO-WEI

2013-01-01

Matrine is one of the main active components that is extracted from the dry roots of Sophora flavescens. The compound has potent antitumor activity in various cancer cell lines. However, the anticancer activity of matrine in colon cancer cells remains unclear. The purpose of the present study was to investigate the effects of matrine on the growth of human colon cancer cells and the expression of the associated proteins. Cancer cell proliferation was measured by 3-(4,5-dimethylthiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. The cell cycle distribution and apoptosis were analyzed by flow cytometry (FCM). The activation of the caspases and the expression of pro-apoptotic and anti-apoptotic factors were examined using western blot analysis. Matrine was shown to significantly inhibit the proliferation of HT29 cells in a dose- and time-dependent manner, and also to reduce the percentage of cells in the G2/M phase, which was most frequently associated with an increase of cells arrested in the G0/G1 phase of the cell cycle. Western blot analysis revealed that matrine induced the activation of caspase-3 and -9 and the release of cytochrome C (Cyto C) from the mitochondria to the cytosol. Furthermore, the pro-apoptotic factor, Bax, was upregulated and the anti-apoptotic factor, Bcl-2, was downregulated, eventually leading to a reduction in the ratio of Bcl-2/Bax proteins. The results demonstrated that matrine inhibits proliferation and induces apoptosis of HT29 human cells in vitro. The induction of apoptosis appears to occur through the upregulation of Bax, the downregulation of Bcl-2, the release of Cyto C from the mitochondria to the cytosol and the activation of caspase-3 and caspase-9, which subsequently trigger major apoptotic cascades. Matrine has potent antitumor activity in HT29 cells and may be used as a novel effective reagent in the treatment of colon cancer. PMID:24137393

Minocycline, a putative neuroprotectant, co-administered with doxorubicin-cyclophosphamide chemotherapy in a xenograft model of triple-negative breast cancer

PubMed Central

Himmel, Lauren E.; Lustberg, Maryam B.; DeVries, A. Courtney; Poi, Ming; Chen, Ching-Shih; Kulp, Samuel K.

2016-01-01

Minocycline is purported to have neuroprotective properties in experimental models of some human neurologic diseases, and has therefore been identified as a putative neuroprotectant for chemotherapy-induced cognitive impairment (CICI) in breast cancer patients. However, because its mechanism of action is believed to be mediated through anti-inflammatory, anti-apoptotic, and anti-oxidant pathways, co-administration of minocycline with chemotherapeutic agents has the potential to reduce the efficacy of anticancer drugs. The objective of this study is to evaluate the effect of minocycline on the activity of the AC chemotherapeutic regimen (Adriamycin [doxorubicin], Cytoxan [cyclophosphamide]) in in vitro and in vivo models of triple-negative breast cancer (TNBC). Clonogenic and methylthiazol tetrazolium (MTT) assays were used to assess survival and viability in two TNBC cell lines treated with increasing concentrations of AC in the presence or absence of minocycline. Biomarkers of apoptosis, cell stress, and DNA damage were evaluated by western blot. The in vivo effects of AC and minocycline, each alone and in combination, were assessed in a xenograft model of TNBC in female athymic nude mice by weekly tumor volume measurement, body and organ weight measurement, and histopathology. Apoptosis and proliferation were characterized by immunohistochemistry in the xenografts tumors. Brains from tumor-bearing mice were evaluated for microglial activation, glial scars, and the proportion of neural progenitor cells. Data from these in vitro and in vivo studies demonstrate that minocycline does not diminish the cytotoxic and tumor-suppressive effects of this chemotherapeutic drug combination in TNBC cells. Moreover, minocycline appeared to prevent the reduction in doublecortin-positive neural progenitor cells observed in AC-treated mice. We posit that minocycline may be useful clinically for its reported neuroprotective activity in breast cancer patients receiving AC without loss of chemotherapeutic efficacy. PMID:27555377

Minocycline, a putative neuroprotectant, co-administered with doxorubicin-cyclophosphamide chemotherapy in a xenograft model of triple-negative breast cancer.

PubMed

Himmel, Lauren E; Lustberg, Maryam B; DeVries, A Courtney; Poi, Ming; Chen, Ching-Shih; Kulp, Samuel K

2016-10-01

Minocycline is purported to have neuroprotective properties in experimental models of some human neurologic diseases, and has therefore been identified as a putative neuroprotectant for chemotherapy-induced cognitive impairment (CICI) in breast cancer patients. However, because its mechanism of action is believed to be mediated through anti-inflammatory, anti-apoptotic, and anti-oxidant pathways, co-administration of minocycline with chemotherapeutic agents has the potential to reduce the efficacy of anticancer drugs. The objective of this study is to evaluate the effect of minocycline on the activity of the AC chemotherapeutic regimen (Adriamycin [doxorubicin], Cytoxan [cyclophosphamide]) in in vitro and in vivo models of triple-negative breast cancer (TNBC). Clonogenic and methylthiazol tetrazolium (MTT) assays were used to assess survival and viability in two TNBC cell lines treated with increasing concentrations of AC in the presence or absence of minocycline. Biomarkers of apoptosis, cell stress, and DNA damage were evaluated by western blot. The in vivo effects of AC and minocycline, each alone and in combination, were assessed in a xenograft model of TNBC in female athymic nude mice by weekly tumor volume measurement, body and organ weight measurement, and histopathology. Apoptosis and proliferation were characterized by immunohistochemistry in the xenografts tumors. Brains from tumor-bearing mice were evaluated for microglial activation, glial scars, and the proportion of neural progenitor cells. Data from these in vitro and in vivo studies demonstrate that minocycline does not diminish the cytotoxic and tumor-suppressive effects of this chemotherapeutic drug combination in TNBC cells. Moreover, minocycline appeared to prevent the reduction in doublecortin-positive neural progenitor cells observed in AC-treated mice. We posit that minocycline may be useful clinically for its reported neuroprotective activity in breast cancer patients receiving AC without loss of chemotherapeutic efficacy. Copyright © 2016 Elsevier GmbH. All rights reserved.

Nitroblue tetrazolium test

MedlinePlus

... infections. Normal value ranges may vary slightly from one lab to another. Talk to your doctor about the meaning of your test results. What Abnormal Results Mean If the sample does not change color when NBT is added, ...

Specific detection of membrane-toxic substances with a conductivity assay.

PubMed

Eich, J; Dürholt, H; Steger-Hartmann, T; Wagner, E

2000-03-01

A conductivity assay that represents a new biotest able to detect the effects of membrane-toxic compounds, e.g., detergents, organic solvents, and radical formers, on various organisms was previously described and developed. The conductivity assay measures ion leakage from cells, tissues, or whole plant and animal organisms whose membrane systems have been damaged by membrane-toxic compounds. In this study the specificity of the conductivity assay for membrane-toxic compounds was tested by comparing the electrolyte efflux from Elodea canadensis leaves during incubation with a well-known detergent (benzalkonium chloride) using different plant physiological and biochemical techniques (photochemical efficiency, plasmolysis capacity, NBT reduction, and electron microscopy of membranes of E. canadensis leaves). The comparison of the different methods proved that the electrolyte loss during benzalkonium chloride incubation determined in the conductivity assay is due to membrane impairment. The observed electrolyte loss correlated with a reduction of photochemical efficiency and a decrease in both plasmolysis and NBT reduction capacity. Furthermore, a disintegration of the plasmalemma could be seen in the electron micrographs. These results indicate that the measured electrolyte loss in the conductivity assay is a specific effect of membrane-toxic compounds. Copyright 2000 Academic Press.

Biological Interactions of Nanomaterials

DTIC Science & Technology

2008-12-01

reagent contains the tetrazolium compound MTS and an electron coupling reagent ( phenazine ethosulfate; PES). The quantity of formazan product as...PES Phenazine ethosulfate PS Polysaccharide RET Rearranged during transfection ROS Reactive oxygen species SD Standard deviation SEM Scanning

Influence of Gold Nanoshell on Hyperthermia of Super Paramagnetic Iron Oxide Nanoparticles (SPIONs)

PubMed Central

Mohammad, Faruq; Balaji, Gopalan; Weber, Andrew; Uppu, Rao M.; Kumar, Challa S. S. R.

2010-01-01

Gold nanoshell around super paramagnetic iron oxide nanoparticles (SPIONs) was synthesized and small angle X-ray scattering (SAXS) analysis suggests a gold coating of approximately 0.4 to 0.5 nm thickness. On application of low frequency oscillating magnetic fields (44 – 430 Hz), a four- to five-fold increase in the amount of heat released with gold-coated SPIONs (6.3 nm size) in comparison with SPIONs (5.4 nm size) was observed. Details of the influence of frequencies of oscillating magnetic field, concentration and solvent on heat generation are presented. We also show that, in the absence of oscillating magnetic field, both SPIONs and SPIONs@Au are not particularly cytotoxic to mammalian cells (MCF-7 breast carcinoma cells and H9c2 cardiomyoblasts) in culture, as indicated by the reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium by viable cells in a phenazine methosulfate-assisted reaction. PMID:21103390

Phagocytic cell function in active brucellosis.

PubMed Central

Ocon, P; Reguera, J M; Morata, P; Juarez, C; Alonso, A; Colmenero, J D

1994-01-01

In this study, we analyzed phagocytic cell function in 51 patients with active brucellosis and its relationship with different clinical, serological, and evolutionary variables. A control group was made up of 30 blood donors of similar geographic extraction, age, and sex, with no previous history of brucellosis or known exposure ot the infection or specific antibodies. The investigations were carried out at the time of diagnosis, at the conclusion of treatment, and after 6 months of follow-up. Polymorphonuclear leukocyte adherence and nitroblue tetrazolium reduction in response to Brucella antigen were significantly increased in the patients at the time of diagnosis with respect to the control group. In contrast, chemotaxis in response to Brucella antigen and phagocytosis were significantly reduced in the patients with respect to the control group. The alterations in phagocytic cell function were greater in patients with bacteremia, with focal forms of the disease, or with a longer diagnostic delay. Most of these initial alterations tended to normalize with treatment, indicating their transient character. PMID:8112863

Long non-coding RNA OIP5-AS1 promotes proliferation of lung cancer cells and leads to poor prognosis by targeting miR-378a-3p.

PubMed

Wang, Maolong; Sun, Xiao; Yang, Yuling; Jiao, Wenjie

2018-06-13

The antisense of the OIP5-AS1 gene is a long non-coding RNA (lncRNA) that is reported to be upregulated and promotes cell proliferation in multiple human cancers; however, its function in lung cancer is unknown. We investigated the regulatory function and underlying mechanisms of OIP5-AS1 in lung cancer. OIP5-AS1 and microRNA (miR)-378a-3p expression were assayed by quantitative real-time PCR, and proliferation-related protein expression was measured by Western blotting. Cell viability was detected using methyl thiazolyl tetrazolium assay. Luciferase reporter assay and RNA immunoprecipitation were used to detect the direct regulation of miR-378a-3p by OIP5-AS1. Nude mice were used to test the function of OIP5-AS1 in vivo. OIP5-AS1 was highly expressed in lung cancer tissues and was correlated with tumor size and tumor growth speed. OIP5-AS1 overexpression increased lung cancer cell proliferation in vitro. Further investigation revealed that OIP5-AS1 functions as a competing endogenous RNA of miR-378a-3p. MiR-378a-3p overexpression inhibited cell proliferation and caused proliferation-associated proteins CDK4 and CDK6 to decrease in A549 cells. Overexpression of wild type OIP5-AS1 led to strong CDK4 and CDK6 expression; however, these two proteins did not change when mutated OIP5-AS1 was upregulated. Finally, in vivo assay showed that the speed of tumor growth was increased and decreased when OIP5-AS1 was upregulated and downregulated, respectively. Our results revealed that OIP5-AS1 acts as a growth-promoting lncRNA in lung cancer by suppressing miR-378a-3p function. OIP5-AS1 and miR-378a-3p interaction may provide a potential target for lung cancer treatment. © 2018 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.

Toxicity of nano- and micro-sized silver particles in human hepatocyte cell line L02

NASA Astrophysics Data System (ADS)

Liu, Pengpeng; Guan, Rongfa; Ye, Xingqian; Jiang, Jiaxin; Liu, Mingqi; Huang, Guangrong; Chen, Xiaoting

2011-07-01

Silver nanoparticles (Ag NPs) previously classified as antimicrobial agents have been widely used in consumers and industrial products, especially food storage material. Ag NPs used as antimicrobial agents may be found in liver. Thus, examination of the ability of Ag NPs to penetrate the liver is warranted. The aim of the study was to determine the optimal viability assay for using with Ag NPs in order to assess their toxicity to liver cells. For toxicity evaluations, cellular morphology, mitochondrial function (3-(4, 5-dimethylazol-2-yl)-2, 5-diphenyl-tetrazolium bromide, MTT assay), membrane leakage of lactate dehydrogenase (lactate dehydrogenase, LDH release assay), Oxidative stress markers (malonaldehyde (MDA), glutathione (GSH) and superoxide dismutase (SOD)), DNA damage (single cell gel eletrophoresis, SCGE assay), and protein damage were assessed under control and exposed conditions (24 h of exposure). The results showed that mitochondrial function decreased significantly in cells exposed to Ag NPs at 25 μg·mL-1. LDH leakage significantly increased in cells exposed to Ag NPs (>= 25 μg mL-1) while micro-sized silver particles tested displayed LDH leakage only at higher doses (100 μg·mL-1). The microscopic studies demonstrated that nanoparticle-exposed cells at higher doses became abnormal in size, displaying cellular shrinkage, and an acquisition of an irregular shape. Due to toxicity of silver, further study conducted with reference to its oxidative stress. The results exhibited significant depletion of GSH level, increase in SOD levels and lead to lipid peroxidation, which suggested that cytotoxicity of Ag NPs in liver cells might be mediated through oxidative stress. The results demonstrates that Ag NPs lead to cellular morphological modifications, LDH leakage, mitochondrial dysfunction, and cause increased generation of ROS, depletion of GSH, lipid peroxidation, oxidative DNA damage and protein damage. Though the exact mechanism behind Ag NPs toxicity is suggested oxidative stress and lipid peroxidation playing an important role in Ag NPs elicited cell membrane disruption, DNA damage, protein damage and subsequent cell death. Our preliminary data suggest that oxidative stress might contribute to Ag NPs cytotoxicity. To reveal whether apoptosis involved in Ag NPs toxicity, further studies are underway.

Correlation between chemotherapy resistance in osteosarcoma patients and PAK5 and Ezrin gene expression

PubMed Central

Liu, Qian; Xu, Bo; Zhou, Wanshan

2018-01-01

The correlation between PAK5 (P21-activated kinase 5) and Ezrin gene expression and chemotherapy resistance of osteosarcoma patients was investigated. The cisplatin (CDDP)-resistance model of osteosarcoma cells SOSP-9607/CDDP was established to detect the cell growth curve. Methyl thiazolyl tetrazolium (MTT) assay was used to detect the drug resistance of cells to chemotherapy drugs. Transwell assay was used to detect the invasive capacity of cells. Semi-quantitative PCR (qPCR) was used to detect the mRNA expression levels in the drug resistance-related genes PAK5 and Ezrin. Western blot analysis was used to detect the protein expression levels in PAK5 and Ezrin. Tumor tissues were taken from the osteosarcoma patients with chemotherapy resistance to detect the expression levels of PAK5 and Ezrin via immunohistochemical detection, and the correlation between PAK5 and Ezrin expressions was studied. The results of MTT assay showed that the growth rate of SOSP-9607 was similar to that of SOSP-9607/CDDP, and the difference was not statistically significant (P>0.05). The sensitivity of SOSP-9607 to CDDP was significantly higher than that of SOSP-9607/CDDP, and the difference was statistically significant (P<0.01). Transwell assay showed that the migration capacity of SOSP-9607/CDDP was significantly better than that of SOSP-9607 (P<0.01), indicating that the drug resistance cell lines of osteosarcoma were constructed successfully. Semi-qPCR and western blot analysis showed that the protein expression levels in PAK5 and Ezrin in SOSP-9607/CDDP were significantly higher than those in SOSP-9607 (P<0.01). The results of immunohistochemistry showed that the expression quantities of PAK5 and Ezrin in osteosarcoma tissues were significantly higher than those in para-tumor tissues (P<0.01). Pearson's correlation analysis showed that expression of PAK5 and Ezrin was positively correlated (r=0.197, P=0.023). The osteosarcoma resistance is closely related to the expression levels of PAK5 and Ezrin genes. Thus, PAK5 and Ezrin genes may affect the tolerance of osteosarcoma patients to chemotherapy drugs during treatment via the synergistic effect. PMID:29391894

Spectrum of primary immunodeficiency disorders in Sri Lanka

PubMed Central

2013-01-01

Background While primary immunodeficiencies (PID has been recognized in the west for decades, recognition has been delayed in the third world. This study attempts to detail the spectrum of PID, the therapy provided, and constraints in the diagnosis and treatment in a middle income country such as Sri Lanka. Methods Nine hundred and forty two patients with recurrent infections and features suggestive of immune deficiency, referred from the entire country in a 4 year period, to the sole immunology unit in Sri Lanka were included. The following tests were performed. Full blood counts, serum Immunoglobulin and complement C3 and C4 levels, functional antibody levels, enumeration of lymphocyte subsets, in vitro and in vivo T cell functional assays,, nitroblue tetrazolium assay to diagnose chronic granulomatous disease, hair shaft assay to diagnose Griscelli syndrome. Sequencing of the common gamma chain to identify x linked severe combined immune deficiency, and X linked agammaglobulinemia was confirmed by assaying for Btk mutations by single sequence conformation polymorphism. HIV/AIDS was excluded in all patients. Results Seventy three patients were diagnosed with a primary immune deficiency. The majority (60.27%) had antibody deficiency. Common variable immune deficiency was the commonest (28.76%), followed by X linked agammaglobulinemia (XLA) (20.54%). Five patients had possible hyper IgM syndrome. Ten patients had severe combined immune deficiency (SCID), including 2 with x linked SCID, in addition to DiGeorge syndrome (2), ataxia telangiectasia (6), autosomal dominant hyper IgE syndrome (2), chronic granulomatous disease (4), leucocyte adhesion deficiency type 1 (2) and Griscelli syndrome (3). Patients with autoinflammatory, innate immune and complement defects could not be identified due to lack of facilities. Conclusions Antibody deficiency is the commonest PID, as in the west.IgA deficiency is rare. Autoinflammatory diseases, innate immune and complement deficiencies could not be identified due to lack of diagnostic facilities. Lack of awareness of PID among adult physicians result in delay in treatment of adult patients. While treatment of antibody deficiencies provided in state hospitals has extended life expectancy, there is no treatment available for severe T cell defects. PMID:24373416

Chemical Composition and In Vitro Cytotoxicity of Essential Oils from Leaves and Flowers of Callistemon citrinus from Western Himalayas.

PubMed

Kumar, Dharmesh; Sukapaka, Mahesh; Babu, G D Kiran; Padwad, Yogendra

2015-01-01

Plant-based traditional system of medicine continues to play an important role in healthcare. In order to find new potent source of bioactive molecules, we studied the cytotoxic activity of the essential oils from the flowers and leaves of Callistemon citrinus. This is the first report on anticancer potential of essential oils of C. citrinus. Cytotoxicity of essential oil was evaluated using sulfo-rhodamine B (SRB) assay against human lung carcinoma (A549), rat glioma (C-6), human colon cancer (Colo-205) and human cervical cancer (SiHa) cells. Apoptosis induction was evaluated by caspase-3/7 activity which was further confirmed by western blotting. Percentage cell apoptosis was determined by Annexin V based dead cell assay followed by DNA content as cell cycle analysis against A549 and C-6 cells. While 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to check the toxicity against normal human peripheral blood mononuclear cells (PBMCs), the immunomodulatory activity on mouse splenocytes was evaluated using SRB assay. The GC and GC-MS analysis of these essential oils revealed high content of α-pinene (32.3%), limonene (13.1%) and α-terpineol (14.6%) in leaf sample, whereas the flower oil was dominated by 1,8-cineole (36.6%) followed by α-pinene (29.7%). The leaf oil contained higher amount of monoterpene hydrocarbons (52.1%) and sesquiterpenoids (14%) as compared to flower oil (44.6% and 1.2%, respectively). However, the flower oil was predominant in oxygenated monoterpenes (43.5%). Although both leaf and flower oils showed highest cytotoxicity on A549 cells (61.4%±5.0 and 66.7%±2.2, respectively), only 100 μg/mL flower oil was significantly active against C-6 cells (69.1%±3.1). Interestingly, no toxicity was recorded on normal cells. Higher concentration of 1,8-cineole and/or synergistic effect of the overall composition were probably responsible for the efficacy of flower and leaf oils against the tested cells. These oils may form potential source of natural anti-cancer compounds and play important role in human health.

Regulation of tumorigenesis and metastasis of hepatocellular carcinoma tumor endothelial cells by microRNA-3178 and underlying mechanism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Wei; Shen, Shiqiang, E-mail: shenshiqiang2014@hotmail.com; Wu, Shanmin

2015-08-28

This study explored the effects of microRNA-3178 (miR-3178) on hepatocellular carcinoma (HCC) tumor endothelial cells (TECs) and on the target mRNA. Real-time polymerase chain reaction (PCR) was performed to detect the differential expression of miR-3178 in hepatic sinusoidal endothelial cells (HSECs) and HCC TECs. Furthermore, HCC TECs were transfected with miR-3178 mimic/inhibitor or their respective negative controls. The expression of miR-3178 before and after transfection was confirmed through RT-PCR. The effects of miR-3178 on the proliferation, apoptosis, cell cycle, invasion, migration, and angiogenesis of HCC TECs were also investigated through methyl thiazol tetrazolium assay, flow cytometry, matrigel invasion assay, transwellmore » migration assay, and tube formation assay. Early growth responsive gene 3 (EGR3), as the putative target of miR-3178, was detected through RT-PCR and Western blot. Compared with HSECs, HCC TECs had lower miR-3178 expression levels (P < 0.001). MiR-3178 mimic inhibited proliferation, arrested cell cycle in G1 phase, and increased apoptosis. The numbers of migrated and invaded cells and capillary-like structures were significantly less in the mimic group than in the other groups. MiR-3178 mimic significantly decreased the mRNA and protein expression levels of EGR3. By contrast, miR-3178 inhibitor induced opposite effects. We conclude that miR-3178 was lowly expressed in HCC TECs, and miR-3178 mimic specifically inhibited the proliferation, migration, invasion, and angiogenesis and promoted the apoptosis and G1 phase arrest of HCC TECs in vitro through the inhibition of EGR3 expression. Thus, miR-3178 might be a critical target in HCC therapy. - Highlights: • MiR-3178 is significantly low-expression in HCC TECs. • MiR-3178 acts as a tumor suppressor to inhibit tumorigenesis and metastasis. • MiR-3178 inhibit angiogenesis of HCC TECs. • EGR3 may be a target gene of miR-3178. • MiR-3178 may have therapeutic application for treatment of HCC.« less

9 CFR 113.28 - Detection of mycoplasma contamination.

Code of Federal Regulations, 2011 CFR

2011-01-01

... following: 1 percent tetrazolium chloride (ml) 5.5 1 percent thallium acetate (ml) 25 Penicillin (units) 500... solution 525,000 units of Penicillin. Dispense 10 ml aliquots into 60×15 mm disposable culture dishes or...

9 CFR 113.28 - Detection of mycoplasma contamination.

Code of Federal Regulations, 2013 CFR

2013-01-01

... following: 1 percent tetrazolium chloride (ml) 5.5 1 percent thallium acetate (ml) 25 Penicillin (units) 500... solution 525,000 units of Penicillin. Dispense 10 ml aliquots into 60×15 mm disposable culture dishes or...

9 CFR 113.28 - Detection of mycoplasma contamination.

Code of Federal Regulations, 2012 CFR

2012-01-01

... following: 1 percent tetrazolium chloride (ml) 5.5 1 percent thallium acetate (ml) 25 Penicillin (units) 500... solution 525,000 units of Penicillin. Dispense 10 ml aliquots into 60×15 mm disposable culture dishes or...

9 CFR 113.28 - Detection of mycoplasma contamination.

Code of Federal Regulations, 2014 CFR

2014-01-01

... following: 1 percent tetrazolium chloride (ml) 5.5 1 percent thallium acetate (ml) 25 Penicillin (units) 500... solution 525,000 units of Penicillin. Dispense 10 ml aliquots into 60×15 mm disposable culture dishes or...

Antioxidant Effects of Herbal Tea Leaves from Yacon (Smallanthus sonchifolius) on Multiple Free Radical and Reducing Power Assays, Especially on Different Superoxide Anion Radical Generation Systems.

PubMed

Sugahara, Shintaro; Ueda, Yuto; Fukuhara, Kumiko; Kamamuta, Yuki; Matsuda, Yasushi; Murata, Tatsuro; Kuroda, Yasuhiro; Kabata, Kiyotaka; Ono, Masateru; Igoshi, Keiji; Yasuda, Shin

2015-11-01

Yacon (Smallanthus sonchifolius), a native Andean plant, has been cultivated as a crop and locally used as a traditional folk medicine for the people suffering from diabetes and digestive/renal disorders. However, the medicinal properties of this plant and its processed foods have not been completely established. This study investigates the potent antioxidative effects of herbal tea leaves from yacon in different free radical models and a ferric reducing model. A hot-water extract exhibited the highest yield of total polyphenol and scavenging effect on 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical among four extracts prepared with hot water, methanol, ethanol, and ethylacetate. In addition, a higher reducing power of the hot-water extract was similarly demonstrated among these extracts. Varying concentrations of the hot-water extract resulted in different scavenging activities in four synthetic free radical models: DPPH radical (EC50 28.1 μg/mL), 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) cation radical (EC50 23.7 μg/mL), galvinoxyl radical (EC50 3.06 μg/mL), and chlorpromazine cation radical (EC50 475 μg/mL). The yacon tea-leaf extract further demonstrated superoxide anion (O2(-)) radical scavenging effects in the phenazine methosulfate-NADH-nitroblue tetrazolium (EC50 64.5 μg/mL) and xanthine oxidase assay systems (EC50 20.7 μg/mL). Subsequently, incubating human neutrophilic cells in the presence of the tea-leaf extract could suppress the cellular O2(-) radical generation (IC50 65.7 μg/mL) in a phorbol 12-myristate 13-acetate-activated cell model. These results support yacon tea leaves may be a good source of natural antioxidants for preventing O2(-) radical-mediated disorders. Yacon has been considered to be a potent alternative food source for patients who require a dietary cure in regional area, while the leaf part has been provided and consumed as an herbal tea in local markets. We demonstrated here potent antioxidative effects of the tea leaves from yacon in different free radical assays, reducing power assay, and cellular superoxide anion radical generation assay. Results support yacon tea leaves may be a good source of natural antioxidants for preventing O2(-) radical-mediated disorders. © 2015 Institute of Food Technologists®

The 15-lipoxygenase inhibitory, antioxidant, antimycobacterial activity and cytotoxicity of fourteen ethnomedicinally used African spices and culinary herbs.

PubMed

Dzoyem, Jean Paul; Kuete, Victor; McGaw, Lyndy J; Eloff, Jacobus N

2014-10-28

Culinary herbs and spices are widely used ethnomedically across Africa. They are traditionally employed in the treatment of several ailments including inflammation disorders, pain alleviation and infectious diseases. Pharmacological studies are necessary to provide a scientific basis to substantiate their traditional use and safety. In this study, the 15-lipoxygenase inhibitory, antioxidant, antimycobacterial and the cytotoxic activities, total phenolic and flavonoid contents of fourteen edible plants were investigated. The 15-lipoxygenase inhibitory activity was evaluated by the ferrous oxidation-xylenol orange (FOX) assay method. The antioxidant activity was determined using free-radical scavenging assays. The antimycobacterial activity was determined by a broth microdilution method against three species of mycobacteria: Mycobacterium smegmatis, Mycobacterium aurum and Mycobacterium fortuitum using tetrazolium violet as growth indicator. The cytotoxicity was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on Vero monkey kidney cells. All the extracts tested had some 15-lipoxygenase inhibitory activity ranging from 32.9 to 78.64%. Adansonia digitata (fruit) had the highest antioxidant capacity (IC₅₀ values of 8.15 μg/mL and 9.16 μg/mL in the DPPH and ABTS assays respectively; TEAC of 0.75 in the FRAP assay) along with the highest amount of total phenolics (237.68 mg GAE/g) and total flavonoids (16.14 mg E/g). There were good correlations between DPPH and ABTS values (R(2) 0.98) and between total phenolics and total flavonoids (R(2) 0.94). Tamarindus indica had significant antimycobacterial activity against Mycobacterium aurum (MIC 78 μg/mL). As could be expected with edible plants, all the extracts had a relatively low cytotoxicity with LC₅₀ values higher than 102 μg/mL with the exception of the two Aframomum species (33 and 74 μg/mL). This study provides scientific support for some of the the traditional uses and the pharmacological activities of the culinary herbs and spices investigated. The results suggest that increasing intake of some of these herbs may be useful in preventing or reducing the progression of lifestyle-related diseases. The diversity of the pharmacological activities of the extract from the fruit of Adansonia digitata suggested that this plant might be valuable for application in human and animal health. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Methanol extract of Codium fragile inhibits tumor necrosis factor-α-induced matrix metalloproteinase-9 and invasiveness of MDA-MB-231 cells by suppressing nuclear factor-κB activation.

PubMed

Dilshara, Matharage Gayani; Jayasooriya, Rajapaksha Gedara Prasad Tharanga; Kang, Chang-Hee; Choi, Yung-Hyun; Kim, Gi-Young

2016-06-01

To evaluate whether the methanol extract of Codium fragile (MECF) regulates tumor necrosis factor-α (TNF-α)-induced invasion of human breast cancer MDA-MB-231 cells by suppressing matrix metalloproteinase-9 (MMP-9). Reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis were performed to analyze the expression of MMP-9 and nuclear factor-κB (NF-κB) subunits, p65 and p50, and IκB in MDA-MB-231 cells. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used for cell viability. MMP-9 activity and invasion were measured by gelatin zymography and a matrigel invasion assay, respectively. NF-κB activity was measured by an electrophoretic mobility shift assay and luciferase activity. MECF had no effect on cell viability up to a concentration of 100 μg/mL in human breast cancer MDA-MB-231 cells regardless of the presence of TNF-α. MDA-MB-231 cells that were stimulated with TNF-α showed a marked increase of invasion compared to the untreated control, whereas pretreatment with MECF downregulated the TNF-α-induced invasion of MDA-MB-231 cells. Additionally, zymography, western blot analysis, and RT-PCR confirmed that MECF decreased TNF-α-induced MMP-9 expression and activity which is a key regulator for cancer invasion. According to an electrophoretic morbidity shift assay, pretreatment with MECF in MDA-MB-231 cells significantly decreased the TNF-α-induced DNA-binding activity of NF-κB, which is an important transcription factor for regulating cancer invasion-related genes such as MMP-9. Furthermore, treatment with MECF sustained the expression of p65 and p50 in response to TNF-α in the cytosolic compartment. The luciferase assay demonstrated that MECF attenuated TNF-α-induced NF-κB luciferase activity. MECF exhibited its anti-invasive capability by downregulating TNF-α-induced MMP-9 expression, resulting from the suppression of NF-κB activity in the human breast cancer cell line MDA-MB-231. Copyright © 2016 Hainan Medical College. Production and hosting by Elsevier B.V. All rights reserved.

Effects of magnolol and honokiol on adhesion, yeast-hyphal transition, and formation of biofilm by Candida albicans.

PubMed

Sun, Lingmei; Liao, Kai; Wang, Dayong

2015-01-01

The first step in infection by Candida albicans is adhesion to host cells or implanted medical devices and this followed by hyphal growth and biofilm formation. Yeast-to-hyphal transition has long been identified as a key factor in fungal virulence. Following biofilm formation, C. albicans is usually less sensitive or insensitive to antifungals. Therefore, development of new antifungals with inhibitory action on adhesion, yeast-hyphal transition and biofilm formation by C. albicans is very necessary. The effects of magnolol and honokiol on hypha growth were investigated using different induction media. Their inhibitory effects were determined using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5- carboxanilide assay, and biofilm thickness and viability were observed by a confocal scanning laser microscope. Mammalian cells were used in adhesion assays. Genes related to hyphae development and cell adhesions were analyzed by real-time reverse transcription-polymerase chain reaction. The exogenous cyclic adenosine monophosphate was used to determine the mechanisms of action of magnolol and honokiol. Caenorhabditis elegans was used as an in vivo model to estimate the antifungal activities of magnolol and honokiol. Magnolol and honokiol inhibited adhesion, the transition from yeast to hypha, and biofilm formation by C. albicans through the Ras1-cAMP-Efg1 pathway. Moreover, magnolol and honokiol prolonged the survival of nematodes infected by C. albicans. Magnolol and honokiol have potential inhibitory effects against biofilm formation by C. albicans. This study provides useful information towards the development of new strategies to reduce the incidence of C. albicans biofilm-associated infection.

Early effects of low dose 12C6+ ion or X-ray irradiation on human peripheral blood lymphocytes

NASA Astrophysics Data System (ADS)

Chen, Yingtai; Li, Yumin; Zhang, Hong; Xie, Yi; Chen, Xuezhong; Ren, Jinyu; Zhang, Xiaowei; Zhu, Zijiang; Liu, Hongliang; Zhang, Yawei

2010-04-01

The aim of this study was to estimate the acute effects of low dose 12C6+ ions or X-ray radiation on human immune function. The human peripheral blood lymphocytes (HPBL) of seven healthy donors were exposed to 0.05 Gy 12C6+ ions or X-ray radiation and cell responses were measured at 24 h after exposure. The cytotoxic activities of HPBL were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT); the percentages of T and NK cells subsets were detected by flow cytometry; mRNA expression of interleukin (IL)-2, tumor necrosis factor (TNF)-α and interferon (IFN)-γ were examined by real time quantitative RT-PCR (qRT-PCR); and these cytokines protein levels in supernatant of cultured cells were assayed by enzyme-linked immunosorbent assays (ELISA). The results showed that the cytotoxic activity of HPBL, mRNA expression of IL-2, IFN-γ and TNF-α in HPBL and their protein levels in supernatant were significantly increased at 24 h after exposure to 0.05 Gy 12C6+ ions radiation and the effects were stronger than observed for X-ray exposure. However, there was no significant change in the percentage of T and NK cells subsets of HPBL. These results suggested that 0.05 Gy high linear energy transfer (LET) 12C6+ radiation was a more effective approach to host immune enhancement than that of low LET X-ray. We conclude that cytokines production might be used as sensitive indicators of acute response to LDI.

Prognostic Value of Soluble Death Receptor Ligands in Patients with Transitional Cell Carcinoma of Bladder.

PubMed

Ben Bahria-Sediki, Islem; Chebil, Mohamed; Sampaio, Carla; Martel-Frachet, Véronique; Cherif, Mohamed; Zermani, Rachida; Rammeh, Soumaya; Ben Ammar Gaaied, Amel; Bettaieb, Ali

2018-05-02

The activation of Fas/Fas ligand (FasL) and DR4-DR5/tumor necrosis factor-related-apoptosis-inducing ligand (TRAIL) pathways in cancer cells triggers apoptosis. The objective of this study was to investigate the prognostic value of soluble FasL (sFasL) and soluble (sTRAIL) in the serum of patients with bladder cancer. The sFasL and sTRAIL levels in the sera of patients with bladder cancer or healthy donors were determined using the enzyme-linked immunosorbent assay. Micro-culture tetrazolium viability assay and Western blot were used to analyze cell cytotoxicity and death receptors protein expression respectively. Whether no difference in sTRAIL levels was seen between patients and controls, the level of sFasL was higher in patients than that in healthy donors. According to, sFasL level was the highest in the serum of patients with superficial stage or low- and medium-grade cancer. Moreover, sFasL in patients with superficial noninvasive bladder tumors or low- and medium-grade cancers was higher than that in patients with invasive carcinomas and high-grade cancers. Patients with high levels of sFasL survive longer than those with low levels, probably related to the cytotoxic potential of FasL preserved in its soluble form. The data suggest that monitoring the level of sFasL and its cytotoxic activity could be a prognostic marker in the follow-up of patients with bladder cancer. © 2018 S. Karger AG, Basel.

Promoting effects of chemical permeation enhancers on insulin permeation across TR146 cell model of buccal epithelium in vitro.

PubMed

Xue, Xiao-yan; Zhou, Ying; Chen, Ying-ying; Meng, Jing-ru; Jia, Min; Hou, Zheng; Bai, Hui; Mao, Xing-gang; Luo, Xiao-xing

2012-04-01

To find potential enhancers for facilitating the buccal delivery of insulin, a TR146 cell-culture model of buccal epithelium, cultured on commercially available insert plates, was used to evaluate the permeability-enhancing effects of several traditional and new types of chemical enhancers, including N-acetyl-L-cysteine (NAC), sodium deoxycholate (SDC), sodium nitroprusside (SNP), reduced glutathione (GSH), glutamine (Gln), chitosan (CS), L-arginine (Arg), 1-dodecylazacycloheptan-2-one (Azone), and soybean lecithin (SPC) (50 and 10 μg/mL respectively). Permeability studies were performed to determine the enhancing effects of these compounds on insulin permeation across the cell-culture model. The enhancing effects of the enhancers were assessed by calculating the apparent permeability coefficients and enhancement ratio. Cytotoxicity of the permeation enhancers at different concentrations was investigated by using the methylthiazolydiphenyl-tetrazolium bromide (MTT) assay. Results showed that 50 μg/mL of NAC, SDC, GSH, CS, Arg, Azone, SPC, SNP, and 10 μg/mL of SNP had a significant enhancing effect on promoting the transport of insulin across the TR146 cell model. MTT assays showed that 50 μg/mL of Gln, Azone, SDC, SNP, Arg, 10 μg/mL SDC, and Arg had obvious toxic effects on TR146 cells. Therefore, NAC, GSH, CS, SPC, and SNP appear to be safe, effective permeability enhancers that promote the transport of insulin across the TR146 cell-culture model of buccal epithelium and may be potential enhancers for buccal delivery of insulin with both low toxicity and high efficiency.

Cytotoxic and Apoptogenic Effects of Cyanidin-3-Glucoside on the Glioblastoma Cell Line.

PubMed

Hosseini, Masoumeh Mansoubi; Karimi, Aliasghar; Behroozaghdam, Mitra; Javidi, Mohammad Amin; Ghiasvand, Saeedeh; Bereimipour, Ahmad; Aryan, Hoda; Nassiri, Farbod; Jangholi, Ehsan

2017-12-01

Glioblastoma multiforme (GBM) is the most prevalent and aggressive primary cerebral tumor. The median survival time is 15 months despite maximum treatment because the tumor is resistant to most therapeutic modalities. Several studies have indicated chemopreventive and chemotherapeutic activity of cyanidin-3-glucoside (C3G) as an anthocyanin component. We aimed to illustrate the cytotoxic and apoptogenic effects of C3G in the U87 cell line (human GBM cell line). Cytotoxic activity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium assay after treatment with C3G at different concentrations in the U87 cell line. Cisplatin was used as a positive control for 24 and 48 hours. The percentage of apoptotic cells was determined using an Annexin V/propidium iodide assay, and the expression of bax, bcl2, and p53 genes was assessed using real-time polymerase chain reaction. Treatment of U87 cells with 40 μg/mL of C3G resulted in 32% apoptotic cells after 24 hours. To further confirm that C3G treatment induced apoptosis in U87 cells, RNA expression of bax, bcl2, and p53 genes was investigated after treatment. Real-time polymerase chain reaction indicated that the expression of bax and p53 increased, whereas the expression of bcl2 decreased. C3G had an apoptogenic effect in the GBM cell line. New information regarding the therapeutic effects of C3G in GBM could ultimately lead to the production of new drugs. Copyright © 2017 Elsevier Inc. All rights reserved.

The construction of a novel kind of non-viral gene delivery vector based on protein as core backbone.

PubMed

Li, D; Kong, Y; Yu, H; Lehtinen, A; Huang, H; Shen, F; Min, L; Zhou, J; Tang, G; Wang, Q

2008-04-01

A novel kind of non-viral gene delivery vector based on transferrin (Tf) as the core component was constructed with high transfection efficiency and low toxicity. The synthesis vector of Tf-PEI600 was confirmed by different physicochemical methods, including (1)H nuclear magnetic resonance, gel permeation chromatography, X-ray and thermogravimetric analysis. The cytotoxicity and gene delivery efficiency of the synthesized vector were verified by in vitro experiments. The agarose gel electrophoresis assay indicated that the novel copolymer Tf-PEI600 could efficiently condense plasmid DNA and the condensed nanoparticles exhibited a spherical shape. As the weight ratio of Tf-PEI600 to DNA reached 15.0, the particle size (about 200 nm) and the zeta potential (about 20 mV) of the nanoparticles became optimal for gene delivery. The methylthiazolyl tetrazolium (MTT) assay showed the cytotoxicity of Tf-PEI600 to be similar to that of PEI600 and much lower than that of PEI25kDa. In gene-delivery experiments with COS-7 cells and HepG2 cells, the Tf-PEI600 showed about a 30- to 53-fold higher efficiency than PEI600 and nearly equal to that of PEI25kDa. These data suggest that Tf-PEI600, with the advantages of low toxicity and high gene-delivery efficiency, might have great prospects in the practice of gene delivery. The core-shell structure of Tf-PEI600 also provided a novel strategy for the construction of non-viral gene delivery vectors.

In Vitro Ultrastructural Changes of MCF-7 for Metastasise Bone Cancer and Induction of Apoptosis via Mitochondrial Cytochrome C Released by CaCO3/Dox Nanocrystals

PubMed Central

Shafiu Kamba, Abdullahi; Ismail, Maznah; Tengku Ibrahim, Tengku Azmi; Zakaria, Zuki Abu Bakar; Hassan Gusau, Lawal

2014-01-01

Bones are the most frequent site for breast cancer cells to settle and spread (metastasise); bone metastasis is considered to have a substantial impact on the quality of patients with common cancers. However, majority of breast cancers develop insensitivity to conventional chemotherapy which provides only palliation and can induce systemic side effects. In this study we evaluated the effect of free Dox and CaCO3/Dox nanocrystal on MCF-7 breast cancer using MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide), neural red, and lactate dehydrogenase colorimetric assays while DNA fragmentation and BrdU genotoxicity were also examined. Apoptogenic protein Bax, cytochrome C, and caspase-3 protein were analysed. Morphological changes of MCF-7 were determined using contrast light microscope and scanning and transmission electron microscope (SEM and TEM). The findings of the analysis revealed higher toxicity of CaCO3/Dox nanocrystal and effective cells killing compared to free Dox, morphological changes such as formation of apoptotic bodies, membrane blebbing, and absent of microvilli as indicated by the SEM analysis while TEM revealed the presence of chromatin condensation, chromosomal DNA fragmentation, cell shrinkage, and nuclear fragmentation. Results of TUNEL assay verified that most of the cells undergoes apoptosis by internucleosomal fragmentation of genomic DNA whereas the extent of apoptotic cells was calculated using the apoptotic index (AI). Therefore, the biobased calcium carbonate nanocrystals such as Dox carriers may serve as an alternative to conventional delivery system. PMID:25028650

Susceptible cytotoxicity to ultraviolet B light in fibroblasts and keratinocytes cultured from autoimmune-prone MRL/Mp-lpr/lpr mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Furukawa, F.; Lyon, M.B.; Norris, D.A.

1989-09-01

The MRL/Mp-lpr/lpr (MRL/l) mouse is an autoimmune model of spontaneous lupus erythematosus (LE), in addition to lupus nephritis. In order to better understand the mechanisms of photosensitivity in LE, in vitro photocytotoxicity was examined by using fibroblasts and keratinocytes cultured from MRL/l mice, control MRL/Mp- +/+ (MRL/n) mice, and normal BALB/c mice. A colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and the acridine orange/ethidium bromide assay were used for determination of cytotoxicity. Fibroblasts cultured from newborn MRL/l mice showed higher susceptibility to single ultraviolet light B (UVB) light irradiation at a dose of 100-500 mJ than those from MRL/n, F1 hybrid ofmore » (MRL/l x MRL/n mice), and BALB/c mice. However, the susceptibility to UVB was not observed in young (1-month-old) and adult (4-month-old) MRL/l mice. UVA light irradiation was not cytotoxic. Keratinocytes cultured from MRL mice showed lower cytotoxicity to UVB irradiation than fibroblasts cultured. However, keratinocytes from newborn MRL/l mice showed higher cytotoxicity to 50 mJ UVB irradiation than cells from MRL/n mice. Syngeneic or allogeneic sera augmented UVB-induced cytotoxicity of fibroblasts cultured. UVB irradiation of spleen cells induced no significant difference of cytotoxicity between MRL/l and MRL/n mice. Based on the results of F1 hybrid of (MRL/l x MRL/n) mice, the susceptibility seemed to be associated with autoimmune traits and to be regulated by genetical background.« less

Tellurium tetrachloride and diphenyl ditelluride cause cytotoxicity in rat hippocampal astrocytes.

PubMed

Roy, Shalini; Hardej, Diane

2011-10-01

Tellurium tetrachloride (TeCl(4)) and diphenyl ditelluride (DPDT) cytotoxicity, was investigated in rat astrocytes. Concentrations of 0.24-250μM (24h) were tested for viability using MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) and trypan blue exclusion. MTT showed significant decreases at all concentrations tested for both compounds. Significant decreases in viability were seen in 1.95-250μM of DPDT and 0.97-250μM of TeCl(4) with trypan blue exclusion. The LC(50) for both compounds was 62.5μM. Light and scanning microscopy confirm toxicity observed at higher concentrations. Thiobarbituric acid reactive substances (TBARs) assay, TUNEL, cytochrome c and caspase release were carried out. No significant increase in TBARS with either agent was observed (15.625-62.5μM). TUNEL and cytochrome c assays demonstrated apoptosis in TeCl(4) treated cells (31.25-125μM). Non-apoptotic cells were observed in DPDT treated cells. Studies of caspase 3/7 and caspase 9 indicated increased activity in TeCl(4) but not in DPDT treated cells. Optical Emission Spectroscopy of DPDT and TeCl(4) treated cells demonstrated significant accumulation of elemental tellurium in all treatment groups (31.25-125μM). We conclude that DPDT and TeCl(4) are cytotoxic to astrocytes. TeCl(4) treated cells die via the intrinsic apoptotic pathway. Accumulation of tellurium occurs with both compounds, but results in different mechanisms of cell death. Copyright © 2011 Elsevier Ltd. All rights reserved.

Cytotoxicity and cytokine expression induced by silorane and methacrylate-based composite resins

PubMed Central

LONGO, Daniele Lucca; PAULA-SILVA, Francisco Wanderley Garcia; FACCIOLI, Lucia Helena; GATÓN-HERNÁNDEZ, Patrícia Maria; de QUEIROZ, Alexandra Mussolino; da SILVA, Léa Assed Bezerra

2016-01-01

ABSTRACT The successful use of composite resins in Dentistry depends on physicochemical properties, but also on the biological compatibility of resins, because of the close association between pulp and dentin. Objective The aim of this study was to evaluate cytotoxicity and cytokine production induced by light-cured or non-light-cured methacrylate-based and silorane composite resins in RAW 264.7 macrophages. Material and Methods Cells were stimulated with the extracts from light-cured or non-light-cured composite resins. After incubation for 24 h, cytotoxicity was assessed with the lactate dehydrogenase (LDH) and methyl thiazolyl tetrazolium (MTT) assays, and total protein was quantified using the Lowry method. TNF-α detection was examined with an enzyme-linked immunosorbent assay (ELISA) conducted with cell supernatants after cell stimulation for 6, 12, and 24 h. Data were analyzed using one-way analysis of variance (ANOVA) and Tukey’s post hoc test (α=0.05). Results KaloreTM and FiltekTM Silorane were cytotoxic with or without light curing (p<0.05) after 24 h of incubation. KaloreTM stimulated the early production of TNF-α in comparison with control (p<0.05), whereas FiltekTM Silorane did not affect TNF-α levels after 6 and 12 h (p>0.05). However, after 24 h FiltekTM Silorane inhibited the production of TNF-α (p<0.05). Conclusions KaloreTM and FiltekTM Silorane were cytotoxic regardless of light curing. The extract obtained from KaloreTM after 15 days of incubation stimulated the production of TNF-α, unlike that obtained from FiltekTM Silorane. PMID:27556204

Combined morphine and limb remote ischemic perconditioning provides an enhanced protection against myocardial ischemia/reperfusion injury by antiapoptosis.

PubMed

Wang, Shi-Yu; Cui, Xin-Long; Xue, Fu-Shan; Duan, Ran; Li, Rui-Ping; Liu, Gao-Pu; Yang, Gui-Zhen; Sun, Chao

2016-05-01

Both morphine and limb remote ischemic perconditioning (RIPer) can protect against myocardial ischemia/reperfusion injury (IRI). This experiment was designed to assess whether combined morphine and limb RIPer could provide and enhanced protection against myocardial IRI in an in vivo rat model. One hundred male Sprague-Dawley rats were randomly allocated to six groups: sham, ischemia/reperfusion (IR), ischemic preconditioning, RIPer, morphine (M), and combined morphine and remote ischemic perconditioning (M + RIPer). Ventricular arrhythmias that occurred during ischemia and early reperfusion were scored, and serum creatine kinase isoenzyme and cardiac troponin I levels were assayed. The infarct size was determined by Evans blue and triphenyl tetrazolium chloride staining. The apoptosis in the myocardial ischemic core, ischemic border, and nonischemic areas was assessed through real-time polymerase chain reaction for Bax and Bcl-2 and with the transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assay. The infarct size, serum cardiac troponin I level, incidence, and score of the arrhythmias during the initial reperfusion were significantly reduced in the M + RIPer group compared with the IR group but did not differ significantly between the ischemic preconditioning and M + RIPer groups. Transferase-mediated deoxyuridine triphosphate-biotin nick end labeling-positive cells were significantly decreased, and the Bcl-2/Bax ratio was significantly increased in the M + RIPer group compared with the IR group. This experiment demonstrates that combined morphine and limb RIPer provides an enhanced protection against myocardial IRI by the Bcl-2-linked apoptotic signaling pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

Ionic extraction of a novel nano-sized bioactive glass enhances differentiation and mineralization of human dental pulp cells.

PubMed

Gong, Weiyu; Huang, Zhiwei; Dong, Yanmei; Gan, Yehua; Li, Shenglin; Gao, Xuejun; Chen, Xiaofeng

2014-01-01

This study aimed to investigate the effects of a novel nano-sized 58S bioactive glass (nano-58S BG) on the odontogenic differentiation and mineralization of human dental pulp cells (hDPCs) in vitro. Extractions were prepared by incubating nano-58S BG, 45S5 BG, or 58S BG particulates in Dulbecco modified Eagle medium at 1% w/v for 24 hours and were filtrated through 0.22-μm filters. The supernatants were used as BG extractions. The hDPCs were cultured in nano-58S BG, 45S5 BG, and 58S BG extractions. The proliferation of hDPCs was evaluated using the methylthiazol tetrazolium assay. Odontogenic differentiation was evaluated based on the real-time polymerase chain reaction of differentiation- and mineralization-related genes, namely, alkaline phosphatase (ALP), collagen type I, dentin sialophosphoprotein (DSPP), and dentin matrix protein 1. The gene expressions were verified using ALP activity assessment, immunocytochemistry staining of osteocalcin and DSPP, and mineralization assay using alizarin red S stain. All BG extractions up-regulated the expression of odontogenic genes, and the most significant enhancement was in the nano-58S BG group. All BG extractions, especially nano-58S, increased ALP activity, osteocalcin and DSPP protein production, and mineralized nodules formation. Compared with regular BG, the novel nano-58S BG can induce the differentiation and mineralization of hDPCs more efficiently and might be a better potential candidate for dentin-pulp complex regeneration. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Inhibition of Survivin Influences the Biological Activities of Canine Histiocytic Sarcoma Cell Lines

PubMed Central

Hoshino, Yuki; Hosoya, Kenji; Okumura, Masahiro

2013-01-01

Canine histiocytic sarcoma (CHS) is an aggressive malignant neoplasm that originates from histiocytic lineage cells, including dendritic cells and macrophages, and is characterized by progressive local infiltration and a very high metastatic potential. Survivin is as an apoptotic inhibitory factor that has major functions in cell proliferation, including inhibition of apoptosis and regulation of cell division, and is expressed in most types of human and canine malignant neoplasms, including melanoma and osteosarcoma. To investigate whether survivin was expressed at high levels in CHS and whether its expression was correlated with the aggressive biological behavior of CHS, we assessed relation between survivin expression and CHS progression, as well as the effects of survivin inhibition on the biological activities of CHS cells. We comparatively analyzed the expression of 6 selected anti-apoptotic genes, including survivin, in specimens from 30 dogs with histiocytic sarcoma and performed annexin V staining to evaluate apoptosis, methylthiazole tetrazolium assays to assess cell viability and chemosensitivity, and latex bead assays to measure changes in phagocytic activities in 4 CHS cell lines and normal canine fibroblasts transfected with survivin siRNA. Survivin gene expression levels in 30 specimens were significantly higher than those of the other 6 genes. After transfection with survivin siRNA, apoptosis, cell growth inhibition, enhanced chemosensitivity, and weakened phagocytic activities were observed in all CHS cell lines. In contrast, normal canine fibroblasts were not significantly affected by survivin knockdown. These results suggested that survivin expression may mediate the aggressive biological activities of CHS and that survivin may be an effective therapeutic target for the treatment of CHS. PMID:24260303

Anticancer activity of Petroselinum sativum seed extracts on MCF-7 human breast cancer cells.

PubMed

Farshori, Nida Nayyar; Al-Sheddi, Ebtesam Saad; Al-Oqail, Mai Mohammad; Musarrat, Javed; Al-Khedhairy, Abdulaziz Ali; Siddiqui, Maqsood Ahmed

2013-01-01

Pharmacological and preventive properties of Petroselinum sativum seed extracts are well known, but the anticancer activity of alcoholic extracts and oil of Petroselinum sativum seeds on human breast cancer cells have not been explored so far. Therefore, the present study was designed to investigate the cytotoxic activities of these extracts against MCF-7 cells. Cells were exposed to 10 to 1000 μg/ml of alcoholic seed extract (PSA) and seed oil (PSO) of Petroselinum sativum for 24 h. Post-treatment, percent cell viability was studied by 3-(4, 5-dimethylthiazol-2yl)-2, 5-biphenyl tetrazolium bromide (MTT) and neutral red uptake (NRU) assays, and cellular morphology by phase contrast inverted microscopy. The results showed that PSA and PSO significantly reduced cell viability, and altered the cellular morphology of MCF-7 cells in a concentration dependent manner. Concentrations of 50 μg/ml and above of PSA and 100 μg/ml and above of PSO were found to be cytotoxic in MCF-7 cells. Cell viability at 50, 100, 250, 500 and 1000 μg/ml of PSA was recorded as 81%, 57%, 33%, 8% and 5%, respectively, whereas at 100, 250, 500, and 1000 μg/ml of PSO values were 90%, 78%, 62%, and 8%, respectively by MTT assay. MCF-7 cells exposed to 250, 500 and 1000 μg/ml of PSA and PSO lost their typical morphology and appeared smaller in size. The data revealed that the treatment with PSA and PSO of Petroselinum sativum induced cell death in MCF-7 cells.

Antiproliferation and induction of cell death of Phaffia rhodozyma (Xanthophyllomyces dendrorhous) extract fermented by brewer malt waste on breast cancer cells.

PubMed

Teo, Ivy Tuang Ngo; Chui, Chung Hin; Tang, Johnny Cheuk On; Lau, Fung Yi; Cheng, Gregory Yin Ming; Wong, Raymond Siu Ming; Kok, Stanton Hon Lung; Cheng, Chor Hing; Chan, Albert Sun Chi; Ho, Kwok Ping

2005-11-01

Astaxanthin has been shown to have antiproliferative activity on breast cancer and skin cancer cells. However, the high cost of production, isolation and purification of purified astaxanthin from natural sources or chemically synthetic methods limit its usage on cancer therapy. We show that astaxanthin could be produced by fermentating the Phaffia rhodozyma (Xanthophyllomyces dendrorhous) yeast cells with brewer malt waste using a 20 L B. Braun fermentor. The percentage composition of astaxanthin from the P. rhodozyma was >70% of total pigment as estimated by the high performance liquid chromatographic analysis. Furthermore, the antiproliferative activity of this P. rhodozyma cell extract (PRE) was demonstrated on breast cancer cell lines including the MCF-7 (estrogen receptor positive) and MDA-MB231 (estrogen receptor negative) by using the [3-(4,5-dimethylthiazol-2-yl)-5-(3-arboxymethoxyphenyl)-2- (4-sulfophenyl)-2H-tetrazolium] (MTS) assay. No apoptotic cell death, but growth inhibitory effect was induced after 48 h of PRE incubation as suggested by morphological investigation. Anchorage-dependent clonogenicity assay showed that PRE could reduce the colony formation potential of both breast cancer cell lines. Cell death was observed from both breast cancer cell lines after incubation with PRE for 6 days. Taken together, our results showed that by using an economic method of brewer malt waste fermentation, we obtained P. rhodozyma with a high yield of astaxanthin and the corresponding PRE could have short-term growth inhibition and long-term cell death activity on breast cancer cells.

Effects of Platinum Nanocolloid in Combination with Gamma Irradiation on Normal Human Esophageal Epithelial Cells.

PubMed

Li, Qiang; Tanaka, Yoshiharu; Saitoh, Yasukazu; Miwa, Nobuhiko

2016-05-01

Our previous study demonstrated that platinum nanocolloid (Pt-nc), combined with lower-dose gamma irradiation at 3, 5, and 7 Gy significantly decreased proliferation and accelerated apoptosis of the human esophageal squamous cell carcinoma-derived cell line KYSE-70. The aim of the present study was to determine, under the same conditions as our previous study where gamma rays combined with Pt-nc were carcinostatic to KYSE-70 cells, if we could induce a radioprotective or the radiation-sensitizing effect on the human normal esophageal epithelial cells (HEEpiC). HEEpiC were treated with various Pt-nc concentrations and then irradiated with various gamma-ray doses. The proliferative status of HEEpiC was evaluated using trypan blue dye-exclusion and WST-8 assays. The cellular and nucleic morphological features were determined using crystal violet and Hoechst 33342 stainings, respectively. The intracellular level of reactive oxygen species (ROS) in HEEpiC was evaluated with a nitro blue tetrazolium (NBT) assay. The apoptotic status was detected with caspase-3, Bax, and Bcl-2 by Western blotting. Either Pt-nc or gamma irradiation could inhibit the growth of HEEpiC; however, their combined use exerted a significant proliferation-inhibitory effect in a Pt-nc dose-dependent manner than gamma irradiation alone. Pt-nc resulted in radiation sensitization rather than radiation protection on HEEpiC in vitro similar to KYSE-70 cells, when Pt-nc was administrated alone or combined with gamma irradiation. Thus, Pt-nc has an inhibitory effect on cell proliferation, a facilitative effect on apoptosis, and a certain degree of toxicity against HEEpiC.

Effect of triptolide on progesterone production from cultured rat granulosa cells.

PubMed

Zhang, J; Jiang, Z; Mu, X; Wen, J; Su, Y; Zhang, L

2012-06-01

Triptolide(CAS 38748-32-2), a major active component of Tripterygium wilfordii Hook F (TWHF), is known to have multiple pharmacological activities. However, studies have also shown that triptolide is highly disrupt to the reproductive system by disrupting normal steroid hormone signaling. In the present study, we investigated the effect of triptolide (5, 10, or 20 nM for 24 h) on progesterone production by rat granulosa cells. Triptolide inhibited both basal and human chorionic gonadotropin (HCG)- and 8-bromo-cAMP-stimulated progesterone production as revealed by RIA assay. Furthermore, the HCG-evoked increase in cellular cAMP content was also inhibited by triptolide, indicating that disruption of the cAMP/PKA signaling pathway may mediate the deleterious effects of triptolide on progesterone regulation. In addition, triptolide inhibited 25-OH-cholesterol-stimulated progesterone production, suggesting that activity of the P450 side chain cleavage (P450scc) enzyme was also be inhibited by triptolide. Western blot and quantitative real-time PCR (qRT-PCR) assays further revealed that triptolide decreased mRNA and protein expression of P450scc and the steroidogenic regulatory (StAR) protein in granulosa cells. In contrast, cell viability tests using 3-(4,5-dimethyl-thiazol-2-yl)-2,5- diphenyl-tetrazolium bromide (MTT) indicated that triptolide did not cause measurable cell death at doses that suppressed steroidogenesis. The reproductive toxicity of triptolide may be caused by disruption of cAMP/PKA-mediated expression of a number of progesterone synthesis enzymes or regulatory proteins, leading to reduced progesterone synthesis and reproductive dysfunction. © Georg Thieme Verlag KG Stuttgart · New York.

Antiproliferative effects of cinobufacini on human hepatocellular carcinoma HepG2 cells detected by atomic force microscopy

PubMed Central

Wu, Qing; Lin, Wei-Dong; Liao, Guan-Qun; Zhang, Li-Guo; Wen, Shun-Qian; Lin, Jia-Ying

2015-01-01

AIM: To investigate the antiproliferative activity of cinobufacini on human hepatocellular carcinoma HepG2 cells and the possible mechanism of its action. METHODS: HepG2 cells were treated with different concentrations of cinobufacini. Cell viability was measured by methylthiazolyl tetrazolium (MTT) assay. Cell cycle distribution was analyzed by flow cytometry (FCM). Cytoskeletal and nuclear alterations were observed by fluorescein isothiocyanate-phalloidin and DAPI staining under a laser scanning confocal microscope. Changes in morphology and ultrastructure of cells were detected by atomic force microscopy (AFM) at the nanoscale level. RESULTS: MTT assay indicated that cinobufacini significantly inhibited the viability of HepG2 cells in a dose-dependent manner. With the concentration of cinobufacini increasing from 0 to 0.10 mg/mL, the cell viability decreased from 74.9% ± 2.7% to 49.41% ± 2.2% and 39.24% ± 2.1% (P < 0.05). FCM analysis demonstrated cell cycle arrest at S phase induced by cinobufacini. The immunofluorescence studies of cytoskeletal and nuclear morphology showed that after cinobufacini treatment, the regular reorganization of actin filaments in HepG2 cells become chaotic, while the nuclei were not damaged seriously. Additionally, high-resolution AFM imaging revealed that cell morphology and ultrastructure changed a lot after treatment with cinobufacini. It appeared as significant shrinkage and deep pores in the cell membrane, with larger particles and a rougher cell surface. CONCLUSION: Cinobufacini inhibits the viability of HepG2 cells via cytoskeletal destruction and cell membrane toxicity. PMID:25624718

[Anti-sense miRNA-21 oligonucleotide inhibits Tb 3.1 human tongue squamous cell carcinoma growth in vitro].

PubMed

Tao, Ying-jie; Ren, Yu; Dong, Jia-bin; Zhang, Lun; Cheng, Jun-ping; Zhou, Xuan

2011-02-01

To investigate the effect of micro RNA-21 (miRNA-21) knocking on the Tb3.1 human tongue squamous cell carcinoma growth. Anti-sense miRNA-21 oligonucleotide was delivered with oligofectamine to suppress Tb 3.1 tongue cancer cell growth in vitro. Real-time polymerase chain reaction (PCR) was conducted to detect the miRNA-21 expression after transfection. Methyl thiazolyl tetrazolium (MTT) assay was used to determine Tb 3.1 cell survival rate. Apoptosis were examined by flow-cytometry. Matrigel matrix and transwell assay were used to determine Tb 3.1 cell colony formation and migration ability. Antigen KI-67 (Ki67), B cell lymphoma (Bcl-2), phosphatase and tensin homolog (PTEN), matrirx metalloproteinase 2 (MMP-2, MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) protein expression in Tb 3.1 cell were measured by Western blotting. miRNA-21 expression was decreased in miRNA-21 antisense oligonucleotide (ASODN) group. The survival rate of Tb 3.1 cells with AS-miRNA-21 transfection was significantly suppressed (F = 27.02, P = 0.00) and early phase apoptosis (F = 26.641, P = 0.001) induced in Tb 3.1 cell. Ki67, Bcl-2, MMP-2 and MMP-9 protein were down regulated while PTEN and TIMP-1 protein expression was increased. Blocking miRNA-21 expression in Tb3.1 cell could suppress cancer cell growth in vitro and miRNA-21 can serve as a novel target candidate for human tongue cancer gene therapy.

Rhubarb Antagonizes Matrix Metalloproteinase-9-induced Vascular Endothelial Permeability

PubMed Central

Cui, Yun-Liang; Zhang, Sheng; Tian, Zhao-Tao; Lin, Zhao-Fen; Chen, De-Chang

2016-01-01

Background: Intact endothelial structure and function are critical for maintaining microcirculatory homeostasis. Dysfunction of the latter is an underlying cause of various organ pathologies. In a previous study, we showed that rhubarb, a traditional Chinese medicine, protected intestinal mucosal microvascular endothelial cells in rats with metastasizing septicemia. In this study, we investigated the effects and mechanisms of rhubarb on matrix metalloproteinase-9 (MMP9)-induced vascular endothelial (VE) permeability. Methods: Rhubarb monomers were extracted and purified by a series of chromatography approaches. The identity of these monomers was analyzed by hydrogen-1 nuclear magnetic resonance (NMR), carbon-13 NMR, and distortionless enhancement by polarization transfer magnetic resonance spectroscopy. We established a human umbilical vein endothelial cell (HUVEC) monolayer on a Transwell insert. We measured the HUVEC permeability, proliferation, and the secretion of VE-cadherin into culture medium using fluorescein isothiocyanate-dextran assay, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay, and enzyme-linked immunosorbent assay, respectively, in response to treatment with MMP9 and/or rhubarb monomers. Results: A total of 21 rhubarb monomers were extracted and identified. MMP9 significantly increased the permeability of the HUVEC monolayer, which was significantly reduced by five individual rhubarb monomer (emodin, 3,8-dihydroxy-1-methyl-anthraquinone-2-carboxylic acid, 1-O-caffeoyl-2-(4-hydroxyl-O-cinnamoyl)-β-D-glucose, daucosterol linoleate, and rhein) or a combination of all five monomers (1 μmol/L for each monomer). Mechanistically, the five-monomer mixture at 1 μmol/L promoted HUVEC proliferation. In addition, MMP9 stimulated the secretion of VE-cadherin into the culture medium, which was significantly inhibited by the five-monomer mixture. Conclusions: The rhubarb mixture of emodin, 3,8-dihydroxy-1-methyl-anthraquinone-2-carboxylic acid, 1-O-caffeoyl-2-(4-hydroxyl-O-cinnamoyl)-β-D-glucose, daucosterol linoleate, and rhein, at a low concentration, antagonized the MMP9-induced HUVEC monolayer permeability by promoting HUVEC proliferation and reducing extracellular VE-cadherin concentrations. PMID:27411464

Edaravone inhibits hypoxia-induced trophoblast-soluble Fms-like tyrosine kinase 1 expression: a possible therapeutic approach to preeclampsia.

PubMed

Zhao, Y; Zheng, Y F; Luo, Q Q; Yan, T; Liu, X X; Han, L; Zou, L

2014-07-01

To investigate the effects of edaravone, a potent free radical scavenger used clinically, on hypoxia-induced trophoblast-soluble Fms-like tyrosine kinase 1 (sFlt-1) expression. A trophoblast cell line (HRT-8/SVneo) impaired by cobalt chloride (CoCl2) was used as the cell model under hypoxic conditions. 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) was used to measure the viability of cells exposed to CoCl2 and edaravone. The levels of intracellular reactive oxygen species (ROS) were analyzed by flow cytometry. mRNA expression of sFlt-1, vascular endothelial growth factor (VEGF), and placental growth factor (PlGF) in trophoblasts was measured by real-time polymerase chain reaction, and the secretion of sFlt-1, VEGF, and PlGF proteins was analyzed by enzyme-linked immunosorbent assays (ELISAs). A human umbilical vein endothelial cell (HUVEC) tube-formation assay was performed to identify the effects of CoCl2 and edaravone on vascular development. CoCl2 treatment caused the loss of trophoblast viability, the formation of ROS, and sFlt-1 mRNA and protein expression in a dose-dependent manner. Pretreatment with edaravone significantly inhibited hypoxia-induced oxidative stress formation and sFlt-1 expression in trophoblasts. Neither PlGF nor VEGF mRNA or protein expression was increased by CoCl2. In the in vitro tube formation assay, edaravone showed a protective role in vascular development under hypoxic conditions. This study demonstrated that hypoxia leading to increased sFlt-1 release in trophoblasts may contribute to the placental vascular formation abnormalities observed in preeclampsia and suggested that the free radical scavenger edaravone could be a candidate for the effective treatment of preeclampsia. Copyright © 2014 Elsevier Ltd. All rights reserved.

Cryptotanshinone induces cell cycle arrest and apoptosis through the JAK2/STAT3 and PI3K/Akt/NFκB pathways in cholangiocarcinoma cells.

PubMed

Ke, Fayong; Wang, Zheng; Song, Xiaoling; Ma, Qiang; Hu, Yunping; Jiang, Lin; Zhang, Yijian; Liu, Yingbin; Zhang, Yong; Gong, Wei

2017-01-01

Cholangiocarcinoma (CCA) is the most common biliary tract malignancy in the world with high resistance to current chemotherapies and extremely poor prognosis. The main objective of this study was to investigate the inhibitory effects of cryptotanshinone (CTS), a natural compound isolated from Salvia miltiorrhiza Bunge , on CCA both in vitro and in vivo and to explore the underlying mechanisms of CTS-induced apoptosis and cell cycle arrest. The anti-tumor activity of CTS on HCCC-9810 and RBE cells was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and colony forming assays. Cell cycle changes were detected by flow cytometric analysis. Apoptosis was detected by annexin V/propidium iodide double staining and Hoechst 33342 staining assays. The efficacy of CTS in vivo was evaluated using a HCCC-9810 xenograft model in athymic nude mice. The expression of key proteins involved in cell apoptosis and signaling pathway in vitro was analyzed by Western blot analysis. CTS induced potent growth inhibition, S-phase arrest, apoptosis, and colony-forming inhibition in HCCC-9810 and RBE cells in a dose-dependent manner. Intraperitoneal injection of CTS (0, 10, or 25 mg/kg) for 4 weeks significantly inhibited the growth of HCCC-9810 xenografts in athymic nude mice. CTS treatment induced S-phase arrest with a decrease of cyclin A1 and an increase of cyclin D1 protein level. Bcl-2 expression was downregulated remarkably, while Bax expression was increased after apoptosis occurred. Additionally, the activation of JAK2/STAT3 and PI3K/Akt/NFκB was significantly inhibited in CTS-treated CCA cells. CTS induced CCA cell apoptosis by suppressing both the JAK2/STAT3 and PI3K/Akt/NFκB signaling pathways and altering the expression of Bcl-2/Bax family, which was regulated by these two signaling pathways. CTS may serve as a potential therapeutic agent for CCA.

Antioxidant and antiproliferative potentials of methanol extract of Xylopia aethiopica (Dunal) A. Rich in PC-3 and LNCaP cells.

PubMed

Adaramoye, Oluwatosin Adekunle; Erguen, Bettina; Nitzsche, Bianca; Höpfner, Michael; Jung, Klaus; Rabien, Anja

2017-07-26

Our previous studies showed that fruit methanol extract from Xylopia aethiopica (MEXA) exhibited antiproliferative activity in human cervical cancer cells via the induction of apoptosis. The present study was designed to assess the antiproliferative, antiangiogenic and antioxidant effects of MEXA on prostate cancer (PCa) cells (PC-3 and LNCaP). PC-3 and LNCaP cells were cultured and treated with MEXA (10, 50 and 100 μg/mL). The sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) and lactate dehydrogenase (LDH) assays were used to evaluate cell viability and cytotoxicity, respectively. DNA fragmentation was determined by cell death detection ELISA plus, and angiogenesis was assessed by chicken chorioallantoic membrane (CAM) assay. The antioxidant activities of MEXA were determined by DPPH and hydroxyl (OH) radicals' scavenging methods as well as through the inhibition of lipid peroxidation (LPO) in rats' liver homogenate. MEXA at 100, 250 and 500 μg/mL scavenged DPPH by 48%, 62%, 70% and OH radical by 39%, 58%, 67%, respectively. MEXA significantly (p<0.05) inhibited LPO in a concentration-dependent manner. In addition, MEXA had antiproliferative effects on PC-3 and LNCaP with IC50 of 62.1 and 73.6 μg/mL, respectively, at 96 h. The LDH assay showed that MEXA had low toxicity in vitro at its IC50 values. The extent of DNA fragmentation by MEXA showed higher values in PC-3 and LNCaP, suggesting the possible induction of apoptosis. In contrast, MEXA did not affect the network of vessels in CAM, thus lacking anti-angiogenic property. These findings suggest that MEXA induces antiproliferative activity in PCa cells through a mechanism that involves apoptosis. Therefore, MEXA may be a potential therapeutic agent for PCa.

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

PubMed

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

2016-04-21

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

Mitomycin C induces apoptosis in cultured corneal fibroblasts derived from type II granular corneal dystrophy corneas

PubMed Central

Choi, Seung-il; Lee, Hyung Keun; Cho, Young Jae

2008-01-01

Purpose The present study investigated the effect of mitomycin C (MMC) on cell viability, apoptosis, and transforming growth factor beta-induced protein (TGFBIp) expression in cultured normal corneal fibroblasts and heterozygote or homozygote granular corneal dystrophy type II (GCD II) corneal fibroblasts. Methods Keratocytes were obtained from normal cornea or from heterozygote or homozygote GCD II patients after lamellar or penetrating keratoplasty. To measure cell viability, corneal fibroblasts were incubated with 0.02% MMC for 3 h, 6 h, and 24 h or with 0%, 0.01%, 0.02%, and 0.04% MMC for 24 h and then tested using lactate dehydrogenase (LDH) and 3-[4,5-demethylthiazol-2,5-diphenyl-2H-tetrazolium bromide] (MTT) assays. To measure apoptosis, cells were analyzed by FACS analysis and annexin V staining. Bcl-xL, Bax, and TGFBI mRNA expression was measured using reverse transcription polymerase chain reaction (RT–PCR) assays. Cellular and media levels of TGFBIp protein were measured by immunoblotting. Results MTT and LDH assays showed that MMC reduced cell viability in all three cell types in a dose-dependent and time-dependent manner (p<0.05). FACS analysis and annexin V staining showed that MMC caused apoptosis with GCD II homozygote cells being most affected. RT–PCR analysis showed that MMC decreased Bcl-xL mRNA expression and increased Bax mRNA expression in all cell types. RT–PCR and immunoblotting analysis showed that MMC reduced TGFBI mRNA levels and cellular and media TGFBIp protein levels in all cell types. Conclusions MMC induced apoptosis, and the effects of MMC were greatest in GCD II homozygote cells. MMC also reduced the production of TGFBIp in all three types of corneal fibroblasts. These findings may explain the additional therapeutic effect of MMC in GCD II patients. PMID:18615204

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

PubMed Central

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

2016-01-01

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

Inhibitory effect of PDGF-BB and serum-stimulated responses in vascular smooth muscle cell proliferation by hinokitiol via up-regulation of p21 and p53.

PubMed

Li, Jiun-Yi; Liu, Chun-Ping; Shiao, Wei-Cheng; Jayakumar, Thanasekaran; Li, Yi-Shin; Chang, Nen-Chung; Huang, Shih-Yi; Hsieh, Cheng-Ying

2018-04-01

Vascular smooth muscle cell (VSMC) proliferation plays a major role in the progression of vascular diseases. In the present study, we established the efficacy and the mechanisms of action of hinokitiol, a tropolone derivative found in Chamaecyparis taiwanensis , Cupressaceae, in relation to platelet-derived growth factor-BB (PDGF-BB) and serum-dependent VSMC proliferation. Primary cultured rat VSMCs were pre-treated with hinokitiol and then stimulated by PDGF-BB (10 ng/ml) or serum (10% fetal bovine serum). Cell proliferation and cytotoxicity were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and lactose dehydrogenase assay, respectively. The degree of DNA synthesis was evaluated by BrdU-incorporation measurements and observed using confocal microscopy. Immunoblotting was utilized to determine the protein level of p-extracellular signal-regulated kinase (ERK) 1/2, p-Akt, p-phosphoinositide 3-kinase (PI3K), p-Janus kinase 2 (JAK2), p-p53, and p21 Cip1 . The promoter activity of p21 and p53 activity were measured by dual luciferase reporter assay. Treatment with hinokitiol (1-10 μM) inhibited PDGF-BB and serum-induced VSMC proliferation and DNA synthesis in a concentration-dependent manner. Cytotoxicity was not observed in hinokitiol-treated VSMCs at the studied concentrations. Pre-incubation of VSMCs with hinokitiol did not alter PDGF-BB-induced phosphorylation of ERK1/2, Akt, PI3K or JAK2. Interestingly, hinokitiol induced promoter activity of p21 and p21 protein expression in VSMCs. Furthermore, hinokitiol augmented p53 protein phosphorylation and subsequently led to enhanced p53 activity. These data suggest that the anti-proliferative effects of hinokitiol in VSMCs may be mediated by activation of p21 and p53 signaling pathways, and it may contribute to the prevention of vascular diseases associated with VSMC proliferation.

Inhibitory effect of PDGF-BB and serum-stimulated responses in vascular smooth muscle cell proliferation by hinokitiol via up-regulation of p21 and p53

PubMed Central

Li, Jiun-Yi; Liu, Chun-Ping; Shiao, Wei-Cheng; Jayakumar, Thanasekaran; Li, Yi-Shin; Chang, Nen-Chung

2018-01-01

Introduction Vascular smooth muscle cell (VSMC) proliferation plays a major role in the progression of vascular diseases. In the present study, we established the efficacy and the mechanisms of action of hinokitiol, a tropolone derivative found in Chamaecyparis taiwanensis, Cupressaceae, in relation to platelet-derived growth factor-BB (PDGF-BB) and serum-dependent VSMC proliferation. Material and methods Primary cultured rat VSMCs were pre-treated with hinokitiol and then stimulated by PDGF-BB (10 ng/ml) or serum (10% fetal bovine serum). Cell proliferation and cytotoxicity were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and lactose dehydrogenase assay, respectively. The degree of DNA synthesis was evaluated by BrdU-incorporation measurements and observed using confocal microscopy. Immunoblotting was utilized to determine the protein level of p-extracellular signal-regulated kinase (ERK) 1/2, p-Akt, p-phosphoinositide 3-kinase (PI3K), p-Janus kinase 2 (JAK2), p-p53, and p21Cip1. The promoter activity of p21 and p53 activity were measured by dual luciferase reporter assay. Results Treatment with hinokitiol (1–10 μM) inhibited PDGF-BB and serum-induced VSMC proliferation and DNA synthesis in a concentration-dependent manner. Cytotoxicity was not observed in hinokitiol-treated VSMCs at the studied concentrations. Pre-incubation of VSMCs with hinokitiol did not alter PDGF-BB-induced phosphorylation of ERK1/2, Akt, PI3K or JAK2. Interestingly, hinokitiol induced promoter activity of p21 and p21 protein expression in VSMCs. Furthermore, hinokitiol augmented p53 protein phosphorylation and subsequently led to enhanced p53 activity. Conclusions These data suggest that the anti-proliferative effects of hinokitiol in VSMCs may be mediated by activation of p21 and p53 signaling pathways, and it may contribute to the prevention of vascular diseases associated with VSMC proliferation. PMID:29765446

Long Noncoding RNA H19 Inhibits Cell Viability, Migration, and Invasion Via Downregulation of IRS-1 in Thyroid Cancer Cells

PubMed Central

Wang, Peng; Xu, Weimin; Liu, Haixia; Bu, Qingao; Sun, Diwen

2017-01-01

Thyroid cancer is a common endocrine gland malignancy which exhibited rapid increased incidence worldwide in recent decades. This study was aimed to investigate the role of long noncoding RNA H19 in thyroid cancer. Long noncoding RNA H19 was overexpressed or knockdown in thyroid cancer cells SW579 and TPC-1, and the expression of long noncoding RNA H19 was detected by real-time polymerase chain reaction. The cell viability, migration, and invasion were determined by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide assay, Transwell assay, and wound healing assay, respectively. Furthermore, cell apoptosis was analyzed by flow cytometry, and expressions of some factors that were related to phosphatidyl inositide 3-kinases/protein kinase B and nuclear factor κB signal pathway were measured by Western blotting. This study revealed that cell viability and migration/invasion of SW579 and TPC-1 were significantly decreased by long noncoding RNA H19 overexpression compared with the control group (P < .05), whereas cell apoptosis was statistically increased (P < .001). Meanwhile, cell viability and migration/invasion were significantly increased after long noncoding RNA H19 knockdown (P < .05). Furthermore, long noncoding RNA H19 negatively regulated the expression of insulin receptor substrate 1 and thus effect on cell proliferation and apoptosis. Insulin receptor substrate 1 regulated the activation of phosphatidyl inositide 3-kinases/AKT and nuclear factor κB signal pathways. In conclusion, long noncoding RNA H19 could suppress cell viability, migration, and invasion via downregulation of insulin receptor substrate 1 in SW579 and TPC-1 cells. These results suggested the important role of long noncoding RNA H19 in thyroid cancer, and long noncoding RNA H19 might be a potential target of thyroid cancer treatment. PMID:29332545

[Effect of NOR1 gene knockdown on the biological behavior of HeLa cells].

PubMed

Tan, Yixin; Li, Wenjuan; Yi, Mei; Wang, Wei; Zheng, Pan; Zhang, Haijing; Xiang, Bo; Li, Guiyuan

2014-08-01

To explore the effect of the oxidored nitro domain containing protein 1 (NOR1) gene knockdown on the biological behavior of HeLa cells in cervical carcinoma. The recombinant plasmids pSUPER-shNOR1-1, pSUPER-shNOR1-2 and pSUPERscramble, which targeted to NOR1 gene, were constructed by pSUPER.neo+GFP vector, transfected into HeLa cells respectively using Lipofectamine 2000 reagent, and followed by G418 selection. The expression level of NOR1 mRNA and protein were determined by RT-PCR and Western blotting, respectively. Methyl thiazolyl tetrazolium (MTT) assay was performed to determine the growth curve of cell viability. The stable transfectants were treated with H₂O₂ and cell apoptosis was determined by Hoechst 33258 staining and terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) assay. The expression levels of Bcl-2, cleaved caspase 9 and poly ADP-ribose polymerase (PARP) were measured by Western blot. NOR1- knockdown HeLa cells were successfully constructed by transfection of pSUPER-shNOR1-1 or pSUPER-shNOR1-2 plasmids into HeLa cells. MTT assay showed that the silence of endogenous NOR1 in HeLa cells could lead to the increase in cell viability and proliferation, and the inhibition of H₂O₂-induced apoptosis compared with the negative control. Western blot showed that the expression level of active caspase 9 and cleaved PARP was inhibited in NOR1-knockdown cells when they were treated with H₂O₂ while the expression level of Bcl-2 protein increased. Silence of endogenous NOR1 facilitates the cell viability and growth of HeLa cells, and attenuates HeLa cells apoptosis induced by H₂O₂, which might be mediated by up-regulation of Bcl-2 level and down-regulation of the cleaved caspase 9 cascade.

Viwithan, a Standardized Withania somnifera Root Extract Induces Apoptosis in Murine Melanoma Cells

PubMed Central

Sudeep, H.V.; Gouthamchandra, K.; Venkatesh, B. J.; Prasad, K. Shyam

2017-01-01

Background: Withania somnifera is an Indian medicinal herb known for the multipotential ability to cure various therapeutic ailments as described in the ayurvedic system of medicine. Objective: In the present study, we have evaluated the antiproliferative activity of a standardized W. somnifera root extract (Viwithan) against different human and murine cancer cell lines. Materials and Methods: The cytotoxicity of Viwithan was determined using thiazolyl blue tetrazolium blue assay and crystal violet staining. The apoptotic changes in B16F1 cells following treatment with Viwithan were observed by acridine orange/ethidium bromide (AO/EB) staining and DNA fragmentation assay. The binding affinity of withanolides in Viwithan with antiapoptotic proteins B-cell lymphoma 2, B-cell lymphoma-extra large, and myeloid cell leukemia 1 (MCL-1) were studied using in silico approach. Results: The half maximal inhibitory concentration (IC50) values of Viwithan against liver hepatocellular carcinoma, Henrietta Lacks cervical carcinoma cells, human colorectal carcinoma cell line, and Ehrlich ascites carcinoma cells were 1830, 968, 2715, and 633 μg/ml, respectively. Interestingly, Viwithan was highly effective against B16F1 cells with an IC50 value of 220 μg/ml after 24 h treatment. The morphological alterations of apoptotic cell death were clearly observed in the AO/EB-stained cells after treatment with Viwithan. Viwithan induced late apoptotic changes in treated B16F1 cells as evident by the ladder formation of fragmented DNA in a time-dependent manner. The findings of molecular docking showed that withanolides present in Viwithan have a more binding affinity with the antiapoptotic proteins, particularly MCL-1. Conclusion: We have reported for the first time that Viwithan with 5% withanolides has a potent cytotoxic effect, particularly against B16F1 murine melanoma cells among the different cancer cell lines tested. SUMMARY The present study reports for the first time that Viwithan, a standardized 5% Withania somnifera root extract, has potent cytotoxicity against B16F1 murine melanoma cellsWe have investigated the in vitro cytotoxicity of Viwithan in different human and murine cancer cells. Interestingly, we found that Viwithan was particularly very effective against B16F1 melanoma cells with a half maximal inhibitory concentration value of 220 μg/mlThe microscopic observations following acridine orange/ethidium bromide staining and DNA fragmentation assays clearly indicated that Viwithan might initiate late apoptosis in B16F1 cellsThe binding affinity of withanolides in Viwithan with antiapoptotic proteins of B-cell lymphoma 2 family was predicted using AutoDock tool. The results from in silico studies indicated a plausible synergistic effect of withanolides attributing to the Viwithan-induced apoptosis through suppression of intrinsic pathway for carcinogenesis. Abbreviations used: MTT: Thiazolyl blue tetrazolium blue; DMSO: Dimethyl sulfoxide; BSA: Bovine serum albumin; DMEM: Dulbecco's minimum essential medium; NCCS: National Centre for Cell Science; PBS: Phosphate-Buffered Saline; HepG2: Liver hepatocellular carcinoma; HeLa: Henrietta Lacks cervical carcinoma cells; HCT-116: Human colorectal carcinoma cell line; EAC: Ehrlich ascites carcinoma cells; IC50: Half maximal inhibitory concentration; AO/EB: Acridine orange/Ethidium bromide; BCL-2: B-cell lymphoma 2; BCL-XL: B-cell lymphoma-extra large; MCL-1: Myeloid cell leukemia 1; PDB: Protein Data Bank; ANOVA: Analysis of variance. PMID:29491636

Viwithan, a Standardized Withania somnifera Root Extract Induces Apoptosis in Murine Melanoma Cells.

PubMed

Sudeep, H V; Gouthamchandra, K; Venkatesh, B J; Prasad, K Shyam

2018-01-01

Withania somnifera is an Indian medicinal herb known for the multipotential ability to cure various therapeutic ailments as described in the ayurvedic system of medicine. In the present study, we have evaluated the antiproliferative activity of a standardized W. somnifera root extract (Viwithan) against different human and murine cancer cell lines. The cytotoxicity of Viwithan was determined using thiazolyl blue tetrazolium blue assay and crystal violet staining. The apoptotic changes in B16F1 cells following treatment with Viwithan were observed by acridine orange/ethidium bromide (AO/EB) staining and DNA fragmentation assay. The binding affinity of withanolides in Viwithan with antiapoptotic proteins B-cell lymphoma 2, B-cell lymphoma-extra large, and myeloid cell leukemia 1 (MCL-1) were studied using in silico approach. The half maximal inhibitory concentration (IC50) values of Viwithan against liver hepatocellular carcinoma, Henrietta Lacks cervical carcinoma cells, human colorectal carcinoma cell line, and Ehrlich ascites carcinoma cells were 1830, 968, 2715, and 633 μg/ml, respectively. Interestingly, Viwithan was highly effective against B16F1 cells with an IC50 value of 220 μg/ml after 24 h treatment. The morphological alterations of apoptotic cell death were clearly observed in the AO/EB-stained cells after treatment with Viwithan. Viwithan induced late apoptotic changes in treated B16F1 cells as evident by the ladder formation of fragmented DNA in a time-dependent manner. The findings of molecular docking showed that withanolides present in Viwithan have a more binding affinity with the antiapoptotic proteins, particularly MCL-1. We have reported for the first time that Viwithan with 5% withanolides has a potent cytotoxic effect, particularly against B16F1 murine melanoma cells among the different cancer cell lines tested. The present study reports for the first time that Viwithan, a standardized 5% Withania somnifera root extract, has potent cytotoxicity against B16F1 murine melanoma cellsWe have investigated the in vitro cytotoxicity of Viwithan in different human and murine cancer cells. Interestingly, we found that Viwithan was particularly very effective against B16F1 melanoma cells with a half maximal inhibitory concentration value of 220 μg/mlThe microscopic observations following acridine orange/ethidium bromide staining and DNA fragmentation assays clearly indicated that Viwithan might initiate late apoptosis in B16F1 cellsThe binding affinity of withanolides in Viwithan with antiapoptotic proteins of B-cell lymphoma 2 family was predicted using AutoDock tool. The results from in silico studies indicated a plausible synergistic effect of withanolides attributing to the Viwithan-induced apoptosis through suppression of intrinsic pathway for carcinogenesis. Abbreviations used: MTT: Thiazolyl blue tetrazolium blue; DMSO: Dimethyl sulfoxide; BSA: Bovine serum albumin; DMEM: Dulbecco's minimum essential medium; NCCS: National Centre for Cell Science; PBS: Phosphate-Buffered Saline; HepG2: Liver hepatocellular carcinoma; HeLa: Henrietta Lacks cervical carcinoma cells; HCT-116: Human colorectal carcinoma cell line; EAC: Ehrlich ascites carcinoma cells; IC50: Half maximal inhibitory concentration; AO/EB: Acridine orange/Ethidium bromide; BCL-2: B-cell lymphoma 2; BCL-XL: B-cell lymphoma-extra large; MCL-1: Myeloid cell leukemia 1; PDB: Protein Data Bank; ANOVA: Analysis of variance.

Buckwheat (Fagopyrum esculentum M.) Sprout Treated with Methyl Jasmonate (MeJA) Improved Anti-Adipogenic Activity Associated with the Oxidative Stress System in 3T3-L1 Adipocytes

PubMed Central

Lee, Young-Jun; Kim, Kui-Jin; Park, Kee-Jai; Yoon, Bo-Ra; Lim, Jeong-Ho; Lee, Ok-Hwan

2013-01-01

Buckwheat sprouts contain various bioactive compounds including rutin which have a number of biological activities. We have previously shown that buckwheat sprouts (TBWE) treated with methyl jasmonate (MeJA) significantly increased the amount of phenolics and the antioxidant activity. The aim of this study was to demonstrate the effect of TBWE on anti-adipogenesis and pro-oxidant enzyme in 3T3-L1 adipocytes. We also evaluated the anti-oxidative activity of TBWE in adipocytes by using the nitroblue tetrazolium assay. Our data showed that TBWE markedly inhibited adipocyte differentiation and ROS production in 3T3-L1 cells compared with control groups. Moreover, TBWE has strongly shown the inhibition of adipogenic transcription factor as well as pro-oxidant enzymes. Together, we demonstrate that the MeJA treatment significantly increased the amount of phenolic compound, resulting in the suppression of adipogenesis and ROS production in the 3T3-L1 cells. These findings indicate that TBWE has the potential for anti-adipogenesis activity with anti-oxidative properties. PMID:23344050

Protective immune responses in guinea pigs and swine induced by a suicidal DNA vaccine of the capsid gene of swine vesicular disease virus.

PubMed

Sun, Shi-Qi; Liu, Xiang-Tao; Guo, Hui-Chen; Yin, Shuang-Hui; Shang, You-Jun; Feng, Xia; Liu, Zai-Xin; Xie, Qing-Ge

2007-03-01

A suicidal DNA vaccine based on a Semliki Forest virus (SFV) replicon was evaluated for the development of a vaccine against swine vesicular disease virus (SVDV). The 1BCD gene of SVDV was cloned and inserted into pSCA1, an SFV DNA-based replicon vector. The resultant plasmid, pSCA/1BCD, was transfected into BHK-21 cells and the antigenicity of the expressed protein was confirmed using an indirect immunofluorescence assay. Immunogenicity was studied in guinea pigs and swine. Animals were injected intramuscularly three times with pSCA/1BCD at regular intervals. Anti-SVDV antibodies were detected by ELISA, the lymphocyte proliferation response was tested by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide method and neutralizing antibodies were measured by microneutralization tests. The data showed that SVDV-specific antibodies, neutralizing antibodies and lymphocyte proliferation were induced in both guinea pigs and swine. Furthermore, after three successive vaccinations with pSCA/1BCD, half of the pigs were protected against challenge with SVDV. These results should encourage further work towards the development of a DNA vaccine against SVDV.

Redox and fungicidal properties of phthalocyanine metal complexes as related to active oxygen.

PubMed

Vol'pin, M E; Novodarova, G N; Krainova NYu; Lapikova, V P; Aver'yanov, A A

2000-10-01

Some chemical and fungicidal effects of 20 phthalocyanines of Co, Fe, Cu, and Al were studied. Under dark conditions, these complexes reduced nitroblue tetrazolium in the presence of KCN, accelerated the autoxidation of ascorbate or hydroquinone and decomposed hydrogen peroxide. In the later reaction, hydroxyl radical was generated as evidenced with the deoxyribose assay. The inhibition by superoxide dismutase and catalase of catalyzed autoxidation of ascorbate suggests the participation of superoxide anion-radical and hydrogen peroxide in the reaction. Most complexes were toxic to the fungus Magnaporthe grisea which causes blast disease of rice. The toxicity was enhanced by light being diminished by antioxidant reagents sequestering active oxygen species. Some complexes (including nontoxic ones), after 1-day contact with a leaf surface of the disease-susceptible rice cultivar, induced the fungitoxicity of leaf diffusate. This toxicity was also light-activated and sensitive to antioxidant reagents. Several complexes, when added to inocula, decreased 2-3 times the frequency of the compatible symptoms of the blast. It is suggested that in planta, the dark redox activity of phthalocyanines along with their photosensitization promote the generation of active oxygen, which damages the parasite and, therefore, favors disease resistance.

Spirolactone and spirothiolactone rhodamine-pyrene probes for detection of Hg2 + with different sensing properties and its application in living cells

NASA Astrophysics Data System (ADS)

Rui, Qing-Qing; Zhou, Yi; Fang, Yuan; Yao, Cheng

2016-04-01

Two new rhodamine B-based fluorescent probes PyRbS and PyRbO containing pyrene moiety were designed and synthesized. Both of the probes showed colorimetric and fluorometric sensing abilities for Hg2 + with high selectivity over other metal ions. The binding analysis using Job's plot suggested 1:1 stoichiometry for the complexes formed for Hg2 +. Compared with PyRbO, the PyRbS showed higher selectivity and sensitivity due to the thiophilic property of Hg2 + ion. The PyRbS exhibited the linear fluorescence quenching to Hg2 + in the range of 0.3 to 4.8 μM (λex = 365 nm) and 0.3 to 5.4 μM (λex = 515 nm), and the detection limit was 0.72 μM. Moreover, ratiometric changes of PyRbS with Hg2 + in absorption spectrum were observed, which could not be obtained in the combination of PyRbO with Hg2 +. In addition, the methyl thiazolyl tetrazolium (MTT) assay demonstrated that RbPyS had low cytotoxicity and was successfully used to monitor intracellular Hg2 + levels in living cells.

Isolation and Characterization of Marine Brevibacillus sp. S-1 Collected from South China Sea and a Novel Antitumor Peptide Produced by the Strain

PubMed Central

Zheng, Lanhong; Yi, Yao; Liu, Jia; Lin, Xiukun; Yang, Kangli; Lv, Mei; Zhou, Xinwen; Hao, Jianhua; Liu, Junzhong; Zheng, Yuan; Sun, Mi

2014-01-01

A Gram-positive, rod-shaped bacterium, designated as S-1, was isolated from a marine sediment sample collected from South China Sea. Phylogenetic analysis based on 16S rRNA gene sequence showed that S-1 belongs to the genus Brevibacillus. A novel cytotoxic peptide was isolated from the fermentation broth of the marine-derived bacterium Brevibacillus sp. S-1, using ion-exchange chromatography and reverse-phase HPLC chromatography. The molecular weight of this peptide was determined as 1570 Da by MALDI-TOF mass spectrometry, and its structure was proposed as a cyclic peptide elucidated by MALDI-TOF/TOF mass spectrometry and de novo sequencing. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay showed that this peptide exhibited cytotoxicity against BEL-7402 human hepatocellular carcinoma cells, RKO human colon carcinoma cells, A549 human lung carcinoma cells, U251 human glioma cells and MCF-7 human breast carcinoma cells. Additionally, SBP exhibited low cytotoxicity against HFL1 human normal fibroblast lung cells. The result suggested that the cytotoxic effect of the peptide is specific to tumor cells. PMID:25372839

Modification of bone graft by blending with lecithin to improve hydrophilicity and biocompatibility.

PubMed

Wang, Y; Cui, F Z; Jiao, Y P; Hu, K; Fan, D D

2008-03-01

Lecithin was blended to improve the hydrophilicity and biocompatibility of bone graft containing poly(l-lactic acid) (PLLA). Solution blending and freeze drying were used to fabricate symmetrical scaffolds containing different percentages of lecithin (lecithin: PLLA = 0, 5, 10 wt%). Scanning electron microscopy showed that the scaffolds maintained the three-dimensional porous structure. A water uptake experiment proved the significant improvement of hydrophilicity of the blend scaffold. With the addition of lecithin, the compressive strength and compressive modulus decreased. When the weight ratio of lecithin to PLLA was up to 10%, the compressive strength was still more than the lower limit of natural cancellous bone. To test the biocompatibility of the scaffolds, cell culture in vitro and subcutaneous implantation in vivo were performed. MC3T3-E1 preosteoblastic cells were cultured on the scaffolds for 7 days. Methylthiazol tetrazolium assay and laser scanning confocal microscopy were used to exhibit proliferation and morphology of the cells. The subcutaneous implantation in rats tested inflammatory response to the scaffolds. The results proved the better biocompatibility and milder inflammatory reactions of the blend scaffold (lecithin: PLLA = 5%) compared with the scaffold without lecithin. The modified scaffold containing lecithin is promising for bone tissue engineering.

Investigation of the genotoxic and cytotoxic effects of widely used neonicotinoid insecticides in HepG2 and SH-SY5Y cells.

PubMed

Şenyildiz, Mine; Kilinc, Adem; Ozden, Sibel

2018-06-01

Neonicotinoids are a relatively new type of insecticide to control a variety of pests. Although they are generally considered to be safe, they can lead to harmful effects on human and environmental health. We aimed to investigate possible effects of common neonicotinoid insecticides (acetamiprid, clothianidin, imidacloprid, thiacloprid, and thiamethoxam) on cytotoxicity and DNA damage in human neuroblastoma (SH-SY5Y) and human hepatocellular carcinoma (HepG2) cells. Our results indicated that 50% of inhibitory concentration values of neonicotinoids are in the range of 0.96 to >4 mM in SH-SY5Y cells and 0.53 to >4 mM in HepG2 cells by the methyl tetrazolium and neutral red uptake tests after 24 and 48 h exposure. We observed significant DNA damage at 500 µM of five neonicotinoids in SHSY-5Y cells, while only imidacloprid, thiametoxam, and thiacloprid showed some alterations in HepG2 cells after 24 h exposure using the alkaline comet assay. In conclusion, neonicotinoid insecticides may induce cytotoxicity and DNA damage in cell cultures; therefore, further studies are needed to better understand the toxicity of neonicotinoids.

Cytotoxic activity of erypogein d from erythrina poeppigiana (leguminosae) against cervical cancer (HeLa), breast cancer (MCF-7) and ovarian cancer (SKOV-3) cells

NASA Astrophysics Data System (ADS)

Herlina, T.; Gaffar, S.; Widowati, W.

2018-05-01

Cancer is the uncontrolled growth of abnormal cells and continues to divide rapidly in the body. Current anticancer treatment usually causes many side effects. Natural products are then explored to be new alternatives for cancer treatment. Flavonoids have been known to possess medicinal properties, including anticancer. This study was performed to observe the cytotoxic activity of isoflavanone compound, erypogein D from Erythrina poeppigiana, toward cervical cancer (HeLa), breast cancer (MCF-7) and ovarian cancer (SKOV-3) cells. The cytotoxic activity of erypogein D was tested using MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxyme-thoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. The percentage of cell mortality was calculated and the IC50 was analyzed using probit analysis. The result showed that cytotoxic activity of the erypogein D against HeLa, SKOV-3, and MCF-7 cells had an IC50 value 225, 70.74, and 30.12 μM, respectively. Based on IC50 value can be concluded that erypogein D is the most cytotoxic to breast cancer MCF-7 cell. However the cytotoxic activity of erypogein D toward MCF7 is moderate.

Synthesis of a novel glucose capped gold nanoparticle as a better theranostic candidate

PubMed Central

Suvarna, Saritha; Das, Ujjal; KC, Sunil; Mishra, Snehasis; Sudarshan, Mathummal; Saha, Krishna Das; Dey, Sanjit; Chakraborty, Anindita; Narayana, Y.

2017-01-01

Gold nanoparticles are predominantly used in diagnostics, therapeutics and biomedical applications. The present study has been designed to synthesize differently capped gold nanoparticles (AuNps) by a simple, one-step, room temperature procedure and to evaluate the potential of these AuNps for biomedical applications. The AuNps are capped with glucose, 2-deoxy-D-glucose (2DG) and citrate using different reducing agents. This is the first report of synthesis of 2DG-AuNp by the simple room temperature method. The synthesized gold nanoparticles are characterized with UV-Visible Spectroscopy, Fourier transform infrared spectroscopy (FTIR), Transmission electron microscopy (TEM) and selected area electron diffraction (SAED), Dynamic light scattering (DLS), and Energy-dispersive X-ray spectroscopy (SEM-EDS). Surface-enhanced Raman scattering (SERS) study of the synthesized AuNps shows increase in Raman signals up to 50 times using 2DG. 3-(4, 5-dimethylthiozol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay has been performed using all the three differently capped AuNps in different cell lines to assess cytotoxcity if any, of the nanoparticles. The study shows that 2DG-AuNps is a better candidate for theranostic application. PMID:28582426

Preparation and characterization of aminoethyl hydroxypropyl starch modified with collagen peptide.

PubMed

Wen, Huigao; Hu, Jin; Ge, Hongyu; Zou, Shengqiong; Xiao, Yao; Li, Ya; Feng, Han; Fan, Lihong

2017-08-01

The preparation of aminoethyl hydroxypropyl starch collagen peptide (AEHPS-COP) was via an enzyme-catalyzed reaction between amino groups in aminoethyl hydroxypropyl starch (AEHPS) and γ-carboxamide groups in collagen peptide (COP) by using microbial transglutaminase (MTGase) as biocatalyst. As an intermediate reactant, AEHPS was synthesized from hydroxypropyl starch (HPS) and 2-chloroethylamine hydrochloride (CEH). The chemical structures of HPS, AEHPS and AEHPS-COP were characterized by Fourier transform infrared spectroscopy (FT-IR) and 13 C nuclear magnetic resonance ( 13 C NMR). The reaction conditions that influenced the degree of substitution (DS) of AEHPS-COP were optimized, which included the reaction temperature, the reaction time, the mass ratio of collagen peptide to aminoethyl hydroxypropyl starch and the pH value. In addition, in vitro antioxidant activities of AEHPS-COP were evaluated through the scavenging activity of hydroxyl and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. Furthermore, the methylthiazol tetrazolium (MTT) assay was applied to investigate the cell viability of AEHPS-COP. The results indicated that the AEHPS-COP exhibited better cell viability to L929 mouse fibroblast cells. Therefore, the AEHPS-COP showed a promising potential application in cosmetic, biomedical and pharmaceutical fields. Copyright © 2017 Elsevier B.V. All rights reserved.

Cell Attachment and Proliferation of Human Adipose-Derived Stem Cells on PLGA/Chitosan Electrospun Nano-Biocomposite

PubMed Central

Razavi, Shahnaz; Karbasi, Saeed; Morshed, Mohammad; Zarkesh Esfahani, Hamid; Golozar, Mohammad; Vaezifar, Sedigheh

2015-01-01

Objective In this study, nano-biocomposite composed of poly (lactide-co-glycolide) (PLGA) and chitosan (CS) were electrospun through a single nozzle by dispersing the CS nano-powders in PLGA solution. The cellular behavior of human adipose derived stem cells (h-ADSCs) on random and aligned scaffolds was then evaluated. Materials and Methods In this experimental study, the PLGA/CS scaffolds were prepared at the different ratios of 90/10, 80/20, and 70/30 (w/w) %. Morphology, cell adhesion and prolif- eration rate of h-ADSCs on the scaffolds were assessed using scanning electron microscope (SEM), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and trypan blue staining respectively. Results H-ADSCs seeded on the matrices indicated that the PLGA/CS composite matrix with aligned nanofibres and higher content of CS nano-powders gave significantly better performance than others in terms of cell adhesion and proliferation rate (P<0.05). Conclusion We found that CS enhanced cell adhesion and proliferation rate, and aligned nanofibers guided cell growth along the longitudinal axis of the nanofibers, which would provide a beneficial approach for tissue engineering. PMID:26464814

Inhibitory effects of indole α-lipoic acid derivatives on nitric oxide production in LPS/IFNγ activated RAW 264.7 macrophages.

PubMed

Karabay, Arzu Zeynep; Koc, Aslı; Gurkan-Alp, A Selen; Buyukbingol, Zeliha; Buyukbingol, Erdem

2015-04-01

Alpha-lipoic acid (α-lipoic acid) is a potent antioxidant compound that has been shown to possess anti-inflammatory effects. RAW 264.7 macrophages produce various inflammatory mediators such as nitric oxide, IL-1β, IL-6 and TNF-alpha upon activation with LPS (Lipopolysaccharide) and IFNγ (interferon gamma). In this study, the effect of 12 synthetic indole α-lipoic acid derivatives on nitric oxide production and iNOS (inducible nitric oxide synthase) protein expression in LPS/IFNγ activated RAW 264.7 macrophages was determined. Cell proliferation, nitric oxide levels and iNOS protein expression were examined with thiazolyl blue tetrazolium blue test, griess assay and western blot, respectively. Our results showed that all of the indole α-lipoic acid derivatives showed significant inhibitory effects on nitric oxide production and iNOS protein levels (p < 0.05). The most active compounds were identified as compound I-4b, I-4e and II-3b. In conclusion, these indole α-lipoic acid derivatives may have the potential for treatment of inflammatory conditions related with high nitric oxide production. Copyright © 2015 John Wiley & Sons, Ltd.

Expression and functional analysis of novel molecule - Latcripin-13 domain from Lentinula edodes C91-3 produced in prokaryotic expression system.

PubMed

Wang, Jia; Zhong, Mintao; Liu, Ben; Sha, Li; Lun, Yongzhi; Zhang, Wei; Li, Xingyun; Wang, Xiaoli; Cao, Jing; Ning, Anhong; Huang, Min

2015-01-25

The shiitake mushroom Lentinula edodes has health benefits and is used to treat various diseases due to its immunomodulatory and antineoplastic properties. In the present study, the Latcripin-13 domain, isolated from L. edodes, was expressed in Escherichia coli Rosetta-gami(DE3) in the form of inclusion bodies. The Latcripin-13 domain was purified by Ni-His affinity chromatography with high purity and refolded by urea gradient dialysis. The product showed biological activity in A549 cells, a human lung cancer cell line, by flow cytometry and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method. The MTT assay and the flow cytometry results revealed that there was a great difference between the Latcripin-13 domain-treated group and the control group (p<0.05). Similarly, cell apoptosis observed by transmission electron microscopy (TEM) supported the flow cytometry results. This work demonstrated that the Latcripin-13 domain can induce apoptosis of A549 cells, which will bring new insights into the development of new antitumor drugs in the future. Copyright © 2014 Elsevier B.V. All rights reserved.

Enantioselective Metabolism and Interference on Tryptophan Metabolism of Myclobutanil in Rat Hepatocytes.

PubMed

Wang, Yao; Qiu, Jing; Zhu, Wentao; Wang, Xinru; Zhang, Ping; Wang, Dezhen; Zhou, Zhiqiang

2015-09-01

Myclobutanil, (RS)-2-(4-chlorophenyl)-2-(1H-1, 2, 4-triazol-1-ylmethyl) hexanenitrile is a widely used triazole fungicide. In this study, enantioselective metabolism and cytotoxicity were investigated in rat hepatocytes by chiral HPLC-MS/MS and the methyl tetrazolium (MTT) assay, respectively. Furthermore, tryptophan metabolism disturbance in rat hepatocytes after myclobutanil exposure was also evaluated by target metabolomics method. The half-life (t1/2) of (+)-myclobutanil was 10.66 h, whereas that for (-)-myclobutanil was 15.07 h. Such results indicated that the metabolic process of myclobutanil in rat hepatocytes was enantioselective with an enrichment of (-)-myclobutanil. For the cytotoxicity research, the calculated EC50 (12 h) values for rac-myclobutanil, (+)- and (-)-myclobutanil were 123.65, 150.65 and 152.60 µM, respectively. The results of tryptophan metabolites profiling showed that the levels of kynurenine (KYN) and XA were both up-regulated compared to the control, suggesting the activation effect of the KYN pathway by myclobutanil and its enantiomers which may provide an important insight into its toxicity mechanism. The data presented here could be useful for the environmental hazard assessment of myclobutanil. © 2015 Wiley Periodicals, Inc.

Antioxidant property of aerial parts and root of Phyllanthus fraternus Webster, an important medicinal plant.

PubMed

Upadhyay, Richa; Chaurasia, Jitendra Kumar; Tiwari, Kavindra Nath; Singh, Karuna

2014-01-01

In present study free radical scavenging potential of aerial parts and root of Phyllanthus fraternus was investigated. Extraction was done in water and ethanol. Total antioxidant capacity was measured by DPPH free radical scavenging method; ethanolic extract of aerial part was most potent in activity with 50% inhibition at 258 μg/mL concentration. Lipid peroxidation (LPO) was measured in terms of thiobarbituric acid-reactive substances (TBARS) by using egg-yolk homogenates as lipid-rich media with EC₅₀ of aerial part (ethanolic) 1522 μg/mL which was found to be most active. Superoxide (SO) radical scavenging activity was measured using riboflavin-light-nitroblue tetrazolium assay. Ethanolic and aqueous extract of both aerial part and root was almost similar in superoxide radical scavenging activity. Reducing power was determined on the basis of Fe³⁺-Fe⁺ transformation in the presence of extract. Total phenolic and flavonoid contents were also measured by spectroscopic method. Results showed that the ethanolic fraction of aerial part is most active towards antioxidant potential and this activity is related to its polyphenolic content and reducing potential. Thus, P. fraternus extract can be used as potent natural antioxidant.

Protective effect of pomegranate seed oil against H2O2 -induced oxidative stress in cardiomyocytes

PubMed Central

Bihamta, Mehdi; Hosseini, Azar; Ghorbani, Ahmad; Boroushaki, Mohammad Taher

2017-01-01

Objective: It has been well documented that oxidative stress is involved in the pathogenesis of cardiac diseases. Previous studies have shown that pomegranate seed oil (PSO) has antioxidant properties. This study was designed to investigate probable protective effects of PSO against hydrogen peroxide (H2O2)-induced damage in H9c2 cardiomyocytes. Materials and Methods: The cells were pretreated 24 hr with PSO 1 hr before exposure to 200 µM H2O2. Cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay. The level of reactive oxygen species (ROS) and lipid peroxidation were measured by fluorimetric methods. Results: H2O2 significantly decreased cell viability which was accompanied by an increase in ROS production and lipid peroxidation and a decline in superoxide dismutase activity. Pretreatment with PSO increased viability of cardiomyocytes and decrease the elevated ROS production and lipid peroxidation. Also, PSO was able to restore superoxide dismutase activity. Conclusion: PSO has protective effect against oxidative stress-induced damage in cardiomyocytes and can be considered as a natural cardioprotective agent to prevent cardiovascular diseases. PMID:28265546

Anti-Dengue Virus Constituents from Formosan Zoanthid Palythoa mutuki

PubMed Central

Lee, Jin-Ching; Chang, Fang-Rong; Chen, Shu-Rong; Wu, Yu-Hsuan; Hu, Hao-Chun; Wu, Yang-Chang; Backlund, Anders; Cheng, Yuan-Bin

2016-01-01

A new marine ecdysteroid with an α-hydroxy group attaching at C-4 instead of attaching at C-2 and C-3, named palythone A (1), together with eight known compounds (2–9) were obtained from the ethanolic extract of the Formosan zoanthid Palythoa mutuki. The structures of those compounds were mainly determined by NMR spectroscopic data analyses. The absolute configuration of 1 was further confirmed by comparing experimental and calculated circular dichroism (CD) spectra. Anti-dengue virus 2 activity and cytotoxicity of five isolated compounds were evaluated using virus infectious system and [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assays, respectively. As a result, peridinin (9) exhibited strong antiviral activity (IC50 = 4.50 ± 0.46 μg/mL), which is better than that of the positive control, 2′CMC. It is the first carotene-like substance possessing anti-dengue virus activity. In addition, the structural diversity and bioactivity of the isolates were compared by using a ChemGPS–NP computational analysis. The ChemGPS–NP data suggested natural products with anti-dengue virus activity locate closely in the chemical space. PMID:27517937

Microbial toxicity of the insensitive munitions compound, 2,4-dinitroanisole (DNAN), and its aromatic amine metabolites.

PubMed

Liang, Jidong; Olivares, Christopher; Field, Jim A; Sierra-Alvarez, Reyes

2013-11-15

2,4-Dinitroanisole (DNAN) is an insensitive munitions compound considered to replace conventional explosives such as 2,4,6-trinitrotoluene (TNT). DNAN undergoes facile microbial reduction to 2-methoxy-5-nitroaniline (MENA) and 2,4-diaminoanisole (DAAN). This study investigated the inhibitory effect of DNAN, MENA, and DAAN toward various microbial targets in anaerobic (acetoclastic methanogens) and aerobic (heterotrophs and nitrifiers) sludge, and the bioluminescent bacterium, Aliivibrio fischeri, used in the Microtox assay. Aerobic heterotrophic and nitrifying batch experiments with DAAN could not be performed because the compound underwent extensive autooxidation in these assays. DNAN severely inhibited methanogens, nitrifying bacteria, and A. fischeri (50% inhibitory concentrations (IC50) ranging 41-57μM), but was notably less inhibitory to aerobic heterotrophs (IC50>390 μM). Reduction of DNAN to MENA and DAAN lead to a marked decrease in methanogenic inhibition (i.e., DNAN>MENA≈DAAN). Reduction of all nitro groups in DNAN also resulted in partial detoxification in assays with A. fischeri. In contrast, reduction of a single nitro group did not alter the inhibitory impact of DNAN toward A. fischeri and nitrifying bacteria given the similar IC50 values determined for MENA and DNAN in these assays. These results indicate that reductive biotransformation could reduce the inhibitory potential of DNAN. Copyright © 2013 Elsevier B.V. All rights reserved.

How can we reduce costs of solid-phase multiplex-bead assays used to determine anti-HLA antibodies?

PubMed

Kamburova, E G; Wisse, B W; Joosten, I; Allebes, W A; van der Meer, A; Hilbrands, L B; Baas, M C; Spierings, E; Hack, C E; van Reekum, F E; van Zuilen, A D; Verhaar, M; Bots, M L; Drop, A C A D; Plaisier, L; Seelen, M A J; Sanders, J S F; Hepkema, B G; Lambeck, A J; Bungener, L B; Roozendaal, C; Tilanus, M G J; Vanderlocht, J; Voorter, C E; Wieten, L; van Duijnhoven, E M; Gelens, M; Christiaans, M H L; van Ittersum, F J; Nurmohamed, A; Lardy, N M; Swelsen, W; van der Pant, K A; van der Weerd, N C; Ten Berge, I J M; Bemelman, F J; Hoitsma, A; van der Boog, P J M; de Fijter, J W; Betjes, M G H; Heidt, S; Roelen, D L; Claas, F H; Otten, H G

2016-09-01

Solid-phase multiplex-bead assays are widely used in transplantation to detect anti-human leukocyte antigen (HLA) antibodies. These assays enable high resolution detection of low levels of HLA antibodies. However, multiplex-bead assays are costly and yield variable measurements that limit the comparison of results between laboratories. In the context of a Dutch national Consortium study we aimed to determine the inter-assay and inter-machine variability of multiplex-bead assays, and we assessed how to reduce the assay reagents costs. Fifteen sera containing a variety of HLA antibodies were used yielding in total 7092 median fluorescence intensities (MFI) values. The inter-assay and inter-machine mean absolute relative differences (MARD) of the screening assay were 12% and 13%, respectively. The single antigen bead (SAB) inter-assay MARD was comparable, but showed a higher lot-to-lot variability. Reduction of screening assay reagents to 50% or 40% of manufacturers' recommendations resulted in MFI values comparable to 100% of the reagents, with an MARD of 12% or 14%, respectively. The MARD of the 50% and 40% SAB assay reagent reductions were 11% and 22%, respectively. From this study, we conclude that the reagents can be reliably reduced at least to 50% of manufacturers' recommendations with virtually no differences in HLA antibody assignments. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Effect of Lapatinib on the Outgrowth of Metastatic Breast Cancer Cells to the Brain

PubMed Central

Gril, Brunilde; Palmieri, Diane; Bronder, Julie L.; Herring, Jeanne M.; Vega-Valle, Eleazar; Feigenbaum, Lionel; Liewehr, David J.; Steinberg, Seth M.; Merino, Maria J.; Rubin, Stephen D.

2008-01-01

Background The brain is increasingly being recognized as a sanctuary site for metastatic tumor cells in women with HER2-overexpressing breast cancer who receive trastuzumab therapy. There are no approved or widely accepted treatments for brain metastases other than steroids, cranial radiotherapy, and surgical resection. We examined the efficacy of lapatinib, an inhibitor of the epidermal growth factor receptor (EGFR) and HER2 kinases, for preventing the outgrowth of breast cancer cells in the brain in a mouse xenograft model of brain metastasis. Methods EGFR-overexpressing MDA-MB-231-BR (231-BR) brain-seeking breast cancer cells were transfected with an expression vector that contained or lacked the HER2 cDNA and used to examine the effect of lapatinib on the activation (ie, phosphorylation) of cell signaling proteins by immunoblotting, on cell growth by the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and on cell migration using a Boyden chamber assay. The outgrowth of large (ie, >50 μm2) and micrometastases was counted in brain sections from nude mice that had been injected into the left cardiac ventricle with 231-BR cells and, beginning 5 days later, treated by oral gavage with lapatinib or vehicle (n = 22–26 mice per treatment group). All statistical tests were two-sided. Results In vitro, lapatinib inhibited the phosphorylation of EGFR, HER2, and downstream signaling proteins; cell proliferation; and migration in 231-BR cells (both with and without HER2). Among mice injected with 231-BR-vector cells, those treated with 100 mg lapatinib/kg body weight had 54% fewer large metastases 24 days after starting treatment than those treated with vehicle (mean number of large metastases per brain section: 1.56 vs 3.36, difference = 1.80, 95% confidence interval [CI] = 0.92 to 2.68, P < .001), whereas treatment with 30 mg lapatinib/kg body weight had no effect. Among mice injected with 231-BR-HER2 cells, those treated with either dose of lapatinib had 50%–53% fewer large metastases than those treated with vehicle (mean number of large metastases per brain section, 30 mg/kg vs vehicle: 3.21 vs 6.83, difference = 3.62, 95% CI = 2.30 to 4.94, P < .001; 100 mg/kg vs vehicle: 3.44 vs 6.83, difference = 3.39, 95% CI = 2.08 to 4.70, P < .001). Immunohistochemical analysis revealed reduced phosphorylation of HER2 in 231-BR-HER2 cell–derived brain metastases from mice treated with the higher dose of lapatinib compared with 231-BR-HER2 cell–derived brain metastases from vehicle-treated mice (P < .001). Conclusions Lapatinib is the first HER2-directed drug to be validated in a preclinical model for activity against brain metastases of breast cancer. PMID:18664652

Effect of lapatinib on the outgrowth of metastatic breast cancer cells to the brain.

PubMed

Gril, Brunilde; Palmieri, Diane; Bronder, Julie L; Herring, Jeanne M; Vega-Valle, Eleazar; Feigenbaum, Lionel; Liewehr, David J; Steinberg, Seth M; Merino, Maria J; Rubin, Stephen D; Steeg, Patricia S

2008-08-06

The brain is increasingly being recognized as a sanctuary site for metastatic tumor cells in women with HER2-overexpressing breast cancer who receive trastuzumab therapy. There are no approved or widely accepted treatments for brain metastases other than steroids, cranial radiotherapy, and surgical resection. We examined the efficacy of lapatinib, an inhibitor of the epidermal growth factor receptor (EGFR) and HER2 kinases, for preventing the outgrowth of breast cancer cells in the brain in a mouse xenograft model of brain metastasis. EGFR-overexpressing MDA-MB-231-BR (231-BR) brain-seeking breast cancer cells were transfected with an expression vector that contained or lacked the HER2 cDNA and used to examine the effect of lapatinib on the activation (ie, phosphorylation) of cell signaling proteins by immunoblotting, on cell growth by the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and on cell migration using a Boyden chamber assay. The outgrowth of large (ie, >50 microm(2)) and micrometastases was counted in brain sections from nude mice that had been injected into the left cardiac ventricle with 231-BR cells and, beginning 5 days later, treated by oral gavage with lapatinib or vehicle (n = 22-26 mice per treatment group). All statistical tests were two-sided. In vitro, lapatinib inhibited the phosphorylation of EGFR, HER2, and downstream signaling proteins; cell proliferation; and migration in 231-BR cells (both with and without HER2). Among mice injected with 231-BR-vector cells, those treated with 100 mg lapatinib/kg body weight had 54% fewer large metastases 24 days after starting treatment than those treated with vehicle (mean number of large metastases per brain section: 1.56 vs 3.36, difference = 1.80, 95% confidence interval [CI] = 0.92 to 2.68, P < .001), whereas treatment with 30 mg lapatinib/kg body weight had no effect. Among mice injected with 231-BR-HER2 cells, those treated with either dose of lapatinib had 50%-53% fewer large metastases than those treated with vehicle (mean number of large metastases per brain section, 30 mg/kg vs vehicle: 3.21 vs 6.83, difference = 3.62, 95% CI = 2.30 to 4.94, P < .001; 100 mg/kg vs vehicle: 3.44 vs 6.83, difference = 3.39, 95% CI = 2.08 to 4.70, P < .001). Immunohistochemical analysis revealed reduced phosphorylation of HER2 in 231-BR-HER2 cell-derived brain metastases from mice treated with the higher dose of lapatinib compared with 231-BR-HER2 cell-derived brain metastases from vehicle-treated mice (P < .001). Lapatinib is the first HER2-directed drug to be validated in a preclinical model for activity against brain metastases of breast cancer.

Rapid assay for microbially reducible ferric iron in aquatic sediments

USGS Publications Warehouse

Lovely, Derek R.; Philips , Elizabeth J.P.

1987-01-01

The availability of ferric iron for microbial reduction as directly determined by the activity of iron-reducing organisms was compared with its availability as determined by a newly developed chemical assay for microbially reducible iron. The chemical assay was based on the reduction of poorly crystalline ferric iron by hydroxylamine under acidic conditions. There was a strong correlation between the extent to which hydroxylamine could reduce various synthetic ferric iron forms and the susceptibility of the iron to microbial reduction in an enrichment culture of iron-reducing organisms. When sediments that contained hydroxylamine-reducible ferric iron were incubated under anaerobic conditions, ferrous iron accumulated as the concentration of hydroxylamine-reducible ferric iron declined over time. Ferrous iron production stopped as soon as the hydroxylamine-reducible ferric iron was depleted. In anaerobic incubations of reduced sediments that did not contain hydroxylamine-reducible ferric iron, there was no microbial iron reduction, even though the sediments contained high concentrations of oxalate-extractable ferric iron. A correspondence between the presence of hydroxylamine-reducible ferric iron and the extent of ferric iron reduction in anaerobic incubations was observed in sediments from an aquifer and in fresh- and brackish-water sediments from the Potomac River estuary. The assay is a significant improvement over previously described procedures for the determination of hydroxylamine-reducible ferric iron because it provides a correction for the high concentrations of solid ferrous iron which may also be extracted from sediments with acid. This is a rapid, simple technique to determine whether ferric iron is available for microbial reduction.

An investigation on the physicochemical properties of the nanostructured [(4-X)PMAT][N(CN)2] ion pairs as energetic and tunable aryl alkyl amino tetrazolium based ionic liquids

NASA Astrophysics Data System (ADS)

Khalili, Behzad; Rimaz, Mehdi

2017-06-01

In this study the different class of tunable and high nitrogen content ionic liquids termed TAMATILs (Tunable Aryl Methyl Amino Tetrazolium based Ionic Liquids) were designed. The physicochemical properties of the nanostructured TAMATILs composed of para substituted phenyl methyl amino tetrazolium cations [(4-X)PMAT]+ (X = H, Me, OCH3, OH, NH2, NO2, F, CN, CHO, CF3, COMe and CO2Me) and dicyanimide anion [N(CN)2]- were fully investigated using M06-2X functional in conjunction with the 6-311++G(2d,2p) basis set. For all of the studied nanostructured ILs the structural parameters, interaction energy, cation's enthalpy of formation, natural charges, charge transfer values and topological properties were calculated and discussed. The substituent effect on the interaction energy and physicochemical properties also is taking into account. The results showed that the strength of interaction has a linear correlation with electron content of the phenyl ring in a way the substituents with electron withdrawing effects lead to make more stable ion pairs with higher interaction energies. Some of the main physical properties of ILs such as surface tension, melting point, critical-point temperature, electrochemical stability and conductivity are discussed and estimated for studying ion pairs using quantum chemical computationally obtained thermochemical data. Finally the enthalpy and Gibbs free energy of formation for twelve nanostructured individual cations with the general formula of [(4-X)PMAT]+ (X = 4-H, 4-Me, 4-OMe, 4-OH, 4-NH2, 4-NO2, 4-F, 4-CN, 4-CHO, 4-CF3, 4-COMe and 4-CO2Me) are calculated.

RESPIRATORY METABOLISM OF NORMAL AND DIVISIONLESS STRAINS OF CANDIDA ALBICANS

PubMed Central

Ward, John M.; Nickerson, Walter J.

1958-01-01

Respiration of a normal strain of Candida albicans was compared with that of a divisionless mutant which has a biochemical lesion such that metabolically generated hydrogen "spills over," during growth, for non-specific dye reduction. This waste is not at expense of growth, since both strains grow at essentially similar rates, nor at expense of respiration, since the mutant reduces oxygen more rapidly than the normal strain. Respiration in both strains is qualitatively similar, and seemingly unique among highly aerobic organisms in that it is not mediated by cytochrome oxidase. In resting cells of both strains, respiration is not only resistant to, but markedly stimulated by, high concentrations of cyanide, carbon monoxide, and azide. In contrast, growth of these yeasts is inhibited by low concentrations of cyanide and azide. Cytochrome oxidase could not be detected in cell-free preparations; reduced cytochrome c was not oxidized by such preparations. Cytochrome bands could not be observed in thick cell suspensions treated with reducing agents. However, incorporation of superoptimal levels of zinc and iron into the culture medium resulted in growth of cells possessing distinct cytochrome bands; respiration of these cells remained insensitive to cyanide, monoxide, and azide, and the bands were maintained in a reduced form on oxygenation. In the divisionless yeast, tetrazolium dyes compete with oxygen for reduction; this is not the case in the normal strain. The firmness with which hydrogen transfer is channeled in the latter for reduction of disulfide bonds (of importance in the division mechanism) and of oxygen, is contrasted with the lack of such control in the mutant. PMID:13514006

Effect of electron beam irradiation on the structural properties of poly(vinyl alcohol) formulations with triphenyl tetrazolium chloride dye (TTC)

NASA Astrophysics Data System (ADS)

Ali, Z. I.; Said, Hossam M.; Ali, H. E.

2006-01-01

Films of poly(vinyl alcohol) (PVA) composites with triphenyl tetrazolium chloride (TTC) dye were prepared and exposed to various radiation doses delivered by accelerated electrons. The results showed that at a low dose of 50 kGy, the colour difference (Δ E*) of PVA/TTC films was increased by ˜10 times of the initial value. However, the change in colour differences did not go systematically with increasing the TTC content, in which the composite with 1.5 wt% displayed higher value than that with 3.5 wt%. The differential scanning calorimetry (DSC) showed that the presence of the TTC dye caused a depression in the melting point ( Tm) and heat of fusion (Δ Hf) of the PVA bulk polymer. However, the thermogravimetric analysis (TGA) showed that the presence of the TTC dye improved the thermal stability of PVA. Also, the tensile strength at break of PVA/TTC composites was improved after electron beam irradiation.

Anticancer Activity of Chloroform Extract and Sub-fractions of Nepeta deflersiana on Human Breast and Lung Cancer Cells: An In vitro Cytotoxicity Assessment.

PubMed

Al-Oqail, Mai M; Al-Sheddi, Ebtesam S; Siddiqui, Maqsood A; Musarrat, Javed; Al-Khedhairy, Abdulaziz A; Farshori, Nida N

2015-10-01

Cancer is one of the major causes of death worldwide. The plant-derived natural products have received considerable attention in recent years due to their diverse pharmacological properties including anticancer effects. Nepeta deflersiana (ND) is used in the folk medicine as antiseptic, carminative, antimicrobial, antioxidant, and for treating rheumatic disorders. However, the anticancer activity of ND chloroform extract has not been explored so far. The present study was aimed to investigate the anticancer activities of chloroform Nepeta deflersiana extract and various sub-fractions (ND-1-ND-15) of ND against human breast cancer cells (MCF-7) and human lung cancer cells (A-549). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and neutral red uptake assays, and cellular morphological alterations using phase contrast light microscope were studied. Cells were exposed with 10-1000 μg/ml of sub-fractions of ND for 24 h. Results showed that selected sub-fractions of the chloroform extract significantly reduced the cell viability of MCF-7 and A-549 cells, and altered the cellular morphology in a concentration-dependent manner. Among the sub-fractions, ND-10 fraction showed relatively higher cytotoxicity compared to other fractions whereas, ND-1 did not cause any cytotoxicity even at higher concentrations. The A-549 cells were found to be more sensitive to growth inhibition by all the extracts as compared to the MCF-7 cells. The present study provides preliminary screening of anticancer activities of chloroform extract and sub-fractions of ND, which can be further used for the development of a potential therapeutic anticancer agent. Nepeta deflersiana extract exhibit cytotoxicity and altered the cellular morphology. Sub-fractions of the chloroform extract of Nepeta deflersiana reduced the cell viability of MCF-7 and A-549 cells. Among the sub-fractions, ND-10 fraction showed relatively higher cytotoxicity. The A-549 cells were found to be more sensitive as compared to the MCF-7 cells. Abbreviations used: MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NRU: Neutral red uptake; DMEM: Dulbecco's modified eagle medium; FBS: Fetal bovine serum; PBS: Phosphate buffer saline; DMSO: Dimethyl sulfoxide.

Anti-cancer Effects of Polyphenolic Compounds in Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor-resistant Non-small Cell Lung Cancer

PubMed Central

Jeong, Hyungmin; Phan, Ai N. H.; Choi, Jong-Whan

2017-01-01

Background: Polyphenolic phytochemicals are natural compounds, easily found in fruits and vegetables. Importantly, polyphenols have been intensively studied as excellent antioxidant activity which contributes to anticancer function of the natural compounds. Lung cancer has been reported to mainly account for cancer-related deaths in the world. Moreover, epidermal growth factor receptor tyrosine kinase inhibitor (TKI) resistance is one of the biggest issues in cancer treatment, especially in nonsmall cell lung cancer (NSCLC). Even though several studies both in preclinical and clinical trials have showed promising therapeutic effects of polyphenolic compounds in anticancer therapy, the function of the natural compounds in TKI-resistant (TKIR) lung cancer remains poorly studied. Objective: The aim of this study is to screen polyphenolic compounds as potential anticancer adjuvants which suppress TKIR lung cancer. Materials and Methods: Colony formation and thiazolyl blue tetrazolium blue assay were performed in the pair-matched TKI-sensitive (TKIS) versus TKIR tumor cell lines to investigate the therapeutic effect of polyphenolic compounds in TKIR NSCLC. Results: Our data show that equol, kaempferol, resveratrol, and ellagic acid exhibit strong anticancer effect in HCC827 panel. Moreover, the inhibitory effect of most of tested polyphenolic compounds was highly selective for TKIR lung cancer cell line H1993 while sparing the TKIS one H2073. Conclusion: This study provides an important screening of potential polyphenolic compounds for drug development to overcome TKI resistance in advanced lung cancer. SUMMARY The study provides an important screening of potential polyphenolic compounds for drug development to overcome tyrosine kinase inhibitor (TKI) resistance in advance lung cancerEquol, kaempferol, resveratrol, and ellagic acid show strong anticancer effect in HCC827 panel, including TKI-sensitive (TKIS) and TKI-resistant clonesThe inhibitory effect of polyphenolic compounds such as equol, kaempferol, resveratrol, ellagic acid, gallic acid, p-Coumaric, and hesperidin is highly selective for TKI-resistant lung cancer cell line H1993 while sparing the TKIS one H2073. Abbreviations used: EGFR: Epidermal growth factor receptor, EMT: Epithelial-to-mesenchymal transition, GTP: Green tea polyphenols, IGF1R: Insulin-like growth factor 1 receptor, MET: Met proto-oncogene, MTT: Thiazolyl blue tetrazolium blue, NSCLC: Non-small cell lung cancer, ROS: Reactive oxygen species, RTK: Receptor tyrosine kinase, STAT3: Signal transducer and activator of transcription 3, TKIR: TKI-resistant, TKIs: Tyrosine kinase inhibitors, TKIS: TKI-sensitive. PMID:29200719

Fluorescence Adherence Inhibition Assay: A Novel Functional Assessment of Blocking Virus Attachment by Vaccine-Induced Antibodies

PubMed Central

Asati, Atul; Kachurina, Olga; Karol, Alex; Dhir, Vipra; Nguyen, Michael; Parkhill, Robert; Kouiavskaia, Diana; Chumakov, Konstantin; Warren, William; Kachurin, Anatoly

2016-01-01

Neutralizing antibodies induced by vaccination or natural infection play a critically important role in protection against the viral diseases. In general, neutralization of the viral infection occurs via two major pathways: pre- and post-attachment modes, the first being the most important for such infections as influenza and polio, the latter being significant for filoviruses. Neutralizing capacity of antibodies is typically evaluated by virus neutralization assays that assess reduction of viral infectivity to the target cells in the presence of functional antibodies. Plaque reduction neutralization test, microneutralization and immunofluorescent assays are often used as gold standard virus neutralization assays. However, these methods are associated with several important prerequisites such as use of live virus requiring safety precautions, tedious evaluation procedure and long assessment time. Hence, there is a need for a robust, inexpensive high throughput functional assay that can be performed rapidly using inactivated virus, without extensive safety precautions. Herein, we report a novel high throughput Fluorescence Adherence Inhibition assay (fADI) using inactivated virus labeled with fluorescent secondary antibodies virus and Vero cells or erythrocytes as targets. It requires only few hours to assess pre-attachment neutralizing capacity of donor sera. fADI assay was tested successfully on donors immunized with polio, yellow fever and influenza vaccines. To further simplify and improve the throughput of the assay, we have developed a mathematical approach for calculating the 50% titers from a single sample dilution, without the need to analyze multi-point titration curves. Assessment of pre- and post-vaccination human sera from subjects immunized with IPOL®, YF-VAX® and 2013–2014 Fluzone® vaccines demonstrated high efficiency of the assay. The results correlated very well with microneutralization assay performed independently by the FDA Center of Biologics Evaluation and Research, with plaque reduction neutralization test performed by Focus Diagnostics, and with hemaglutination inhibition assay performed in-house at Sanofi Pasteur. Taken together, fADI assay appears to be a useful high throughput functional immunoassay for assessment of antibody-related neutralization of the viral infections for which pre-attachment neutralization pathway is predominant, such as polio, influenza, yellow fever and dengue. PMID:26863313

Effects of β-glucan polysaccharide revealed by the dominant lethal assay and micronucleus assays, and reproductive performance of male mice exposed to cyclophosphamide

PubMed Central

Oliveira, Rodrigo Juliano; Pesarini, João Renato; Sparça Salles, Maria José; Nakamura Kanno, Tatiane Yumi; dos Santos Lourenço, Ana Carolina; da Silva Leite, Véssia; da Silva, Ariane Fernanda; Matiazi, Hevenilton José; Ribeiro, Lúcia Regina; Mantovani, Mário Sérgio

2014-01-01

β-glucan is a well-known polysaccharide for its chemopreventive effect. This study aimed to evaluate the chemopreventive ability of β-glucan in somatic and germ cells through the dominant lethal and micronucleus assays, and its influence on the reproductive performance of male mice exposed to cyclophosphamide. The results indicate that β-glucan is capable of preventing changes in DNA in both germ cells and somatic ones. Changes in germ cells were evaluated by the dominant lethal assay and showed damage reduction percentages of 46.46% and 43.79% for the doses of 100 and 150 mg/kg. For the somatic changes, evaluated by micronucleus assay in peripheral blood cells in the first week of treatment, damage reduction percentages from 80.63–116.32% were found. In the fifth and sixth weeks, the percentage ranged from 10.20–52.54% and −0.95–62.35%, respectively. Besides the chemopreventive efficiency it appears that the β-glucan, when combined with cyclophosphamide, is able to improve the reproductive performance of males verified by the significant reduction in rates of post-implantation losses and reabsorption in the mating of nulliparous females with males treated with cyclophosphamide. PMID:24688298

Effects of β-glucan polysaccharide revealed by the dominant lethal assay and micronucleus assays, and reproductive performance of male mice exposed to cyclophosphamide.

PubMed

Oliveira, Rodrigo Juliano; Pesarini, João Renato; Sparça Salles, Maria José; Nakamura Kanno, Tatiane Yumi; Dos Santos Lourenço, Ana Carolina; da Silva Leite, Véssia; da Silva, Ariane Fernanda; Matiazi, Hevenilton José; Ribeiro, Lúcia Regina; Mantovani, Mário Sérgio

2014-03-01

β-glucan is a well-known polysaccharide for its chemopreventive effect. This study aimed to evaluate the chemopreventive ability of β-glucan in somatic and germ cells through the dominant lethal and micronucleus assays, and its influence on the reproductive performance of male mice exposed to cyclophosphamide. The results indicate that β-glucan is capable of preventing changes in DNA in both germ cells and somatic ones. Changes in germ cells were evaluated by the dominant lethal assay and showed damage reduction percentages of 46.46% and 43.79% for the doses of 100 and 150 mg/kg. For the somatic changes, evaluated by micronucleus assay in peripheral blood cells in the first week of treatment, damage reduction percentages from 80.63-116.32% were found. In the fifth and sixth weeks, the percentage ranged from 10.20-52.54% and -0.95-62.35%, respectively. Besides the chemopreventive efficiency it appears that the β-glucan, when combined with cyclophosphamide, is able to improve the reproductive performance of males verified by the significant reduction in rates of post-implantation losses and reabsorption in the mating of nulliparous females with males treated with cyclophosphamide.

Reduction of cell viability induced by IFN-alpha generates impaired data on antiviral assay using Hep-2C cells.

PubMed

de Oliveira, Edson R A; Lima, Bruna M M P; de Moura, Wlamir C; Nogueira, Ana Cristina M de A

2013-12-31

Type I interferons (IFNs) exert an array of important biological functions on the innate immune response and has become a useful tool in the treatment of various diseases. An increasing demand in the usage of recombinant IFNs, mainly due to the treatment of chronic hepatitis C infection, augmented the need of quality control for this biopharmaceutical. A traditional bioassay for IFN potency assessment is the cytopathic effect reduction antiviral assay where a given cell line is preserved by IFN from a lytic virus activity using the cell viability as a frequent measure of end point. However, type I IFNs induce other biological effects such as cell-cycle arrest and apoptosis that can influence directly on viability of many cell lines. Here, we standardized a cytopathic effect reduction antiviral assay using Hep-2C cell/mengovirus combination and studied a possible impact of cell viability variations caused by IFN-alpha 2b on responses generated on the antiviral assay. Using the four-parameter logistic model, we observed less correlation and less linearity on antiviral assay when responses from IFN-alpha 2b 1000 IU/ml were considered in the analysis. Cell viability tests with MTT revealed a clear cell growth inhibition of Hep-2C cells under stimulation with IFN-alpha 2b. Flow cytometric cell-cycle analysis and apoptosis assessment showed an increase of S+G2 phase and higher levels of apoptotic cells after treatment with IFN-alpha 2b 1000 IU/ml under our standardized antiviral assay procedure. Considering our studied dose range, we also observed strong STAT1 activation on Hep-2C cells after stimulation with the higher doses of IFN-alpha 2b. Our findings showed that the reduction of cell viability driven by IFN-alpha can cause a negative impact on antiviral assays. We assume that the cell death induction and the cell growth inhibition effect of IFNs should also be considered while employing antiviral assay protocols in a quality control routine and emphasizes the importance of new approaches for IFN potency determination. Copyright © 2013 Elsevier B.V. All rights reserved.

Voltage-Gated Na+ Channels are Modulated by Glucose and Involved in Regulating Cellular Insulin Content of INS-1 Cells.

PubMed

Chen, Chong; Wang, Songhua; Hu, Qingjuan; Zeng, Lvming; Peng, Hailong; Liu, Chao; Huang, Li-Ping; Song, Hao; Li, Yuping; Yao, Li-Hua; Meng, Wei

2018-01-01

Islet beta cells (β-cells) are unique cells that play a critical role in glucose homeostasis by secreting insulin in response to increased glucose levels. Voltage-gated ion channels in β-cells, such as K+ and Ca2+ channels, contribute to insulin secretion. The response of voltage-gated Na+ channels (VGSCs) in β-cells to the changes in glucose levels remains unknown. This work aims to determine the role of extracellular glucose on the regulation of VGSC. The effect of glucose on VGSC currents (INa) was investigated in insulin-secreting β-cell line (INS-1) cells of rats using whole-cell patch clamp techniques, and the effects of glucose on insulin content and cell viability were determined using Enzyme-Linked Immunosorbent Assay (ELISA) and Methylthiazolyldiphenyl-tetrazolium Bromide (MTT) assay methods respectively. Our results show that extracellular glucose application can inhibit the peak of INa in a concentration-dependent manner. Glucose concentration of 18 mM reduced the amplitude of INa, suppressed the INa of steady-state activation, shifted the steady-state inactivation curves of INa to negative potentials, and prolonged the time course of INa recovery from inactivation. Glucose also enhanced the activity-dependent attenuation of INa and reduced the fraction of activated channels. Furthermore, 18 mM glucose or low concentration of tetrodotoxin (TTX, a VGSC-specific blocker) partially inhibited the activity of VGSC and also improved insulin synthesis. These results revealed that extracellular glucose application enhances the insulin synthesis in INS-1 cells and the mechanism through the partial inhibition on INa channel is involved. Our results innovatively suggest that VGSC plays a vital role in modulating glucose homeostasis. © 2018 The Author(s). Published by S. Karger AG, Basel.

Osthole suppresses the proliferation and accelerates the apoptosis of human glioma cells via the upregulation of microRNA-16 and downregulation of MMP-9.

PubMed

Lin, Kai; Gao, Zhiyu; Shang, Bin; Sui, Shaohua; Fu, Qiang

2015-09-01

Osthole (7-methoxy-8-isoamyl alkenyl coumarin) has been reported to exhibit marked anticancer effects on several types of cancer. The expression levels of matrix metalloproteinase-9 (MMP-9) are closely associated with the pathogenesis of glioma. Furthermore, it is reported that the upregulation of microRNA‑16 (miR‑16) by the MMP‑9 signaling pathway can restrain the proliferation of cancer cells. To examine whether osthole increases the anticancer effect on human glioma cells in the present study, the common glioma cell line, U87, was treated with osthole at concentrations of 0, 50, 100 and 200 µΜ. The effects of osthole on cell viability were determined using a 3‑(4,5‑dimethylthiazol‑2‑thiazolyl)‑2,5‑diphenyl‑tetrazolium bromide assay. The rate of cellular apoptosis was analyzed by measuring the activity of caspase‑3 and using flow cytometry. The expression of MMP‑9 was determined using gelatin zymography assays and the expression of miR‑16 was determined using reverse transcription‑quantitative polymerase chain reaction. The results demonstrated that osthole significantly suppressed the proliferation and accelerated the apoptosis of the U87 cells. Furthermore, increased expression levels of miR‑16 and reduced protein expression levels of MMP‑9 were found in the U87 cells. In addition, miR‑16 was found to regulate the expression of MMP‑9 in the U87 cells through transfection of miR‑16 precursor and anti‑miR‑16 into the U87 cells. In conclusion, these observations indicated that osthole suppressed the proliferation and accelerated the apoptosis of human glioma cells through upregulation of the expression of miR‑16 and downregulation of the expression of MMP-9.

Effects of Magnolol and Honokiol on Adhesion, Yeast-Hyphal Transition, and Formation of Biofilm by Candida albicans

PubMed Central

Sun, Lingmei; Liao, Kai; Wang, Dayong

2015-01-01

Background The first step in infection by Candida albicans is adhesion to host cells or implanted medical devices and this followed by hyphal growth and biofilm formation. Yeast-to-hyphal transition has long been identified as a key factor in fungal virulence. Following biofilm formation, C. albicans is usually less sensitive or insensitive to antifungals. Therefore, development of new antifungals with inhibitory action on adhesion, yeast-hyphal transition and biofilm formation by C. albicans is very necessary. Methods The effects of magnolol and honokiol on hypha growth were investigated using different induction media. Their inhibitory effects were determined using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5- carboxanilide assay, and biofilm thickness and viability were observed by a confocal scanning laser microscope. Mammalian cells were used in adhesion assays. Genes related to hyphae development and cell adhesions were analyzed by real-time reverse transcription-polymerase chain reaction. The exogenous cyclic adenosine monophosphate was used to determine the mechanisms of action of magnolol and honokiol. Caenorhabditis elegans was used as an in vivo model to estimate the antifungal activities of magnolol and honokiol. Results and conclusions Magnolol and honokiol inhibited adhesion, the transition from yeast to hypha, and biofilm formation by C. albicans through the Ras1-cAMP-Efg1 pathway. Moreover, magnolol and honokiol prolonged the survival of nematodes infected by C. albicans. Magnolol and honokiol have potential inhibitory effects against biofilm formation by C. albicans. General Significance This study provides useful information towards the development of new strategies to reduce the incidence of C. albicans biofilm-associated infection. PMID:25710475

Prostate-Specific Natural Health Products (Dietary Supplements) Radiosensitize Normal Prostate Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hasan, Yasmin; Schoenherr, Diane; Martinez, Alvaro A.

Purpose: Prostate-specific health products (dietary supplements) are taken by cancer patients to alleviate the symptoms linked with poor prostate health. However, the effect of these agents on evidence-based radiotherapy practice is poorly understood. The present study aimed to determine whether dietary supplements radiosensitized normal prostate or prostate cancer cell lines. Methods and Materials: Three well-known prostate-specific dietary supplements were purchased from commercial sources available to patients (Trinovin, Provelex, and Prostate Rx). The cells used in the study included normal prostate lines (RWPE-1 and PWR-1E), prostate tumor lines (PC3, DU145, and LNCaP), and a normal nonprostate line (HaCaT). Supplement toxicity wasmore » assessed using cell proliferation assays [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and cellular radiosensitivity using conventional clonogenic assays (0.5-4Gy). Cell cycle kinetics were assessed using the bromodeoxyuridine/propidium iodide pulse-labeling technique, apoptosis by scoring caspase-3 activation, and DNA repair by assessing gammaH2AX. Results: The cell growth and radiosensitivity of the malignant PC3, DU145, and LNcaP cells were not affected by any of the dietary prostate supplements (Provelex [2mug/mL], Trinovin [10mug/mL], and Prostate Rx [50 mug/mL]). However, both Trinovin (10mug/mL) and Prostate Rx (6mug/mL) inhibited the growth rate of the normal prostate cell lines. Prostate Rx increased cellular radiosensitivity of RWPE-1 cells through the inhibition of DNA repair. Conclusion: The use of prostate-specific dietary supplements should be discouraged during radiotherapy owing to the preferential radiosensitization of normal prostate cells.« less

Piper betel leaf extract: anticancer benefits and bio-guided fractionation to identify active principles for prostate cancer management.

PubMed

Paranjpe, Rutugandha; Gundala, Sushma R; Lakshminarayana, N; Sagwal, Arpana; Asif, Ghazia; Pandey, Anjali; Aneja, Ritu

2013-07-01

Plant extracts, a concoction of bioactive non-nutrient phytochemicals, have long served as the most significant source of new leads for anticancer drug development. Explored for their unique medicinal properties, the leaves of Piper betel, an evergreen perennial vine, are a reservoir of phenolics with antimutagenic, antitumor and antioxidant activities. Here, we show that oral feeding of betel leaf extract (BLE) significantly inhibited the growth of human prostate xenografts implanted in nude mice compared with vehicle-fed controls. To gain insights into the 'active principles', we performed a bioactivity-guided fractionation of methanolic BLE employing solvents of different polarity strengths using classical column chromatography. This approach yielded 15 fractions, which were then pooled to 10 using similar retention factors on thin-layer chromatographs. Bioactivity assays demonstrated that one fraction in particular, F2, displayed a 3-fold better in vitro efficacy to inhibit proliferation of prostate cancer cells than the parent BLE. The presence of phenols, hydroxychavicol (HC) and chavibetol (CHV), was confirmed in F2 by nuclear magnetic resonance, high-performance liquid chromatography and mass spectroscopy. Further, the HC containing F2 subfraction was found to be ~8-fold more potent than the F2 subfraction that contained CHV, in human prostate cancer PC-3 cells as evaluated by the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide assay. Removing CHV from F2 remarkably decreased the IC50 of this fraction, indicating that HC is perhaps the major bioactive constituent, which is present to an extent of 26.59% in BLE. These data provide evidence that HC is a potential candidate for prostate cancer management and warrants further preclinical evaluation.

Evaluation of the antiplasmodial and leishmanicidal potential of Myrciaria dubia (Myrtaceae) extract.

PubMed

Correia, Vanessa Carolina de Sena; Lima, Nathália Oliveira; Oliveira, Flávio Augusto de Souza; Santos, Ana Paula de Azevedo Dos; Teles, Carolina Bioni Garcia; Oliveira, Waldesse Piragé de; Pimenta, And Raphael Sanzio

2016-01-01

Malaria and leishmaniasis are prevalent in tropical regions, which have environmental characteristics that are highly favorable to protozoa and vectors of these diseases; the transmission of these infections in sub-tropical regions, although recognized, represents only a small fraction of cases. Plants are constantly being used in the search for and acquisition of new drugs, and many compounds derived from them have been used to combat various diseases. In this study, we evaluated the action of the dichloromethanolic extract of Myrciaria dubia leaves against the protozoa Plasmodium falciparum, Leishmania amazonensis, Leishmania braziliensis, and Leishmania chagasi through bioassays. The extract from M. dubia was tested for its anti-P. falciparum activity in an anti-histidine-rich protein II immunosorbent assay. The antileishmanial assays were performed using the resazurin method, while cytotoxicity against human hepatoma (HepG2) strain was determined using the colorimetric MTT [3-(4, 5-dimethyl-2- thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide] method. The M. dubia extract presented a half-maximal inhibitory concentration equal to 2.35 (1.05)μg/mL for P. falciparum, 190.73 (6.41) μg/mL for L. amazonensis, and greater than equal to 200µg/mL for L. chagasi and L. braziliensis strains. The cytotoxic concentration for 50% of the cells was above 500μg/mL for HepG2, indicating no toxicity and greater selectivity against parasites. The results obtained indicate the presence of antiplasmodial and leishmanicidal bioactive compounds in the dichloromethanolic extracts of M. dubia leaves, and point towards future studies to elucidate the mechanism of action for each physiological effect.

Picfeltarraenin IA inhibits lipopolysaccharide-induced inflammatory cytokine production by the nuclear factor-κB pathway in human pulmonary epithelial A549 cells.

PubMed

Shi, Rong; Wang, Qing; Ouyang, Yang; Wang, Qian; Xiong, Xudong

2016-02-01

The present study aimed to investigate the effect of picfeltarraenin IA (IA) on respiratory inflammation by analyzing its effect on interleukin (IL)-8 and prostaglandin E2 (PGE2) production. The expression of cyclooxygenase 2 (COX2) in human pulmonary adenocarcinoma epithelial A549 cells in culture was also examined. Human pulmonary epithelial A549 cells and the human monocytic leukemia THP-1 cell line were used in the current study. Cell viability was measured using a methylthiazol tetrazolium assay. The production of IL-8 and PGE2 was investigated using an enzyme-linked immunosorbent assay. The expression of COX2 and nuclear factor-κB (NF-κB)-p65 was examined using western blot analysis. Treatment with lipopolysaccharide (LPS; 10 µg/ml) resulted in the increased production of IL-8 and PGE2, and the increased expression of COX2 in the A549 cells. Furthermore, IA (0.1-10 µmol/l) significantly inhibited PGE2 production and COX2 expression in cells with LPS-induced IL-8, in a concentration-dependent manner. The results suggested that IA downregulates LPS-induced COX2 expression, and inhibits IL-8 and PGE2 production in pulmonary epithelial cells. Additionally, IA was observed to suppress the expression of COX2 in THP-1 cells, and also to regulate the expression of COX2 via the NF-κB pathway in the A549 cells, but not in the THP-1 cells. These results indicate that IA regulates LPS-induced cytokine release in A549 cells via the NF-κB pathway.

THE EFFECT OF TUNGSTATE NANOPARTICLES ON REACTIVE OXYGEN SPECIES AND CYTOTOXICITY IN RAW 264.7 MOUSE MONOCYTE MACROPHAGE CELLS

PubMed Central

Dunnick, Katherine M.; Badding, Melissa A.; Schwegler-Berry, Diane; Patete, Jonathan M.; Koenigsmann, Christopher; Wong, Stanislaus S.; Leonard, Stephen S.

2015-01-01

Due to their unique size, surface area, and chemical characteristics, nanoparticles’ use in consumer products has increased. However, the toxicity of nanoparticle (NP) exposure during the manufacturing process has not been fully assessed. Tungstate NP are used in numerous products, including but not limited to scintillator detectors and fluorescent lighting. As with many NP, no apparent toxicity studies have been completed with tungstate NP. The hypothesis that tungstate NP in vitro exposure results in reactive oxygen species (ROS) formation and cytotoxicity was examined. Differences in toxicity based on tungstate NP size, shape (sphere vs. wire), and chemical characteristics were determined. RAW 264.7 mouse monocyte macrophages were exposed to tungstate NP, and ROS formation was assessed via electron spin resonance (ESR), and several assays including hydrogen peroxide, intracellular ROS, and Comet. Results showed ROS production induced by tungstate nanowire exposure, but this exposure did not result in oxidative DNA damage. Nanospheres showed neither ROS nor DNA damage following cellular exposure. Cells were exposed over 72 h to assess cytotoxicity using an MTT (tetrazolium compound) assay. Results showed that differences in cell death between wires and spheres occurred at 24 h but were minimal at both 48 and 72 h. The present results indicate that tungstate nanowires are more reactive and produce cell death within 24 h of exposure, whereas nanospheres are less reactive and did not produce cell death. Results suggest that differences in shape may affect reactivity. However, regardless of the differences in reactivity, in general both shapes produced mild ROS and resulted in minimal cell death at 48 and 72 h in RAW 264.7 cells. PMID:25208664

Tim-3 facilitates osteosarcoma proliferation and metastasis through the NF-κB pathway and epithelial-mesenchymal transition.

PubMed

Feng, Z M; Guo, S M

2016-09-02

The aim of this study was to investigate the expression of T-cell immunoglobulin mucin domain molecule-3 (Tim-3) in osteosarcoma tissues, and analyze its effect on cell proliferation and metastasis in an osteosarcoma cell line. Tim-3 mRNA and protein expression in osteosarcoma tissue was detected by reverse transcriptase-polymerase chain reaction and immunohistochemistry, respectively. Additionally, the cell viability, apoptosis rate, and invasive ability of the osteosarcoma cell line MG-63 were tested using the methyl thiazolyl tetrazolium assay, Annexin V-propidium iodide flow cytometry, and a Transwell assay, respectively, following Tim-3 interference using small interfering RNA (siRNA). We also analyzed the expression of Snail, E-cadherin, vimentin, and nuclear factor (NF)-kB in the cells by western blot. We observed that Tim-3 mRNA and protein was significantly overexpressed in osteosarcoma tissues, compared to the adjacent normal tissue (P < 0.01). Moreover, MG-63 cells transfected with the Tim-3 siRNA presented lower cell viability, a greater number of apoptotic cells, and decreased invasive ability (P < 0.01), compared to control cells. Additionally, we observed a decrease in Snail and vimentin expression, an increase in the E-cadherin level, and an increase in NF-kB p65 phosphorylation (P < 0.01) in Tim-3 siRNA-transfected MG-63 cells. Based on these results, we concluded that Tim-3 is highly expressed in osteosarcoma tissue. Moreover, we speculated that interfering in Tim-3 expression could significantly suppress osteosarcoma cell (MG-63) proliferation and metastasis via the NF-kB/Snail signaling pathway and epithelial-mesenchymal transition.

Mast cells exert pro-inflammatory effects of relevance to the pathophyisology of tendinopathy.

PubMed

Behzad, Hayedeh; Sharma, Aishwariya; Mousavizadeh, Rouhollah; Lu, Alex; Scott, Alex

2013-01-01

We have previously found an increased mast cell density in tendon biopsies from patients with patellar tendinopathy compared to controls. This study examined the influence of mast cells on basic tenocyte functions, including production of the inflammatory mediator prostaglandin E2 (PGE2), extracellular matrix remodeling and matrix metalloproteinase (MMP) gene transcription, and collagen synthesis. Primary human tenocytes were stimulated with an established human mast cell line (HMC-1). Extracellular matrix remodeling was studied by culturing tenocytes in a three-dimensional collagen lattice. Survival/proliferation was assessed with the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assay. Levels of mRNA for COX-2, COL1A1, MMP1, and MMP7 were determined by quantitative real-time polymerase chain reaction (qPCR). Cox-2 protein level was assessed by Western blot analysis and type I procollagen was detected by immunofluorescent staining. PGE2 levels were determined using an enzyme-linked immunosorbent assay (ELISA). Mast cells stimulated tenocytes to produce increased levels of COX-2 and the pro-inflammatory mediator PGE2, which in turn decreased COL1A1 mRNA expression. Additionally, mast cells reduced the type I procollagen protein levels produced by tenocytes. Transforming growth factor beta 1 (TGF-β1) was responsible for the induction of Cox-2 and PGE2 by tenocytes. Mast cells increased MMP1 and MMP7 transcription and increased the contraction of a three-dimensional collagen lattice by tenocytes, a phenomenon which was blocked by a pan-MMP inhibitor (Batimastat). Our data demonstrate that mast cell-derived PGE2 reduces collagen synthesis and enhances expression and activities of MMPs in human tenocytes.

Treatment of Near-Infrared Photodynamic Therapy Using a Liposomally Formulated Indocyanine Green Derivative for Squamous Cell Carcinoma

PubMed Central

Maruyama, Tetsuro; Akutsu, Yasunori; Suganami, Akiko; Tamura, Yutaka; Fujito, Hiromichi; Ouchi, Tomoki; Akanuma, Naoki; Isozaki, Yuka; Takeshita, Nobuyoshi; Hoshino, Isamu; Uesato, Masaya; Toyota, Taro; Hayashi, Hideki; Matsubara, Hisahiro

2015-01-01

Introduction Photodynamic therapy (PDT) is a less invasive option for cancer treatment that has evolved through recent developments in nanotechnology. We have designed and synthesized a novel liposome system that includes an indocyanine green (ICG) derivative, ICG-C18, in its bilayer. In addition to its use as an optical imager to visualize blood, lymphatic, and bile flow, ICG has also been used as an optical sensitizer. In the present report, we evaluate the use of our novel liposome system, LP-ICG-C18, in PDT for squamous cell carcinoma in an autologous murine model. Materials and Methods An excitation pulse beam (300 μJ/pulse) of a single band (800 nm) was used for sensitization. The cytotoxicity of the photodynamic therapy was evaluated in terms of cellular morphology changes, methyl thiazolyl tetrazolium (MTT) assay results, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining. We tested the enhanced permeability and retention effect of LP-ICG-C18 in tumor-bearing C3H/He mice using a near-infrared fluorescence imaging system and fluorescence microscopy. We also examined the antitumor effect of PDT by measuring tumor volume in tumor-bearing mice. Results Cell death and apoptosis were only observed in the PDT group receiving LP-ICG-C18. LP-ICG-C18 itself had no cytotoxic activity and showed good biocompatibility. LP-ICG-C18 accumulated on the tumor 24 hours after injection and was retained for approximately 3 weeks. Tumor cell apoptosis following PDT with LP-ICG-C18 was also observed under optical microscopy, MTT assay, and TUNEL staining. Conclusion These findings suggest that LP-ICG-C18 may be an effective intervening material in PDT for malignant disease. PMID:25850029