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Sample records for proliferation mtt assay

  1. (MTT) dye reduction assay.

    African Journals Online (AJOL)

    to inhibit proliferation of HeLa cells was determined using the 3443- dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) dye reduction assay. Extracts from roots of Agathisanthemum bojeri, Synaptolepis kirkii and Zanha africana and the leaf extract of Physalis peruviana at a concentration of 10 pg/ml inhibited cell ...

  2. Evaluation of a MTT assay in measurement of radiosensitizing effect

    International Nuclear Information System (INIS)

    Higuchi, Keiko; Mitsuhashi, Norio; Saitoh, Jun-ichi; Maebayashi, Katsuya; Sakurai, Hideyuki; Akimoto, Tetsuo; Niibe, Hideo

    1999-01-01

    The usefulness of a MTT assay by measuring the radiosensitizing effect of caffeine on rat yolk sac tumor cell line with a mutant-type p53 in vitro was evaluated. A rat yolk sac tumor cell line with a mutant-type p53, NMT-1R, was used in this study. The radiosensitivity of NMT-1R with or without caffeine was measured with a MTT assay. The results were compared with those by a clonogenic assay. Caffeine at a concentration of 2.0 mM which released radiation-induced G 2 block demonstrated a radiosensitizing effect, but caffeine at a concentration of 0.5 mM did not. The radiosensitizing effect of caffeine measured by a MTT assay correlated with that measured by a clonogenic assay. A MTT assay was useful to measure radiosensitivity and/or a radiosensitizing effect in vitro. (author)

  3. The citotoxicity of calcium hydroxide intracanal dressing by MTT assay

    Directory of Open Access Journals (Sweden)

    Nanik Zubaidah

    2007-12-01

    Full Text Available Calcium hydroxide had been used as the intracanal dressing in endodontic treatment due to its high alkaline and high antimicrobial capacity. It also be able to dissolve the necrotic tissue, prevent the root resorbtion and regenerate a new hard tissue. The aim of this study is to identify the concentration of calcium hydroxide that has the lowest citotoxicity. There are 5 groups, each group had 8 samples with different concentration of calcium hydroxide. Group I: 50%, Group II: 55%, Group III: 60%, Group IV: 65% and Group V: 70%. The citotoxicity test by using enzymatic assay of MTT [3-(4.5- dimethylthiazol-2yl ]-2.5 diphenyl tetrazolium bromide, against fibroblast cell (BHK-21. The result of susceptibility test was showed by the citotoxicity detection of the survive cell of fibroblast that was measured spectrophotometrically using 595 nm beam. The data was analyzed using One-Way ANOVA test with significant difference α = 0.05 and subsequently LSD test. The result showed that in concentration 50%, 55%, 60%, 65%, and 70% calcium hydroxide had low toxicity, but calcium hydroxide 60%, had the lowest toxicity.

  4. A study on the toxicity of three radiosensitizers on retinoblastoma cells by MTT assay

    International Nuclear Information System (INIS)

    Yi Xianjin; Jin Yizun; Ding Li; Ni Zhou; Wang Wenji

    1994-01-01

    The toxicity of three radiosensitizers BSO, CM and RSU-1069 on retinoblastoma cells was determined and the efficiency of in vitro MTT assay on drug-screening for retinoblastoma was also evaluated. The results showed that the MTT assay is very useful. The toxicity of radiosensitizers on retinoblastoma cells is dependent on cell line characteristics, drug concentration and time of exposure to it

  5. The optimal condition of performing MTT assay for the determination of radiation sensitivity

    International Nuclear Information System (INIS)

    Hong, Semie; Kim, Il Han

    2001-01-01

    The measurement of radiation survival using a clonogenic assay, the established standard, can be difficult and time consuming. In this study, We have used the MTT assay, based on the reduction of a tetrazolium salt to a purple formazan precipitate by living cells, as a substitution for clonogenic assay and have examined the optimal condition for performing this assay in determination of radiation sensitivity. Four human cancer cell lines - PCI-1, SNU-1066, NCI-H63O and RKO cells have been used. For each cell line, a clonogenic assay and a MTT assay using Premix WST-1 solution, which is one of the tetrazolium salts and does not require washing or solubilization of the precipitate were carried out after irradiation of 0, 2, 4, 6, 8, 10 Gy, For clonogenic assay, cells in 25 cm 2 flasks were irradiated after overnight incubation and the resultant colonies containing more than 50 cells were scored after culturing the cells for 10-14 days, For MTT assay, the relationship between absorbance and cell number, optimal seeding cell number, and optimal timing of assay was determined. Then, MTT assay was performed when the irradiated cells had regained exponential growth or when the non-irradiated cells had undergone four or more doubling times. There was minimal variation in the values gained from these two methods with the standard deviation generally less than 5%, and there were no statistically significant differences between two methods according to t-test in low radiation dose (below 6 Gy). The regression analyses showed high linear correlation with the R 2 value of 0.975-0.992 between data from the two different methods. The optimal cell numbers for MTT assay were found to be dependent on plating efficiency of used cell line. Less than 300 cells/well were appropriate for cells with high plating efficiency (more than 30%). For cells with low plating efficiency (less than 30%), 500 cells/well or more were appropriate for assay. The optimal time for MTT assay was alter 6

  6. Chemosensitivity testing of primary human renal cell carcinoma by a tetrazolium based microculture assay (MTT).

    Science.gov (United States)

    Mickisch, G; Fajta, S; Keilhauer, G; Schlick, E; Tschada, R; Alken, P

    1990-01-01

    MTT staining procedures have been used in chemosensitivity testing of established cell lines of human and other sources as well as of human leukaemias, but only limited information on its application in primary solid human tumors is presently available. We have evaluated MTT staining in primary human Renal Cell Carcinomas (RCCs), studied various factors interfering with the optimal use, and finally applied it in subsequent chemosensitivity testing. The method depends on the conversion of a water-soluble tetrazolium salt (MTT) to a purple colored formazan precipitate, a reaction effected by enzymes active only in living cells. Single cell suspensions of RCCs were obtained either by enzymatic dispersion or by mechanical dissagregation, filtered through gauze, and purified by Ficoll density centrifugation. Tests were carried out in 96-well microculture plates. 10(4) viable tumor cells per well at 4 h incubation time with 20 micrograms MTT/100 microliters total medium volume yielded best results. Formazan crystals were dissolved with DMSO, and the plates were immediately measured on a microculture plate reader at 540 nm. Under these criteria, linearity of the system could be demonstrated. For chemosensitivity testing, cells were continuously exposed to a number of drugs prior to the MTT staining procedure. Reproducibility of results was assessed and confirmed by culturing RCCs in flasks additionally, resubmitting them after 1, 2, and 4 weeks to the MTT assay. We conclude that the semiautomated MTT assay offers a valid, rapid, reliable and simple method to determine the degree of chemoresistance in primary human RCCs.

  7. Neutral Red versus MTT assay of cell viability in the presence of copper compounds.

    Science.gov (United States)

    Gomez Perez, Mariela; Fourcade, Lyvia; Mateescu, Mircea Alexandru; Paquin, Joanne

    2017-10-15

    Copper is essential for numerous physiological functions, and copper compounds may display therapeutic as well as cytotoxic effects. The MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay is a standard test largely used in cytotoxicity studies. This report shows that low micromolar levels of copper compounds such as Cu(II)Urea 2 , Cu(II)Ser 2 and CuCl 2 can interfere with the MTT assay making improper the detection of formazan product of MTT reduction. Comparatively, the Neutral Red assay appears to be sensitive and showing no interference with these compounds. The lactate dehydrogenase alternative assay cannot be used because of inhibitory effect of these copper compounds on the enzyme activity. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. In vitro Cell Viability by CellProfiler® Software as Equivalent to MTT Assay.

    Science.gov (United States)

    Gasparini, Luciana S; Macedo, Nayana D; Pimentel, Elisângela F; Fronza, Marcio; Junior, Valdemar L; Borges, Warley S; Cole, Eduardo R; Andrade, Tadeu U; Endringer, Denise C; Lenz, Dominik

    2017-07-01

    This study evaluated in vitro cell viability by the colorimetric MTT stands for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay compared to image analysis by CellProfiler ® software. Hepatoma (Hepa-1c1c7) and fibroblast (L929) cells were exposed to isolated substances, camptothecin, lycorine, tazettine, albomaculine, 3-epimacronine, trispheridine, galanthine and Padina gymnospora , Sargassum sp. methanolic extract, and Habranthus itaobinus Ravenna ethyl acetate in different concentrations. After MTT assay, cells were stained with Panotic dye kit. Cell images were obtained with an inverted microscope equipped with a digital camera. The images were analyzed by CellProfiler ® . No cytotoxicity at the highest concentration analyzed for 3-epimacronine, albomaculine, galanthine, trispheridine, P. gymnospora extract and Sargassum sp. extract where detected. Tazettine offered cytotoxicity only against the Hepa1c1c7 cell line. Lycorine, camptothecin, and H. itaobinus extract exhibited cytotoxic effects in both cell lines. The viability methods tested were correlated demonstrated by Bland-Atman test with normal distribution with mean difference between the two methods close to zero, bias value 3.0263. The error was within the limits of the confidence intervals and these values had a narrow difference. The correlation between the two methods was demonstrated by the linear regression plotted as R 2 . CellProfiler ® image analysis presented similar results to the MTT assay in the identification of viable cells, and image analysis may assist part of biological analysis procedures. The presented methodology is inexpensive and reproducible. In vitro cell viability assessment with MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay may be replaced by image analysis by CellProfiler ® . The viability methods

  9. Genotoxic and cytotoxic potential of whole plant extracts of Kalanchoe laciniata by Ames and MTT assay.

    Science.gov (United States)

    Sharif, Ali; Akhtar, Muhammad Furqan; Akhtar, Bushra; Saleem, Ammara; Manan, Maria; Shabbir, Maryam; Ashraf, Muneeb; Peerzada, Sohaib; Ahmed, Shoaib; Raza, Moosa

    2017-01-01

    Lack of data on safety of herbal medicines have endangered human health and life. The present study evaluated the genotoxic and mutagenic effect of Kalanchoe laciniata to access the safety and usefulness of the medicinal plant. Aqua-methanolic and n-hexane extracts of K. laciniata were evaluated for the genotoxic potential using Ames assay and cytotoxicity was evaluated using MTT assay. Ames assay was conducted using two strains of Salmonella typhimurium TA-100 and TA-102 whereas MTT assay was performed on baby hamster kidney cell line BHK-21. Aqua-methanolic extract of K. laciniata exhibited significant mutagenicity when exposed to TA-102 strain with a mutagenic index of 50.66 and 54.74 at maximum dose 150 mg/plate. The extract was also mutagenic to TA-100 strain but to a lesser extent. M.I of n-hexane extract was 12.15 and 15.51 for TA-100 and TA-102 respectively. n-hexane extract was mutagenic but little difference was observed between results of two strains. Both extracts were found to be cytotoxic with an IC 50 of 321.9 and 638.5 µg/mL for aqua-methanolic and n-hexane extracts respectively. On the basis of results it was concluded that aqua-methanolic and n-hexane extracts of K. laciniata possess mutagenic and cytotoxic potential. It is suggested to explore the plant further to evaluate its safety in rodents and other species.

  10. An efficient and economical MTT assay for determining the antioxidant activity of plant natural product extracts and pure compounds.

    Science.gov (United States)

    Liu, Yunbao; Nair, Muraleedharan G

    2010-07-23

    Antioxidants scavenge free radicals, singlet oxygen, and electrons in cellular redox reactions. The yellow MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] is reduced to a purple formazan by mitochondrial enzymes. NADPH is the basis of established in vitro cell viability assays. An antioxidant assay has been developed utilizing the redox reaction between MTT and selected natural product extracts and purified compounds. This simple, fast, and inexpensive MTT antioxidant assay is comparable with the lipid peroxidation inhibitory assay and can be mechanized to achieve high throughput.

  11. Cytotoxic Effect of Iron Oxide Nanoparticles on Mouse Embryonic Stem Cells by MTT Assay

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    Homa Mohseni Kouchesfehani

    2016-07-01

    Full Text Available Background: Despite the wide range of applications, there is a serious lack of information on the impact of the nanoparticles on human health and the environment. The present study was done to determine the range of dangerous concentrations of iron oxide nanoparticle and their effects on mouse embryonic stem cells. Methods: Iron oxide nanoparticles with less than 20 nanometers diameter were encapsulated by a PEG-phospholipid. The suspension of iron oxide nanoparticles was prepared using the culture media and cell viability was determined by MTT assay. Results: MTT assay was used to examine the cytotoxicity of iron oxide nanoparticle s. Royan B1 cells were treated with medium containing different concentrations (10, 20, 30, 40, 50, and 60µg/ml of the iron oxide nanoparticle. Cell viability was determined at 12 and 24 hours after treatment which showed significant decreases when concentration and time period increased. Conclusion: The main mechanism of nanoparticles action is still unknown, but in vivo and in vitro studies in different environments suggest that they are capable of producing reactive oxygen species (ROS. Therefore, they may have an effect on the concentration of intracellular calcium, activation of transcription factors, and changes in cytokine. The results of this study show that the higher concentration and duration of treatment of cells with iron oxide nanoparticles increase the rate of cell death.

  12. In vitro effects of piracetam on the radiosensitivity of hypoxic cells (adaptation of MTT assay to hypoxic conditions)

    International Nuclear Information System (INIS)

    Gheuens, E.E.O.; Bruijn, E.A. de; Van der Heyden, S.; Van Oosterom, A.T.; Lagarde, P.; Pooter, C.M.J. de; Chomy, F.

    1995-01-01

    This paper describes the adaptation of the MTT assay to hypoxic conditions in order to test the in vitro effect of piracetam on hypoxic cells and particularly on the radiosensitivity of hypoxic cells since this drug has shown clinical effect on acute and chronic hypoxia. The V79 cell line was selected by reference to preliminary hypoxic experiments using clonogenic assay and euoxic experiments using clonogenic and MTT assays. Cell growth and survival in our hypoxic conditions were assessed using MTT assay with an enclosure and special 48-well plates both made of glass. Growth curves on glass plates after 1-hour exposure to nitrogen versus air were comparable, so there is no bias effect due to gas composition. Survival curves using MTT versus reference clonogenic assay were comparable after radiation exposure in eu- and hypoxic conditions, and confirm the validity of our original technique for creating hypoxia. The Oxygen Enhancement Ratio was of about 3 for 1-hour hypoxic exposure. Piracetam gave no cytotoxic effect up to 10 mM of piracetam. Growth curves after continuous drug exposure and 1-hour euoxic versus hypoxic exposure gave no cytotoxic effect up to 10 mM of piracetam. Survival curves after continuous drug exposure to 10 mM of piracetam gave no significant effect on the radiosensitivity of hypoxic V79 cells using MTT or clonogenic assay. (author). 32 refs., 6 figs

  13. The use of intermediate electron acceptors to enhance MTT bioreduction in a microculture tetrazolium assay for human growth hormone.

    Science.gov (United States)

    Goodwin, C J; Holt, S J; Downes, S; Marshall, N J

    1996-01-01

    We contrast the effects of three intermediate electron acceptors (IEAs) on the highly quantitative ESTA bioassay for human growth hormone. This is a microculture tetrazolium assay based upon the in vitro reduction of the tetrazolium salt MTT, by Nb2 cells which have been activated with hGH. Each of the IEAs influenced MTT-formazan production in a distinctive manner. The two quinonoids, namely menadione and co-enzyme Q0 markedly increased the MTT-formazan produced by hormone activated Nb2 cells and thereby amplified the response of our bioassay for human growth hormone (hGH). The exceptionally low bioassay baseline which is characteristic of the unstimulated Nb2 cells when only MTT is added was retained in the presence of CoQ0, but was greatly increased by menadione. Phenazine methosulphate, which is the most widely used redox intermediary in microculture tetrazolium assays, also increased the baseline, but had only a minimal additional effect on MTT reduction by activated Nb2 cells. We conclude that CoQ0 is the preferred IEA for this ESTA bioassay for hGH.

  14. In vitro effects of piracetam on the radiosensitivity of hypoxic cells (adaptation of MTT assay to hypoxic conditions); Effets in vitro du piracetam sur la radiosensibilite des cellules hypoxiques (adapatation du test au MTT aux conditions d`hypoxie)

    Energy Technology Data Exchange (ETDEWEB)

    Gheuens, E.E.O.; Bruijn, E.A. de; Van der Heyden, S.; Van Oosterom, A.T. [Universitaire Instelling Antwerpen, Antwerp (Belgium); Lagarde, P. [Universitaire Instelling Antwerpen, Antwerp (Belgium)]|[Institut Bergonie, 33 - Bordeaux (France); Pooter, C.M.J. de [Universitaire Instelling Antwerpen, Antwerp (Belgium)]|[Hopital de Middelheim, Anvers (Belgium); Chomy, F. [Institut Bergonie, 33 - Bordeaux (France)

    1995-12-31

    This paper describes the adaptation of the MTT assay to hypoxic conditions in order to test the in vitro effect of piracetam on hypoxic cells and particularly on the radiosensitivity of hypoxic cells since this drug has shown clinical effect on acute and chronic hypoxia. The V79 cell line was selected by reference to preliminary hypoxic experiments using clonogenic assay and euoxic experiments using clonogenic and MTT assays. Cell growth and survival in our hypoxic conditions were assessed using MTT assay with an enclosure and special 48-well plates both made of glass. Growth curves on glass plates after 1-hour exposure to nitrogen versus air were comparable, so there is no bias effect due to gas composition. Survival curves using MTT versus reference clonogenic assay were comparable after radiation exposure in eu- and hypoxic conditions, and confirm the validity of our original technique for creating hypoxia. The Oxygen Enhancement Ratio was of about 3 for 1-hour hypoxic exposure. Piracetam gave no cytotoxic effect up to 10 mM of piracetam. Growth curves after continuous drug exposure and 1-hour euoxic versus hypoxic exposure gave no cytotoxic effect up to 10 mM of piracetam. Survival curves after continuous drug exposure to 10 mM of piracetam gave no significant effect on the radiosensitivity of hypoxic V79 cells using MTT or clonogenic assay. (author). 32 refs., 6 figs.

  15. The MTT assays of bovine retinal pericytes and human microvascular endothelial cells on DLC and Si-DLC-coated TCPS.

    Science.gov (United States)

    Okpalugo, T I T; McKenna, E; Magee, A C; McLaughlin, J; Brown, N M D

    2004-11-01

    MTT (Tetrazolium)-assay suggests that diamond-like carbon (DLC) and silicon-doped DLC (Si-DLC) films obtained under appropriate deposition parameters are not toxic to bovine retinal pericytes, and human microvascular endothelial cells (HMEC). The observed frequency distributions of the optical density (OD) values indicative of cell viability are near Gaussian-normal distribution. One-way ANOVA indicates that at 0.05 levels the population means are not significantly different for the coated and control samples. The observed OD values depend on the cell line (cell growth/metabolic rate), possibly cell cycle stage, the deposition parameters-bias voltage, ion energy, pressure, argon precleaning, and the dopant. For colored thin films like DLC with room temperature photoconductivity and photoelectric effects, it is important to account for the OD contribution from the coating itself. MTT assay, not surprisingly, seems not to be highly sensitive to interfacial cellular interaction resulting from the change in the film's nanostructure, because the tetrazolium metabolism is mainly intracellular and not interfacial. The thin films were synthesized by 13.56 MHz RF-PECVD using argon and acetylene as source gases, with tetramethylsilane (TMS) vapor introduced for silicon doping. This study could be relevant to biomedical application of the films in the eye, peri-vascular, vascular compartments, and for cell-tissue engineering. (c) 2004 Wiley Periodicals, Inc.

  16. Cytotoxicity test of 40, 50 and 60% citric acid as dentin conditioner by using MTT assay on culture cell line

    Directory of Open Access Journals (Sweden)

    Christian Khoswanto

    2008-09-01

    Full Text Available Background: Open dentin is always covered by smear layer, therefore before restoration is performed, cavity or tooth which has been prepared should be clean from dirt. The researchers suggested that clean dentin surface would reach effective adhesion between resin and tooth structure, therefore dentin conditioner like citric acid was used to reach the condition. Even though citric acid is not strong acid but it can be very erosive to oral mucous. Several requirements should be fulfilled for dental product such as non toxic, non irritant, biocompatible and should not have negative effect against local, systemic or biological environment. Cytotoxicity test was apart of biomaterial evaluation and needed for standard screening. Purpose: This study was to know the cytotoxicity of 40, 50, 60% citric acid as dentin conditioner using MTT assay. Method: This study is an experimental research using the Post-Test Only Control Group Design. Six samples of each 40, 50 and 60% citric acid for citotoxicity test using MTT assay. The density of optic formazan indicated the number of living cells. All data were statistically analyzed by one way ANOVA. Result: The percentage of living cells in 40, 50 and 60% citric acid were 95.14%, 93.42% and 93.14%. Conclusion: Citric acid is non toxic and safe to be used as dentine conditioner.

  17. (MTT) dye reduction assay.

    African Journals Online (AJOL)

    P. 0 Box 6501 3, Dar Es Salaam, Tanzania. Laboratory of Pharmaceutical Biology and Phytopharmacology, Faculty of Pharmacy, Catholic. University of Leuven, Belgium. Thirty-three aqueous methanolic extracts obtained from thirty plant species, belonging to seventeen families were screened for cytotoxic activity against ...

  18. Design, synthesis, and validation of an in vitro platform peptide-whole cell screening assay using MTT reagent

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    Sahar Ahmed

    2017-05-01

    Full Text Available An in vitro platform to perform peptide screening against different cancer cell lines was designed. The strategy for this screening relied on the design and detection of high-affinity cancer-targeting peptides based on the sequences of NGR and P160. Evaluation of the best binding peptides was performed via incubation of the peptide array-bounded cells with MTT reagent, which is reduced to purple formazan in living cells and further quantified using an Elispot and Kodak imager. For proof of concept, a peptide library (132 spots, and 66 different peptides was designed, synthesized, and screened against different cancer cell lines. The current strategy assists in the identification of positive and negative peptides as well as the relative binding between positive ones. Better binding peptide sequences of the NGR motif were demonstrated to show up to a 2.6-fold increase in CD13+ cell lines with insignificant binding to CD13− ones. Comparable results were observed for P160 peptide sequences, to which different peptides had increased binding, with an up to 3-fold increase relative to the native P160 peptide. Based on our results, new peptide sequences for cancer targeting were identified, and the developed strategy was applied to two different peptide libraries.

  19. Cucurbitacin B inhibits proliferation, induces G2/M cycle arrest and autophagy without affecting apoptosis but enhances MTT reduction in PC12 cells

    Directory of Open Access Journals (Sweden)

    Chuanhong Wu

    2016-03-01

    Full Text Available In the present study, the effect of cucurbitacin B (a natural product with anti-cancer effect was studied on PC12 cells. It significantly reduced the cell number, changed cell morphology and inhibited colony formation while MTT results showed increased cell viability. Cucurbitacin B treatment increased activity of succinode hydrogenase. No alteration in the integrity of mem-brane, the release of lactic dehydrogenase, the mitochondrial membrane potential, and the expression of apoptotic proteins suggested that cucurbitacin B did not induce apoptosis. The cell cycle was remarkably arrested at G2/M phase. Furthermore, cucurbitacin B induced autophagy as evidence by accumulation of autophagic vacuoles and the increase of LC3II. In addition, cucurbitacin B up-regulated the expression of p-beclin-1, p-ULK1, p-Wee1, p21 and down-regulated p-mTOR, p-p70S6K, CDC25C, CDK1, Cyclin B1. In conclusion, cucurbitacin B inhibited PC12 proliferation but caused MTT pitfall. Cucurbitacin B induced G2/M cell cycle arrest, autophagy, but not the apoptosis in PC12 cells.

  20. Assessment of cosmetic ingredients in the in vitro reconstructed human epidermis test method EpiSkin™ using HPLC/UPLC-spectrophotometry in the MTT-reduction assay.

    Science.gov (United States)

    Alépée, N; Hibatallah, J; Klaric, M; Mewes, K R; Pfannenbecker, U; McNamee, P

    2016-06-01

    Cosmetics Europe recently established HPLC/UPLC-spectrophotometry as a suitable alternative endpoint detection system for measurement of formazan in the MTT-reduction assay of reconstructed human tissue test methods irrespective of the test system involved. This addressed a known limitation for such test methods that use optical density for measurement of formazan and may be incompatible for evaluation of strong MTT reducer and/or coloured chemicals. To build on the original project, Cosmetics Europe has undertaken a second study that focuses on evaluation of chemicals with functionalities relevant to cosmetic products. Such chemicals were primarily identified from the Scientific Committee on Consumer Safety (SCCS) 2010 memorandum (addendum) on the in vitro test EpiSkin™ for skin irritation testing. Fifty test items were evaluated in which both standard photometry and HPLC/UPLC-spectrophotometry were used for endpoint detection. The results obtained in this study: 1) provide further support for Within Laboratory Reproducibility of HPLC-UPLC-spectrophotometry for measurement of formazan; 2) demonstrate, through use a case study with Basazol C Blue pr. 8056, that HPLC/UPLC-spectrophotometry enables determination of an in vitro classification even when this is not possible using standard photometry and 3) addresses the question raised by SCCS in their 2010 memorandum (addendum) to consider an endpoint detection system not involving optical density quantification in in vitro reconstructed human epidermis skin irritation test methods. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. An investigation of compositions and effects of local herbal Glycyrrhiza glabra and Mentha pulegium extracts on Helicobacter pylori and cell Line of stomach cancer (AGS by MTT assays

    Directory of Open Access Journals (Sweden)

    Fereshteh Salmani Jamaat1

    2017-07-01

    Full Text Available Background & Aim:According to globally development of stomach cancer especially in Ardabil, Iran, as the second major cause of mortality throughout the world, increased drug-resistant bacteria including Helicobacter pylori as the most important risk factors for stomach cancer, and side effects of antibiotics and chemical drugs normally used to treat. Experimental: The current research was conducted to investigate the anticancer and antimicrobial effects of native herbs of liquorice (Glycyrrhiza glabra and pennyroyal (Mentha pulegium extractions for finding a solution with the lowest complications in control or treatment of stomach cancer.The extractions were firstly obtained using Soxhlet and methanol solvent and then their compounds were determined by GC/MS. Antimicrobial activity, MIC and MBC of the extractions were assessed respectively using agar diffusion and broth dilution test and the anticancer effect on stomach cancer (cell line AGS was assessed by MTT assay. H. pylori ATCC 26695 was respectively revived and purified on Brucella broth containing 7% citrated horse serum and Columbia agar. Results: The analysis showed that liquorice extract contains 15 compositions, mainly consists of 26.48% Nonane, 23.38% Ethylcyclohexan, 8.29% 3-Bromodecane, 10.31% trans-2-Heptenal, 8.93% 9-Octadecenamide and 4.68% β-pregna and pennyroyal extract contains 17 compositions, mainly including 3.36% Camphor, 22.79% Pulegone, 4.92% Paramenth-3-n8-l, 8.06% Menthoforan, 7.54% Cis-Isopulegon and 24.58% α-Selinene. The bacteria were resistant or semi-sensitive to common antibiotics, whereas had considerable sensitivity to herbal extracts and liquorice showed almost three times more antibacterial effect. Pennyroyal extract had no cytotoxic effects, but the anticancer effect was observed in liquorice extract with optimal concentration of 25 μg/ml after 48 hours. Recommended applications/industries: In conclusion, liquorice extract due to the significant health

  2. Chemosensitivity and radiosensitivity of small cell lung cancer cell lines studied by a newly developed 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) hybrid assay

    International Nuclear Information System (INIS)

    Hida, T.; Ueda, R.; Takahashi, T.; Watanabe, H.; Kato, T.; Suyama, M.; Sugiura, T.; Ariyoshi, Y.

    1989-01-01

    The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) hybrid assay was developed by technically combining the human tumor clonogenic assay and the MTT assay to make the most of both assays. This assay was able to estimate the in vitro growth of cultured cell lines and of tumor cells in pleural effusion, suggesting the possibility of its use for assessment of chemosensitivity and radiosensitivity of fresh tumor samples. Multiple cell lines [including morphological and/or phenotypical in vitro converters and cisplatin (CDDP)-resistant lines] were established from three patients with small cell lung cancer at different stages of the disease. Chemosensitivity of these cell lines to four commonly used chemotherapeutic drugs was tested by the MTT hybrid assay. SK1 and SK3 lines were established from Patient S. K. before and after chemotherapy and radiotherapy, respectively. SK3/CDDP, a CDDP-resistant line derived from the SK3 line, was 30-fold more resistant to CDDP [50% inhibiting dose (IC50), 21.5 micrograms/ml] than the SK1 line. In Patient M. O., MOA2/CDDP, a CDDP-resistant line derived from MOA2 (an in vitro converter from the MO line), was 41-fold more resistant to CDDP (IC50, 37 micrograms/ml) than the parent MO line. From Patient T. M., TM1 and TM2 lines were established before and after chemotherapy, respectively. The latter showed 6-fold more resistance to CDDP than the former. Chemosensitivity of these lines to three other drugs, 4-hydroperoxycyclophosphamide, Adriamycin, and etoposide, suggested cross-resistance between CDDP and 4-hydroperoxycyclophosphamide. Radiosensitivity study was also carried out with the MTT hybrid assay. The MOA2 line was more resistant [Do, 3.0 Gy; extrapolation number (n), 4.0] than the parental MO line (Do, 1.6 Gy; n, 2.1). There was no clear difference in radiosensitivity between the cell lines established before and after radiation therapy in Patient S. K

  3. Use of HPLC/UPLC-spectrophotometry for detection of formazan in in vitro Reconstructed human Tissue (RhT)-based test methods employing the MTT-reduction assay to expand their applicability to strongly coloured test chemicals.

    Science.gov (United States)

    Alépée, N; Barroso, J; De Smedt, A; De Wever, B; Hibatallah, J; Klaric, M; Mewes, K R; Millet, M; Pfannenbecker, U; Tailhardat, M; Templier, M; McNamee, P

    2015-06-01

    A number of in vitro test methods using Reconstructed human Tissues (RhT) are regulatory accepted for evaluation of skin corrosion/irritation. In such methods, test chemical corrosion/irritation potential is determined by measuring tissue viability using the photometric MTT-reduction assay. A known limitation of this assay is possible interference of strongly coloured test chemicals with measurement of formazan by absorbance (OD). To address this, Cosmetics Europe evaluated use of HPLC/UPLC-spectrophotometry as an alternative formazan measurement system. Using the approach recommended by the FDA guidance for validation of bio-analytical methods, three independent laboratories established and qualified their HPLC/UPLC-spectrophotometry systems to reproducibly measure formazan from tissue extracts. Up to 26 chemicals were then tested in RhT test systems for eye/skin irritation and skin corrosion. Results support that: (1) HPLC/UPLC-spectrophotometry formazan measurement is highly reproducible; (2) formazan measurement by HPLC/UPLC-spectrophotometry and OD gave almost identical tissue viabilities for test chemicals not exhibiting colour interference nor direct MTT reduction; (3) independent of the test system used, HPLC/UPLC-spectrophotometry can measure formazan for strongly coloured test chemicals when this is not possible by absorbance only. It is therefore recommended that HPLC/UPLC-spectrophotometry to measure formazan be included in the procedures of in vitro RhT-based test methods, irrespective of the test system used and the toxicity endpoint evaluated to extend the applicability of these test methods to strongly coloured chemicals. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Gas Chromatography, GC/Mass Analysis and Bioactivity of Essential Oil from Aerial Parts of Ferulago trifida: Antimicrobial, Antioxidant, AChE Inhibitory, General Toxicity, MTT Assay and Larvicidal Activities.

    Science.gov (United States)

    Tavakoli, Saeed; Vatandoost, Hassan; Zeidabadinezhad, Reza; Hajiaghaee, Reza; Hadjiakhoondi, Abbas; Abai, Mohammad Reza; Yassa, Narguess

    2017-09-01

    We aimed to investigate different biological properties of aerial parts essential oil of Ferulago trifida Boiss and larvicidal activity of its volatile oils from all parts of plant. Essential oil was prepared by steam distillation and analyzed by Gas chromatography and GC/Mass. Antioxidant, antimicrobial, cytotoxic effects and AChE inhibitory of the oil were investigated using DPPH, disk diffusion method, MTT assay and Ellman methods. Larvicidal activity of F. trifida essential oil against malaria vector Anopheles stephensi was carried out according to the method described by WHO. In GC and GC/MS analysis, 58 compounds were identified in the aerial parts essential oil, of which E-verbenol (9.66%), isobutyl acetate (25.73%) and E-β-caryophyllene (8.68%) were main compounds. The oil showed (IC 50 = 111.2μg/ml) in DPPH and IC 50 = 21.5 mg/ml in the investigation of AChE inhibitory. Furthermore, the oil demonstrated toxicity with (LD 50 = 1.1μg/ml) in brine shrimp lethality test and with (IC 50 = 22.0, 25.0 and 42.55 μg/ml) on three cancerous cell lines (MCF-7, A-549 and HT-29) respectively. LC 50 of stem, root, aerial parts, fruits, and flowers essential oils against larvae of An. stephensi were equal with 10.46, 22.27, 20.50, 31.93 and 79.87ppm respectively. In antimicrobial activities, essential oil was effective on all specimens except Escherichia coli , Aspergillus niger and Candida albicans. The essential oil showed moderate antioxidant activity, strong antimicrobial properties and good toxic effect in brine shrimp test and MTT assay on three cancerous cell lines.

  5. Gas Chromatography, GC/Mass Analysis and Bioactivity of Essential Oil from Aerial Parts of Ferulago trifida: Antimicrobial, Antioxidant, AChE Inhibitory, General Toxicity, MTT assay and Larvicidal Activities

    Directory of Open Access Journals (Sweden)

    Saeed Tavakoli

    2017-10-01

    Full Text Available Background: We aimed to investigate different biological properties of aerial parts essential oil of Ferulago trifida Boiss and larvicidal activity of its volatile oils from all parts of plant.Methods: Essential oil was prepared by steam distillation and analyzed by Gas chromatography and GC/Mass. Anti­oxidant, antimicrobial, cytotoxic effects and AChE inhibitory of the oil were investigated using DPPH, disk diffusion method, MTT assay and Ellman methods. Larvicidal activity of F. trifida essential oil against malaria vector Anoph­eles stephensi was carried out according to the method described by WHO.Results: In GC and GC/MS analysis, 58 compounds were identified in the aerial parts essential oil, of which E-ver­benol (9.66%, isobutyl acetate (25.73% and E-β-caryophyllene (8.68% were main compounds. The oil showed (IC50= 111.2µg/ml in DPPH and IC50= 21.5 mg/ml in the investigation of AChE inhibitory. Furthermore, the oil demonstrated toxicity with (LD50= 1.1µg/ml in brine shrimp lethality test and with (IC50= 22.0, 25.0 and 42.55 µg/ml on three cancerous cell lines (MCF-7, A-549 and HT-29 respectively. LC50 of stem, root, aerial parts, fruits, and flowers essential oils against larvae of An. stephensi were equal with 10.46, 22.27, 20.50, 31.93 and 79.87ppm respectively. In antimicrobial activities, essential oil was effective on all specimens except Escherichia coli, Asper­gillus niger and Candida albicans.Conclusion: The essential oil showed moderate antioxidant activity, strong antimicrobial properties and good toxic effect in brine shrimp test and MTT assay on three cancerous cell lines.

  6. Microscopic analysis of MTT stained boar sperm cells

    African Journals Online (AJOL)

    tulyasys

    2015-06-08

    2H-tetrazolium bromide is widely used for assessment of cytotoxicity, cell viability, and proliferation studies in cell biology (van Meerloo et al., 2011;. Stockert et al., 2012). The stain is abbreviated as MTT.

  7. Evaluation of royal jelly as an alternative to fetal bovine serum in cell culture using cell proliferation assays and live cell imaging.

    Science.gov (United States)

    Musa, Marahaini; Nasir, Nurul Fatihah Mohamad; Thirumulu, Kannan Ponnuraj

    2014-01-01

    Royal jelly is a nutritious substance produced by the young nurse bees and contains significant amounts of proteins which are important for cell growth and proliferation. The aim of this study was to evaluate the effect of royal jelly as an alternative to fetal bovine serum (FBS) in cell culture using cell proliferation assays and live cell imaging. MRC-5 cells were treated with various concentrations of royal jelly extract in MTT assay. The control groups were comprised of Alpha-Minimal Essential Medium (α-MEM) alone and α-MEM with 10% FBS. Subsequently, the cell proliferation was studied for 10 days using Alamar Blue assay and live cell imaging from 48 to 72 h. The population doubling time (PDT) was determined using trypan blue assay after live cell imaging. In MTT assay, 0.156 and 0.078 mg/ml of royal jelly produced higher cell viability compared to positive control group but were not significantly different (P > 0.05). In the Alamar Blue assay, 0.156 and 0.078 mg/ml of royal jelly produced greater percentage of reduction at day 3 even though no significant difference was found (P > 0.05). Based on live cell imaging, the PDT for positive, negative, 0.156 and 0.078 mg/ml of royal jelly groups were 29.09, 62.50, 41.67 and 41.67 h respectively. No significant difference was found in the PDT between all the groups (P > 0.05). Royal jelly does not exhibit similar ability like FBS to facilitate cell growth under the present test conditions.

  8. Localization of MTT formazan in lipid droplets. An alternative hypothesis about the nature of formazan granules and aggregates

    Directory of Open Access Journals (Sweden)

    G Diaz

    2009-08-01

    Full Text Available MTT (3-(4, 5-dimethyl-2-thiazolyl-2, 5-dihphenyltetrazolium bromide assay is a widely used method to assess cell viability and proliferation. MTT is readily taken up by cells and enzymatically reduced to formazan, a dark compound which accumulates in cytoplasmic granules. Formazan is later eliminated by the cell by a mechanisms often indicated as exocytosis, that produces characteristic needle-like aggregates on the cell surface. The shape of formazan aggregates and the rate of exocytosis change in the presence of bioactive amyloid b peptides (Ab and cholesterol. Though the cellular mechanisms involved in MTT reduction have been extensively investigated, the exact nature of formazan granules and the process of exocytosis are still obscure. Using Nile Red, which stains differentially neutral and polar lipids, and a fluorescent analog of cholesterol (NBD-cholesterol, we found that formazan localized in lipid droplets, consistent with the lipophilic nature of formazan. However, formazan granules and aggregates were also found to form after killing cells with paraformaldehyde fixation. Moreover, formazan aggregates were also obtained in cell-free media, using ascorbic acid to reduce MTT. The density and shape of formazan aggregates obtained in cell-free media was sensitive to cholesterol and Ab. In cells, electron microscopy failed to detect the presence of secretory vesicles, but revealed unusual fibers of 50 nm of diameter extending throughout the cytoplasm. Taken together, these findings suggest that formazan efflux is driven by physico-chemical interactions at molecular level without involving higher cytological mechanisms.

  9. Curcumin and Viscum album Extract Decrease Proliferation and Cell Viability of Soft-Tissue Sarcoma Cells: An In Vitro Analysis of Eight Cell Lines Using Real-Time Monitoring and Colorimetric Assays.

    Science.gov (United States)

    Harati, K; Behr, B; Daigeler, A; Hirsch, T; Jacobsen, F; Renner, M; Harati, A; Wallner, C; Lehnhardt, M; Becerikli, M

    2017-01-01

    The cytostatic effects of the polyphenol curcumin and Viscum album extract (VAE) were assessed in soft-tissue sarcoma (STS) cells. Eight human STS cell lines were used: fibrosarcoma (HT1080), liposarcoma (SW872, T778, MLS-402), synovial sarcoma (SW982, SYO1, 1273), and malignant fibrous histiocytoma (U2197). Primary human fibroblasts served as control cells. Cell proliferation, viability, and cell index (CI) were analyzed by BrdU assay, MTT assay, and real-time cell analysis (RTCA). As indicated by BrdU and MTT, curcumin significantly decreased the cell proliferation of five cell lines (HT1080, SW872, SYO1, 1273, and U2197) and the viability of two cell lines (SW872 and SW982). VAE led to significant decreases of proliferation in eight cell lines (HT1080, SW872, T778, MLS-402, SW982, SYO1, 1293, and U2197) and reduced viability in seven STS lines (HT1080, SW872, T778, MLS-402, SW982, SYO1, and 1273). As indicated by RTCA for 160 h, curcumin decreased the CI of all synovial sarcoma cell lines as well as T778 and HT1080. VAE diminished the CI in most of the synovial sarcoma (SW982, SYO1) and liposarcoma (SW872, T778) cell lines as well as HT1080. Primary fibroblasts were not affected adversely by the two compounds in RTCA. Curcumin and VAE can inhibit the proliferation and viability of STS cells.

  10. Clonality analysis of lymphoid proliferations using the BIOMED-2 clonality assays: a single institution experience

    Science.gov (United States)

    Kokovic, Ira; Novakovic, Barbara Jezersek; Cerkovnik, Petra; Novakovic, Srdjan

    2014-01-01

    Background Clonality determination in patients with lymphoproliferative disorders can improve the final diagnosis. The aim of our study was to evaluate the applicative value of standardized BIOMED-2 gene clonality assay protocols for the analysis of clonality of lymphocytes in a group of different lymphoid proliferations. Materials and methods. With this purpose, 121 specimens from 91 patients with suspected lymphoproliferations submitted for routine diagnostics from January to December 2011 were retrospectively analyzed. According to the final diagnosis, our series comprised 32 cases of B-cell lymphomas, 38 cases of non-Hodgkin’s T-cell lymphomas and 51 cases of reactive lymphoid proliferations. Clonality testing was performed using the BIOMED-2 clonality assays. Results The determined sensitivity of the TCR assay was 91.9%, while the sensitivity of the IGH assay was 74.2%. The determined specificity of the IGH assay was 73.3% in the group of lymphomas and 87.2% in the group of reactive lesions. The determined specificity of the TCR assay was 62.5% in the group of lymphomas and 54.3% in the group of reactive lesions. Conclusions In the present study, we confirmed the utility of standardized BIOMED-2 clonality assays for the detection of clonality in a routine diagnostical setting of non-Hodgkin’s lymphomas. Reactions for the detection of the complete IGH rearrangements and reactions for the detection of the TCR rearrangements are a good choice for clonality testing of a wide range of lymphoid proliferations and specimen types while the reactions for the detection of incomplete IGH rearrangements have not shown any additional diagnostic value. PMID:24991205

  11. Cytotoxicity of ferrite particles by MTT and agar diffusion methods for hyperthermic application

    International Nuclear Information System (INIS)

    Kim, Dong-Hyun; Lee, Se-Ho; Kim, Kyoung-Nam; Kim, Kwang-Mahn; Shim, In-Bo; Lee, Yong-Keun

    2005-01-01

    We investigated the cytotoxicity of the prepared various ferrites (Fe-, Li-, Ni/Zn/Cu-, Ba-, Sr-, Co-, Co/Ni-ferrites) using MTT assay as well as agar diffusion method. Their cytotoxicity was compared with that of alginate-encapsulated ferrites. In the MTT assay, Fe 3 O 4 and SrFe 12 O 19 ferrite showed the highest cell viability of 90%. Alginate-encapsulated Ba-ferrite was ranked mildly cytotoxic, whereas their ferrite particles were ranked cytotoxic

  12. Evaluation of different toxicity assays applied to proliferating cells and to stratified epithelium in relation to permeability enhancement with glycocholate

    DEFF Research Database (Denmark)

    Eirheim, Heidi Ugelstad; Bundgaard, Christoffer; Nielsen, Hanne Mørck

    2004-01-01

    The purpose of the present study was to evaluate different toxicity assays for use on proliferating buccal TR146 cells and on stratified TR146 epithelium and to compare these results to the permeability enhancing effect of glycocholate (GC). Both the proliferating cells and the epithelium were...... across the epithelium concurrent with a decrease in the transepithelial electrical resistance (TEER) was also determined. The robustness of the epithelium was significantly higher than that of the proliferating cells (P...

  13. Dissecting the T Cell Response: Proliferation Assays vs. Cytokine Signatures by ELISPOT

    Directory of Open Access Journals (Sweden)

    Magdalena Tary-Lehmann

    2012-05-01

    Full Text Available Chronic allograft rejection is in part mediated by host T cells that recognize allogeneic antigens on transplanted tissue. One factor that determines the outcome of a T cell response is clonal size, while another is the effector quality. Studies of alloimmune predictors of transplant graft survival have most commonly focused on only one measure of the alloimmune response. Because differing qualities and frequencies of the allospecific T cell response may provide distinctly different information we analyzed the relationship between frequency of soluble antigen and allo-antigen specific memory IFN-g secreting CD4 and CD8 T cells, their ability to secrete IL-2, and their proliferative capacity, while accounting for cognate and bystander proliferation. The results show proliferative responses primarily reflect on IL-2 production by antigen-specific T cells, and that proliferating cells in such assays entail a considerable fraction of bystander cells. On the other hand, proliferation (and IL-2 production did not reflect on the frequency of IFN-γ producing memory cells, a finding particularly accentuated in the CD8 T cell compartment. These data provide rationale for considering both frequency and effector function of pre-transplant T cell reactivity when analyzing immune predictors of graft rejection.

  14. Dissecting the T Cell Response: Proliferation Assays vs. Cytokine Signatures by ELISPOT

    Science.gov (United States)

    Anthony, Donald D.; Milkovich, Kimberly A.; Zhang, Wenji; Rodriguez, Benigno; Yonkers, Nicole L.; Tary-Lehmann, Magdalena; Lehmann, Paul V.

    2012-01-01

    Chronic allograft rejection is in part mediated by host T cells that recognize allogeneic antigens on transplanted tissue. One factor that determines the outcome of a T cell response is clonal size, while another is the effector quality. Studies of alloimmune predictors of transplant graft survival have most commonly focused on only one measure of the alloimmune response. Because differing qualities and frequencies of the allospecific T cell response may provide distinctly different information we analyzed the relationship between frequency of soluble antigen and allo-antigen specific memory IFN-γ secreting CD4 and CD8 T cells, their ability to secrete IL-2, and their proliferative capacity, while accounting for cognate and bystander proliferation. The results show proliferative responses primarily reflect on IL-2 production by antigen-specific T cells, and that proliferating cells in such assays entail a considerable fraction of bystander cells. On the other hand, proliferation (and IL-2 production) did not reflect on the frequency of IFN-γ producing memory cells, a finding particularly accentuated in the CD8 T cell compartment. These data provide rationale for considering both frequency and effector function of pre-transplant T cell reactivity when analyzing immune predictors of graft rejection. PMID:24710419

  15. Cytotoxicity of ferrite particles by MTT and agar diffusion methods for hyperthermic application

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Dong-Hyun [Brain Korea 21 Project for Medical Science, Yonsei University College of Dentistry, Seoul 120-752 (Korea, Republic of); Department and Research Institute of Dental Biomaterials and Bioengineering, Yonsei University College of Dentistry, Seoul 120-752 (Korea, Republic of); Lee, Se-Ho [Brain Korea 21 Project for Medical Science, Yonsei University College of Dentistry, Seoul 120-752 (Korea, Republic of); Department and Research Institute of Dental Biomaterials and Bioengineering, Yonsei University College of Dentistry, Seoul 120-752 (Korea, Republic of); Kim, Kyoung-Nam [Brain Korea 21 Project for Medical Science, Yonsei University College of Dentistry, Seoul 120-752 (Korea, Republic of); Department and Research Institute of Dental Biomaterials and Bioengineering, Yonsei University College of Dentistry, Seoul 120-752 (Korea, Republic of); Kim, Kwang-Mahn [Brain Korea 21 Project for Medical Science, Yonsei University College of Dentistry, Seoul 120-752 (Korea, Republic of); Department and Research Institute of Dental Biomaterials and Bioengineering, Yonsei University College of Dentistry, Seoul 120-752 (Korea, Republic of); Shim, In-Bo [Department of Electronic Physics, Kookmin University, Seoul 136-702 (Korea, Republic of); Lee, Yong-Keun [Brain Korea 21 Project for Medical Science, Yonsei University College of Dentistry, Seoul 120-752 (Korea, Republic of) and Department and Research Institute of Dental Biomaterials and Bioengineering, Yonsei University College of Dentistry, Seoul 120-752 (Korea, Republic of)]. E-mail: leeyk@yumc.yonsei.ac.kr

    2005-05-15

    We investigated the cytotoxicity of the prepared various ferrites (Fe-, Li-, Ni/Zn/Cu-, Ba-, Sr-, Co-, Co/Ni-ferrites) using MTT assay as well as agar diffusion method. Their cytotoxicity was compared with that of alginate-encapsulated ferrites. In the MTT assay, Fe{sub 3}O{sub 4} and SrFe{sub 12}O{sub 19} ferrite showed the highest cell viability of 90%. Alginate-encapsulated Ba-ferrite was ranked mildly cytotoxic, whereas their ferrite particles were ranked cytotoxic.

  16. Evaluation of estrogenic potential of flavonoids using a recombinant yeast strain and MCF7/BUS cell proliferation assay.

    Directory of Open Access Journals (Sweden)

    Flávia A Resende

    Full Text Available Phytoestrogens are of interest because of their reported beneficial effects on many human maladies including cancer, neurodegeneration, cardiovascular disease and diabetes. Furthermore, there is a search for compounds with estrogenic activity that can replace estrogen in hormone replacement therapy during menopause, without the undesirable effects of estrogen, such as the elevation of breast cancer occurrence. Thus, the principal objective of this study was to assess the estrogenic activity of flavonoids with different hydroxylation patterns: quercetin, kaempferol, luteolin, fisetin, chrysin, galangin, flavone, 3-hydroxyflavone, 5-hydroxyflavone and 7-hydroxyflavone via two different in vitro assays, the recombinant yeast assay (RYA and the MCF-7 proliferation assay (E-screen, since the most potent phytoestrogens are members of the flavonoid family. In these assays, kaempferol was the only compound that showed ERα-dependent transcriptional activation activity by RYA, showing 6.74±1.7 nM EEQ, besides acting as a full agonist for the stimulation of proliferation of MCF-7/BUS cells. The other compounds did not show detectable levels of interaction with ER under the conditions used in the RYA. However, in the E-screen assay, compounds such as galangin, luteolin and fisetin also stimulated the proliferation of MCF-7/BUS cells, acting as partial agonists. In the evaluation of antiestrogenicity, the compounds quercetin, chrysin and 3-hydroxyflavone significantly inhibited the cell proliferation induced by 17-β-estradiol in the E-screen assay, indicating that these compounds may act as estrogen receptor antagonists. Overall, it became clear in the assay results that the estrogenic activity of flavonoids was affected by small structural differences such as the number of hydroxyl groups, especially those on the B ring of the flavonoid.

  17. Limit-dilution assay and clonal expansion of all T cells capable of proliferation

    International Nuclear Information System (INIS)

    Chen, W.-F.; Wilson, A.; Scollay, R.; Shortman, K.

    1982-01-01

    A limit-dilution microculture system is presented in which almost all mature T cells, cultured at a level of about 1 cell/well, grow and expand to clones averaging 60,000 cells over an 8-9 day period. Cloning efficiency is 70-100%, so the set of expanded clones is representative of the starting T-cell population. T cells of all Lyt phenotypes form clones of progeny cells. The system involves culture in flat-bottom microtitre trays, in the presence of concanavalin A as the initiating stimulus, together with appropriately irradiated spleen filler cells and a supplementary source of soluble T cell growth factors. The resultant clones may be screened for cytolytic function, as described in the accompanying paper. The system may be used to assay the level of T cells capable of expansion or precursor function (PTL-p) by using [ 3 H]TdR uptake as a readout for the presence or absence of proliferating clones. Analysis of the frequency of positive cultures shows a good fit to the expected Poisson distribution, with no evidence of complicating suppressor or helper effects. (Auth.)

  18. Limit-dilution assay and clonal expansion of all T cells capable of proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Chen, W.F.; Wilson, A.; Scollay, R.; Shortman, K. (Walter and Eliza Hall Inst. of Medical Research, Parkville (Australia))

    1982-08-13

    A limit-dilution microculture system is presented in which almost all mature T cells, cultured at a level of about 1 cell/well, grow and expand to clones averaging 60,000 cells over an 8-9 day period. Cloning efficiency is 70-100%, so the set of expanded clones is representative of the starting T-cell population. T cells of all Lyt phenotypes form clones of progeny cells. The system involves culture in flat-bottom microtitre trays, in the presence of concanavalin A as the initiating stimulus, together with appropriately irradiated spleen filler cells and a supplementary source of soluble T cell growth factors. The resultant clones may be screened for cytolytic function, as described in the accompanying paper. The system may be used to assay the level of T cells capable of expansion or precursor function (PTL-p) by using (/sup 3/H)TdR uptake as a readout for the presence or absence of proliferating clones. Analysis of the frequency of positive cultures shows a good fit to the expected Poisson distribution, with no evidence of complicating suppressor or helper effects.

  19. Understanding colonization and proliferation potential of endophytes and pathogen in planta via plating, polymerase chain reaction and ergosterol assay.

    Science.gov (United States)

    Chow, Yiing Yng; Rahman, Sadequr; Ting, Adeline Su Yien

    2017-01-01

    This study aimed to establish the colonization behavior and proliferation potential of three endophytes and one pathogen Ganoderma boninense (Gb) introduced into oil palm ramets (host model). The endophytes selected were Diaporthe phaseolorum (WAA02), Trichoderma asperellum (T2), and Penicillium citrinum (BTF08). Ramets were first inoculated with 100 mL of fungal cells (10 6  cfu mL - 1 ) via soil drenching. For the next 7 days, ramets were sampled and subjected to three different assays to detect and identify fungal colonization, and establish their proliferation potential in planta . Plate assay revealed the presence of endophytes in root, stem and leaf tissues within 7 days after inoculation. Polymerase Chain Reaction (PCR) detected and identified the isolates from the plant tissues. The ergosterol assay (via high performance liquid chromatography, HPLC) confirmed the presence of endophytes and Gb in planta . The increase in ergosterol levels throughout 49 days was however insignificant, suggesting that proliferation may be absent or may occur very slowly in planta . This study strongly suggests that the selected endophytes could colonize the host upon inoculation, but proliferation occurs at a slower rate, which may subsequently influence the biocontrol expression of endophytes against the pathogen.

  20. Understanding colonization and proliferation potential of endophytes and pathogen in planta via plating, polymerase chain reaction, and ergosterol assay

    Directory of Open Access Journals (Sweden)

    Yiing Yng Chow

    2017-01-01

    Full Text Available This study aimed to establish the colonization behavior and proliferation potential of three endophytes and one pathogen Ganoderma boninense (Gb introduced into oil palm ramets (host model. The endophytes selected were Diaporthe phaseolorum (WAA02, Trichoderma asperellum (T2, and Penicillium citrinum (BTF08. Ramets were first inoculated with 100 mL of fungal cells (106 cfu mL−1 via soil drenching. For the next 7 days, ramets were sampled and subjected to three different assays to detect and identify fungal colonization, and establish their proliferation potential in planta. Plate assay revealed the presence of endophytes in root, stem and leaf tissues within 7 days after inoculation. Polymerase Chain Reaction (PCR detected and identified the isolates from the plant tissues. The ergosterol assay (via high-performance liquid chromatography, HPLC confirmed the presence of endophytes and Gb in planta. The increase in ergosterol levels throughout 49 days was however insignificant, suggesting that proliferation may be absent or may occur very slowly in planta. This study strongly suggests that the selected endophytes could colonize the host upon inoculation, but proliferation occurs at a slower rate, which may subsequently influence the biocontrol expression of endophytes against the pathogen.

  1. Cyquant cell proliferation assay as a fluorescence-based method for in vitro screening of antimalarial activity.

    Science.gov (United States)

    Sriwilaijaroen, Nongluk; Kelly, Jane Xu; Riscoe, Michael; Wilairat, Prapon

    2004-12-01

    The appearance of drug resistant parasites and the absence of an effective vaccine have resulted in the need for new effective antimalarial drugs. Consequently, a convenient method for in vitro screening of large numbers of antimalarial drug candidates has become apparent. The CyQUANT cell proliferation assay is a highly sensitive fluorescence-based method for quantitation of cell number by measuring the strong fluorescence produced when green GR dye binds to nucleic acids. We have applied the CyQUANT assay method to evaluate the growth of Plasmodium falciparum D6 strain in culture. The GR-nucleic acid fluorescence linearly correlated with percent parasitemia at both 0.75 or 1 percent hematocrit with the same correlation coefficient of r2 = 0.99. The sensitivity of P. falciparum D6 strain to chloroquine and to 3,6-bis-omega-diethylaminoamyloxyxanthone, a novel antimalarial, determined by the CyQUANT assay were comparable to those obtained by the traditional [3H]-ethanolamine assay: IC50 value of chloroquine was 54 nM and 51 nM by the CyQUANT and [3H]-ethanolamine assay, respectively; IC50 value for 3,6-bis-omega-diethylaminoamyloxyxanthone was 254 nM and 223 nM by the CyQUANT and [3H]-ethanolamine assay, respectively. This procedure requires no radioisotope, uses simple equipment, and is an easy and convenient procedure, with no washing and harvesting steps. Moreover, all procedures can be set up continuously and thus, the CyQUANT assay is suitable in automatic high through-put drug screening of antimalarial drugs.

  2. Effect of borax on immune cell proliferation and sister chromatid exchange in human chromosomes

    OpenAIRE

    Pongsavee Malinee

    2009-01-01

    Abstract Background Borax is used as a food additive. It becomes toxic when accumulated in the body. It causes vomiting, fatigue and renal failure. Methods The heparinized blood samples from 40 healthy men were studied for the impact of borax toxicity on immune cell proliferation (lymphocyte proliferation) and sister chromatid exchange in human chromosomes. The MTT assay and Sister Chromatid Exchange (SCE) technic were used in this experiment with the borax concentrations of 0.1, 0.15, 0.2, 0...

  3. Proliferation assay of mouse embryonic stem (ES) cells exposed to atmospheric-pressure plasmas at room temperature

    International Nuclear Information System (INIS)

    Miura, Taichi; Hirano, Kazumi; Ogura, Chika; Ikeguchi, Masamichi; Seki, Atsushi; Nishihara, Shoko; Ando, Ayumi; Kanazawa, Tatsuya; Hamaguchi, Satoshi

    2014-01-01

    Proliferation assays of mouse embryonic stem (ES) cells have been performed with cell culture media exposed to atmospheric-pressure plasmas (APPs), which generate reactive species in the media at room temperature. It is found that serum in cell culture media functions as a scavenger of highly reactive species and tends to protect cells in the media against cellular damage. On the other hand, if serum is not present in a cell culture medium when it is exposed to APP, the medium becomes cytotoxic and cannot be detoxified by serum added afterwards. Plasma-induced cytotoxic media hinder proliferation of mouse ES cells and may even cause cell death. It is also shown by nuclear magnetic resonance spectroscopy that organic compounds in cell culture media are in general not significantly modified by plasma exposure. These results indicate that if there is no serum in media when they are exposed to APPs, highly reactive species (such as OH radicals) generated in the media by the APP exposure are immediately converted to less reactive species (such as H 2 O 2 ), which can no longer readily react with serum that is added to the medium after plasma exposure. This study has clearly shown that it is these less reactive species, rather than highly reactive species, that make the medium cytotoxic to mouse ES cells. (paper)

  4. A simple method to measure cell viability in proliferation and cytotoxicity assays

    Directory of Open Access Journals (Sweden)

    Ricardo Carneiro Borra

    2009-09-01

    Full Text Available Resazurin dye has been broadly used as indicator of cell viability in several types of assays for evaluation of the biocompatibility of medical and dental materials. Mitochondrial enzymes, as carriers of diaphorase activities, are probably responsible for the transference of electrons from NADPH + H+ to resazurin, which is reduced to resorufin. The level of reduction can be quantified by spectrophotometers since resazurin exhibits an absorption peak at 600 ηm and resorufin at 570 ηm wavelengths. However, the requirement of a spectrophotometer and specific filters for the quantification could be a barrier to many laboratories. Digital cameras containing red, green and blue filters, which allow the capture of red (600 to 700 ηm and green (500 to 600 ηm light wavelengths in ranges bordering on resazurin and resorufin absorption bands, could be used as an alternative method for the assessment of resazurin and resorufin concentrations. Thus, our aim was to develop a simple, cheap and precise method based on a digital CCD camera to measure the reduction of resazurin. We compared the capability of the CCD-based method to distinguish different concentrations of L929 and normal Human buccal fibroblast cell lines with that of a conventional microplate reader. The correlation was analyzed through the Pearson coefficient. The results showed a strong association between the measurements of the method developed here and those made with the microplate reader (r² = 0.996; p < 0.01 and with the cellular concentrations (r² = 0.965; p < 0.01. We concluded that the developed Colorimetric Quantification System based on CCD Images allowed rapid assessment of the cultured cell concentrations with simple equipment at a reduced cost.

  5. Quantification of cellular viability for the MTT method

    International Nuclear Information System (INIS)

    Altanes, M.

    2001-01-01

    In the last years, the scientists have been given to the task of finding new biomaterials whose biocompatibility with the human body allow them to substitute parts of the organism, as the bones and the junctures and also that they are little rejected. In the following work it was evaluated and quantified by the method of the MTT, a colorimetric, quick, simple and economic technical, the possible citohepatic effect of a watery extract of a biomaterials of polymeric origin (acrilamide and metacrilic acid) obtained for technical of gamma irradiation, consistent in culture medium 199 and veal serum, in cells Vero. In order to compare the answer of the analysis material, they were used the controls negative (polyethylene of high molecular weight), and positive (Sulphate of Streptomycin), just as they indicate it the established norms, being achieved the waited result of each one of them. In the observation made to the optic microscope, after 24 and 48 hours of contact between the cells and the extract of the analysis material, they were not perceived indicative of damage or cellular lysis, or morphologic changes in the structure of cells that it indicated us the possible cytotoxic effect of this biomaterials, therefore the qualitative analysis was not enough for the determination of the cytotoxic effect of the analysis sample

  6. In Vitro Endothelial Cell Proliferation Assay Reveals Distinct Levels of Proangiogenic Cytokines Characterizing Sera of Healthy Subjects and of Patients with Heart Failure

    Directory of Open Access Journals (Sweden)

    Rebecca Voltan

    2014-01-01

    Full Text Available Although myocardial angiogenesis is thought to play an important role in heart failure (HF, the involvement of circulating proinflammatory and proangiogenic cytokines in the pathogenesis and/or prognosis of HF has not been deeply investigated. By using a highly standardized proliferation assay with human endothelial cells, we first demonstrated that sera from older (mean age 52±7.6 years; n=46 healthy donors promoted endothelial cell proliferation to a significantly higher extent compared to sera obtained from younger healthy donors (mean age 29±8.6 years; n=20. The promotion of endothelial cell proliferation was accompanied by high serum levels of several proangiogenic cytokines. When we assessed endothelial cell proliferation in response to HF patients’ sera, we observed that a subset of sera (n=11 promoted cell proliferation to a significantly lesser extent compared to the majority of sera (n=18. Also, in this case, the difference between the patient groups in the ability to induce endothelial cell proliferation correlated to significant (P<0.05 differences in serum proangiogenic cytokine levels. Unexpectedly, HF patients associated to the highest endothelial proliferation index showed the worst prognosis as evaluated in terms of subsequent cardiovascular events in the follow-up, suggesting that high levels of circulating proangiogenic cytokines might be related to a worse prognosis.

  7. Evaluation of rapid MTT tube method for detection of drug susceptibility of mycobacterium tuberculosis to rifampicin and isoniazid

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    Raut U

    2008-01-01

    Full Text Available Purpose: To evaluate MTT method for detection of drug resistance to rifampicin and isoniazid in M.tuberculosis . This method utilises the ability of viable mycobacterial cells to reduce MTT( 3-4,5-dimethylthiazol-2-yl-2, 5-diphenyl tetrazolium bromide. Methods: The method was standardised with known resistant and sensitive strains of M.tuberculosis and was then extended to 50 clinical isolates. An inoculum of 10 7 cfu/mL was prepared in Middlebrook 7H9 medium supplemented with oleic acid, albumin, dextrose and catalase. For each drug three tubes were used, one with INH(0.2μg/mL or RIF(1μg/mL, another as inoculum control and third as blank control. These were incubated at 37°C for four and seven days respectively for RIF and INH after which MTT assay was performed. Results were read visually and by colorimeter at 570 nm. Relative optical density unit (RODU of 0.2 was taken as cut off. Results were compared with drug sensitivity obtained by proportion method using LJ medium. Results: For rifampicin, concordance with proportion method was 90% by visual and 94% by RODU. Sensitivity and specificity was 86.8% and 100% respectively by visual method and 95.2% and 87.5% respectively by RODU. For Isoniazid, concordance was 94% and sensitivity and specificity was 94.7 and 91.7% respectively by both visual and RODU. Conclusions: MTT assay proved to be rapid and cheap method for performing drug sensitivity of M.tuberculosis

  8. Monitoring T-Cell Responses in Translational Studies: Optimization of Dye-Based Proliferation Assay for Evaluation of Antigen-Specific Responses

    Directory of Open Access Journals (Sweden)

    Anja Ten Brinke

    2017-12-01

    Full Text Available Adoptive therapy with regulatory T cells or tolerance-inducing antigen (Ag-presenting cells is innovative and promising therapeutic approach to control undesired and harmful activation of the immune system, as observed in autoimmune diseases, solid organ and bone marrow transplantation. One of the critical issues to elucidate the mechanisms responsible for success or failure of these therapies and define the specificity of the therapy is the evaluation of the Ag-specific T-cell responses. Several efforts have been made to develop suitable and reproducible assays. Here, we focus on dye-based proliferation assays. We highlight with practical examples the fundamental issues to take into consideration for implementation of an effective and sensitive dye-based proliferation assay to monitor Ag-specific responses in patients. The most critical points were used to design a road map to set up and analyze the optimal assay to assess Ag-specific T-cell responses in patients undergoing different treatments. This is the first step to optimize monitoring of tolerance induction, allowing comparison of outcomes of different clinical studies. The road map can also be applied to other therapeutic interventions, not limited to tolerance induction therapies, in which Ag-specific T-cell responses are relevant such as vaccination approaches and cancer immunotherapy.

  9. Quantifying rates of cell migration and cell proliferation in co-culture barrier assays reveals how skin and melanoma cells interact during melanoma spreading and invasion.

    Science.gov (United States)

    Haridas, Parvathi; Penington, Catherine J; McGovern, Jacqui A; McElwain, D L Sean; Simpson, Matthew J

    2017-06-21

    Malignant spreading involves the migration of cancer cells amongst other native cell types. For example, in vivo melanoma invasion involves individual melanoma cells migrating through native skin, which is composed of several distinct subpopulations of cells. Here, we aim to quantify how interactions between melanoma and fibroblast cells affect the collective spreading of a heterogeneous population of these cells in vitro. We perform a suite of circular barrier assays that includes: (i) monoculture assays with fibroblast cells; (ii) monoculture assays with SK-MEL-28 melanoma cells; and (iii) a series of co-culture assays initiated with three different ratios of SK-MEL-28 melanoma cells and fibroblast cells. Using immunostaining, detailed cell density histograms are constructed to illustrate how the two subpopulations of cells are spatially arranged within the spreading heterogeneous population. Calibrating the solution of a continuum partial differential equation to the experimental results from the monoculture assays allows us to estimate the cell diffusivity and the cell proliferation rate for the melanoma and the fibroblast cells, separately. Using the parameter estimates from the monoculture assays, we then make a prediction of the spatial spreading in the co-culture assays. Results show that the parameter estimates obtained from the monoculture assays lead to a reasonably accurate prediction of the spatial arrangement of the two subpopulations in the co-culture assays. Overall, the spatial pattern of spreading of the melanoma cells and the fibroblast cells is very similar in monoculture and co-culture conditions. Therefore, we find no clear evidence of any interactions other than cell-to-cell contact and crowding effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Immunoradiometric assay of a novel proliferation marker: tumour polypeptide specific antigen in breast cancer management in north India

    International Nuclear Information System (INIS)

    Phanna-Hazra, P.; Sharma, U.; Gupta, K.K.; Bhatnagar, V.; Idnani, R.; Gangwar, P.K.; Hazra, D.K.

    2004-01-01

    Tissue Polypeptide Specific Antigen (TPS) is a novel tumour marker defined by the monoclonal antibody M3 ,discovered by Bjorklund,which is claimed to be a proliferation marker, and belonging to the cytokeratin 8-18 family. M3 defines this antigen in particular out of a group of Tissue Polypeptide antigens (TPA) charecterised by the same Swedish group and was claimed to be a pancarcinoma antigen reportedly being elevated in several different cancers in the European literature.Pancarcinoma markers are of interest in relation to cancer detection as well as for assessing therapy and prognosis.Pancarcinoma antigens are also of interest for radiobioconjugate immunotargetting both for diagnosis as well as for therapy. There were no reports on this marker in an Indian population and therefore this study was initiated at two institutions in Meerut and Agra, both in the northern state of Uttar Pradesh. In addition to 250 healthy controls, 288 cases of breast cancer were studied .In addituion benign disorders were studied: breast fibroadenosis 16, breast fibro adenoma 5, breast abscess 4. TPS levels were determined by an immunoradiometric assay. In controls all but 4 had values less than 80 Units/1,the upper normal level quoted by Bjorklund.The relationship of the serum levels of the markers to histological grade and anatomical stage of the tumour were studied. In addition several of the cases underwent therapy with chemotherapy /radiotherapy/surgery or combinations thereof. The response to treatment during follow up was categorized as Complete emission(CR),Partial Remission (PR)Stable Disease(SD), Progressive Disease(PD)and recurrence (R) by the respective clinicians using standard criteria. In the cancer subjects there was a close correlation of TPS elevation with anatomical stage. 10 cases belonged to stage 1 with TPS levels 176.1+-103.94 Units/L ,61 to stage II(TPS levels 206.45 +-168.23).,79 to stage Ilia (TPS 251.5+-168.53),83 to stage IIIB (TPS 537.35+- 691.71),and 55

  11. The Effects of NDRG2 Overexpression on Cell Proliferation and Invasiveness of SW48 Colorectal Cancer Cell Line.

    Science.gov (United States)

    Golestan, Ali; Mojtahedi, Zahra; Ghalamfarsa, Ghasem; Hamidinia, Maryam; Takhshid, Mohammad Ali

    2015-09-01

    Colorectal cancer (CRC) is one of the most common causes of cancer-related death in the world. The expression of N-myc downstream-regulated gene 2 (NDRG2) is down-regulated in CRC. The aim of this study was to investigate the effect of NDRG2 overexpression on cell proliferation and invasive potential of SW48 cells. SW48 cells were transfected with a plasmid overexpressing NDRG2. After stable transfection, the effect of NDRG2 overexpression on cell proliferation was evaluated by MTT assay. The effects of NDRG2 overexpression on cell migration, invasion and cell motility and matrix metalloproteinase 9 (MMP9) activities were also investigated using matrigel transwell assay, wound healing assay and gelatin zymography, respectively. MTT assay showed that overexpression of NDRG2 caused attenuation of SW48 cell proliferation. Transwell and wound healing assay revealed that NDRG2 overexpression led to inhibition of migration, invasion, and motility of SW48 cells. The overexpression of NDRG2 also reduced the activity of secreted MMP-9. The results of this study suggest that NDRG2 overexpression inhibits proliferation and invasive potential of SW48 cells, which likely occurs via suppression of MMP-9 activity.

  12. Mutagenicity of the peroxisome proliferators clofibrate, Wyeth 14,643 and di-2-ethylhexyl phthalate in the lacZ plasmid-based transgenic mouse mutation assay

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    Boerrigter Michaël

    2004-01-01

    Full Text Available Abstract Background Peroxisome proliferators are considered rodent carcinogens that are putative human non-carcinogens based on the presumed absence of direct genetic toxicity in rodent and human cells and the resistance of human cells to the induction of peroxisomes by peroxisome proliferators. The highly sensitive lacZ plasmid-based transgenic mouse mutation assay was employed to investigate the mutagenicity of several peroxisome proliferators based on several lines of evidence suggesting that these agents may in fact exert a genotoxic effect. Methods Male and female lacZ-plasmid based transgenic mice were treated at 4 months of age with 6 doses of 2,333 mg di-2-ethylhexyl phthalate (DHEP, 200 mg Wyeth-14,643, or 90 mg clofibrate per kg of bodyweight, respectively, over a two-week period. Control animals were treated with the respective vehicles only (35% propyl glycol for DEHP and Wyeth-14,643 treatment controls and sterile water for clofibrate treatment controls. The mutant frequency in liver, kidney and spleen DNA was determined as the proportion of retrieved mutant and wild-type lacZ plasmids expressed in Escherichia Coli C host cells employing a positive selection system for mutant plasmids. Results Exposure to DEHP or Wyeth-14,643 significantly increased the mutant frequency in liver, but not in kidney or spleen, of both female and male mice. Treatment with clofibrate did not lead to an increased mutant frequency in any of the organs studied. Conclusion The results indicate that some peroxisome proliferators display an organ-specific mutagenicity in lacZ plasmid-based transgenic mice consistent with historical observations of organ- and compound-specific carcinogenicity.

  13. Quantitative Validation of the Presto Blue Metabolic Assay for Online Monitoring of Cell Proliferation in a 3D Perfusion Bioreactor System.

    Science.gov (United States)

    Sonnaert, Maarten; Papantoniou, Ioannis; Luyten, Frank P; Schrooten, Jan Ir

    2015-06-01

    As the fields of tissue engineering and regenerative medicine mature toward clinical applications, the need for online monitoring both for quantitative and qualitative use becomes essential. Resazurin-based metabolic assays are frequently applied for determining cytotoxicity and have shown great potential for monitoring 3D bioreactor-facilitated cell culture. However, no quantitative correlation between the metabolic conversion rate of resazurin and cell number has been defined yet. In this work, we determined conversion rates of Presto Blue, a resazurin-based metabolic assay, for human periosteal cells during 2D and 3D static and 3D perfusion cultures. Our results showed that for the evaluated culture systems there is a quantitative correlation between the Presto Blue conversion rate and the cell number during the expansion phase with no influence of the perfusion-related parameters, that is, flow rate and shear stress. The correlation between the cell number and Presto Blue conversion subsequently enabled the definition of operating windows for optimal signal readouts. In conclusion, our data showed that the conversion of the resazurin-based Presto Blue metabolic assay can be used as a quantitative readout for online monitoring of cell proliferation in a 3D perfusion bioreactor system, although a system-specific validation is required.

  14. Quantitative Validation of the Presto Blue™ Metabolic Assay for Online Monitoring of Cell Proliferation in a 3D Perfusion Bioreactor System

    Science.gov (United States)

    Sonnaert, Maarten; Papantoniou, Ioannis; Luyten, Frank P.

    2015-01-01

    As the fields of tissue engineering and regenerative medicine mature toward clinical applications, the need for online monitoring both for quantitative and qualitative use becomes essential. Resazurin-based metabolic assays are frequently applied for determining cytotoxicity and have shown great potential for monitoring 3D bioreactor-facilitated cell culture. However, no quantitative correlation between the metabolic conversion rate of resazurin and cell number has been defined yet. In this work, we determined conversion rates of Presto Blue™, a resazurin-based metabolic assay, for human periosteal cells during 2D and 3D static and 3D perfusion cultures. Our results showed that for the evaluated culture systems there is a quantitative correlation between the Presto Blue conversion rate and the cell number during the expansion phase with no influence of the perfusion-related parameters, that is, flow rate and shear stress. The correlation between the cell number and Presto Blue conversion subsequently enabled the definition of operating windows for optimal signal readouts. In conclusion, our data showed that the conversion of the resazurin-based Presto Blue metabolic assay can be used as a quantitative readout for online monitoring of cell proliferation in a 3D perfusion bioreactor system, although a system-specific validation is required. PMID:25336207

  15. Predictive value of the flow cytometric PCNA - assay (proliferating cell nuclear antigen) in head and neck tumors after accelerated-hyperfractionated radiochemotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Wenz, F; Lohr, F; Rudat, V; Dietz, A; Flentje, M; Wannenmacher, M

    1995-07-01

    Purpose/Objective: Proliferation of surviving tumor cells during fractionated radiotherapy may limit tumor control, especially in rapidly proliferating tumors. It has been widely accepted, that this may play a major role in head and neck tumors. Several methods for the assessment of tumor proliferation have been developed, however, most of them are either laborious, invasive or potentially toxic. Today, the gold standard is the flow cytometric BrdUrd assay. We present a flow cytometric method for detection of PCNA, which is an intranuclear proliferation associated protein, in solid human head and neck tumors and how these data correlate with outcome. Materials and Methods: Pretherapeutic biopsies of 20 inoperable patients with squamous cell carcinoma of the head and neck (T3-4N2M0) were examined. The tissue was disaggregated with pepsin/HCl, antibody staining was performed using the clone PC10. Biparametric flow cytometry was performed after a FITC conjugated secondary antibody and propidiumjodine staining was applied. The PCNA-index (i.e. percentage PCNA-positive cells), the DNA-index and the S-phase fraction (SPF, euploid tumors only) were determined. The therapy consisted of combined accelerated-hyperfractionated radiochemotherapy (66 Gy in 5 wks, concomittant boost of 1.6 Gy/d in wks 4+5, Carboplatin in wks 1+5). The median follow-up time was 14 mths (5 - 28), the clinical partners (V.R., A.D.) were 'blinded' towards the PCNA-values. Results: 13 patients suffered from disease progession and 11 died. The actuarial median survival and disease free survival (DFS) were 14.4 and 10.7 mths, respectively. The PCNA-values ranged from 3.2 to 70% (median 9%), there were 7 aneuploid and 13 euploid tumors. SFP in the euploid tumors ranged from 4 to 14.5% (median 10.5%). Neither SFP nor ploidy had a significant influence on the outcome. The patients were divided according to their PCNA-value in higher (n=10) and lower (n=10) than the median. The survival and DFS were 13

  16. A chimera embryo assay reveals a decrease in embryonic cellular proliferation induced by sperm from X-irradiated male mice

    International Nuclear Information System (INIS)

    Obasaju, M.F.; Wiley, L.M.; Oudiz, D.J.; Raabe, O.; Overstreet, J.W.

    1989-01-01

    Male mice were divided into three experimental groups and a control group. Mice in the experimental groups received one of three doses of acute X irradiation (1.73, 0.29, and 0.05 Gy) and together with the control unirradiated mice were then mated weekly to unirradiated female mice for a 9-week experimental period. Embryos were recovered from the weekly matings at the four-cell stage and examined by the chimera assay for proliferative disadvantage. Aggregation chimeras were constructed of embryos from female mice mated to irradiated males (experimental embryos) and embryos from females mated to unexposed males (control embryos) and contained either one experimental embryo and one control embryo (heterologous chimera) or two control embryos (control chimera). The control embryo in heterologous chimeras and either embryo in control chimeras were prelabeled with the vital dye fluorescein isothiocyanate (FITC), and the chimeras were cultured for 40 h and viewed under phase-contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution from the FITC-labeled embryo. Experimental and control embryos that were cultured singly were also examined for embryo cell number at the end of the 40-h culture period. In control chimeras, the mean ratio of the unlabeled cells:total chimera cell number (henceforth referred to as ''mean ratio'') was 0.50 with little or no weekly variation over the 9-week experimental period. During Weeks 4-7, the mean ratios of heterologous chimeras differed significantly from the mean ratio of control chimeras with the greatest differences occurring during Week 7 (0.41 for chimeras of 0.05 Gy dose group, 0.40 for chimeras of the 0.29 Gy dose group, and 0.17 for chimeras of the 1.73 Gy dose group)

  17. Fluoride Stimulates the Proliferation of Osteoclasts in vitro by Upregulating MCM3

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    Shengbin Bai

    2016-06-01

    Full Text Available We have previously shown that the expression of the minichromosome maintenance protein 3 (MCM3 gene was upregulated in lymphocytes of patients with skeletal fluorosis. We speculated that increased MCM3 expression may be contribute to osteopathy in patients with skeletal fluorosis. Here, we investigated the effect of fluoride on the proliferation of osteoclasts derived from RAW264.7 cells and the involvement of MCM3. Our MTT assays showed that 0.25 mM NaF markedly stimulated the proliferation of RAW264.7 cells. The RT-PCR and immunoblotting assays revealed that 0.25 mM NaF upregulated MCM3 expression in RAW264.7 cells. The MTT assays additionally demonstrated that stimulation with MCM3 potentiated the effect of fluorine on the proliferation of RAW264.7 cells. These results demonstrated that fluoride at clinical relevant concentration upregulates MCM3 expression in osteoclasts in vitro. We are currently conducting a series of experiments to examine whether increased MCM3 in osteoclasts indeed contributes to osteopathy in skeletal fluorosis.

  18. Assessment of Nili-Ravi buffalo ( Bubalus bubalis ) semen by MTT ...

    African Journals Online (AJOL)

    The study was conducted to examine and validate the MTT test to assess the sperm viability of Nili-Ravi buffalo bulls and compare the efficiency of the test with the supra-vital staining technique (eosin-nigrosine) and hypo-osmotic swelling test. Fresh semen samples from breeding Nili-Ravi buffalo bulls (n = 20) were ...

  19. Research on the promoting role of apelin-13 in proliferation, migration and capillary-like tube formation of RF/6A cells

    Directory of Open Access Journals (Sweden)

    Kun-Peng Xie

    2017-05-01

    Full Text Available AIM: To investigate the effects of apelin-13 on proliferation, migration and capillary-like tube formation of a monkey choroid / retinal endothelial cell line, RF/6A, to clarify whether apelin-13 could promote retinal angiogenesis in vitro.METHODS: RF/6A cells in good conditions were administrated with DMSO(the control group, apelin-13 at 0.1μmol/L(low dose groupor apelin-13 at 1μmol/L(high dose group. Cell proliferation, migration and capillary-like tube formation were detected by using the MTT assay, scratch assay and matrigel assay, respectively, at 24h after plating the cells. RESULTS: Cell proliferation was promoted in both low and high dose apelin-13 groups compared to the control cells(PPPCONCLUSION: Apelin-13 could obviously promote the angiogenesis capacity of RF/6A cells, suggesting that apelin-13 was an important pro-angiogenic factor in retinal endothelial cells.

  20. MiR-155 promotes cell proliferation and inhibits apoptosis by PTEN signaling pathway in the psoriasis.

    Science.gov (United States)

    Xu, Longjiang; Leng, Hong; Shi, Xin; Ji, Jiang; Fu, Jinxiang; Leng, Hong

    2017-06-01

    MicroRNAs (miRNAs) have been demonstrated to contribute to malignant progression in psoriasis development. The purposes of the study was to evaluated the effects of miRNA-155 on cell proliferation, migration and apoptosis in psoriasis development via PTEN singaling pathway and identify its direct target protein. Quantitative real-time RT-PCR (qRT-PCR) was performed to examine the level of miR-155 in psoriasis cells, miR-155 was downregulated in a psoriasis cell line Hacat by transfected with small interfering RNA (siRNA), respectively. Cell survival was detected by the MTT assay and colony formation assay. Cell migration and invasion were measured via wound-healing assayand transwell assay. In addition, cell cycle and apoptosis about psoriasis cells was measured by flow cytometry. In this study, qRT-PCR assay showed that the expressions of miR-155 mRNA in psoriasis tissues were significantly higher than that in normal tissues. The assays about cell growth and proliferation showed that miR-155 knockdown led to a significant decrease in cell proliferation which was determined by MTT assay and colony formation assay compared to those of Lv-NC cells. Flow cytometry analysis showed that depletion of miR-155 could cause cell cycle change and the number of apoptotic cells was significantly increased in Lv-miR155 cells compared with control cells. In addition, the expression of several apoptosis-related factors were dramatically changed, such as PTEN, PIP 3 , AKT, p-AKT, Bax and Bcl-2. Our findings indicate that down-regulation of miR-155 significantly inhibits proliferation, migration, invasion and promotes apoptosis through PTEN singaling pathway in psoriasis cells. miR-155 might function as an oncogene miRNA in the progress of psoriasis. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  1. Cytotoxicity of dental alloys, metals, and ceramics assessed by millipore filter, agar overlay, and MTT tests.

    Science.gov (United States)

    Sjögren, G; Sletten, G; Dahl, J E

    2000-08-01

    Biocompatibility of dental materials is dependent on the release of elements from the materials. In addition, the composition, pretreatment, and handling of the materials influence the element release. This study evaluated the cytotoxicity of dental alloys, metals, and ceramics, with specific emphasis on the effects of altering the composition and the pretreatment. By using cells from a mouse fibroblast cell line and the agar overlay test, Millipore filter test, and MTT test, cytotoxicity of various metals, metal alloys, and ceramics for dental restoration were studied. Effects of altering the composition of a high noble gold alloy and of pretreatment of a ceramic-bonding alloy were also studied. In addition, the release of elements into the cell culture medium by the materials studied was measured using an inductively coupled plasma optical emission spectrophotometer. The results of the MTT test were analyzed statistically using ANOVA and Scheffé test at a significance level of P filter tests. For the MTT test, no significant differences were observed between these materials and controls, with the exception of JS C-gold and unalloyed titanium. The modified materials were ranked from "mildly cytotoxic" to "moderately cytotoxic" in the agar overlay and Millipore filter tests and from "noncytotoxic" to "moderately cytotoxic" in the MTT test. Thus, cytotoxicity was related to the alloy composition and treatment. The release of Cu and Zn seemed to be important for the cytotoxic effect. Alterations in the composition and the pretreatment can greatly influence the cytotoxicity, and the results stress the importance of carefully following the manufacturers' instructions when handling dental materials.

  2. Effects of cyclic stretch on proliferation of mesenchymal stem cells and their differentiation to smooth muscle cells

    International Nuclear Information System (INIS)

    Ghazanfari, Samane; Tafazzoli-Shadpour, Mohammad; Shokrgozar, Mohammad Ali

    2009-01-01

    Bone marrow mesenchymal stem cells (MSCs) are capable of differentiating into a variety of cell types such as vascular smooth muscle cells (SMCs). In this study, we investigated influence of cyclic stretch on proliferation of hMSCs for different loading conditions, alignment of actin filaments, and consequent differentiation to SMCs. Isolated cells from bone marrow were exposed to cyclic stretch utilizing a customized device. Cell proliferation was examined by MTT assay, alignment of actin fibers by a designed image processing code, and cell differentiation by fluorescence staining. Results indicated promoted proliferation of hMSCs by cyclic strain, enhanced by elevated strain amplitude and number of cycles. Such loading regulated smooth muscle α-actin, and reoriented actin fibers. Cyclic stretch led to differentiation of hMSCs to SMCs without addition of growth factor. It was concluded that applying appropriate loading treatment on hMSCs could enhance proliferation capability, and produce functional SMCs for engineered tissues.

  3. Upregulation of miR-3607 promotes lung adenocarcinoma proliferation by suppressing APC expression.

    Science.gov (United States)

    Lin, Yong; Gu, Qiangye; Sun, Zongwen; Sheng, Baowei; Qi, Congcong; Liu, Bing; Fu, Tian; Liu, Cun; Zhang, Yan

    2017-11-01

    Lung cancer is the leading cause of worldwide cancer-related deaths, although many drugs and new therapeutic approaches have been used, the 5-years survival rate is still low for lung cancer patients. microRNAs have been shown to regulate lung cancer initiation and development, here we studied the role of miR-3607 in lung cancer cell proliferation. We found miR-3607 was upregulated in lung cancer tissues and cells, miR-3607 overexpression promoted lung cancer cell A549 proliferation determined by MTT assay, colony formation assay, anchorage-independent growth ability assay and bromodeoxyuridine incorporation assay, while the opposite phenotypes were shown when miR-3607 was knocked down. Predicted analysis suggested a Wnt signaling pathway regulator adenomatous polyposis coli (APC) was the target of miR-3607, miR-3607 could directly bind to the 3'UTR of APC, and promoted Cyclin D1 and c-Myc expression which can be suppressed by APC. Double knockdown of miR-3607 and APC copied the phenotypes of miR-3607 overexpression, suggesting miR-3607 promoted lung cancer cell A549 proliferation by targeting APC. In conclusion, our study suggested miR-3607 contributes to lung cancer cell proliferation by inhibiting APC. Copyright © 2017. Published by Elsevier Masson SAS.

  4. Biocompatibility of various hydoxyapatite scaffolds evaluated by proliferation of rat’s bone marrow mesenchymal stem cells: an in vitro study

    Directory of Open Access Journals (Sweden)

    Achmad F. Kamal

    2013-12-01

    Full Text Available Background: Scaffold (biomaterial biocompatibility test should be performed in vitro prior to in vivo stem cell application in animal or clinical trial. These test consists of direct and indirect toxicity test (MTT assay [3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide]. Those tests were used to identify cell morphological changes, cell-substrate adhesion impairment, and reduction in cell proliferation activity.Methods: The tested scaffolds were hydroxyapatite-calcium sulphate (HA-CaSO4 (scaffold I, nano-particular HA paste (scaffold II, synthetic HA granule (scaffold III, bovine HA granule (scaffold IV, and morsellized bovine xenograft (scaffold V. Direct contact toxicity test and MTT assay [3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide] were performed on those groups. In direct contact toxicity test, we put granules of various scaffolds within plates and incubated together with mesenchymal stem cells (MSCs. In MTT assay we included phenol 20 mg/mL and 100 mg/mL group as positive control. Morphology, cell adhesion impairment, and cell growth were monitored daily until day-7. Cells counting in the direct contact toxicity test was conducted on day-7.Results: There were no changes on 24 hours observation after direct contact. On day-7, an impairment of cell adhesion to plastic substrates, changes in cell morphology, and cell death were observed, especially in scaffold I, scaffold II, and scaffold V. In MTT assay, only scaffold I, phenol 20 mg/mL, and phenol 100 mg/mL showed more than 50% inhibition at 24-hour and 7-day-observation. Extracts from scaffold II, III, IV, and V did not affect the viability and proliferation of bone marrow MSCs (inhibition value < 50%. Scaffold II, III, IV and V were proven non-cytotoxic and have good biocompatibility in vitro,  no statistical significant differences were observed among the scaffold groups (p > 0.05.Conclusion: We understand which scaffold was nontoxic or the least toxic to

  5. Isolation of Melittin from Iranian Honey Bee Venom and Investigation of Its Effect on Proliferation of Cervical Cancer- HeLa Cell Line

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    K Pooshang Bagheri

    2013-06-01

    Full Text Available Introduction: Cervical cancer is the second prevalent cancer in developing countries and the sixth prevalent cancer in USA. Since conventional treatment methods are associated with detrimental side effects, searching for new drugs using natural ingredients is very important. Previous studies have shown that melittin (main component of honey bee venom has anticancer properties along with the effect on cell membrane and activation of apoptosis. In this study, inhibitory effects of melittin on the viability and proliferation of cervical cancer cell line (HeLa was investigated. Methods: Melittin was purified from honeybee venom using reversed-phase HPLC method. Then, biological activity of melittin was examined by hemolytic activity analysis on the red blood cells. In order to investigate whether melittin inhibits proliferation of HeLa cell, MTT assay was performed. HeLa cells were plated in a 96-well plate and treated with serially diluted concentrations of melittin for 12 and 24 hours. The viability of the cells was measured via MTT assay at 540nm. Results: Melittin showed a strong hemolytic activity (HD50=0.5 µg/ml which can be reduced by FBS(HD50=2 µg/ml. Results of MTT assay indicated that melittin shows cytotoxic effect on cervical cancer cells with IC50 = 1.2 ug/ml at 12h incubation period. Conclusion: In this study, biological activity of melittin and inhibitory effect of FBS on hemolysis were determined via hemolytic activity analysis. MTT assay indicated that melittin induced cytotoxic effects in a dose dependent manner on cervical cancer cells and it also revealed dependence on incubation time as well.

  6. Effect of borax on immune cell proliferation and sister chromatid exchange in human chromosomes.

    Science.gov (United States)

    Pongsavee, Malinee

    2009-10-30

    Borax is used as a food additive. It becomes toxic when accumulated in the body. It causes vomiting, fatigue and renal failure. The heparinized blood samples from 40 healthy men were studied for the impact of borax toxicity on immune cell proliferation (lymphocyte proliferation) and sister chromatid exchange in human chromosomes. The MTT assay and Sister Chromatid Exchange (SCE) technic were used in this experiment with the borax concentrations of 0.1, 0.15, 0.2, 0.3 and 0.6 mg/ml. It showed that the immune cell proliferation (lymphocyte proliferation) was decreased when the concentrations of borax increased. The borax concentration of 0.6 mg/ml had the most effectiveness to the lymphocyte proliferation and had the highest cytotoxicity index (CI). The borax concentrations of 0.15, 0.2, 0.3 and 0.6 mg/ml significantly induced sister chromatid exchange in human chromosomes (P Borax had effects on immune cell proliferation (lymphocyte proliferation) and induced sister chromatid exchange in human chromosomes. Toxicity of borax may lead to cellular toxicity and genetic defect in human.

  7. Effect of borax on immune cell proliferation and sister chromatid exchange in human chromosomes

    Directory of Open Access Journals (Sweden)

    Pongsavee Malinee

    2009-10-01

    Full Text Available Abstract Background Borax is used as a food additive. It becomes toxic when accumulated in the body. It causes vomiting, fatigue and renal failure. Methods The heparinized blood samples from 40 healthy men were studied for the impact of borax toxicity on immune cell proliferation (lymphocyte proliferation and sister chromatid exchange in human chromosomes. The MTT assay and Sister Chromatid Exchange (SCE technic were used in this experiment with the borax concentrations of 0.1, 0.15, 0.2, 0.3 and 0.6 mg/ml. Results It showed that the immune cell proliferation (lymphocyte proliferation was decreased when the concentrations of borax increased. The borax concentration of 0.6 mg/ml had the most effectiveness to the lymphocyte proliferation and had the highest cytotoxicity index (CI. The borax concentrations of 0.15, 0.2, 0.3 and 0.6 mg/ml significantly induced sister chromatid exchange in human chromosomes (P Conclusion Borax had effects on immune cell proliferation (lymphocyte proliferation and induced sister chromatid exchange in human chromosomes. Toxicity of borax may lead to cellular toxicity and genetic defect in human.

  8. The effects of CD147 on the cell proliferation, apoptosis, invasion, and angiogenesis in glioma.

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    Yin, Haoyuan; Shao, Ying; Chen, Xuan

    2017-01-01

    To analyze the effects of extracellular matrix metalloproteinase inducer (CD147) on glioma proliferation, apoptosis, invasion, and angiogenesis. Tissue samples were obtained from 101 glioma cases while normal brain tissues were obtained from 30 brain injury cases. Immunohistochemical assay was performed to detect the expressions of CD147, CD34, and VEGF in tissue samples. QRT-PCR was performed to detect the relative expression of CD147 mRNA in human glioma cell lines. CD147 siRNA was transfected into glioma cell line U251. Cell proliferation, apoptosis, invasion, and angiogenesis were tested by MTT, flow cytometry, Transwell assay, and vasculogenic mimicry assay, respectively. Expressions of relative proteins were analyzed with western blot. CD147 was positively expressed with the percentage of 0, 37.5, 44.8, 67.9, and 85.7 % in normal tissues and glioma tissues with WHO grades I-IV, respectively, and the scores of MVDand VEGF were associated with the expression of CD147. CD147 was significantly upregulated in the human glioma cell lines (P CD147 suppressed cell proliferation, blocked cell cycle, induced apoptosis, inhibited cell invasion and angiogenesis in glioma cells in vitro. The expression of CD147 was significantly associated with WHO tumor grade and angiogenesis; silencing of CD147 contributed to inhibition of glioma proliferation, invasion, and angiogenesis. Our study provided firm evidence that CD 147 is a potential glioma target for anti-angiogenic therapies.

  9. Synergistic Effects of SAM and Selenium Compounds on Proliferation, Migration and Adhesion of HeLa Cells.

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    Sun, Licui; Zhang, Jianxin; Yang, Qiu; Si, Yang; Liu, Yiqun; Wang, Qin; Han, Feng; Huang, Zhenwu

    2017-08-01

    To determine the antitumor activities and molecular mechanism of selenium compounds in HeLa cells. Western blotting was used to detect ERK and AKT activation in HeLa cells induced by selenium compounds selenomethionine (SeMet), methylselenocysteine (MeSeCys) and methylseleninic acids (MeSeA). Using MTT, wound-healing and Matrigel adhesion assays, the antitumor effects of SAM and selenium compounds were evaluated in HeLa cells. MeSeA inhibited ERK and AKT signaling pathways and suppressed the proliferation (peffects compared to the other treatments. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  10. Effect of photobiomodulation on viability and proliferation of stem cells from exfoliated deciduous teeth under different nutritional conditions

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    Morato de Souza, Letícia; Guilherme Roque Rinco, Ugo; Aparecida Tavares Aguiar, Daniela; Aparecido de Almeida Junior, Luciano; Cosme-Silva, Leopoldo; Marchini Oliveira, Thais; Teixeira Marques, Nádia Carolina; Thiemy Sakai, Vivien

    2018-02-01

    This study aimed to evaluate the effect of different doses of low-level laser irradiation on the viability and proliferation of stem cells from exfoliated deciduous teeth (SHED) cultured under nutritional deficit (cellular stress) or regular nutritional conditions. SHED underwent irradiation by a red laser between 1.2 and 6.2 J cm-2. Prior to the irradiation, all groups received culture medium (MEMα, Eagle’s minimum essential medium alpha modification) supplemented with 1% of fetal bovine serum (FBS) for 1 h. After the irradiation, cells received MEMα supplemented with 10% of FBS (regular nutrition) or 1% of FBS (nutritional deficit). Cell viability and proliferation were respectively determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet assays 6 and 24 h after irradiation (P  cell viability and proliferation of SHED after laser irradiation, except for 1.2 J cm-2.

  11. Melatonin regulates CRE-dependent gene transcription underlying osteoblast proliferation by activating Src and PKA in parallel.

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    Tao, Lin; Zhu, Yue

    2018-01-01

    Several studies have indicated a relationship between melatonin and idiopathic scoliosis, including our previous work which demonstrated that melatonin can inhibit osteoblast proliferation; however, the mechanism remains unclear. Here, we utilized a MTT assay to show that melatonin significantly reduces osteoblast proliferation in a concentration-and time-dependent manner. Through a combination of techniques, including real-time PCR, MTT assays, immunofluorescence, and luciferase assays, we confirmed that melatonin-induced changes in phosphorylated cAMP response element-binding protein (CREB) reduced transcriptional activity in a melatonin receptor-dependent manner. Surprisingly, treatment of osteoblasts with the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor PD98059 up-regulated other cascades upstream of CREB. We next treated cells with PKA and Src inhibitors and observed that melatonin can also activate the protein kinase A (PKA) and Src pathways. To examine whether Src is upstream from the cAMP-PKA pathway, we measured cAMP levels in response to melatonin with and without a Src inhibitor (PP2) and found that PP2 had no additional effect. Therefore, the transcription-dependent mechanisms involved in CREB phosphorylation, along with melatonin, activated Src via a parallel signaling pathway that was separate from that of PKA. Finally, we transfected osteoblasts with lentiviral CREB short hairpin (sh) RNAs and found a decrease in the expression of proliferating cell nuclear antigen (PCNA) and osteoblast proliferation. These results suggest that CREB and PCNA are downstream targets of melatonin signaling, and that the down-regulation of CREB, which is regulated via PKA and Src pathways, contributes to the melatonin-induced inhibition of osteoblast proliferation.

  12. [Knockdown of NEDD9 inhibits the proliferation, invasion and migration of esophageal carcinoma EC109 cells].

    Science.gov (United States)

    Zhang, Wen; Li, Shaojun; Zhao, Yunlong; Guo, Nannan; Li, Yingjie

    2016-12-01

    Objective To observe the expression of the neural precursor cell expressed, developmentally down-regulated 9 (NEDD9) in esophageal cancer, to investigate the impact of decreased expression of NEDD9 on invasion and migration, and to explicit the function of NEDD9 in EC109 human esophageal cancer cell line. Methods Immunohistochemical staining was used to detect the expression of NEDD9 in human esophageal cancer tissues and paracancerous normal tissues. RNA interfering (RNAi) was used to knockdown NEDD9 in EC109 cells. The interference efficiency was detected by reverse transcription PCR (RT-PCR) and Western blot analysis. Cell proliferation was determined by MTT assay and the invasion and migration abilities of EC109 cells were monitored by Transwell TM assay. The protein levels of proliferating cell nuclear antigen (PCNA), Bax and Bcl-2 were tested by Western blotting. Results The positive expression rate of NEDD9 in esophageal carcinoma tissues was significantly higher compared with that in the paracancerous tissues. After NEDD9 expression was successfully downregulated in EC109 cells by siRNA, the proliferation, invasion and migration rates in transfection group were significantly lower than those in control group; meanwhile, the expression of Bcl-2 was reduced and Bax expression was enhanced. Conclusion The protein expression level of NEDD9 is higher in esophageal carcinoma tissues than that in adjacent normal tissues. Knockdown of NEDD9 expression can restrain the proliferation, invasion and migration of EC109 cells.

  13. Proliferation and osteogenic differentiation of human periodontal ligament cells on akermanite and β-TCP bioceramics

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    L Xia

    2011-07-01

    Full Text Available The purpose of this study was to investigate the effects of akermanite as compared to β-TCP on attachment, proliferation, and osteogenic differentiation of human periodontal ligament cells (hPDLCs. Scanning electron microscopy (SEM and actin filament labeling were used to reveal attachment and growth of hPDLCs seeded on β-TCP and akermanite ceramic. Cell proliferation was tested by lactic acid production and MTT analysis, while osteogenic differentiation was assayed by alkaline phosphatase (ALP expression and real-time polymerase chain reaction (PCR analysis on markers of osteopontin (OPN, dentin matrix acidic phosphoprotein-1 (DMP-1, and osteocalcin (OCN, and further detected by enzyme-linked immunosorbent analysis (ELISA analysis for OCN expression. Besides, the ions released from akermanite and their effect on hPDLCs was also measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES, MTT analysis, ALP expression and real-time PCR analysis. hPDLCs attached well on both ceramics, but showed better spreading on akermanite. hPDLCs proliferated more rapidly on akermanite than β-TCP. Importantly, osteogenic differentiation of hPDLCs was enhanced on akermanite compared to β-TCP. Besides, Ca, Mg and Si ions were released from akermanite, while only Ca ions were released from β-TCP. Moreover, more pronounced proliferation and higher osteogenic gene expression for hPDLCs cultured with akermanite extract were detected as compared to cells cultured on akermanite. Therefore, akermanite ceramic showed an enhanced effect on proliferation and osteogenic differentiation of hPDLCs, which might be attributed to the release of ions containing Ca, Mg and Si from the material. It is suggested that akermanite ceramics may serve as a potential material for periodontal bone regeneration.

  14. The miR-383-LDHA axis regulates cell proliferation, invasion and glycolysis in hepatocellular cancer

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    Zhixiong Fang

    2017-02-01

    Full Text Available Objective(s: To explore the correlation between expression patterns and functions of miR-383 and LDHA in hepatocellular cancer (HCC. Materials and Methods: We detected the expression of miR-383 and LDHA in 30 HCC tissues and their matched adjacent normal tissues using qRT-PCR. Then we performed MTT assay, foci formation assay, transwell migration assay, glucose uptake assay and lactate production assay to explore the function of miR-383 in cell proliferation, invasion and glycolysis in HCC cell lines. Luciferase reporter assay was used to explore whether LDHA was a target gene of miR-383. Western blot and qRT-PCR were used to further confirm LDHA was targeted by miR-383. Then the above functional experiments were repeated to see whether the function of LDHA could be inhibited by miR-383. Results: The results of qRT-PCR showed that miR-383 was down-regulated in HCC tissues compared with their matched adjacent normal tissues. Functional experiments showed that overexpression of miR-383 significantly suppressed cell proliferation, invasion and glycolysis. Luciferase reporter assay showed LDHA was a target gene of miR-383 and expression of LDHA was inversely correlated with that of miR-383 in HCC. Besides, increased cell proliferation, invasion and glycolysis triggered by LDHA could be inhibited by overexpression of miR-383 in HCC cell lines. Conclusion: Our study proved that miR-383 is down-regulated in HCC and acts as a tumor suppressor through targeting LDHA. Targeting the miR-383-LDHA axis might be a promising strategy in HCC treatment.

  15. [miR-25 promotes cell proliferation by targeting RECK in human cervical carcinoma HeLa cells].

    Science.gov (United States)

    Qiu, Gang; Fang, Baoshuan; Xin, Guohong; Wei, Qiang; Yuan, Xiaoye; Wu, Dayong

    2015-01-01

    To investigate the effect of miR-25 on the proliferation of human cervical carcinoma HeLa cells and its association with reversion-inducing cysteine-rich protein with Kazal motifs (RECK). The recombinant plasmids of pcDNATM6.2-GW-pre-miR-25, pmirGLO-RECK-WT, pmirGLO-RECK-MT and anti-miR-25 were constructed, and their transfection efficiencies into HeLa cells were identified by real-time quantitative PCR (qRT-PCR). The potential proliferation-stimulating function of miR-25 was analyzed by MTT assay in HeLa cells. Furthermore, the target effect of miR-25 on the RECK was determined by dual-luciferase reporter assay system, qRT-PCR and Western blotting. Sequence analysis demonstrated that the recombinant plasmids of pcDNATM6.2-GW-pre-miR-25 and pmirGLO-RECK-WT, pmirGLO-RECK-MT were successfully constructed, and qRT-PCR revealed that the transfection efficiencies of pre-miR-25 and anti-miR-25 were desirable in HeLa cells. MTT assay showed that miR-25 over-expression promoted the proliferation of HeLa cells. In addition, the luciferase activity was significantly reduced in HeLa cells cotransfected with pre-miR-25 and RECK-WT. The qRT-PCR and Western blotting indicated that the expression level of RECK was up-regulated in HeLa cells transfected with anti-miR-25 at the transcriptional and posttranscriptional levels. miR-25 could promote cell proliferation by targeting RECK in HeLa cells.

  16. Role of PD 0332991 on the Proliferation and Apoptosis of Vascular Endothelial Cells

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    Chenlong ZHAO

    2018-05-01

    Full Text Available Background and objective Angiogenesis is an important process in the development of tumor. PD 0332991, a cell cycle inhibitor, can specifically inhibit CD4/6 phosphorylation and cell cycle progression. In xeongraft mice models, PD 0332991 treated mice had significantly decreased angiogenesis and vascular density compared with the control group, but the mechanism remains unknown. The purpose of this study is to investigate the role and molecular mechanism of PD 0332991 on vascular endothelial cells. Methods EA.hy926 cells, a kind of vascular endothelial cell, were used as the research model. The effects of PD 0332991 on the activity and proliferation of EA.hy926 cells were detected by the MTT, EdU assays. Wound-healing assays and transwell assays were used to determine the effects of PD 0332991 on the mobility of EA.hy926. The influence of PD 0332991 on cell cycle and apoptosis of endothelial cells was tested by flow cytometry, and the Western blot was applied to observe the expression of cell cycle related proteins in EA.hy926 cells treated by PD 0332991. Results PD 0332991 significantly inhibited the proliferation and mobility of EA.hy926 cells, caused cell cycle arrest and apoptosis. At the same time, PD 0332991 inhibited the expression of CDK4/6 and phosphorylation of Rb, and thus inhibited the cell cycle progression of EA.hy926 cells. Conclusion PD 0332991 can inhibit the proliferation and activity of endothelial cells and induces apoptosis.

  17. [Effects of selenium compounds on proliferation, migration and adhesion of HeLa cells].

    Science.gov (United States)

    Sun, Licui; Lu, Jiaxi; Wang, Qin; Liu, Yiqun; Han, Feng; Yang, Yanhua; Zhang, Hongkun; Huang, Zhenwu

    2015-03-01

    To explore the effects of methylseleninic acid (MeSeA), selenomethionine (SeMet) and methylselenocysteine (MeSeCys) on proliferation, migration and adhesion of HeLa cells. HeLa cells were cultured and treated with MeSeA, SeMet and MeSeCys for 12 - 72 h respectively. MTT assay, healing assay and in vitro cell Matrigel adhesion assay were used to detect the proliferation, migration and adhesion of HeLa cells. Compared to the control group, the proliferation of HeLa cells was remarkably inhibited by MeSeA (P HeLa cells in MeSeA group was inhibited by 34% (P HeLa cells with inhibitions of 18% and 13% was in SeMet group in 4 h and 8 h. The inhibitions of HeLa cell migration in MeSeCys group was 28% (P HeLa cells in the MeSeA group, the SeMet group as well as the MeSeCys group were inhibited by 36% (P HeLa cell were effectively inhibited by MeSeA, while the adhesive function of HeLa cell was remarkably inhibited by MeSeCys.

  18. Effect of Interlukin-1β on proliferation of gastric epithelial cells in culture

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    Beales Ian LP

    2002-04-01

    Full Text Available Abstract Background Helicobacter pylori is the main risk factor for the development of non-cardia gastric cancer. Increased proliferation of the gastric mucosa is a feature of H. pylori infection. Mucosal interkeukin-1β production is increased in H. pylori infection and IL-1β genotypes associated with increased pro-inflammatory activity are risk factors for the development of gastric cancer. The effect of IL-1β on gastric epithelial cell proliferation has been examined in this study. Methods AGS cells were cultured with IL-1β. DNA synthesis was assed by [3H]thymidine incorporation and total viable cell numbers by MTT assay. Results IL-1β dose dependently increased DNA synthesis and cell numbers. The enhanced proliferation was blocked by interleukin-1 receptor antagonist. Addition of neutralising antibody to GM-CSF reduced IL-1β-stimulated proliferation by 31 ± 4 %. GM-CSF alone significantly stimulated proliferation. Addition or neutralisation of IL-8 had no effect on basal or IL-1β-stimulated proliferation. The tyrosine kinase inhibitor genistein completely blocked IL-1β-stimulated proliferation and inhibition of the extracellular signal related kinase pathway with PD 98059 inhibited IL-1β stimulated proliferation by 58 ± 5 %. Conclusions IL-1β stimulates proliferation in gastric epithelial cells. Autocrine stimulation by GM-CSF contributes to this proliferative response. Signalling via tyrosine kinase activity is essential to the mitogenic response to IL-1β. The extracellular signal related kinase pathway is involved in, but not essential to downstream signalling. IL-1β may contribute to the hyperproliferation seen in H. pylori- infected gastric mucosa, and be involved in the carcinogenic process.

  19. Luteolin Inhibits Angiotensin II-Stimulated VSMC Proliferation and Migration through Downregulation of Akt Phosphorylation

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    Tongda Xu

    2015-01-01

    Full Text Available Luteolin is a naturally occurring flavonoid found in many plants that possesses cardioprotective properties. The purpose of this study was to elucidate the effect of luteolin on vascular smooth muscle cells (VSMCs proliferation and migration induced by Angiotensin II (Ang II and to investigate the mechanism(s of action of this compound. Rat VSMCs were cultured in vitro, and the proliferation and migration of these cells following Ang II stimulation were monitored. Different doses of luteolin were added to VSMC cultures, and the proliferation and migration rate were observed by MTT and Transwell chamber assays, respectively. In addition, the expressions of p-Akt (308, p-Akt (473, and proliferative cell nuclear antigen (PCNA in VSMCs were monitored by Western blotting. This study demonstrated that luteolin has an inhibitory effect on Ang II-induced VSMC proliferation and migration. Further, the levels of p-Akt (308, p-Akt (473, and PCNA were reduced in VSMCs treated with both Ang II and luteolin compared to VSMCs treated with only Ang II. These findings strongly suggest that luteolin inhibits Ang II-stimulated proliferation and migration of VSMCs, which is partially due to downregulation of the Akt signaling pathway.

  20. Indirubin inhibits cell proliferation, migration, invasion and angiogenesis in tumor-derived endothelial cells

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    Li Z

    2018-05-01

    Full Text Available Zhuohong Li, Chaofu Zhu, Baiping An, Yu Chen, Xiuyun He, Lin Qian, Lan Lan, Shijie Li Department of Oncology, The Affiliated Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, Sichuan, China Purpose: Hepatocellular carcinoma is one of the most predominant malignancies with high fatality rate and its incidence is rising at an alarming rate because of its resistance to radio- and chemotherapy. Indirubin is the major active anti-tumor ingredient of a traditional Chinese herbal medicine. The present study aimed to analyze the effects of indirubin on cell proliferation, migration, invasion, and angiogenesis of tumor-derived endothelial cells (Td-EC. Methods: Td-EC were derived from human umbilical vein endothelial cells (HUVEC by treating HUVEC with the conditioned medium of human liver cancer cell line HepG2. Cell proliferation, migration, invasion, and angiogenesis were assessed by MTT, wound healing, in vitro cell invasion, and in vitro tube formation assay. Results: Td-EC were successfully obtained from HUVEC cultured with 50% culture supernatant from serum-starved HepG2 cells. Indirubin significantly inhibited Td-EC proliferation in a dose- and time-dependent manner. Indirubin also inhibited Td-EC migration, invasion, and angiogenesis. However, indirubin’s effects were weaker on HUVEC than Td-EC. Conclusion: Indirubin significantly inhibited Td-EC proliferation, migration, invasion, and angiogenesis. Keywords: indirubin, Td-EC, proliferation, migration, invasion, angiogenesis

  1. [Role of connective tissue growth factor (CTGF) in proliferation and migration of pancreatic cancer cells].

    Science.gov (United States)

    Bai, Yu-chun; Kang, Quan; Luo, Qing; Wu, Dao-qi; Ye, Wei-xia; Lin, Xue-mei; Zhao, Yong

    2011-10-01

    To explore the expression of connective tissue growth factor (CTGF) in pancreatic cancer and its influence on the proliferation and migration of cancer cells. The expression of CTGF in pancreatic cell line PANC-1 cells was analyzed by real-time PCR and in pancreatic carcinoma (50 cases) tissues by immunohistochemistry. The ability of proliferation and migration in vitro of PANC-1 cells was tested by MTT assay, scratch test and Boyden chamber test after the CTGF gene was overexpressed by Ad5-CTGF or silenced with Ad5-siCTGF transfection. CTGF was overexpressed in both pancreatic cancer cells and tissues. Overxpression of CTGF leads to increased proliferation and migration of PANC-1 cells. The CTGF-transfected PANC-1 cells showed apparent stronger proliferation ability and scratch-repair ability than that of empty vector controls. The results of Boyden chamber test showed that there were 34 cells/field (200× magnificantion) of the CTGF-transfected overexpressing cells, much more than the 11 cells/field of the empty vector control cells; and 6 cells/microscopic field of the Ad5-siCTGF-transfected silenced cells, much less than the 15 cells/field of the control cells. CTGF is overexpressed in both pancreatic cancer cells in vitro and in vivo, indicating that it may play an important role in the cell proliferation and migration in pancreatic cancer.

  2. Effects of Src on Proliferation and Invasion of Lung Cancer Cells

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    Rui ZHENG

    2011-04-01

    Full Text Available Background and objective It has been proven that Src played pivotal roles in carcinogenesis, cancer progression and metastasis. The aim of this study is to explore the roles of Src phosphorylation on lung cancer cells. Methods Western blot and immunoprecipitation was used to detect the expression and phosphorylation of Src in lung cancer cells. MTT and Boyden chamber assay was used to examine the effects of inhibition of Src phosphorylation on proliferation and invasion of lung cancer cells in vitro, respectively. Results pp60src was expressed in all lung cancer cell lines in this study. All 5 non-small cell lung cancer (NSCLC cell lines had increased autophosphorylated tyrosine-418, while nearly no phosphorylated Src in small cell lung cancer SBC5 cell line was detected. The effect of inhibition of Src tyrosine kinase on cell proliferation varied among the lung cancer cell lines. Submicromolar Src tyrosine kinase inhibitor (≤1 μM remarkably suppressed the proliferation of PC-9 and A549 cells in a dose dependent manner (P < 0.05, while the same concentration of Src tyrosine kinase inhibitor had no significant effect on proliferation of H226, PC14PE6 and RERFLCOK cells. Invasiveness of lung cancer cells was significantly suppressed by Src tyrosine kinase in a dose-dependent manner (P < 0.05. Conclusion Phosphorylation of Src, but not over-expression, plays a pivotal role in proliferation and invasion of NSCLC cell lines in vitro.

  3. Short time administration of antirheumatic drugs - Methotrexate as a strong inhibitor of osteoblast's proliferation in vitro

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    Annussek Tobias

    2012-09-01

    Full Text Available Abstract Introduction Due to increasing use of disease modifying antirheumatic drugs (DMARDs as first line therapy in rheumatic diseases, dental and maxillofacial practitioner should be aware of drug related adverse events. Especially effects on bone-metabolism and its cells are discussed controversially. Therefore we investigate the in vitro effect of short time administration of low dose methotrexate (MTX on osteoblasts as essential part of bone remodelling cells. Methods Primary bovine osteoblasts (OBs were incubated with various concentrations of MTX, related to tissue concentrations, over a period of fourteen days by using a previously established standard protocol. The effect on cell proliferation as well as mitochondrial activity was assessed by using 3-(4, 5-dimethylthiazol-2-yl 2, 5-diphenyltetrazolium bromide (MTT assay, imaging and counting of living cells. Additionally, immunostaining of extracellular matrix proteins was used to survey osteogenic differentiation. Results All methods indicate a strong inhibition of osteoblast`s proliferation by short time administration of low dose MTX within therapeutically relevant concentrations of 1 to 1000nM, without affecting cell differentiation of middle-stage differentiated OBs in general. More over a significant decrease of cell numbers and mitochondrial activity was found at these MTX concentrations. The most sensitive method seems to be the MTT-assay. MTX-concentration of 0,01nM and concentrations below had no inhibitory effects anymore. Conclusion Even low dose methotrexate acts as a potent inhibitor of osteoblast’s proliferation and mitochondrial metabolism in vitro, without affecting main differentiation of pre-differentiated osteoblasts. These results suggest possible negative effects of DMARDs concerning bone healing and for example osseointegration of dental implants. Especially the specifics of the jaw bone with its high vascularisation and physiological high tissue metabolism

  4. Attachment, proliferation and differentiation of BMSCs on gas-jet/electrospun nHAP/PHB fibrous scaffolds

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    Guan Donghua; Chen Zhiqing; Huang Chunpeng; Lin Yinghe

    2008-01-01

    In this study, poly(3-hydroxybutyrate) (PHB)-based scaffolds containing nanosized hydroxyapatite (nHAP) were manufactured by gas-jet/electrospinning. The morphologies of the scaffolds were characterized. The effect of the scaffolds on attachment, proliferation and differentiation of the bone marrow stroma cells (BMSCs) were accessed by using scanning electron microscopy (SEM), methylthiazol tetrazolium (MTT) assay and alkaline phosphatase (ALP) activity. The results show that the gas-jet/electrospun scaffolds possess an extracellular matrix-like topography. In vitro studies describe that the scaffolds have positive effects on attachment, proliferation and differentiation of BMSCs in vitro. It can be concluded that the scaffolds combing the unique structural features generated by gas-jet/electrospinning with functional factors, have the potential to be used in bone tissue engineering

  5. [Inhibitory effect of 17-AAG combined with paclitaxel on proliferation of esophageal squamous cell carcinoma Eca-109 cells in vitro].

    Science.gov (United States)

    Chen, Size; Chen, Xuemei; Li, Yuqi; Yang, Shu; Mo, Xianyi; Zhang, Fan; Mo, Kailan; Ding, Ying

    2015-06-01

    To investigate the effect of 17-AAG combined with paclitaxel (PTX) on the proliferation and apoptosis of esophageal squamous cell carcinoma cell line Eca-109 in vitro. Eca-109 cells were treated with 17-AAG and PTX either alone or in combination. The proliferation of Eca-109 cells was detected by MTT assay, and the cell cycle changes and cell apoptosis were determined by flow cytometry. Compared with the control group, both 17-AAG and PTX significantly inhibited the proliferation of Eca-109 cells. A combined treatment of the cells with 0.5 µmol/L PTX and 0.625 µmol/L 17-AAG produced an obviously stronger inhibitory effect on the cell proliferation than either of the agents used alone (PAAG and PTX used alone caused Eca-109 cell cycle arrest in G2/M phase and S phase, respectively, and their combined use caused cell cycle arrest in both G2/M and S phases. The cell apoptosis rates of Eca-109 cells treated with 17-AAG, PTX and their combination were 4.52%, 10.91%, and 29.88%, respectively, all significantly higher than that in the control group (1.32%); the combined treatment resulted in a distinct apoptotic peak that was significantly higher than that caused by either of the agents alone. 17-AAG and PTX can inhibit cell proliferation and promote apoptosis of Eca-109 cells, and their combination produces stronger effects in inhibiting cell proliferation and increasing cell apoptosis.

  6. Triptolide inhibits TGF-β1-induced cell proliferation in rat airway smooth muscle cells by suppressing Smad signaling

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    Chen, Ming; Lv, Zhiqiang; Huang, Linjie [Department of Respiratory Medicine, Sun Yat-Sen Memorial Hospital, Institute for Respiratory disease of Sun Yat-sen University, Sun Yat-sen University, Guangzhou, Guangdong Province 510120 (China); Zhang, Wei [Department of Geratology, the Second People' s Hospital of Shenzhen, Shenzhen 518000 (China); Lin, Xiaoling; Shi, Jianting; Zhang, Wei; Liang, Ruiyun [Department of Respiratory Medicine, Sun Yat-Sen Memorial Hospital, Institute for Respiratory disease of Sun Yat-sen University, Sun Yat-sen University, Guangzhou, Guangdong Province 510120 (China); Jiang, Shanping, E-mail: shanpingjiang@126.com [Department of Respiratory Medicine, Sun Yat-Sen Memorial Hospital, Institute for Respiratory disease of Sun Yat-sen University, Sun Yat-sen University, Guangzhou, Guangdong Province 510120 (China)

    2015-02-15

    Background: We have reported that triptolide can inhibit airway remodeling in a murine model of asthma via TGF-β1/Smad signaling. In the present study, we aimed to investigate the effect of triptolide on airway smooth muscle cells (ASMCs) proliferation and the possible mechanism. Methods: Rat airway smooth muscle cells were cultured and made synchronized, then pretreated with different concentration of triptolide before stimulated by TGF-β1. Cell proliferation was evaluated by MTT assay. Flow cytometry was used to study the influence of triptolide on cell cycle and apoptosis. Signal proteins (Smad2, Smad3 and Smad7) were detected by western blotting analysis. Results: Triptolide significantly inhibited TGF-β1-induced ASMC proliferation (P<0.05). The cell cycle was blocked at G1/S-interphase by triptolide dose dependently. No pro-apoptotic effects were detected under the concentration of triptolide we used. Western blotting analysis showed TGF-β1 induced Smad2 and Smad3 phosphorylation was inhibited by triptolide pretreatment, and the level of Smad7 was increased by triptolide pretreatment. Conclusions: Triptolide may function as an inhibitor of asthma airway remodeling by suppressing ASMCs proliferation via negative regulation of Smad signaling pathway. - Highlights: • In this study, rat airway smooth muscle cells were cultured and made synchronized. • Triptolide inhibited TGF-β1-induced airway smooth muscle cells proliferation. • Triptolide inhibited ASMCs proliferation via negative regulation of Smad signaling pathway.

  7. [Inhibitory effect of diclofenac sodium on the proliferation of rabbit corneal epithelial cells in vitro].

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    Wu, Ningling; Du, Zhiyu

    2010-11-01

    To investigate the inhibitory effect of diclofenac sodium on rabbit corneal epithelial cells (RCECs) in vitro and explore its pharmacological mechanism. The fresh rabbit cornea was cultured to obtain the primary RCECs, and RCECs of passage 2 were used in this research. The cells were divided into experimental groups, the cells in which were incubated with different concentrations (18.18, 27.27, 36.36, 45.45, 54.55 μg/ml) of diclofenac sodium, and control group. The effect of diclofenac sodium on the proliferation of cells was measured by methyl thiazolyl thiazolium (MTT) assay 24, 48 and 72 h after incubation. While the RCECs were divided into experimental groups, the cells in which were incubated with 9 and 12.5 μg/ml diclofenac sodium, and control group. The cell cycle and apoptotic rate were observed by flow cytometer. MTT assay showed that diclofenac sodium had obvious inhibitory effect on RCECs, and the inhibition rate was increasing along with the increasing concentration of diclofenac sodium and the incubation time(Pdiclofenac sodium, the cells in G0/G1 phase were obviously increased, and the apoptosis cusp and apoptotic rate were increased. Diclofenac sodium exerts significant inhibitory effect on RCECs in a dosage-dependent manner, and it may function by inducing cell apoptosis and ceasing cell cycles.

  8. Upregulation of CPE promotes cell proliferation and tumorigenicity in colorectal cancer

    International Nuclear Information System (INIS)

    Liang, Xing-Hua; He, Wen-guang; Huang, Yan-Nian; Zeng, Xian-Cheng; Li, Ling-ling; Wu, Geng-Gang; Xie, Yi-Cheng; Zhang, Guang-Xian; Chen, Wei; Yang, Hai-Feng; Liu, Qi-Long; Li, Wen-Hong

    2013-01-01

    Colorectal cancer (CRC) is one of the most common cancers worldwide and a leading cause of cancer related death. Although the mortality rate of CRC is decreasing, finding novel targets for its therapy remains urgent. Carboxypeptidase E (CPE), a member of the pro-protein convertases, which are involved in the maturation of protein precursors, has recently been reported as elevated in many types of cancer. However, its role and mechanisms in tumor progression are poorly understood. In the present study, we investigated expression of CPE in CRC cell lines and tumor tissues using Western blot and real-time qRT-PCR. Plasmids for overexpression and depletion of CPE were constructed and analyzed by Western blot, MTT and colony formation assays and bromodeoxyuridine incorporation assays. The relative expression of p21, p27, and cyclin D1 were analyzed by Real-time qRT-PCR in the indicated cells. Our study showed that CPE was significantly upregulated in CRC cell lines and tumor tissues. MTT and colony formation assays indicated that overexpression of CPE enhanced cell growth rates. BrdU incorporation and flow-cytometry assays showed that ectopic expression of CPE increased the S-phase fraction cells. Soft agar assay proved enhanced tumorigenicity activity in CPE over-expressing CRC cells. Further studies of the molecular mechanisms of CPE indicated that is promoted cell proliferation and tumorigenicity through downregulation of p21 and p27, and upregulation of cyclin D1. Taken together, these data suggest that CPE plays an important role in cell cycle regulation and tumorigenicity, and may serve as a potential target for CRC therapeutics

  9. [Effects of metformin on human oral cancer KB cell proliferation and apoptosis in vitro].

    Science.gov (United States)

    Wang, Fang; Xu, Jincheng; Xia, Fei; Liu, Zhe; Zhao, Surong; Liu, Hao; Jiang, Zhiwen

    2014-02-01

    To investigate the effects of metformin on the proliferation and apoptosis of human oral cancer cell line KB in vitro. Human oral cancer cell line KB was exposed to different doses of metformin (0, 1.25, 2.5, 5, 10, and 20 mmol/L), and the changes in cell viability were detected using MTT assay. Colony formation of the cells was observed following an 8-day metformin exposure. The changes in mitochondrial membrane potential were measured by JC-1 assay, and PI staining was used to observe the cell apoptosis. Western blotting was employed to detect the changes in the protein expressions of GRP78 and activated caspase-3. Metformin exposure caused time- and dose-dependent suppression of KB cell proliferation, and exposure to 5 mmol/L metformin for 24, 48 and 72 h resulted in cell survival rates of 68.0%, 36.9%, and 14.5%, respectively. Metformin significantly inhibited KB cell colony formation. Exposure of the cells to increased concentrations of metformin gradually increased the apoptotic rate and decreased mitochondrial membrane potential. Metformin caused an initial up-regulation followed by a down-regulation of GRP78 expression in KB cells and increased the expression of activated caspase-3. Metformin can inhibit the proliferation and induce apoptosis of KB cells, the mechanism of which may involve the activation of the mitochondrial apoptotic pathway and endoplasmic reticulum stress.

  10. Effect of leptin on proliferation and apoptosis of cholangiocarcinoma QBC939 cells

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    DAI Kai

    2013-03-01

    Full Text Available ObjectiveTo determine whether leptin can exert anti-proliferative and pro-apoptotic effects on human cholangiocarcinoma cells and to investigate the underlying molecular mechanisms. MethodsHuman cholangiocarcinoma QBC939 cells were cultured and treated with different concentrations of leptin. Changes in the proliferation rate were measured by the MTT assay. Changes in cell cycle and in the apoptosis incidence rate were detected by flow cytometry. Changes in cyclin D1, bax and bcl-2 gene expression were detected by measuring mRNA levels by real-time quantitative reverse transcription-polymerase chain reaction (qPCR. Changes in caspase-3 protease activity were detected by fluorometric assay. ResultsLeptin treatment significantly increased the proliferation rate of QBC939 cells in a dose- and time-dependent manner. Compared to untreated QBC939 cells, leptin treatment led to significantly more G0/G1 to S phase transition and significantly lower apoptosis rate. In addition, leptin-treated QBC939 cells showed enhanced mRNA expression of cyclin D1 and bcl-2, but decreased mRNA expression of bax. The leptin treatment also led to decreased caspase-3 activity. ConclusionLeptin promotes S to G0/G1 phase transition and proliferation, but inhibits apoptosis, of human cholangiocarcinoma cells in vitro.

  11. Fluvastatin inhibits AGE-induced cell proliferation and migration via an ERK5-dependent Nrf2 pathway in vascular smooth muscle cells.

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    Ae-Rang Hwang

    Full Text Available Advanced glycation endproduct (AGE-induced vascular smooth muscle cell (VSMC proliferation and reactive oxygen species (ROS production are emerging as important mechanisms of diabetic vasculopathy, but little is known about the molecular mechanism responsible for the antioxidative effects of statins on AGEs. It has been reported that statins exert pleiotropic effects on the cardiovascular system due to decreases in AGE-induced cell proliferation, migration, and vascular inflammation. Thus, in the present study, the authors investigated the molecular mechanism by which statins decrease AGE-induced cell proliferation and VSMC migration. In cultured VSMCs, statins upregulated Nrf2-related antioxidant gene, NQO1 and HO-1, via an ERK5-dependent Nrf2 pathway. Inhibition of ERK5 by siRNA or BIX02189 (a specific ERK5 inhibitor reduced the statin-induced upregulations of Nrf2, NQO1, and HO-1. Furthermore, fluvastatin was found to significantly increase ARE promoter activity through ERK5 signaling, and to inhibit AGE-induced VSMC proliferation and migration as determined by MTT assay, cell counting, FACS analysis, a wound scratch assay, and a migration chamber assay. In addition, AGE-induced proliferation was diminished in the presence of Ad-CA-MEK5α encoding a constitutively active mutant form of MEK5α (an upstream kinase of ERK5, whereas depletion of Nrf2 restored statin-mediated reduction of AGE-induced cell proliferation. Moreover, fluvastatin suppressed the protein expressions of cyclin D1 and Cdk4, but induced p27, and blocked VSMC proliferation by regulating cell cycle. These results suggest statin-induced activation of an ERK5-dependent Nrf2 pathway reduces VSMC proliferation and migration induced by AGEs, and that the ERK5-Nrf2 signal module be viewed as a potential therapeutic target of vasculopathy in patients with diabetes and complications of the disease.

  12. [Lentivirus-mediated shRNA silencing of LAMP2A inhibits the proliferation of multiple myeloma cells].

    Science.gov (United States)

    Li, Lixuan; Li, Jia

    2015-05-01

    To study the effects of lentivirus-mediated short hairpin RNA (shRNA) silencing of lysosome-associated membrane protein type 2A (LAMP2A) expression on the proliferation of multiple myeloma cells. The constructed shRNA lentiviral vector was applied to infect human multiple myeloma cell line MM.1S, and stable expression cell line was obtained by puromycin screening. Western blotting was used to verify the inhibitory effect on LAMP2A protein expression. MTT assay was conducted to detect the effect of knocked-down LAMP2A on MM.1S cell proliferation, and the anti-tumor potency of suberoylanilide hydroxamic acid (SAHA) against the obtained MM.1S LAMP2A(shRNA) stable cell line. Lactate assay was performed to observe the impact of low LAMP2A expression on cell glycolysis. The stable cell line with low LAMP2A expression were obtained with the constructed human LAMP2A-shRNA lentiviral vector. Down-regulation of LAMP2A expression significantly inhibited MM.1S cell proliferation and enhanced the anti-tumor activity of SAHA. Interestingly, decreased LAMP2A expression also inhibited MM.1S cell lactic acid secretion. Down-regulation of LAMP2A expression could inhibit cell proliferation in multiple myeloma cells.

  13. A recombined fusion protein PTD-Grb2-SH2 inhibits the proliferation of breast cancer cells in vitro.

    Science.gov (United States)

    Yin, Jikai; Cai, Zhongliang; Zhang, Li; Zhang, Jian; He, Xianli; Du, Xilin; Wang, Qing; Lu, Jianguo

    2013-03-01

    The growth factor receptor bound protein 2 (Grb2) is one of the affirmative targets for cancer therapy, especially for breast cancer. In this study, we hypothesized the Src-homology 2 (SH2) domain in Grb2 may serve as a competitive protein-binding agent to interfere with the proliferation of breast cancer cells in vitro. We designed, constructed, expressed and purified a novel fusion protein containing the protein transduction domain (PTD) and Grb2-SH2 domain (we named it after PTD-Grb2-SH2). An immunofluorescence assay was used to investigate the location of PTD-Grb2-SH2 in cells. MTT assay and EdU experiments were applied to detect the proliferation of breast cancer cells. The ultra-structure was observed using transmission electron microscopy. Flow cytometry was used to determine the cytotoxicity of PTD-Grb2-SH2 on cell proliferation. We successfully obtained the PTD-Grb2-SH2 fusion protein in soluble form using a prokaryotic expression system. The new fusion protein successfully passed through both the cellular and nuclear membranes of breast cancer cells. The MTT assay showed that PTD-Grb2-SH2 exhibited significant toxicity to breast cancer cells in a dose- and time-dependent manner in vitro. EdU identified the decreased proliferation rates in treated MDA-MB-231 and SK-BR-3 cells. Observation by transmission electron microscopy and flow cytometry further confirmed the cytotoxicity as apoptosis. Our results show that the HIV1-TAT domain is a useful tool for transporting a low molecular weight protein across the cell membrane in vitro. The PTD-Grb2-SH2 may be a novel agent for breast cancer therapy.

  14. TFF1 inhibits proliferation and induces apoptosis of gastric cancer cells in vitro

    Directory of Open Access Journals (Sweden)

    Yanli Ge

    2012-05-01

    Full Text Available Trefoil Factor Family (TFF plays an essential role in the intestinal epithelial restitution, but the relationship between TFF1 and gastric cancer (GC is still unclear. The present study aimed to determine the role of TFF1 in repairing gastric mucosa and in the pathogenesis of GC.The TFF1 expression in different gastric mucosas was measured with immunohistochemistry. Then, siRNA targeting TFF1 or plasmids expressing TFF1 gene were transfected into BGC823 cells, SGC7901 cells and GES-1 cells. The cell proliferation was detected with MTT assay and apoptosis and cell cycle measured by flow cytometry.From normal gastric mucosa to mucosa with dysplasia and to gastric cancer, the TFF1 expression had a decreasing trend. Down-regulation of TFF1 expression significantly reduced the apoptosis of three cell lines and markedly facilitated their proliferation but had no significant effect on cell cycle. Over-expression of TFF1 could promote apoptosis of three cell lines and inhibit proliferation but had no pronounced effect on cell cycle. TFF1 can inhibit proliferation and induce apoptosis of GC cells in vitro.

  15. [Inhibition effects of black rice pericarp extracts on cell proliferation of PC-3 cells].

    Science.gov (United States)

    Jiang, Weiwei; Yu, Xudong; Ren, Guofeng

    2013-05-01

    To observe the inhibitive effects of black rice pericarp extracts on cell proliferation of human prostate cancer cell PC-3 and to explore its effecting mechanism. The black rice pericarp extract was used to treat the PC-3 cells. The inhibitory effect of black rice pericarp extract on cells proliferation of PC-3 was tested by MTT method. Cell apoptosis rates and cell cycle were measured by flow cytometric assay (FCM). Western blot was used to study the protein expression levels of p38, p-p38, JNK, p-JNK. A dose-dependent and time-dependent proliferation inhibition of black rice pericarp extract was demonstrated in PC-3. The most prominent experiment condition was inhibitory concentration with 300microg/ml and treated for 72 h. The experiment result of flow cytometry analysis demonstrates that the apoptosis rate of PC-3 cells increased along with the increasing of black rice pericarp extract concentration, and a G1-S cell cycle arrest was induced in a dose-dependent manner. After PC-3 cell was treated with black rice pericarp extract for 72 h, the expressions of p-p38, p-JNK protein increased. Black rice pericarp extract could inhibit proliferation, change the cell cycle distributions and induce apoptosis in human prostatic cancer cell PC-3. Its inhibitory effect may be through promoting activation of the JNK, p38 signaling pathway. These results suggest that black rice pericarp extract maybe has an inhibitory effect on prostatic cancer.

  16. Effect of punicalagin on proliferation of porcine ovarian granulosa cells in vitro

    Directory of Open Access Journals (Sweden)

    Dagmara Packová

    2016-12-01

    Full Text Available Punicalagin is a major component responsible for pomegranate's (Punica granatum antioxidant properties. Punicalagin is the predominant ellagitannin of Punica granatum and present in two isomeric forms: punicalagin α and β. Punicalagin is metabolised to ellagic acid (antioxidant and microorganisms present in colon can metabolize ellagic acid to urolithins. The aim of in vitro study was to examine the effect of punicalagin on mitochondrial activity and markers of proliferation in porcine ovarian granulosa cells. The cells were cultivated during 24h without (control group and with various doses (0.01, 0.1, 1, 10 and 100 μg*ml-1 of pomegranate compound – punicalagin. MTT assay and immunocytochemistry were used in this study. Stimulatory influence of punicalagin on the mitochondrial activity of ovarian granulosa cells at concentrations 1 μg*ml-1 was found. Punicalagin (at 1 μg*ml-1 had a significant (P < 0.05 impact on the presence of proliferative markers cyclin B1 (increase and PCNA - proliferating cell nuclear antigen (decrease in porcine ovarian granulosa cells. These results suggest dose-dependent effect of punicalagin on cell proliferation. Further verification of possible role of punicalagin in proliferation is therefore needed.

  17. Effects of Voltage-Gated K+ Channel on Cell Proliferation in Multiple Myeloma

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    Wei Wang

    2014-01-01

    Full Text Available Objective. To study the effects and underlying mechanisms of voltage-gated K+ channels on the proliferation of multiple myeloma cells. Methods. RPMI-8226 MM cell line was used for the experiments. Voltage-gated K+ currents and the resting potential were recorded by whole-cell patch-clamp technique. RT-PCR detected Kv channel mRNA expression. Cell viability was analyzed with MTT assay. Cell counting system was employed to monitor cell proliferation. DNA contents and cell volume were analyzed by flow cytometry. Results. Currents recorded in RPMI-8226 cells were confirmed to be voltage-gated K+ channels. A high level of Kv1.3 mRNA was detected but no Kv3.1 mRNA was detected in RPMI-8226 cells. Voltage-gated K+ channel blocker 4-aminopyridine (4-AP (2 mM depolarized the resting potential from −42 ± 1.7 mV to −31.8 ± 2.8 mV (P0.05. Conclusions. In RPMI-8226, voltage-gated K+ channels are involved in proliferation and cell cycle progression its influence on the resting potential and cell volume may be responsible for this process; the inhibitory effect of the voltage-gated K+ channel blocker on RPMI-8226 cell proliferation is a phase-specific event.

  18. Hydrogen sulfide lowers proliferation and induces protective autophagy in colon epithelial cells.

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    Ya C Wu

    Full Text Available Hydrogen sulfide (H(2S is a gaseous bacterial metabolite that reaches high levels in the large intestine. In the present study, the effect of H(2S on the proliferation of normal and cancerous colon epithelial cells was investigated. An immortalized colon epithelial cell line (YAMC and a panel of colon cancer cell lines (HT-29, SW1116, HCT116 were exposed to H(2S at concentrations similar to those found in the human colon. H(2S inhibited normal and cancerous colon epithelial cell proliferation as measured by MTT assay. The anti-mitogenic effect of H(2S was accompanied by G(1-phase cell cycle arrest and the induction of the cyclin-dependent kinase inhibitor p21(Cip. Moreover, exposure to H(2S led to features characteristic of autophagy, including increased formation of LC3B(+ autophagic vacuoles and acidic vesicular organelles as determined by immunofluorescence and acridine orange staining, respectively. Abolition of autophagy by RNA interference targeting Vps34 or Atg7 enhanced the anti-proliferative effect of H(2S. Further mechanistic investigation revealed that H(2S stimulated the phosphorylation of AMP-activated protein kinase (AMPK and inhibited the phosphorylation of mammalian target of rapamycin (mTOR and S6 kinase. Inhibition of AMPK significantly reversed H(2S-induced autophagy and inhibition of cell proliferation. Collectively, we demonstrate that H(2S inhibits colon epithelial cell proliferation and induces protective autophagy via the AMPK pathway.

  19. Activity of selenium on cell proliferation, cytotoxicity, and apoptosis and on the expression of CASP9, BCL-XL and APC in intestinal adenocarcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Mauro, M.O., E-mail: mari.mauro@hotmail.com [Graduate Program in Biological Sciences (Cell and Molecular Biology), Institute of Biosciences, UNESP, Rio Claro (SP) (Brazil); Sartori, Daniele [General Biology Department, State University of Londrina (UEL), Londrina (Brazil); Oliveira, Rodrigo Juliano [Coordination of Open and Distance Education, Graduate Program in Animal Science, Federal University of Mato Grosso do Sul (UFMS), Campo Grande (MS) (Brazil); Ishii, Priscila Lumi [Graduate Program in Biological Sciences (Cell and Molecular Biology), Institute of Biosciences, UNESP, Rio Claro (SP) (Brazil); Mantovani, Mario Sergio [General Biology Department, State University of Londrina (UEL), Londrina (Brazil); Ribeiro, Lucia Regina [Graduate Program in Biological Sciences (Cell and Molecular Biology), Institute of Biosciences, UNESP, Rio Claro (SP) (Brazil)

    2011-10-01

    Intestinal cancers are correlated with diet. Thus, determining and understanding nutrient-genome interactions is important. The present work assessed the action of the oligoelement selenium on cell proliferation, cytotoxicity, and in situ apoptosis induction and on the expression CASP9, BCL-XL and APC genes in intestinal adenocarcinoma cells (HT29). HT29 cells were cultured and treated with selenium at concentrations of 5, 50 and 500 ng/mL with or without the damage-inducing agent doxorubicin. These cells were then evaluated for cytotoxicity (MTT), cell proliferation and in situ apoptosis induction. To evaluate gene expression, only the cells treated with 500 ng/mL of selenium were used. RNA was extracted from these cells, and the expressions of CASP9, BCL-XL and APC were analyzed by the RT-PCR method. The GAPDH gene was used as a reference gene. The MTT assay showed that selenium was not cytotoxic at any of the concentrations tested. The cell proliferation assay showed that selenium did not interfere with cell proliferation at the three concentrations tested. In contrast, when the three concentrations were combined with doxorubicin, a significant decrease in the proliferation rate was observed. The apoptosis rate was significantly increased in the selenium (500 ng/mL) and doxorubicin group. CASP9 expression was increased and BCL-XL expression decreased in the selenium (500 ng/mL) and doxorubicin group. APC was significantly increased in the selenium group alone. These results show that selenium increases apoptosis, especially when it is associated with a damage-inducing agent. Also, selenium has an important role in the expression of the APC gene, which is related to cell cycle regulation.

  20. Activity of selenium on cell proliferation, cytotoxicity, and apoptosis and on the expression of CASP9, BCL-XL and APC in intestinal adenocarcinoma cells

    International Nuclear Information System (INIS)

    Mauro, M.O.; Sartori, Daniele; Oliveira, Rodrigo Juliano; Ishii, Priscila Lumi; Mantovani, Mario Sergio; Ribeiro, Lucia Regina

    2011-01-01

    Intestinal cancers are correlated with diet. Thus, determining and understanding nutrient-genome interactions is important. The present work assessed the action of the oligoelement selenium on cell proliferation, cytotoxicity, and in situ apoptosis induction and on the expression CASP9, BCL-XL and APC genes in intestinal adenocarcinoma cells (HT29). HT29 cells were cultured and treated with selenium at concentrations of 5, 50 and 500 ng/mL with or without the damage-inducing agent doxorubicin. These cells were then evaluated for cytotoxicity (MTT), cell proliferation and in situ apoptosis induction. To evaluate gene expression, only the cells treated with 500 ng/mL of selenium were used. RNA was extracted from these cells, and the expressions of CASP9, BCL-XL and APC were analyzed by the RT-PCR method. The GAPDH gene was used as a reference gene. The MTT assay showed that selenium was not cytotoxic at any of the concentrations tested. The cell proliferation assay showed that selenium did not interfere with cell proliferation at the three concentrations tested. In contrast, when the three concentrations were combined with doxorubicin, a significant decrease in the proliferation rate was observed. The apoptosis rate was significantly increased in the selenium (500 ng/mL) and doxorubicin group. CASP9 expression was increased and BCL-XL expression decreased in the selenium (500 ng/mL) and doxorubicin group. APC was significantly increased in the selenium group alone. These results show that selenium increases apoptosis, especially when it is associated with a damage-inducing agent. Also, selenium has an important role in the expression of the APC gene, which is related to cell cycle regulation.

  1. WNT5A inhibits human dental papilla cell proliferation and migration

    International Nuclear Information System (INIS)

    Peng, L.; Ye, L.; Dong, G.; Ren, L.B.; Wang, C.L.; Xu, P.; Zhou, X.D.

    2009-01-01

    WNT proteins are a large family of cysteine-rich secreted molecules that are linked to both canonical and non-canonical signal pathways, and have been implicated in oncogenesis and tissue development. Canonical WNT proteins have been proven to play critical roles in tooth development, while little is known about the role of non-canonical WNT proteins such as WNT5A. In this study, WNT5A was localized to human dental papilla tissue and human dental papilla cells (HDPCs) cultured in vitro, using immunochemistry and RT-PCR. Recombinant adenovirus encoding full-length Wnt5a cDNA was constructed to investigate the biological role of WNT5A on HDPCs. The BrdU incorporation assay, the MTT assay and flow cytometric analysis showed that over-expression of Wnt5a strongly inhibited the proliferation of HDPCs in vitro. Wound healing and transwell migration assays indicated that over-expression of WNT5A reduced migration of HDPCs. In conclusion, our results showed that WNT5A negatively regulates both proliferation and migration of HDPCs, suggesting its important role in odontogenesis via controlling the HDPCs.

  2. The effect of stem cell factor on proliferation of human endometrial CD146+ cells

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    Mehri Fayazi

    2016-07-01

    Full Text Available Background: Stem cell factor (SCF is a transcriptional factor which plays crucial roles in normal proliferation, differentiation and survival in a range of stem cells. Objective: The aim of the present study was to examine the proliferation effect of different concentrations of SCF on expansion of human endometrial CD146+ cells. Materials and Methods: In this experimental study, total populations of isolated human endometrial suspensions after fourth passage were isolated by magnetic activated cell sorting (MACS into CD146+ cells. Human endometrial CD146+ cells were karyotyped and tested for the effect of SCF on proliferation of CD146+ cells, then different concentrations of 0, 12.5, 25, 50 and 100 ng/ml was carried out and mitogens-stimulated endometrial CD146+ cells proliferation was assessed by MTT assay. Results: Chromosomal analysis showed a normal metaphase spread and 46XX karyotype. The proliferation rate of endometrial CD146P + P cells in the presence of 0, 12.5, 25, 50 and 100 ng/ml SCF were 0.945±0.094, 0.962±0.151, 0.988±0.028, 1.679±0.012 and 1.129±0.145 respectively. There was a significant increase in stem/ stromal cell proliferation following in vitro treatment by 50 ng/ml than other concentrations of SCF (p=0.01. Conclusion: The present study suggests that SCF could have effect on the proliferation and cell survival of human endometrial CD146P+P cells and it has important implications for medical sciences and cell therapies

  3. Effects of mineral trioxide aggregate, BiodentineTM and calcium hydroxide on viability, proliferation, migration and differentiation of stem cells from human exfoliated deciduous teeth

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    Leandro Borges Araújo

    2018-02-01

    Full Text Available Abstract Objective: The aim of the study was to evaluate the effects of the capping materials mineral trioxide aggregate (MTA, calcium hydroxide (CH and BiodentineTM (BD on stem cells from human exfoliated deciduous teeth (SHED in vitro. Material and Methods: SHED were cultured for 1 – 7 days in medium conditioned by incubation with MTA, BD or CH (1 mg/mL, and tested for viability (MTT assay and proliferation (SRB assay. Also, the migration of serum-starved SHED towards conditioned media was assayed in companion plates, with 8 μm-pore-sized membranes, for 24 h. Gene expression of dentin matrix protein-1 (DMP-1 was evaluated by reverse-transcription polymerase chain reaction. Regular culture medium with 10% FBS (without conditioning and culture medium supplemented with 20% FBS were used as controls. Results: MTA, CH and BD conditioned media maintained cell viability and allowed continuous SHED proliferation, with CH conditioned medium causing the highest positive effect on proliferation at the end of the treatment period (compared with BD and MTA (p<0.05. In contrast, we observed increased SHED migration towards BD and MTA conditioned media (compared with CH (p<0.05. A greater amount of DMP-1 gene was expressed in MTA group compared with the other groups from day 7 up to day 21. Conclusion: Our results show that the three capping materials are biocompatible, maintain viability and stimulate proliferation, migration and differentiation in a key dental stem cell population.

  4. Recombinant Escherichia coli Trx-JZTX-III represses the proliferation of mouse hepatocellular carcinoma cells through induction of cell cycle arrest.

    Science.gov (United States)

    Sun, Mei-Na; Zhao, Xue-Jiao; Zhao, Han-Dong; Zhang, Wei-Guang; Li, Feng-Lan; Chen, Ming-Zi; Li, Hui; Li, Guangchao

    2013-06-01

    The aim of the present study was to investigate the effects of recombinant Escherichia coli (E. coli) Trx-jingzhaotoxin (JZTX)-III on cell growth in the mouse hepatocellular carcinoma (HCC) cell line Hepa1-6. The JZTX-III gene sequence was synthesized and cloned into the pET-32a(+) vector to construct the recombinant fusion protein Trx-JZTX-III, which was subsequently purified. Hepa1-6 cells were treated with 0 to 1,000-µg/ml concentrations of Trx-JZTX-III; this was demonstrated to affect cell viability, as determined by the 3-(4,5-dimethylthiazol‑2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assay. The expression of the proliferating cell nuclear antigen (PCNA) protein was investigated using western blot analysis. A colony formation assay was used to determine Hepa1-6 cell proliferation, and the migration ability of cells was determined using a wound‑healing assay. Additionally, flow cytometry was employed to observe changes in the cell cycle. The MTT assay and quantification of PCNA expression indicated that recombinant E. coli Trx-JZTX-III significantly repressed the proliferation of Hepa1-6 cells. Colony formation and the migration of malignant cells was inhibited following treatment with recombinant E. coli Trx-JZTX-III. Flow cytometry showed that recombinant E. coli Trx-JZTX-III induced G0/G1 cell cycle arrest. In conclusion, recombinant E. coli Trx-JZTX-III functions as a tumor suppressor drug in mouse HCC and its underlying mechanism may involve the induction of G0/G1 cell cycle arrest.

  5. MicroRNA-144 inhibits hepatocellular carcinoma cell proliferation

    Indian Academy of Sciences (India)

    MiR-144 was shown to besignificantly down-regulated in HCC tissues and cell lines. Subsequently, overexpression of miR-144 was transfectedinto HCC cell lines so as to investigate its biological function, including MTT, colony formation, and transwell assays.Gain of function assay revealed miR-144 remarkably inhibited ...

  6. Effects of exogenous vitamins A, C, and E and NADH supplementation on proliferation, cytokines release, and cell redox status of lymphocytes from healthy aged subjects.

    Science.gov (United States)

    Bouamama, Samia; Merzouk, Hafida; Medjdoub, Amel; Merzouk-Saidi, Amel; Merzouk, Sid Ahmed

    2017-06-01

    Aging is an inevitable biological event that is associated with immune alterations. These alterations are related to increased cellular oxidative stress and micronutrient deficiency. Antioxidant supplementation could improve these age-related abnormalities. The aim of this study was to determine in vitro effects of vitamin A, vitamin C, vitamin E, and nicotinamide adenine dinucleotide (NADH) on T cell proliferation, cytokine release, and cell redox status in the elderly compared with young adults. Peripheral blood lymphocytes were isolated using a density gradient of Histopaque. They were cultured in vitro and stimulated with concanavalin A in the presence or absence of vitamins. Cell proliferation was determined by conducting MTT assays, and based on interleukin-2 and interleukin-4 secretions. Cell oxidant/antioxidant balance was assessed by assaying reduced glutathione (GSH), malondialdehyde, carbonyl protein levels, and catalase activity. The present study demonstrated that T-lymphocyte proliferation was decreased with aging and was associated with cytokine secretion alterations, GSH depletion, and intracellular oxidative stress. In the elderly, vitamin C, vitamin E, and NADH significantly improved lymphocyte proliferation and mitigated cellular oxidative stress, whereas vitamin A did not affect cell proliferation or cell redox status. In conclusion, vitamin C, vitamin E, and NADH supplementation improved T-lymphocytes response in the elderly, and could contribute to the prevention of age-related immune alterations. Consumption of food items containing these vitamins is recommended, and further investigation is necessary to evaluate the effect of vitamin supplementation in vivo.

  7. Synergistic effects of rmhTRAIL and 17-AAG on the proliferation and apoptosis of multiple myeloma cells.

    Science.gov (United States)

    Wang, Jing; Li, Yun; Sun, Wei; Liu, Jing; Chen, Wenming

    2018-03-22

    This study aimed to investigate synergistic effects of recombinant mutant human tumor necrosis factor-related apoptosis-inducing ligand (rmhTRAIL) and heat-shock protein 90 (HSP90) inhibitor (geldanamycin derivative 17 -allylamino- 17-demethoxy -geldanamycin, 17-AAG) on the proliferation and apoptosis of multiple myeloma (MM) cells. MTT assays evaluated inhibitory effects of rmhTRAIL and 17-AAG in different concentrations and treatment durations on the proliferation of RPMI8226 and U266 cells. The half maximal inhibitory concentration was calculated using OriginPro7.5. Synergistic effects of rmhTRAIL and 17-AAG on apoptosis of MM cells were detected using flow cytometry at 24 and 48 h post-treatment. To evaluate synergistic effects of rmhTRAIL and 17-AAG, the Q-value was calculated using King's formula. rmhTRAIL exhibited significant inhibitory effects on the proliferation of RPMI8226 cells in a dose- and time-dependent manner (>50%), whereas U266 cells were not sensitive to rmhTRAIL (80%). Significant synergistic effects of rmhTRAIL and 17-AAG on the proliferation of RPMI8226 cells were revealed (Q-value > 1.15), whereas synergistic effects were not evident on the proliferation of U266 cells (Q-value effects on apoptosis of RPMI8226 and U266 cells (Q-value > 1.15). The combined application of rmhTRAIL and 17-AAG revealed favorable synergistic effects in the treatment of MM.

  8. RNAi-mediated silencing of CD147 inhibits tumor cell proliferation, invasion and increases chemosensitivity to cisplatin in SGC7901 cells in vitro

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    Zhu Chan

    2010-06-01

    Full Text Available Abstract Background CD147 is a widely distributed cell surface glycoprotein that belongs to the Ig superfamily. CD147 has been implicated in numerous physiological and pathological activities. Enriched on the surface of many tumor cells, CD147 promotes tumor growth, invasion, metastasis and angiogenesis and confers resistance to some chemotherapeutic drugs. In this study, we investigated the possible role of CD147 in the progression of gastric cancer. Methods Short hairpin RNA (shRNA expressing vectors targeting CD147 were constructed and transfected into human gastric cancer cells SGC7901 and CD147 expression was monitored by quantitative realtime RT-PCR and Western blot. Cell proliferation, the activities of MMP-2 and MMP-9, the invasive potential and chemosensitivity to cisplatin of SGC7901 cells were determined by MTT, gelatin zymography, Transwell invasion assay and MTT, respectively. Results Down-regulation of CD147 by RNAi approach led to decreased cell proliferation, MMP-2 and MMP-9 activities and invasive potential of SGC7901 cells as well as increased chemosensitivity to cisplatin. Conclusion CD147 involves in proliferation, invasion and chemosensitivity of human gastric cancer cell line SGC7901, indicating that CD147 may be a promising therapeutic target for gastric cancer.

  9. [Knock-down of ZEB1 inhibits the proliferation, invasion and migration of gastric cancer cells].

    Science.gov (United States)

    Chen, Dengyu; Chu, Yifan; Zheng, Qingwei; Xu, Zhiben; Zhou, Ping; Li, Sheng

    2017-08-01

    Objective To down-regulate the expression of zinc-finger E-box binding homeobox 1 (ZEB1) gene by shRNA, and investigate its effect on invasion, migration and proliferation, as well as the related gene expressions of lncRNA HOTAIR and E-cadherin in human gastric cancer BGC823 cells. Methods RNA interfering (RNAi) was used to knock down ZEB1 in gastric cancer BGC823 cells. The recombinant plasmid shZEB1 was constructed and transfected into the gastric cancer BGC823 cells by Lipofectamine TM 2000, and the stably transfected cells were isolated by G418 selection and limited dilution. The expression of ZEB1 mRNA and protein was detected by real-time quantitative PCR and Western blot analysis. Cell proliferation was determined by MTT assay, and the invasion and migration abilities of BGC823 cells were monitored by Transwell TM invasion assay and wound healing assay, respectively. The expressions of lncRNA HOTAIR and E-cadherin mRNA were detected by real-time quantitative PCR. Results After ZEB1 expression was successfully down-regulated in BGC823 cells by siRNA, the proliferation, invasion and migration rates in shZEB1 transfection group were significantly lower than those in control group; meanwhile, the expression of lncRNA HOTAIR was reduced and E-cadherin expression was enhanced. Conclusion Knock-down of ZEB1 expression by RNA interference can decease lncRNA HOTAIR expression and restrain cell proliferation, invasion and migration in gastric cancer BGC823 cells.

  10. The GAS5/miR-222 Axis Regulates Proliferation of Gastric Cancer Cells Through the PTEN/Akt/mTOR Pathway.

    Science.gov (United States)

    Li, Yanhua; Gu, Junjiao; Lu, Hong

    2017-12-01

    Several lines of evidence have indicated that growth arrest-specific transcript 5 (GAS5) functions as a tumor suppressor and is aberrantly expressed in multiple cancers. GAS5 was found to be downregulated in gastric cancer (GC) tissues, and ectopic expression of GAS5 inhibited GC cell proliferation. The present study aimed to explore the underlying mechanisms of GAS5 involved in GC cell proliferation. GAS5 and miR-222 expressions in GC cell lines were estimated by quantitative real-time polymerase chain reaction. The effects of GAS5 and miR-222 on GC cell proliferation were assessed by MTT assay and 5-bromo-2-deoxyuridine (BrdU) incorporation assays. The interaction between GAS5 and miR-222 was confirmed by luciferase reporter assay and RNA immunoprecipitation assay. The protein levels of the phosphatase and tensin homolog (PTEN), phosphorylated protein kinase B (Akt) (p-Akt), Akt, phosphorylated mammalian target of rapamycin (mTOR) (p-mTOR), and mTOR were determined by western blot. GAS5 was downregulated and miR-222 was upregulated in GC cells. GAS5 directly targeted and suppressed miR-222 expression. GAS5 overexpression and miR-222 inhibition suppressed cell proliferation, increased PTEN protein level and decreased p-Akt and p-mTOR protein levels in GC cells while GAS5 knockdown and miR-222 overexpression exhibited the opposite effects. Moreover, mechanistic analyses revealed that GAS5 regulated GC cell proliferation through the PTEN/Akt/mTOR pathway by negatively regulating miR-222. GAS5/miR-222 axis regulated proliferation of GC cells through the PTEN/Akt/mTOR pathway, which facilitated the development of lncRNA-directed therapy against this deadly disease.

  11. Effects of voluntary exercise on the viability, proliferation and BDNF levels of bone marrow stromal cells in rat pups born from morphine- dependent mothers during pregnancy.

    Science.gov (United States)

    Haydari, Sakineh; Safari, Manouchehr; Zarbakhsh, Sam; Bandegi, Ahmad Reza; Miladi-Gorji, Hossein

    2016-11-10

    This study was designed to investigate whether free access to a running wheel during pregnancy in morphine-dependent mothers would influence the viability, proliferation and BDNF levels of bone marrow stromal cells in rat pups. Pregnant rats were made dependent by chronic administration of morphine in drinking water simultaneously with free access to a running wheel. Male pups are weaned at 21days of birth and their bones marrows were aspirated from the femurs and tibias and also the bone marrow stromal cells (BMSCs) cultured. MTT assay was used to determine cell viability and proliferation rate. The level of BDNF was measured in the supernant of BMSCs culture by ELISA. The sedentary morphine-dependent mothers' pups showed a significant increase in the percentage cell viability and proliferation rate and also a significant decrease in the BDNF protein levels in BMSCs. The rat pups borne from exercising the control and morphine-dependent mothers exhibited an increase in the percentage viability, proliferation rate and BDNF levels of the BMSCs. This study showed that maternal exercise during pregnancy in morphine-dependent and non-dependent mothers, with increasing of BDNF levels increased the proliferation and viability of BMSCs in the rat pups. Also, chronic administration of morphine during pregnancy was able to increase the proliferation and viability of BMSCs in the rat pups. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  12. Effects of Calophyllum inophyllum fruit extract on the proliferation and morphological characteristics of human breast cancer cells MCF-7

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    Shanmugapriya

    2016-04-01

    Full Text Available Objective: To evaluate the antiproliferative activity of Calophyllum inophyllum (C. inophyllum fruit extract against human breast cancer cells MCF-7. Methods: The cytotoxic effect of C. inophyllum fruit extract against MCF-7 cancer cells was evaluated through MTT and CyQuant assays for 24 h and the morphological investigation of treated MCF-7 cells was observed under optical microscope using Giemsa staining. Results: The cytotoxic effect of C. inophyllum fruit extract against MCF-7 cancer cells was evaluated through MTT and CyQuant assays simultaneously for 24 h after treatment, which demonstrated the inhibition of cell viability with the IC50 values of 19.63 µg/mL and 27.54 µg/mL, respectively. The preliminary time-based morphological investigation of MCF-7 cells treated with the IC 50 value (23.59 µg/mL of C. inophyllum fruit extract was observed under an optical microscopy via Giemsa staining, which exhibited prominent histological characteristics of apoptosis. Conclusions: This study clearly proved that the proliferation of human breast cancer cell MCF-7 was inhibited by C. inophyllum fruit extract resulted from the induction of apoptosis in MCF-7 cells.

  13. Attachment and proliferation of human osteoblast-like cells (MG-63) on laser-ablated titanium implant material

    International Nuclear Information System (INIS)

    Györgyey, Ágnes; Ungvári, Krisztina; Kecskeméti, Gabriella; Kopniczky, Judit; Hopp, Béla; Oszkó, Albert; Pelsöczi, István; Rakonczay, Zoltán; Nagy, Katalin; Turzó, Kinga

    2013-01-01

    Demand is increasing for shortening the long (3–6 months) osseointegration period to rehabilitate patients' damaged chewing apparatus in as short a time as possible. For dental implants, as for biomaterials in general, the bio- and osseointegration processes can be controlled at molecular and cellular levels by modification of the implant surface. One of the most promising of such surface modifications is laser ablation, as demonstrated by our previous results [46]. Commercially pure (CP4) sand-blasted, acid-etched titanium disks (Denti® System Ltd., Hungary) were irradiated with a KrF excimer laser (248 nm, fluence 0.4 J/cm 2 , FWHM 18 ns, 2000 pulses), or with a Nd:YAG laser (532 nm, 1.3 J/cm 2 , 10 ns, 200 pulses) then examined by SEM, AFM, and XPS. In vitro attachment (24 h) and proliferation (72 h) of MG-63 osteoblast cells were investigated via dimethylthiazol-diphenyl tetrazolium bromide (MTT), alamarBlue (AB) assays alkaline phosphatase quantification (ALP) and SEM. SEM and AFM revealed significant changes in morphology and roughness. XPS confirmed the presence of TiO 2 on each sample; after Nd:YAG treatment a reduced state of Ti (Ti 3+ ) was also observed. MTT, AB and ALP measurements detected an increase in the number of cells between the 24- and 72 hour observations; however, laser treatment did not affect cell attachment and proliferation significantly. - Highlights: • CP4 titanium implant surfaces were modified with Nd:YAG and KrF excimer laser. • SEM and AFM revealed significant changes in morphology and roughness. • XPS confirmed the presence of TiO 2 on each sample; after Nd:YAG treatment a reduced state of Ti (Ti 3+ ) was found. • Cell proliferation experiments detected an increased number of MG-63 cells between the 24 h and 72 h observations. • Laser treatments neither disturbed, nor enhanced MG-63 cell attachment and proliferation significantly

  14. Up-regulation of hypoxia inducible factor-1α by cobalt chloride correlates with proliferation and apoptosis in PC-2 cells

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    Dai Zhi-Jun

    2012-03-01

    Full Text Available Abstract Background The exact mechanism of the effects of hypoxia on the proliferation and apoptosis in carcinoma cells is still conflicting. This study investigated the variation of hypoxia-inducible factor-1α(HIF-1α expression and the apoptosis effect of hypoxia stimulated by cobalt chloride (CoCl2 in pancreatic cancer PC-2 cells. Methods PC-2 cells were cultured with different concentration (50-200 μmol/L of CoCl2 after 24-120 hours to simulate hypoxia in vitro. The proliferation of PC-2 cells was examined by MTT assay. The cellular morphology of PC-2 cells were observed by light inverted microscope and transmission electron microscope(EM. The expression of HIF-1α on mRNA and protein level was measured by semi-quantitive RT-PCR and Western blot analysis. Apoptosis of PC-2 cells were demonstrated by flow cytometry with Annexin V-FITC/PI double staining. Results MTT assay showed that the proliferation of PC-2 cells were stimulated in the first 72 h, while after treated over 72 h, a dose- dependent inhibition of cell growth could be observed. By using transmission electron microscope, swollen chondrosomes, accumulated chromatin under the nuclear membrane and apoptosis bodies were observed. Flow cytometer(FCM analysis showed the apoptosis rate was correlated with the dosage of CoCl2. RT-PCR and Western blot analysis indicated that hypoxia could up-regulate the expression of HIF-1α on both mRNA and protein levels. Conclusion Hypoxic microenvironment stimulated by CoCl2 could effectively induce apoptosis and influence cell proliferation in PC-2 cells, the mechanism could be related to up-expression of HIF-1α.

  15. Lactoferrin promote primary rat osteoblast proliferation and differentiation via up-regulation of insulin-like growth factor-1 expression.

    Science.gov (United States)

    Hou, Jian-ming; Wu, Man; Lin, Qing-ming; Lin, Fan; Xue, Ying; Lan, Xu-hua; Chen, En-yu; Wang, Mei-li; Yang, Hai-yan; Wang, Feng-xiong

    2014-08-01

    The aim of this study was to explore the effect of lactoferrin (LF) in primary fetal rat osteoblasts proliferation and differentiation and investigate the underlying molecular mechanisms. Primary rat osteoblasts were obtained from the calvarias of neonatal rats. Osteoblasts were treated with LF (0.1-1000 μg/mL), or OSI-906 [a selective inhibitor of insulin-like growth factor 1 (IGF-1) receptor and insulin receptor]. The IGF-1 was then knocked down by small hairpin RNA (shRNA) technology and then was treated with recombinant human IGF-1 or LF. Cell proliferation and differentiation were measured by MTT assay and alkaline phosphatase (ALP) assay, respectively. The expression of IGF-1 and IGF binding protein 2 (IGFBP2) mRNA were analyzed using real-time PCR. LF promotes the proliferation and differentiation of osteoblasts in a certain range (1-100 μg/mL) in time- and dose-dependent manner. The mRNA level of IGF-1 was significantly increased, while the expression of IGFBP2 was suppressed by LF treatment. Knockdown of IGF-1 by shRNA in primary rat osteoblast dramatically decreased the abilities of proliferation and differentiation of osteoblasts and blocked the proliferation and differentiation effect of LF in osteoblasts. OSI906 (5 μM) blocked the mitogenic and differentiation of LF in osteoblasts. Proliferation and differentiation of primary rat osteoblasts in response to LF are mediated in part by stimulating of IGF-1 gene expression and alterations in the gene expression of IGFBP2.

  16. Advanced Glycation End Products Inhibit the Proliferation of Human Umbilical Vein Endothelial Cells by Inhibiting Cathepsin D

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    Yuan Li

    2017-02-01

    Full Text Available We aimed to investigate the effect of advanced glycation end products (AGEs on the proliferation and migration ability of human umbilical vein endothelial cells (HUVECs. Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT assay, real-time cell analyzer and 5-Ethynyl-2′-deoxyuridine (EdU staining. Cell migration was detected by wound-healing and transwell assay. AGEs significantly inhibited the proliferation and migration of HUVECs in a time-and dose-dependent way. Western blotting revealed that AGEs dramatically increased the expression of microtubule-associated protein 1 light chain 3 (LC3 II/I and p62. Immunofluorescence of p62 and acridine orange staining revealed that AGEs significantly increased the expression of p62 and the accumulation of autophagic vacuoles, respectively. Chloroquine (CQ could further promote the expression of LC3 II/I and p62, increase the accumulation of autophagic vacuoles and promote cell injury induced by AGEs. In addition, AGEs reduced cathepsin D (CTSD expression in a time-dependent way. Overexpression of wild-type CTSD significantly decreased the ratio of LC 3 II/I as well as p62 accumulation induced by AGEs, but overexpression of catalytically inactive mutant CTSD had no such effects. Only overexpression of wild-type CTSD could restore the proliferation of HUVECs inhibited by AGEs. However, overexpression of both wild-type CTSD and catalytically inactive mutant CTSD could promote the migration of HUVECs inhibited by AGEs. Collectively, our study found that AGEs inhibited the proliferation and migration in HUVECs and promoted autophagic flux, which in turn played a protective role against AGEs-induced cell injury. CTSD, in need of its catalytic activity, may promote proliferation in AGEs-treated HUVECs independent of the autophagy-lysosome pathway. Meanwhile, CTSD could improve the migration of AGEs-treated HUVECs regardless of its enzymatic activity.

  17. Advanced Glycation End Products Inhibit the Proliferation of Human Umbilical Vein Endothelial Cells by Inhibiting Cathepsin D.

    Science.gov (United States)

    Li, Yuan; Chang, Ye; Ye, Ning; Dai, Dongxue; Chen, Yintao; Zhang, Naijin; Sun, Guozhe; Sun, Yingxian

    2017-02-17

    We aimed to investigate the effect of advanced glycation end products (AGEs) on the proliferation and migration ability of human umbilical vein endothelial cells (HUVECs). Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay, real-time cell analyzer and 5-Ethynyl-2'-deoxyuridine (EdU) staining. Cell migration was detected by wound-healing and transwell assay. AGEs significantly inhibited the proliferation and migration of HUVECs in a time-and dose-dependent way. Western blotting revealed that AGEs dramatically increased the expression of microtubule-associated protein 1 light chain 3 (LC3) II/I and p62. Immunofluorescence of p62 and acridine orange staining revealed that AGEs significantly increased the expression of p62 and the accumulation of autophagic vacuoles, respectively. Chloroquine (CQ) could further promote the expression of LC3 II/I and p62, increase the accumulation of autophagic vacuoles and promote cell injury induced by AGEs. In addition, AGEs reduced cathepsin D (CTSD) expression in a time-dependent way. Overexpression of wild-type CTSD significantly decreased the ratio of LC 3 II/I as well as p62 accumulation induced by AGEs, but overexpression of catalytically inactive mutant CTSD had no such effects. Only overexpression of wild-type CTSD could restore the proliferation of HUVECs inhibited by AGEs. However, overexpression of both wild-type CTSD and catalytically inactive mutant CTSD could promote the migration of HUVECs inhibited by AGEs. Collectively, our study found that AGEs inhibited the proliferation and migration in HUVECs and promoted autophagic flux, which in turn played a protective role against AGEs-induced cell injury. CTSD, in need of its catalytic activity, may promote proliferation in AGEs-treated HUVECs independent of the autophagy-lysosome pathway. Meanwhile, CTSD could improve the migration of AGEs-treated HUVECs regardless of its enzymatic activity.

  18. Colon cancer proliferating desulfosinigrin in wasabi (Wasabia japonica).

    Science.gov (United States)

    Weil, Marvin J; Zhang, Yanjun; Nair, Muraleedharan G

    2004-01-01

    A reduced incidence of different types of cancer has been linked to consumption of Brassica vegetables, and there is evidence that glucosinolates (GSLs) and their hydrolysis products play a role in reducing cancer risk. Wasabi (Wasabia japonica) and horseradish (Armoracia rusticana), both Brassica vegetables, are widely used condiments both in Japanese cuisine and in the United States. Desulfosinigrin (DSS) (1) was isolated from a commercially available wasabi powder and from fresh wasabi roots. Sinigrin (2) was isolated from horseradish roots. DSS and sinigrin were evaluated for their inhibitory effects on cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzymes, on lipid peroxidation, and on the proliferation of human colon (HCT-116), breast (MCF-7), lung (NCIH460), and central nervous system (CNS, SF-268) cancer cell lines. DSS did not inhibit COX enzymes or lipid peroxidation at 250 microg/ml. Sinigrin inhibited lipid peroxidation by 71% at 250 microg/ml. However, DSS promoted the growth of HCT-116 (colon) and NCI H460 (lung) human cancer cells as determined by the MTT assay in a concentration-dependent manner. At 3.72 microg/ml, a 27% increase in the number of viable human HCT-116 colon cancer cells was observed; the corresponding increases at 7.50 and 15 microg/ml were 42 and 69%, respectively. At 60 microg/ml, DSS doubled the number of HCT-16 colon cancer cells. For NCI H460 human lung cancer cells, DSS at 60 microg/ml increased the cell number by 20%. Sinigrin showed no proliferating effect on the tumor cells tested. This is the first report of the tumor cell-proliferating activity by a desulfoglucosinolate, the biosynthetic precursor of GSLs found in Brassica spp.

  19. Intermittent hypoxia reduces microglia proliferation and induces DNA damage in vitro

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    Song Liu

    2016-05-01

    Full Text Available Objective(s:Intermittent hypoxia (IH, caused by obstructive sleep apnea (OSA, could cause hippocampus or neuron damage through multiple signaling pathways, while the underlying mechanisms are still unclear. Thus, the present study aimed to explore the effect of IH on the biological functions of microglia cells. Materials and Methods:Cell proliferation of BV2 cells after exposure to IH were observed by MTT assay and then DNA damage was detected by comet assay. RNA-sequencing assay was performed in cells under IH condition and normal conditions to find out the differentially expressed genes, which were further confirmed by reverse transcriptase polymerase chain reaction (RT-PCR and Western blot assay. Results:As results, IH inhibited the proliferation of BV2 cells, as well as caused DNA damage. RNA-sequencing assay revealed 4 differentially expressed genes (p21, Cyclin D1, Cyclin E2, and Gadd45α which were associated with the network of P53 signaling pathways in BV2 cells, among which, p21 and Gadd45α were dramatically increased while Cyclin D1 and Cyclin E2 were both decreased significantly. Moreover, inflammatory factors including IL-6, TNF-α and iNOS were significantly up-regulated in microglia cells under IH conditions for 8 hr. Conclusion:Our results indicated that IH could inhibit cyclin D1 and cyclin E2 expression via initiating multiple P53 pathways, which further blocked cell cycle transition and attenuated proliferative capability of BV2 cells. Meanwhile, IH activated inflammation reactions in BV2 cells. Present study elaborate the effects of IH on biological functions of microglia and provide theoretical foundation for further study on new therapy methods for OSA.

  20. Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin inhibits the proliferation of ARPE-19 cells

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    Wang Lin

    2010-04-01

    Full Text Available Abstract Background The antiproliferative effect of the Hsp90 inhibitor 17-AAG (17-allylamino-17-demethoxygeldanamycin on human retinal pigment epithelial cells is investigated. Methods MTT and flow cytometry were used to study the antiproliferative effects of the 17-AAG treatment of ARPE-19 cells. 2D gel electrophoresis (2-DE and mass spectrometry were applied to detect the altered expression of proteins, which was verified by real-time PCR. Gene Ontology analysis and Ingenuity Pathway Analysis (IPA were utilized to analyze the signaling pathways, cellular location, function, and network connections of the identified proteins. And SOD assay was employed to confirm the analysis. Results 17-AAG suppressed the proliferation of ARPE-19 cells by inducing cell cycle arrest and apoptosis. Proteomic analysis revealed that the expression of 94 proteins was altered by a factor of more than 1.5 following exposure to 17-AAG. Of these 94, 87 proteins were identified. Real-time PCR results indicated that Hsp90 and Hsp70, which were not identified by proteomic analysis, were both upregulated upon 17-AAG treatment. IPA revealed that most of the proteins have functions that are related to oxidative stress, as verified by SOD assay, while canonical pathway analysis revealed glycolysis/gluconeogenesis. Conclusions 17-AAG suppressed the proliferation of ARPE-19 cells by inducing cell cycle arrest and apoptosis, and possibly by oxidative stress.

  1. Nicotine promotes proliferation and collagen synthesis of chondrocytes isolated from normal human and osteoarthritis patients.

    Science.gov (United States)

    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.

  2. Kaempferol suppresses bladder cancer tumor growth by inhibiting cell proliferation and inducing apoptosis.

    Science.gov (United States)

    Dang, Qiang; Song, Wenbin; Xu, Defeng; Ma, Yanmin; Li, Feng; Zeng, Jin; Zhu, Guodong; Wang, Xinyang; Chang, Luke S; He, Dalin; Li, Lei

    2015-09-01

    The effects of the flavonoid compound, kaempferol, which is an inhibitor of cancer cell proliferation and an inducer of cell apoptosis have been shown in various cancers, including lung, pancreatic, and ovarian, but its effect has never been studied in bladder cancer. Here, we investigated the effects of kaempferol on bladder cancer using multiple in vitro cell lines and in vivo mice studies. The MTT assay results on various bladder cancer cell lines showed that kaempferol enhanced bladder cancer cell cytotoxicity. In contrast, when analyzed by the flow cytometric analysis, DNA ladder experiment, and TUNEL assay, kaempferol significantly was shown to induce apoptosis and cell cycle arrest. These in vitro results were confirmed in in vivo mice studies using subcutaneous xenografted mouse models. Consistent with the in vitro results, we found that treating mice with kaempferol significant suppression in tumor growth compared to the control group mice. Tumor tissue staining results showed decreased expressions of the growth related markers, yet increased expressions in apoptosis markers in the kaempferol treated group mice tissues compared to the control group mice. In addition, our in vitro and in vivo data showed kaempferol can also inhibit bladder cancer invasion and metastasis. Further mechanism dissection studies showed that significant down-regulation of the c-Met/p38 signaling pathway is responsible for the kaempferol mediated cell proliferation inhibition. All these findings suggest kaempferol might be an effective and novel chemotherapeutic drug to apply for the future therapeutic agent to combat bladder cancer. © 2014 Wiley Periodicals, Inc.

  3. Swelling-induced chloride current in glioblastoma proliferation, migration, and invasion.

    Science.gov (United States)

    Wong, Raymond; Chen, Wenliang; Zhong, Xiao; Rutka, James T; Feng, Zhong-Ping; Sun, Hong-Shuo

    2018-01-01

    Glioblastoma (GBM) remains as the most common and aggressive brain tumor. The survival of GBM has been linked to the aberrant activation of swelling-induced chloride current I Cl,swell . In this study, we investigated the effects of I Cl,swell on cell viability, proliferation, and migration in the human GBM cell lines, U251 and U87, using a combination of patch clamp electrophysiology, MTT, colony formation, wound healing assays and Western immunoblotting. First, we showed that the specific inhibitor of I Cl,swell , DCPIB, potently reduced the I Cl,swell in U87 cells. Next, in both U87 and U251 cells, we found that DCPIB reduced GBM viability, proliferation, colony formation, migration, and invasion. In addition, our Western immunoblot assay showed that DCPIB-treated U251 cells had a reduction in JAK2, STAT3, and Akt phosphorylation, thus, suggesting that DCPIB potentially suppresses GBM functions through inhibition of the JAK2/STAT3 and PI3K/Akt signaling pathways. Therefore, the I Cl,swell may be a potential drug target for GBM. © 2017 Wiley Periodicals, Inc.

  4. Kalanchoe tubiflora extract inhibits cell proliferation by affecting the mitotic apparatus.

    Science.gov (United States)

    Hsieh, Yi-Jen; Yang, Ming-Yeh; Leu, Yann-Lii; Chen, Chinpiao; Wan, Chin-Fung; Chang, Meng-Ya; Chang, Chih-Jui

    2012-09-10

    Kalanchoe tubiflora (KT) is a succulent plant native to Madagascar, and is commonly used as a medicinal agent in Southern Brazil. The underlying mechanisms of tumor suppression are largely unexplored. Cell viability and wound-healing were analyzed by MTT assay and scratch assay respectively. Cell cycle profiles were analyzed by FACS. Mitotic defects were analyzed by indirect immunofluoresence images. An n-Butanol-soluble fraction of KT (KT-NB) was able to inhibit cell proliferation. After a 48 h treatment with 6.75 μg/ml of KT, the cell viability was less than 50% of controls, and was further reduced to less than 10% at higher concentrations. KT-NB also induced an accumulation of cells in the G2/M phase of the cell cycle as well as an increased level of cells in the subG1 phase. Instead of disrupting the microtubule network of interphase cells, KT-NB reduced cell viability by inducing multipolar spindles and defects in chromosome alignment. KT-NB inhibits cell proliferation and reduces cell viability by two mechanisms that are exclusively involved with cell division: first by inducing multipolarity; second by disrupting chromosome alignment during metaphase. KT-NB reduced cell viability by exclusively affecting formation of the proper structure of the mitotic apparatus. This is the main idea of the new generation of anti-mitotic agents. All together, KT-NB has sufficient potential to warrant further investigation as a potential new anticancer agent candidate.

  5. [Effects of icotinib hydrochloride on the proliferation and apoptosis of human lung cancer cell lines].

    Science.gov (United States)

    Ma, Li; Han, Xiao-hong; Wang, Shuai; Wang, Jian-fei; Shi, Yuan-kai

    2012-09-25

    To explore the effects of icotinib on the proliferation and apoptosis of various lung cancer cell lines. Human lung cancer cell lines HCC827, H1650, H1975, A549 and human epidermal cancer cell line A431 were treated in vitro with icotinib or gefitinib at a concentration gradient of 0 - 40 µmol/L. Their proliferation effects were analyzed by the thiazolyl blue (MTT) assay and the apoptotic effects detected by flow cytometer. The downstream signaling proteins were detected by Western blot. The median inhibitory concentrations (IC(50)) of icotinib for A431 and HCC827 cell lines were (0.04 ± 0.02) and (0.15 ± 0.06) µmol/L respectively. No significant differences existed between the inhibitions of gefitinib and icotinib on A431, HCC827, H1650, H1975 and A549 cell lines (all P > 0.05). Compared with H1650, H1975 and A549 cell lines, icotinib significantly inhibited A431 (P = 0.009, 0.005 and 0.000) and HCC827 (P = 0.001, 0.001 and 0.000) cell lines. And it lowered the expressions of p-AKT, p-ERK and survivin protein expression through the inhibited activity of p-EGFR protein. Icotinib can arrest the proliferation of lung adenocarcinoma cells with EGFR mutation or over-expression by inhibiting the signal pathways of AKT-ERK and survivin.

  6. Silencing of CXCR4 inhibits tumor cell proliferation and neural invasion in human hilar cholangiocarcinoma.

    Science.gov (United States)

    Tan, Xin-Yu; Chang, Shi; Liu, Wei; Tang, Hui-Huan

    2014-03-01

    To evaluate the expression of CXC motif chemokine receptor 4 (CXCR4) in the tissues of patients with hilar cholangiocarcinoma (hilar-CCA) and to investigate the cell proliferation and frequency of neural invasion (NI) influenced by RNAi-mediated CXCR4 silencing. An immunohistochemical technique was used to detect the expression of CXCR4 in 41 clinical tissues, including hilar-CCA, cholangitis, and normal bile duct tissues. The effects of small interference RNA (siRNA)-mediated CXCR4 silencing were detected in the hilar-CCA cell line QBC939. Cell proliferation was determined by MTT. Expression of CXCR4 was monitored by quantitative real time polymerase chain reaction and Western blot analysis. The NI ability of hilar-CCA cells was evaluated using a perineural cell and hilar-CCA cell coculture migration assay. The expression of CXCR4 was significantly induced in clinical hilar-CCA tissue. There was a positive correlation between the expression of CXCR4 and lymph node metastasis/NI in hilar-CCA patients (philar-CCA. CXCR4 is involved in the invasion and proliferation of human hilar-CCA cell line QBC939, indicating that CXCR4 could be a promising therapeutic target for hilar-CCA.

  7. The Ihh signal is essential for regulating proliferation and hypertrophy of cultured chicken chondrocytes.

    Science.gov (United States)

    Ma, R S; Zhou, Z L; Luo, J W; Zhang, H; Hou, J F

    2013-10-01

    The Indian hedgehog (Ihh) signal plays a vital role in regulating proliferation and hypertrophy of chondrocytes. To investigate its function in postnatal chicken (Gallus gallus) chondrocytes, cyclopamine was used to inhibit Ihh signaling. The MTT and ALP assays revealed the downgrade-proliferation and upgrade-differentiation of chondrocytes. To further elucidate the mechanism, the mRNA expression levels of Ihh, parathyroid hormone related protein (PTHrP), Gli-2, Bcl-2, Bone Morphogenetic Protein 6 (BMP-6), type X collagen (Col X) and type II collagen (Col II) were detected by quantitative real-time RT-PCR analysis, and the protein expressions of Ihh, Col X, and Col II were determined using Western blot analysis. After the Ihh signal was blocked, chondrocytes demonstrated high expression levels of PTHrP and Col X and low levels of Gli-2, BMP-6, Bcl-2 and Col II although Ihh expression was increased. Based on these results, the Ihh signal is essential for balancing chicken chondrocyte proliferation and hypertrophy, and the regulatory function of PTHrP acts in an Ihh-dependent manner. Furthermore, BMP-6 and Bcl-2 played roles in maintaining the development of chondrocytes and may be downstream regulatory factors of Ihh signaling. © 2013.

  8. Inhibitory efficacy of bufadienolides on Na+,K+-pump activity versus cell proliferation.

    Science.gov (United States)

    Xu, Yinfang; Liu, Xuan; Schwarz, Silvia; Hu, Lihong; Guo, Dean; Gu, Quanbao; Schwarz, Wolfgang

    2016-07-01

    Bufadienolides are cytotoxic drugs that may form the basis for anticancer agents. Due to structural and functional similarity to cardiotonic glycosides, application is restricted. We, therefore, investigated correlation of their putative anticancer effects with inhibition of Na + ,K + pumps. The natural bufalin and three derivatives were tested. The anticancer effects of the drugs were checked by observing their inhibitory effects on proliferation of rat liver cancer cells using MTT assay. Inhibition of Na + ,K + -pump was determined by measuring pump-mediated current of rat α1/β1 and α2/β1 Na + ,K + pumps expressed in Xenopus oocytes. All tested bufadienolides inhibited cell proliferation and Na + ,K + pump activity. An activity coefficient A =100x IC 50 Na,K pump /IC 50 pr o liferation was used to describe drug effectivity as anticancer drug. Natural bufalin exhibited lowest effectivity on cell proliferation, and also the A value for rat α1 isoform was the lowest (0.08), the α2 isoform was much less sensitive ( A =1.00). The highest A values were obtained for the BF238 derivative with A =0.88 and 2.64 for the α1 and α2 isoforms, respectively. Therefore, we suggest that search for bufalin derivatives with high anticancer effect and low affinity for both Na + ,K + pump isoforms may be a promising strategy for development of anticancer drugs.

  9. Effects of salinomycin and 17-AAG on proliferation of human gastric cancer cells in vitro

    Science.gov (United States)

    Zhang, Zuwen; Zhao, Jumei; Mi, Zhikuan; Pang, Qiuxia; Wang, Aihong; Chen, Meini; Liu, Xiaobin; Wei, Xiaoli; Liu, Tao

    2017-01-01

    The aim of the present study was to investigate the effects and mechanisms of 17-AAG combined with salinomycin treatment on proliferation and apoptosis of the SGC-7901 gastric cancer cell line. An MTT assay was used to detect the proliferation of SGC-7901 cells. Morphological alterations of cells were observed under inverted phase-contrast and fluorescence microscopes. Cell cycle and apoptosis were assessed by flow cytometry analysis. The protein expression of nuclear factor (NF)-κB p65 and Fas-ligand (L) were evaluated by immunocytochemistry. Salinomycin with a concentration range of 1–32 µmol/l was demonstrated to inhibit growth of SGC-7901 cells effectively, affect the morphology and apoptosis rate of cells, and arrest SGC-7901 cells in S phase. Furthermore, salinomycin significantly increased the protein expression of Fas-L and decreased the protein expression of NF-κB p65. The alterations in SGC-7901 cells co-treated with salinomycin and 17-AAG were more significant compared with cells treated with one drug only. In conclusion, the individual use of salinomycin and combined use with 17-AAG may significantly inhibit SGC-7901 gastric cancer cell proliferation and induce cell apoptosis. The potential mechanisms may be associated with upregulation of Fas-L and downregulation of NF-κB. These results provide a basis for the potential use of salinomycin in gastric cancer treatment. PMID:28627587

  10. [Effects of nicotine on bone marrow stromal cells proliferation and differentiation of chondrocyte in vitro].

    Science.gov (United States)

    Ying, Xiao-zhou; Peng, Lei; Cheng, Shao-wen; Chen, Qing-yu; Zhang, Wei; Kou, Dong-quan; Shen, Yue

    2011-11-01

    To examine the effects of various concentration of nicotine on bone marrow stromal cells (BMSCs) proliferation and differentiation of cartilaginous in vitro. BMSCs was obtained from femoral bone and tibia of New-Zealand albino rabbit. The cells of the 3rd generation were used in study. Different concentration of nicotine (0, 1 x 10(-7), 1 x 10(-6), 1 x 10(-5) M) were added into BMSCs. BMSCs proliferation was analyzed by MTT assay at the 1, 4, 7, 14 days. The expression of collagen type II and aggrecan as the marker genes of cartilaginous differentiation from BMSCs were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Microscope showed that BMSCs transformed from round to fusiform shape. The concentration of nicotine in 1 x 10(-7), 1 x 10(-6) M had a significant positive effect on cell proliferation and the expression of type II collagen in a time-dependent manner when supplemented in commonly used induction media (P<0.05). Concentrations of nicotine in 1 x 10(-7) can promote the expression of aggrecan at the 7th day after induction,and in 1 x 10(-5) M may inhibit the expression of type II collagen and aggrecan. It was implied that local application of nicotine at an appropriate concentration may be a promising approach for enhancing cartilaginous differentiation capacity of BMSCs in cartilage tissue engineering.

  11. Efficient inhibition of fibroblast proliferation and collagen expression by ERK2 siRNAs

    International Nuclear Information System (INIS)

    Li, Fengfeng; Fan, Cunyi; Cheng, Tao; Jiang, Chaoyin; Zeng, Bingfang

    2009-01-01

    Transforming growth factor-β1 and fibroblast growth factor-2 play very important roles in fibroblast proliferation and collagen expression. These processes lead to the formation of joint adhesions through the SMAD and MAPK pathways, in which ERK2 is supposed to be crucial. Based on these assumptions, lentivirus (LV)-mediated small interfering RNAs (siRNAs) targeting ERK2 were used to suppress the proliferation and collagen expression of rat joint adhesion tissue fibroblasts (RJATFs). Among four siRNAs examined, siRNA1 caused an 84% reduction in ERK2 expression (p < 0.01) and was selected as the most efficient siRNA for use in this study. In subsequent experiments, significant downregulation of types I and III collagen were observed by quantitative RT-PCR and Western blot analyses. MTT assays and flow cytometry revealed marked inhibition of RJATF proliferation, but no apoptosis. In conclusion, LV-mediated ERK2 siRNAs may represent novel therapies or drug targets for preventing joint adhesion formation.

  12. LncRNA Taurine-Upregulated Gene 1 Promotes Cell Proliferation by Inhibiting MicroRNA-9 in MCF-7 Cells.

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    Zhao, Xiao-Bo; Ren, Guo-Sheng

    2016-12-01

    This study was designed to investigate the role of taurine-upregulated gene 1 ( TUG1 ) in MCF-7 breast cancer cells and the molecular mechanism involved in the regulation of microRNA-9 (miR-9). The expression of TUG1 in breast cancer tissues and cells was evaluated using quantitative reverse transcription polymerase chain reaction. Cell viability was examined using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay; cell cycle progression and apoptosis were analyzed using flow cytometry. A dual luciferase reporter assay was used to detect the relationship between TUG1 and miR-9. The expression of methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) was measured by western blot. Higher expression of TUG1 was observed in breast cancer tissues and cell lines than in the corresponding controls. TUG1 knockdown reduced proliferation, suppressed cell cycle progression, and promoted apoptosis of MCF-7 cells. The dual luciferase reporter assay showed that TUG1 could negatively regulate the expression of miR-9. MiR-9 inhibition abrogated the effect of TUG1 knockdown on the proliferation, cell cycle progression, and apoptosis of MCF-7 cells. TUG1 positively regulated the expression of MTHFD2 in breast cancer cells. TUG1 knockdown was significantly associated with decreased cell proliferation and it promoted apoptosis of breast cancer cells through the regulation of miR-9.

  13. Splenocyte proliferation, NK cell activation and cytokines production by extract of Scrophularia variegata; an in vitro study on mice spleen cells

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    A. Azadmehr

    2016-10-01

    Full Text Available Background and objectives:Scrophularia variegata M. Beib. (Scrophulariaceae is a medicinal plant, used for various inflammatory diseases in Iranian Traditional Medicine. In the present study, we evaluated the immune modulation and antioxidant effects of the hydroalcoholic extract of S.  variegata. Methods: The splenocytes were harvested from the spleen of Balb/c mice and were cultured. The splenocyte proliferation, NK cell activity, cytokines production and antioxidant effects were evaluated by MTT assay, enzyme- linked immunosorbent assay (ELISA and DPPH assay, respectively. Results: The S. variegata extract significantly increased splenocyte proliferation. The results indicated that the extract increased NK cell cytotoxicity of Yac-1 tumor cells and at the concentration of 50-200 µg/mL significantly increased IFN-γ and IL-2 cytokines, although the level of IL-4 cytokine was significantly reduced. The antioxidant activity was observed in the extract with IC50 302.34±0.11 μg/mL.Conclusion: The increasing in the splenocyte proliferation, anti-tumor NK cell cytotoxicity and cytokine secretion were indicated as potent immunomodulatory effects. These results suggest that S. variegata could be considered in the treatment of immunopathological disorders such as allergy and cancer; however, future studies are necessary.

  14. miR-32 inhibits proliferation, epithelial–mesenchymal transition, and metastasis by targeting TWIST1 in non-small-cell lung cancer cells

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    Li L

    2016-03-01

    Full Text Available Lei Li,1,* Dapeng Wu2,* 1Department of Pneumology, 2Department of Radiotherapy, Huaihe Hospital of Henan University, Kaifeng, Henan, People’s Republic of China *These authors contributed equally to this work Background: By analyzing published microRNA microarray studies, miR-32 was found to be markedly reduced in non-small-cell lung cancer (NSCLC tissues compared with that in nontumor tissues. However, little is known about its role and molecular mechanism involved in NSCLC development and progression. Here, we report the effect of miR-32 on NSCLC cell proliferation, epithelial–mesenchymal transition (EMT, and metastasis. Methods: Quantitative real-time PCR was performed to detect the expression level of miR-32 in primary NSCLC cases and cell lines. miR-32-overexpressing H1299 and A549 cells were constructed by lipofection transfection. MTT, transwell chamber, and Western blot assays were used to assess the effect of miR-32 on proliferation, EMT, and metastasis of NSCLC cells, respectively. Target prediction and luciferase reporter assays were performed to investigate the targets of miR-32. Tumor formation assay in vivo was performed to investigate the antitumor effect of miR-32. Results: An inverse correlation existed between miR-32 expression level and NSCLC cell proliferation, EMT, and metastasis, and upregulation of miR-32 repressed NSCLC cell proliferation, EMT, and metastasis. Moreover, we identified and validated that TWIST1 was a direct target of miR-32, and miR-32 regulated NSCLC cell proliferation, EMT, and metastasis, at least in part via modulation of TWIST1. The animal experiments showed that overexpression of miR-32 inhibited the growth of NSCLC tumors in vivo. Keywords: non-small-cell lung cancer, miR-32, TWIST1, proliferation, EMT, nude mice

  15. The role of Sep (O-phosphoserine) tRNA: Sec (selenocysteine) synthase (SEPSECS) in proliferation, apoptosis and hormone secretion of trophoblast cells.

    Science.gov (United States)

    Zhao, H-D; Zhang, W-G; Sun, M-N; Duan, Q-F; Li, F-L; Li, H

    2013-11-01

    To investigate whether Sep (O-phosphoserine) tRNA: Sec (selenocysteine) synthase (SEPSECS), which plays an essential role in the synthesis of selenoprotein, affects proliferation, apoptosis and hormone secretion of human trophoblast cells. Human trophoblast JEG-3 cells were divided into four groups: control group, SEPSECS silenced-expression group, empty vector group and SEPSECS over-expression group. Over-expression and silenced-expression were achieved by transfection with plasmid DNA or RNA oligonucleotide, respectively. 3-[4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide (MTT) and colony formation assays were performed to investigate cell proliferation, while apoptosis was tested by annexin V-FITC, PI double staining and caspases-3 activation assays, enzyme-linked immunosorbent assay (ELISA) was used to determine the level of progesterone (PG) and human chorionic gonadotropin (hCG). SEPSECS silenced-expression clearly inhibited proliferation of JEG-3 cells (p < 0.05), significantly induced cell apoptosis (p < 0.01) and reduced the production of PG and hCG (p < 0.05). On the contrary, SEPSECS over-expression significantly promoted both cell proliferation (p < 0.01) and secretion of PG and hCG (p < 0.05). SEPSECS significantly affects proliferation, apoptosis and hormone secretion of human trophoblast cells, suggesting that a potential relationship exists among SEPSECS, cell proliferation, apoptosis and hormone production of human placental trophoblast cells. Furthermore, this may provide a clue to uncover the relationship between selenium and human placental in association with an emphasis on the importance of selenium adequacy during pregnancy. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Using Yeast to Easily Determine Mitochondrial Functionality with 1-(4,5-Dimethylthiazol-2-yl)-3,5-Diphenyltetrazolium Bromide (MTT) Assay

    Science.gov (United States)

    Sanchez, Norma Silvia; Konigsberg, Mina

    2006-01-01

    Mitochondrial respiration is an important topic in biochemical courses, but at the same time it is a very arduous and challenging subject for the students to comprehend. In teaching laboratories, experiments are also difficult to perform because mitochondrial isolation as well as oxygen consumption determinations require expensive equipment,…

  17. Blocking Ihh signaling pathway inhibits the proliferation and promotes the apoptosis of PSCs.

    Science.gov (United States)

    Xu, Kai; Guo, Fengjing; Zhang, Shuwei; Liu, Cheng; Wang, Feixiong; Zhou, Zhiguo; Chen, Anmin

    2009-02-01

    The roles of Indian hedgehog (Ihh) signaling pathway in the proliferation and apoptosis of precartilaginous stem cells (PSCs) were investigated. PSCs, labeled with fibroblast growth factor receptor 3 (FGFR-3), were isolated from neonatal rats by immunomagnetic separation. After identification with FGFR-3 and Col II, the cells were incubated with different concentrations of cyclopamine (cyclo), the specific inhibitor of Ihh signaling pathway. The morphologic changes of the cells were observed under the inverted phase contrast microscope. The mRNA expression levels of Ihh, parathyroid hormonerelated peptide (PTHrP), protein Patched (Ptch), Bcl-2 and p21 were detected by RT-PCR. The protein expression levels of Ihh and Ptch were measured by Western blot. MTT assay was used to examine the effects of cyclo on proliferation of PSCs. Apoptosis rate of PSCs was examined by Annexin V/PI assay of flow cytometric analyses. After PSCs were incubated with cyclo, obvious morphologic changes were observed as compared with the control group. The mRNA expression levels of PTHrP, Ptch and Bcl-2 were decreased to varying degrees in a cyclo dose-dependent manner. However, the expression levels of Ihh and p21 mRNA were increased. The protein expression of Ptch and Ihh had the same change as the mRNA expression. Meanwhile, cyclo could obviously inhibit the proliferation and promote the apoptosis of PSCs. The results indicated that Ihh signaling pathway plays an important role in regulating the proliferation and apoptosis of PSCs, which is probably mediated by Bcl-2 and p21.

  18. [Inhibitory effect of baicalein on the proliferation and invasion of osteosarcoma cells and mechanism].

    Science.gov (United States)

    Tang, Zhibin; Li, Chun; Chen, Zhiwei

    2015-03-01

    To explore the effect of baicalein on the proliferation and invasion of osteosarcoma cells and its related mechanism. Osteosarcoma MG-63 cells that were cultured in vitro were respectively treated with 20 μL culture medium (control group), dehydrated alcohol (0 μmol/L baicalein group), 100 and 200 μmol/L baicalein solution for 48 hours. Cell proliferation was analyzed by MTT assay. The cell invasion ability was detected using Transwell(TM) invasion assay. The expression of ezrin mRNA was examined by real-time quantitative PCR. The expressions of ezrin protein and p-ezrin protein were measured using Western blotting. Apoptosis index (AI) was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The inhibitory rates of cell proliferation significantly increased in 100 and 200 μmol/L baicalein groups as compared with 0 μmol/L baicalein group. Moreover, that was higher in 200 μmol/L baicalein group than in 100 μmol/L baicalein group. In comparison with control and 0 μmol/L baicalein groups, the mean cell numbers of permeated membrane and levels of ezrin mRNA, ezrin protein and p-ezrin protein gradually decreased, but AI was gradually elevated with the increase of baicalein concentrations, whereas there was no significant difference in these indicators between 0 μmol/L baicalein group and control group. Baicalein can inhibit the proliferation and invasion of osteosarcoma MG-63 cells. The mechanism may be associated with the inhibited expression and activity of ezrin protein and the promoted tumor cell apoptosis.

  19. Fenofibrate inhibited pancreatic cancer cells proliferation via activation of p53 mediated by upregulation of LncRNA MEG3

    International Nuclear Information System (INIS)

    Hu, Duanmin; Su, Cunjin; Jiang, Min; Shen, Yating; Shi, Aiming; Zhao, Fenglun; Chen, Ruidong; Shen, Zhu; Bao, Junjie; Tang, Wen

    2016-01-01

    There is still no suitable drug for pancreatic cancer treatment, which is one of the most aggressive human tumors. Maternally expressed gene 3 (MEG3), a LncRNA, has been suggested as a tumor suppressor in a range of human tumors. Studies found fenofibrate exerted anti-tumor roles in various human cancer cell lines. However, its role in pancreatic cancer remains unknown. The present study aimed to explore the impacts of fenofibrate on pancreatic cancer cell lines, and to investigate MEG3 role in its anti-tumor mechanisms. We used MTT assay to determine cells proliferation, genome-wide LncRNA microarray analysis to identify differently expressed LncRNAs, siRNA or pCDNA-MEG3 transfection to interfere or upregulate MEG3 expression, western blot to detect protein levels, real-time PCR to determine MEG3 level. Fenofibrate significantly inhibited proliferation of pancreatic cancer cells, increased MEG3 expression and p53 levels. Moreover, knockdown of MEG3 attenuated cytotoxicity induced by fenofibrate. Furthermore, overexpression of MEG3 induced cells death and increased p53 expression. Our results indicated fenofibrate inhibited pancreatic cancer cells proliferation via activation of p53 mediated by upregulation of MEG3. - Highlights: • We found that fenofibrate suppressed proliferation of pancreatic cancer cells. • We found fenofibrate increased LncRNA-MEG3 expression and p53 level in PANC-1 cells. • Inhibition of MEG3 expression attenuated anti-tumor effects of fenofibrate.

  20. [Effect of DOT1L gene silence on proliferation of acute monocytic leukemia cell line THP-1].

    Science.gov (United States)

    Zhang, Yu-Juan; Li, Hua-Wen; Chang, Guo-Qiang; Zhang, Hong-Ju; Wang, Jian; Lin, Ya-Ni; Zhou, Jia-Xi; Li, Qing-Hua; Pang, Tian-Xiang

    2013-08-01

    This study was aimed to investigate the influence of short hairpin RNA (shRNA) on proliferation of human leukemia cell line THP-1. The shRNA targeting the site 732-752 of DOT1L mRNA was designed and chemically synthesized, then a single-vector lentiviral, tet-inducible shRNA-DOT1L system (Plko-Tet-On) was generated. Thereafter, the THP-1 cells with lentivirus were infected to create stable cell line with regulatable shRNA expression. The expression of DOT1L in the THP-1 cell line was assayed by RT-PCR. Effect of shRNA-DOT1L on the proliferation of THP-1 cells was detected with MTT method,and the change of colony forming potential of THP-1 cells was analyzed by colony forming unit test. Cell cycle distribution was tested by flow cytometry. The results indicated that the expression of DOT1L was statistically lower than that in the control groups. The proliferation and colony forming capacity of THP-1 cells were significantly inhibited. The percentage of cells at G0/G1 phase increased in THP-1/shRNA cells treated with Dox while the percentage of cells at S phase significantly decreased as compared with that in the control group. It is concluded that the shRNA targeting DOT1L can effectively inhibit the proliferation of acute monocytic leukemia cell line THP-1.

  1. PDGF stimulation of Mueller cell proliferation: Contributions of c-JNK and the PI3K/Akt pathway

    International Nuclear Information System (INIS)

    Moon, Sang Woong; Chung, Eun Jee; Jung, Sun-Ah; Lee, Joon H.

    2009-01-01

    Platelet-derived growth factor (PDGF) has a critical role in proliferative vitreoretinopathy (PVR) as a chemoattractant and mitogen for retinal pigment epithelial cells and retinal glial cells. Here, we investigated the potential effects of PDGF on the proliferation of Mueller cells and the intracellular signaling pathway mediating these changes. PDGF induced Mueller cell proliferation and increased phosphorylation of the PDGF receptor (PDGFR), as shown by an MTT assay and immunoprecipitation analyses. Both effects were blocked by JNJ, a PDGFR-selective tyrosine kinase inhibitor. PDGF also stimulated phosphorylation of c-JNK and Akt. PDGF-induced Mueller cell proliferation was significantly reduced by pre-treatment with SP600125 and LY294002, inhibitors of c-JNK and Akt phosphorylation, respectively. Our findings collectively indicate that PDGF-stimulated Mueller cell proliferation occurs via activation of the c-JNK and PI3K/Akt signaling pathways. These data provide useful information in establishing the role of Mueller cells in the development of proliferative vitreoretinopathy.

  2. Fenofibrate inhibited pancreatic cancer cells proliferation via activation of p53 mediated by upregulation of LncRNA MEG3

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Duanmin [Department of Gastroenterology, The Second Affiliated Hospital of Soochow University, Suzhou 215004 (China); Su, Cunjin [Department of Pharmacy, The Second Affiliated Hospital of Soochow University, Suzhou 215004 (China); Jiang, Min [Department of Breast Surgery, The First Affiliated Hospital of Soochow University, Suzhou 215004 (China); Shen, Yating [Department of Gastroenterology, The Second Affiliated Hospital of Soochow University, Suzhou 215004 (China); Shi, Aiming; Zhao, Fenglun [Department of Pharmacy, The Second Affiliated Hospital of Soochow University, Suzhou 215004 (China); Chen, Ruidong [Department of Gastroenterology, The Second Affiliated Hospital of Soochow University, Suzhou 215004 (China); Shen, Zhu [Department of Pharmacy, The Second Affiliated Hospital of Soochow University, Suzhou 215004 (China); Bao, Junjie, E-mail: baojjsdfey@sina.com [Department of Pharmacy, The Second Affiliated Hospital of Soochow University, Suzhou 215004 (China); Tang, Wen, E-mail: sztangwen@163.com [Department of Gastroenterology, The Second Affiliated Hospital of Soochow University, Suzhou 215004 (China)

    2016-03-04

    There is still no suitable drug for pancreatic cancer treatment, which is one of the most aggressive human tumors. Maternally expressed gene 3 (MEG3), a LncRNA, has been suggested as a tumor suppressor in a range of human tumors. Studies found fenofibrate exerted anti-tumor roles in various human cancer cell lines. However, its role in pancreatic cancer remains unknown. The present study aimed to explore the impacts of fenofibrate on pancreatic cancer cell lines, and to investigate MEG3 role in its anti-tumor mechanisms. We used MTT assay to determine cells proliferation, genome-wide LncRNA microarray analysis to identify differently expressed LncRNAs, siRNA or pCDNA-MEG3 transfection to interfere or upregulate MEG3 expression, western blot to detect protein levels, real-time PCR to determine MEG3 level. Fenofibrate significantly inhibited proliferation of pancreatic cancer cells, increased MEG3 expression and p53 levels. Moreover, knockdown of MEG3 attenuated cytotoxicity induced by fenofibrate. Furthermore, overexpression of MEG3 induced cells death and increased p53 expression. Our results indicated fenofibrate inhibited pancreatic cancer cells proliferation via activation of p53 mediated by upregulation of MEG3. - Highlights: • We found that fenofibrate suppressed proliferation of pancreatic cancer cells. • We found fenofibrate increased LncRNA-MEG3 expression and p53 level in PANC-1 cells. • Inhibition of MEG3 expression attenuated anti-tumor effects of fenofibrate.

  3. Effect of growth factors (BMP-4/7 & bFGF on proliferation & osteogenic differentiation of bone marrow stromal cells

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    Shaohui Yuan

    2013-01-01

    Full Text Available Background & objectives: BMP (bone morphogenetic protein-4/7 and bFGF (basic fibroblast growth factor significantly promote the osteogenic activity and the proliferation of rabbit BMSCs (bone marrow stromal cells, respectively. However, their synergistic effects on the proliferation and the differentiation of BMSCs remain unclear. In the present study, the effects of bFGF and BMP-4/7 were investigated on the proliferation and the differentiation of rat BMSCs in vitro. Methods: BMSCs were isolated from New Zealand white rabbits and cultured to the third passage. The samples were divided into five groups according to the material implanted: (A 80 ng/ml BMP-4/7; (B 80 ng/ml bFGF; (C 30 ng/ml BMP-4/7 and 30 ng/ml bFGF; (D 50 ng/ml BMP-4/7 and 50 ng/ml bFGF; and (E 80 ng/ml BMP-4/7 and 80 ng/ml bFGF. Cell proliferation was analyzed using methyl thiazolyl tetrazolium (MTT assay. Alkaline phosphatase activity and osteocalcin (OC dynamics were also measured. Results: BMP-4/7 alone significantly (P<0.05 promoted the proliferation of BMSCs. At the same time, it also promoted or inhibited the osteogenic differentiation of BMSCs. The synergistic effects of BMP-4/7 and bFGF significantly promoted both the proliferation and the osteogenic differentiation of BMSCs. The treatment of the synergistic effects was dose and time dependent. Interpretation & conclusions: A rational combination of BMP-4/7 and bFGF can promote the proliferation and the osteogenic differentiation of BMSCs. In addition, the synergistic functions are effective.

  4. Homeobox A7 increases cell proliferation by up-regulation of epidermal growth factor receptor expression in human granulosa cells

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    Yanase Toshihiko

    2010-06-01

    Full Text Available Abstract Background Homeobox (HOX genes encode transcription factors, which regulate cell proliferation, differentiation, adhesion, and migration. The deregulation of HOX genes is frequently associated with human reproductive system disorders. However, knowledge regarding the role of HOX genes in human granulosa cells is limited. Methods To determine the role of HOXA7 in the regulation and associated mechanisms of cell proliferation in human granulosa cells, HOXA7 and epidermal growth factor receptor (EGFR expressions were examined in primary granulosa cells (hGCs, an immortalized human granulosa cell line, SVOG, and a granulosa tumor cell line, KGN, by real-time PCR and Western blotting. To manipulate the expression of HOXA7, the HOXA7 specific siRNA was used to knockdown HOXA7 in KGN. Conversely, HOXA7 was overexpressed in SVOG by transfection with the pcDNA3.1-HOAX7 vector. Cell proliferation was measured by the MTT assay. Results Our results show that HOXA7 and EGFR were overexpressed in KGN cells compared to hGCs and SVOG cells. Knockdown of HOXA7 in KGN cells significantly decreased cell proliferation and EGFR expression. Overexpression of HOXA7 in SVOG cells significantly promoted cell growth and EGFR expression. Moreover, the EGF-induced KGN proliferation was abrogated, and the activation of downstream signaling was diminished when HOXA7 was knocked down. Overexpression of HOXA7 in SVOG cells had an opposite effect. Conclusions Our present study reveals a novel mechanistic role for HOXA7 in modulating granulosa cell proliferation via the regulation of EGFR. This finding contributes to the knowledge of the pro-proliferation effect of HOXA7 in granulosa cell growth and differentiation.

  5. Estrogen receptor α enhances the transcriptional activity of ETS-1 and promotes the proliferation, migration and invasion of neuroblastoma cell in a ligand dependent manner

    International Nuclear Information System (INIS)

    Cao, Peng; Feng, Fan; Dong, Guofu; Yu, Chunyong; Feng, Sizhe; Song, Erlin; Shi, Guobing; Liang, Yong; Liang, Guobiao

    2015-01-01

    It is well known that estrogen receptor α (ERα) participates in the pathogenic progress of breast cancer, hepatocellular carcinoma and head and neck squamous cell carcinoma. In neuroblastoma cells and related cancer clinical specimens, moreover, the ectopic expression of ERα has been identified. However, the detailed function of ERα in the proliferation of neuroblastoma cell is yet unclear. The transcriptional activity of ETS-1 (E26 transformation specific sequence 1) was measured by luciferase analysis. Western blot assays and Real-time RT-PCR were used to examine the expression of ERα, ETS-1 and its targeted genes. The protein-protein interaction between ERα and ETS-1 was determined by co-IP and GST-Pull down assays. The accumulation of ETS-1 in nuclear was detected by western blot assays, and the recruitment of ETS-1 to its targeted gene’s promoter was tested by ChIP assays. Moreover, SH-SY5Y cells’ proliferation, anchor-independent growth, migration and invasion were quantified using the MTT, soft agar or Trans-well assay, respectively. The transcriptional activity of ETS-1 was significantly increased following estrogen treatment, and this effect was related to ligand-mediated activation of ERα. The interaction between the ERα and ETS-1 was identified, and enhancement of ERα activation would up-regulate the ETS-1 transcription factor activity via modulating its cytoplasm/nucleus translocation and the recruitment of ETS-1 to its target gene’s promoter. Furthermore, treatment of estrogen increased proliferation, migration and invasion of neuroblastoma cells, whereas the antagonist of ERα reduced those effects. In this study, we provided evidences that activation of ERα promoted neuroblastoma cells proliferation and up-regulated the transcriptional activity of ETS-1. By investigating the role of ERα in the ETS-1 activity regulation, we demonstrated that ERα may be a novel ETS-1 co-activator and thus a potential therapeutic target in human

  6. Peroxisome Proliferator-Activated Receptor- Is a Potent Target for Prevention and Treatment in Human Prostate and Testicular Cancer

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    Masahide Matsuyama

    2008-01-01

    Full Text Available Peroxisome proliferator-activated receptor- (PPAR- is a ligand-activated transcriptional factor belonging to steroid receptor superfamily. PPAR- plays a role in both adipocyte differentiation and tumorigenesis. Up to date, PPAR- is expressed in various cancer tissues, and PPAR- ligand induces growth arrest of these cancer cells. In this study, we examined the expression of PPAR- in prostate cancer (PC and testicular cancer (TC by RT-PCR and immunohistochemistry, and we also examined the effect of PPAR- ligand in these cells by MTT assay, hoechest staining, and flow cytometry. PPAR- expression was significantly more extensive and intense in malignant tissues than in normal tissues. PPAR- ligand induced the reduction of malignant cell viability through apoptosis. These results demonstrated that the generated PPAR- in PC and TC cells might play an important role in the tumorigenesis. PPAR- may become a new target in the treatment of PC and TC.

  7. Girdin/GIV is upregulated by cyclic tension, propagates mechanical signal transduction, and is required for the cellular proliferation and migration of MG-63 cells

    International Nuclear Information System (INIS)

    Hu, Jiang-Tian; Li, Yan; Yu, Bing; Gao, Guo-Jie; Zhou, Ting; Li, Song

    2015-01-01

    To explore how Girdin/GIV is regulated by cyclic tension and propagates downstream signals to affect cell proliferation and migration. Human osteoblast-like MG-63 cells were exposed to cyclic tension force at 4000 μstrain and 0.5 Hz for 6 h, produced by a four-point bending system. Cyclic tension force upregulated Girdin and Akt expression and phosphorylation in cultured MG-63 cells. Girdin and Akt each promoted the phosphorylation of the other under stimulated tension. In vitro MTT and transwell assays showed that Girdin and Akt are required for cell proliferation and migration during cellular quiescence. Moreover, STAT3 was determined to be essential for Girdin expression under stimulated tension force in the physiological condition, as well as for osteoblast proliferation and migration during quiescence. These findings suggest that the STAT3/Girdin/Akt pathway activates in osteoblasts in response to mechanical stimulation and may play a significant role in triggering osteoblast proliferation and migration during orthodontic treatment. - Highlights: • Tension force upregulates Girdin and Akt expression and phosphorylation. • Girdin and Akt promotes the phosphorylation of each other under tension stimulation. • Girdin and Akt are required for MG-63 cell proliferation and migration. • STAT3 is essential for Girdin expression after application of the tension forces

  8. Girdin/GIV is upregulated by cyclic tension, propagates mechanical signal transduction, and is required for the cellular proliferation and migration of MG-63 cells

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    Hu, Jiang-Tian; Li, Yan; Yu, Bing; Gao, Guo-Jie; Zhou, Ting; Li, Song, E-mail: song_li59@126.com

    2015-08-21

    To explore how Girdin/GIV is regulated by cyclic tension and propagates downstream signals to affect cell proliferation and migration. Human osteoblast-like MG-63 cells were exposed to cyclic tension force at 4000 μstrain and 0.5 Hz for 6 h, produced by a four-point bending system. Cyclic tension force upregulated Girdin and Akt expression and phosphorylation in cultured MG-63 cells. Girdin and Akt each promoted the phosphorylation of the other under stimulated tension. In vitro MTT and transwell assays showed that Girdin and Akt are required for cell proliferation and migration during cellular quiescence. Moreover, STAT3 was determined to be essential for Girdin expression under stimulated tension force in the physiological condition, as well as for osteoblast proliferation and migration during quiescence. These findings suggest that the STAT3/Girdin/Akt pathway activates in osteoblasts in response to mechanical stimulation and may play a significant role in triggering osteoblast proliferation and migration during orthodontic treatment. - Highlights: • Tension force upregulates Girdin and Akt expression and phosphorylation. • Girdin and Akt promotes the phosphorylation of each other under tension stimulation. • Girdin and Akt are required for MG-63 cell proliferation and migration. • STAT3 is essential for Girdin expression after application of the tension forces.

  9. Proliferation risks

    International Nuclear Information System (INIS)

    Carchon, R.

    1998-09-01

    The report gives an overview of different aspects related to safeguards of fissile materials. Existing treaties including the Non-Proliferation Treaty, and the Tlatelolco and the Rarotonga Treaties are discussed. An overview of safeguards systems for the control of fissile materials as well as the role of various authorities is given. An overall overview of proliferation risks, the physical protection of fissile materials and the trade in fissile materials is given. Finally, the status in problem countries and de facto nuclear weapon states is discussed

  10. Asiaticoside induces cell proliferation and collagen synthesis in human dermal fibroblasts

    Directory of Open Access Journals (Sweden)

    Linda Yulianti

    2015-08-01

    Full Text Available Asiatiocoside, a saponin component isolated from Centella asiatica can improve wound healing by promoting the proliferation of human dermal fibroblasts (HDF and synthesis of collagen. The skin-renewing cells and type I and III collagen synthesis decrease with aging, resulting in the reduction of skin elasticity and delayed wound healing. Usage of natural active compounds from plants in wound healing should be evaluated and compared to retinoic acid as an active agent that regulates wound healing. The aim of this study was to compare and evaluate the effect of asiaticoside and retinoic acid to induce greater cell proliferation and type I and III collagen synthesis in human dermal fibroblast. Methods Laboratory experiments were conducted using human dermal fibroblasts (HDF isolated from human foreskin explants. Seven passages of HDF were treated with asiaticoside and retinoic acid at several doses and incubated for 24 and 48 hours. Cell viability in all groups was tested with the MTT assay to assess HDF proliferation. Type I and III collagen synthesis was examined using the respective ELISA kits. Analysis of variance was performed to compare the treatment groups. Results Asiaticoside had significantly stronger effects on HDF proliferation than retinoic acid (p<0.05. The type III collagen production was significantly greater induction with asiaticoside compared to retinoic acid (p<0.05. Conclusion Asiaticoside induces HDF proliferation and type I and III collagen synthesis in a time- and dose-dependent pattern. Asiaticoside has a similar effect as retinoic acid on type I and type III collagen synthesis.

  11. TW-01, a piperazinedione-derived compound, inhibits Ras-mediated cell proliferation and angioplasty-induced vascular restenosis

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Chao-Feng [The Ph.D. Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei 11031, Taiwan (China); Department of Medicine, MacKay Medical College, New Taipei City, Taiwan (China); Division of Cardiology, Department of Internal Medicine, MacKay Memorial Hospital, Taipei, Taiwan (China); Division of Cardiology, Department of Internal Medicine, Shuang Ho Hospital, Taipei Medical University, New Taipei City, Taiwan (China); Huang, Han-Li [The Ph.D. Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei 11031, Taiwan (China); Peng, Chieh-Yu [Chinese Medicine Research and Development Center, China Medical University Hospital, Taichung 404, Taiwan (China); School of Pharmacy, College of Pharmacy, China Medical University, Taichung 404, Taiwan (China); Lee, Yu-Ching [The Center of Translational Medicine, Taipei Medical University, Taipei, Taiwan (China); Ph.D. Program for Biotechnology in Medicine, Taipei Medical University, Taipei, Taiwan (China); Wang, Hui-Po [College of Pharmacy, Taipei Medical University, Taipei 11031, Taiwan (China); Teng, Che-Ming [College of Pharmacy, Taipei Medical University, Taipei 11031, Taiwan (China); Pharmacological Institute, College of Medicine, National Taiwan University, Taipei 100, Taiwan (China); Pan, Shiow-Lin, E-mail: slpan@tmu.edu.tw [The Ph.D. Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei 11031, Taiwan (China); Department of Pharmacology, College of Medicine, Taipei Medical University, Taipei 10031, Taiwan (China)

    2016-08-15

    Purpose: Vascular smooth muscle cell (VSMC) proliferation plays a critical role in the pathogenesis of atherosclerosis and restenosis. This study investigated piperazinedione derived compound TW-01-mediated inhibitory effects on VSMC proliferation and intimal hyperplasia. Methods: Cell proliferation was determined using [{sup 3}H]-thymidine incorporation and MTT assay; cell cycle distribution was measured using flow cytometry; proteins and mRNA expression were determined using western blotting and RT-PCR analyses; DNA binding activity of nuclear factor-κB (NF-κB), as measured using enzyme-linked immunosorbent assays (ELISA); in vivo effects of TW-01 were determined using balloon angioplasty in the rat. Results: TW-01 significantly inhibited cell proliferation. At the concentrations used, no cytotoxic effects were observed. Three predominant signaling pathways were inhibited by TW-01: (a) extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) activation and its downstream effectors of c-fos, c-jun, and c-myc; (b) DNA binding activity of nuclear factor-κB (NF-κB); and, (c) Akt/protein kinase B (PKB) and cell cycle progression. Furthermore, TW-01 also inhibited Ras activation, a shared upstream event of each of these signaling cascades. In vascular injury studies, oral administration of TW-01 significantly suppressed intimal hyperplasia induced by balloon angioplasty. Conclusion: The present study suggests that TW-01 might be a potential candidate for atherosclerosis treatment. - Highlights: • TW-01significantly inhibits vascular smooth muscle cell proliferation. • TW-01 inhibits ERK, Akt and Ras pathway and DNA binding activity of NF-κB. • TW-01 significantly suppresses intimal hyperplasia induced by balloon angioplasty. • TW-01 might be a potential candidate for atherosclerosis treatment.

  12. TW-01, a piperazinedione-derived compound, inhibits Ras-mediated cell proliferation and angioplasty-induced vascular restenosis

    International Nuclear Information System (INIS)

    Lin, Chao-Feng; Huang, Han-Li; Peng, Chieh-Yu; Lee, Yu-Ching; Wang, Hui-Po; Teng, Che-Ming; Pan, Shiow-Lin

    2016-01-01

    Purpose: Vascular smooth muscle cell (VSMC) proliferation plays a critical role in the pathogenesis of atherosclerosis and restenosis. This study investigated piperazinedione derived compound TW-01-mediated inhibitory effects on VSMC proliferation and intimal hyperplasia. Methods: Cell proliferation was determined using [ 3 H]-thymidine incorporation and MTT assay; cell cycle distribution was measured using flow cytometry; proteins and mRNA expression were determined using western blotting and RT-PCR analyses; DNA binding activity of nuclear factor-κB (NF-κB), as measured using enzyme-linked immunosorbent assays (ELISA); in vivo effects of TW-01 were determined using balloon angioplasty in the rat. Results: TW-01 significantly inhibited cell proliferation. At the concentrations used, no cytotoxic effects were observed. Three predominant signaling pathways were inhibited by TW-01: (a) extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) activation and its downstream effectors of c-fos, c-jun, and c-myc; (b) DNA binding activity of nuclear factor-κB (NF-κB); and, (c) Akt/protein kinase B (PKB) and cell cycle progression. Furthermore, TW-01 also inhibited Ras activation, a shared upstream event of each of these signaling cascades. In vascular injury studies, oral administration of TW-01 significantly suppressed intimal hyperplasia induced by balloon angioplasty. Conclusion: The present study suggests that TW-01 might be a potential candidate for atherosclerosis treatment. - Highlights: • TW-01significantly inhibits vascular smooth muscle cell proliferation. • TW-01 inhibits ERK, Akt and Ras pathway and DNA binding activity of NF-κB. • TW-01 significantly suppresses intimal hyperplasia induced by balloon angioplasty. • TW-01 might be a potential candidate for atherosclerosis treatment.

  13. Far infrared radiation promotes rabbit renal proximal tubule cell proliferation and functional characteristics, and protects against cisplatin-induced nephrotoxicity.

    Science.gov (United States)

    Chiang, I-Ni; Pu, Yeong-Shiau; Huang, Chao-Yuan; Young, Tai-Horng

    2017-01-01

    Far infrared radiation, a subdivision of the electromagnetic spectrum, is beneficial for long-term tissue healing, anti-inflammatory effects, growth promotion, sleep modulation, acceleration of microcirculation, and pain relief. We investigated if far infrared radiation is beneficial for renal proximal tubule cell cultivation and renal tissue engineering. We observed the effects of far infrared radiation on renal proximal tubules cells, including its effects on cell proliferation, gene and protein expression, and viability. We also examined the protective effects of far infrared radiation against cisplatin, a nephrotoxic agent, using the human proximal tubule cell line HK-2. We found that daily exposure to far infrared radiation for 30 min significantly increased rabbit renal proximal tubule cell proliferation in vitro, as assessed by MTT assay. Far infrared radiation was not only beneficial to renal proximal tubule cell proliferation, it also increased the expression of ATPase Na+/K+ subunit alpha 1 and glucose transporter 1, as determined by western blotting. Using quantitative polymerase chain reaction, we found that far infrared radiation enhanced CDK5R1, GNAS, NPPB, and TEK expression. In the proximal tubule cell line HK-2, far infrared radiation protected against cisplatin-mediated nephrotoxicity by reducing apoptosis. Renal proximal tubule cell cultivation with far infrared radiation exposure resulted in better cell proliferation, significantly higher ATPase Na+/K+ subunit alpha 1 and glucose transporter 1 expression, and significantly enhanced expression of CDK5R1, GNAS, NPPB, and TEK. These results suggest that far infrared radiation improves cell proliferation and differentiation. In HK-2 cells, far infrared radiation mediated protective effects against cisplatin-induced nephrotoxicity by reducing apoptosis, as indicated by flow cytometry and caspase-3 assay.

  14. Effects of hydroxyapatite nanoparticles on proliferation and apoptosis of human breast cancer cells (MCF-7)

    International Nuclear Information System (INIS)

    Meena, Ramovatar; Kesari, Kavindra Kumar; Rani, Madhu; Paulraj, R.

    2012-01-01

    The study aimed to correlate cell proliferation inhibition with oxidative stress and p53 protein expression in cancerous cells. Hydroxyapatite (HAP) (Ca 10 (PO 4 ) 6 (OH) 2 ) is the essential component of inorganic composition in human bone. It has been found to have obvious inhibitory function on growth of many kinds of tumor cells and its nanoparticle has stronger anti-cancerous effect than macromolecule microparticles. Human breast cancer cells (MCF-7) were cultured and treated with HAP nanoparticles at various concentrations. Cells viability was detected with MTT colorimetric assay. The morphology of the cancerous cells was performed by transmission electron microscopy and the expression of a cell apoptosis related gene (p53) was determined by ELISA assay and flow cytometry (FCM). The intracellular reactive oxygen species (ROS) level in HAP exposed cells was measured by H 2 DCFDA staining. DNA damage was measured by single-cell gel electrophoresis assay. The statistical analysis was done by one way ANOVA. The cellular proliferation inhibition rate was significantly (p < 0.05) increasing in a dose-dependent manner of HAP nanoparticles. Cell apoptotic characters were observed after MCF-7 cells were treated by HAP nanoparticles for 48 h. Moreover, ELISA assay and FCM shows a dose-dependent activation of p53 in MCF-7 cells treated with nanoHAP. These causative factors of the above results may be justified by an overproduction of ROS. In this study, a significant (p < 0.05) increase in the level of intracellular ROS in HAP-treated cells was observed. This study shows that HAP inhibits the growth of human breast cancer MCF-7 cells as well as induces cell apoptosis. This study shows that HAP NPs Induce the production of intracellular reactive oxygen species and activate p53, which may be responsible for DNA damage and cell apoptosis.

  15. [Effects of sintered bone modified with surface mineralization/P24 peptide composite biomaterial on the adhesion, proliferation and osteodifferentiation of MC3T3-E1 cells].

    Science.gov (United States)

    Li, Jingfeng; Zheng, Qixin; Guo, Xiaodong; Chen, Liaobin

    2014-10-01

    In the present research, the effects of sintered bone modified with surface mineralization/P24 peptide composite biomaterials on the adhesion, proliferation and osteodifferentiation of MC3T3-E1 cells were investigated. The experiments were divided into three groups due to biomaterials used: Group A (composite materials of sintered bone modified with surface mineralization and P24, a peptide of bone morphogenetic protein-2); Group B (sintered bone modified with surface mineralization) and Group C (sintered bone only). The three groups were observed by scanning electron microscopy (SEM) before the experiments, respectively. Then MC3T3-E1 cells were cultured on the surfaces of the three kinds of material, respectively. The cell adhesion rate was assessed by precipitation method. The proliferative ability of MC3T3-E1 cells were measured with MTT assay. And the ALP staining and measurement of alkaline phosphatase (ALP) activity were performed to assess the differentiation of cells into osteoblasts. The SEM results showed that the materials in the three groups retained the natural pore structure and the pore sizes were in the range between 200-850 μm. The adhesive ratio measurements and MTT assay suggested that adhesion and proliferation of MC3T3-E1 cells in Group A were much higher than those in Group B and Group C (P bone modified with surface mineralization/P24 composite material was confirmed to improve the adhesion rate and proliferation and osteodifferentiation of MC3T3-E1 cells, and maintained their morphology.

  16. Comparison of the effect of activated or non-activated PRP in various concentrations on osteoblast and fibroblast cell line proliferation.

    Science.gov (United States)

    Vahabi, Surena; Yadegari, Zahra; Mohammad-Rahimi, Hossein

    2017-09-01

    Platelet-rich plasma (PRP) contains growth factors which positively affect cell proliferation, cell differentiation, chemotaxis and intracellular matrix synthesis. All these processes are involved in wound healing and tissue regeneration; thus, PRP as a source of growth factors can be used in periodontal regenerative therapies. The purpose of the present study was to assess the effect of various concentrations of activated and non-activated PRP on proliferation of osteoblasts and fibroblasts in vitro. PRP was obtained from three healthy volunteers. 75, 50, 25, and 10% concentrations of f PRP were prepared by dilution in Dulbecco's modified Eagle's medium. In activated PRP groups, PRP concentrations were activated by adding calcium gluconate. Human gingival fibroblast (HGF) cell line and MG-63 (osteosarcoma) human osteoblast-like cell line were used in the study. The MTT proliferation assay was used to assess the effect of different types of PRP concentrates on proliferation of HGF and MG-63 cells, in 24, 48 and 72 h. After 24, 48, and 72 h, the proliferation rate of both cell lines was higher in the positive control group, except in 72 h in HGF cell lines, that 10% non-activated PRP group and 10 and 25% activated PRP groups has higher proliferation rate than the positive control group, which it was not significant. Proliferation rate in cells with 10% activated PRP was highest among samples containing PRP. The current study failed to show the significant effect of activated or non-activated PRP on proliferation of HGFs or MG-63 osteoblast-like cells. However, our results showed that activated PRP had a greater effect than non-activated PRP.

  17. Mesenchymal Stem Cell Conditioned Medium Promotes Proliferation and Migration of Alveolar Epithelial Cells under Septic Conditions In Vitro via the JNK-P38 Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Jie Chen

    2015-11-01

    Full Text Available Background/Aims: Mesenchymal stem cell (MSC based therapies may be useful for treating acute respiratory distress syndrome (ARDS, but the underlying mechanisms are incompletely understood. We investigated the impact of human umbilical cord Wharton's jelly-derived MSC (hUC-MSC secreted factors on alveolar epithelial cells under septic conditions and determined the relevant intracellular signaling pathways. Methods: Human alveolar epithelial cells (AEC and primary human small airway epithelial cells (SAEC were subjected to lipopolysaccharide (LPS with or without the presence of hUC-MSC-conditioned medium (CM. Proliferation and migration of AEC and SAEC were determined via an MTT assay, a wound healing assay and a transwell migration assay (only for AEC. Protein phosphorylation was determined by western blot and the experiments were repeated in presence of small-molecule inhibitors. The hMSC-secretory proteins were identified by LC-MS/MS mass spectrometry. Results: MSC-CM enhanced proliferation and migration. Activation of JNK and P38, but not ERK, was required for the proliferation and migration of AEC and SAEC. Pretreatment of AEC or SAEC with SP600125, an inhibitor of JNK1 or SB200358, an inhibitor of P38, significantly reduced cell proliferation and migration. An array of proteins including TGF-beta receptor type-1, TGF-beta receptor type-2, Ras-related C3 botulinum toxin substrate 1 and Ras-related C3 botulinum toxin substrate 2 which influencing the proliferation and migration of AEC and SAEC were detected in MSC-CM. Conclusion: Our data suggest MSC promote epithelial cell repair through releasing a repertoire of paracrine factors via activation of JNK and P38 MAPK.

  18. Advanced glycation end products promote the proliferation and migration of primary rat vascular smooth muscle cells via the upregulation of BAG3.

    Science.gov (United States)

    Li, Cunshu; Chang, Ye; Li, Yuan; Chen, Shuang; Chen, Yintao; Ye, Ning; Dai, Dongxue; Sun, Yingxian

    2017-05-01

    The present study was aimed to investigate the role of reactive oxygen species (ROS) on advanced glycation end product (AGE)-induced proliferation and migration of vascular smooth muscle cells (VSMCs) and whether Bcl-2‑associated athanogene 3 (BAG3) is involved in the process. Primary rat VSMCs were extracted and cultured in vitro. Cell viability was detected by MTT assay and cell proliferation was detected by EdU incorporation assay. Cell migration was detected by wound healing and Transwell assays. BAG3 was detected using qPCR and western blot analysis. Transcriptional and translational inhibitors (actinomycin D and cycloheximide, respectively) were used to study the effect of AGEs on the expression of BAG3 in VSMCs. Lentiviral plasmids containing short hairpin RNA (shRNA) against rat BAG3 or control shRNA were transduced into VSMCs. Cellular ROS were detected by 2',7'-dichlorofluorescein diacetate (DCFH-DA) staining. Mitochondrial membrane potential was detected by tetramethylrhodamine methyl ester (TMRE) staining. AGEs significantly increased the expression of BAG3 in a dose-and time-dependent manner. Furthermore, AGEs mainly increased the expression of BAG3 mRNA by increasing the RNA synthesis rather than inhibiting the RNA translation. BAG3 knockdown reduced the proliferation and migration of VSMCs induced by AGEs. BAG3 knockdown reduced the generation of ROS and sustained the mitochondrial membrane potential of VSMCs. Reduction of ROS production by N-acetylcysteine (NAC), a potent antioxidant, also reduced the proliferation and migration of VSMCs. On the whole, the present study demonstrated for the first time that AGEs could increase ROS production and promote the proliferation and migration of VSMCs by upregulating BAG3 expression. This study indicated that BAG3 should be considered as a potential target for the prevention and/or treatment of vascular complications of diabetes.

  19. Nuclear proliferation

    International Nuclear Information System (INIS)

    Stencel, S.

    1978-01-01

    The terms and reactions to President Carter's nuclear policy, culminating in the 1978 Nuclear Non-Proliferation Act, are reviewed and analyzed. The new law increases restrictions on nuclear exports, encourages continued use of light water reactors in preference to plutonium-fueled reactors, and emphasizes technical solutions to proliferation problems. Critics of the law point out that it will hurt U.S. trade unfairly, that other countries do not have as many fuel options as the U.S. has, and that nuclear sales have as many political and economic as technical solutions. Compromise areas include new international safety guidelines, the possibility of an international nuclear fuel bank, and a willingness to consider each case on its merits. 21 references

  20. Transient fluctuations of intracellular zinc ions in cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yuan [Division of Human Nutrition, Department of Preventive Medicine and Community Health, The University of Texas Medical Branch, Galveston, TX 77555 (United States); Maret, Wolfgang, E-mail: womaret@utmb.edu [Division of Human Nutrition, Department of Preventive Medicine and Community Health, The University of Texas Medical Branch, Galveston, TX 77555 (United States); Department of Anesthesiology, The University of Texas Medical Branch, Galveston, TX 77555 (United States)

    2009-08-15

    Zinc is essential for cell proliferation, differentiation, and viability. When zinc becomes limited for cultured cells, DNA synthesis ceases and the cell cycle is arrested. The molecular mechanisms of actions of zinc are believed to involve changes in the availability of zinc(II) ions (Zn{sup 2+}). By employing a fluorescent Zn{sup 2+} probe, FluoZin-3 acetoxymethyl ester, intracellular Zn{sup 2+} concentrations were measured in undifferentiated and in nerve growth factor (NGF)-differentiated rat pheochromocytoma (PC12) cells. Intracellular Zn{sup 2+} concentrations are pico- to nanomolar in PC12 cells and are higher in the differentiated than in the undifferentiated cells. When following cellular Zn{sup 2+} concentrations for 48 h after the removal of serum, a condition that is known to cause cell cycle arrest, Zn{sup 2+} concentrations decrease after 30 min but, remarkably, increase after 1 h, and then decrease again to about one half of the initial concentration. Cell proliferation, measured by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, decreases after both serum starvation and zinc chelation. Two peaks of Zn{sup 2+} concentrations occur within one cell cycle: one early in the G1 phase and the other in the late G1/S phase. Thus, fluctuations of intracellular Zn{sup 2+} concentrations and established modulation of phosphorylation signaling, via an inhibition of protein tyrosine phosphatases at commensurately low Zn{sup 2+} concentrations, suggest a role for Zn{sup 2+} in the control of the cell cycle. Interventions targeted at these picomolar Zn{sup 2+} fluctuations may be a way of controlling cell growth in hyperplasia, neoplasia, and diseases associated with aberrant differentiation.

  1. Radiation-induced VEGF-C expression and endothelial cell proliferation in lung cancer

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yu-Hsuan [National Taiwan University Hospital, Department of Oncology, Taipei (China); National Taiwan University, Pharmacological Institute, College of Medicine, Taipei (China); Pan, Shiow-Lin; Wang, Jing-Chi; Teng, Che-Ming [National Taiwan University, Pharmacological Institute, College of Medicine, Taipei (China); Kuo, Sung-Hsin [National Taiwan University Hospital, Department of Oncology, Taipei (China); National Taiwan University College of Medicine, Department of Internal Medicine, Taipei (China); Cheng, Jason Chia-Hsien [National Taiwan University Hospital, Department of Oncology, Taipei (China); National Taiwan University College of Medicine, Graduate Institute of Clinical Medicine, Taipei (China)

    2014-12-15

    The present study was undertaken to investigate whether radiation induces the expression of vascular endothelial growth factor C (VEGF-C) through activation of the PI3K/Akt/mTOR pathway,subsequently affecting endothelial cells. Radiotherapy-induced tumor micro-lymphatic vessel density (MLVD) was determined in a lung cancer xenograft model established in SCID mice. The protein expression and phosphorylation of members of the PI3K/Akt/mTOR pathway and VEGF-C secretion and mRNA expression in irradiated lung cancer cells were assessed by Western blot analysis, enzyme-linked immunosorbent assays (ELISAs), and reverse transcriptase-polymerase chain reaction (RT-PCR). Moreover, specific chemical inhibitors were used to evaluate the role of the PI3K/Akt/mTOR signaling pathway. Conditioned medium (CM) from irradiated control-siRNA or VEGF-C-siRNA-expressing A549 cells was used to evaluate the proliferation of endothelial cells by the MTT assay. Radiation increased VEGF-C expression in a dose-dependent manner over time at the protein but not at the mRNA level. Radiation also up-regulated the phosphorylation of Akt, mTOR, 4EBP, and eIF4E, but not of p70S6K. Radiation-induced VEGF-C expression was down-regulated by LY294002 and rapamycin (both p < 0.05). Furthermore, CM from irradiated A549 cells enhanced human umbilical vein endothelial cell (HUVEC) and lymphatic endothelial cell (LEC) proliferation, which was not observed with CM from irradiated VEGF-C-siRNA-expressing A549 cells. Radiation-induced activation of the PI3K/Akt/mTOR signaling pathway increases VEGF-C expression in lung cancer cells, thereby promoting endothelial cell proliferation. (orig.) [German] Die vorliegende Studie untersucht, ob die Strahlung die Expression von VEGF-C (vascular endothelial growth factor C) mittels Aktivierung des PI3K/Akt/mTOR-Signalwegs induziert und anschliessend die endothelialen Zellen beeinflusst. Die durch Strahlentherapie induzierte Mikrolymphgefaessdichte (MLVD) im Tumor wurde in

  2. Basic Fibroblast Growth Factor Stimulates the Proliferation of Bone Marrow Mesenchymal Stem Cells in Giant Panda (Ailuropoda melanoleuca).

    Science.gov (United States)

    Wang, Jun-Jie; Liu, Yu-Liang; Sun, Yuan-Chao; Ge, Wei; Wang, Yong-Yong; Dyce, Paul W; Hou, Rong; Shen, Wei

    2015-01-01

    It has been widely known that the giant panda (Ailuropoda melanoleuca) is one of the most endangered species in the world. An optimized platform for maintaining the proliferation of giant panda mesenchymal stem cells (MSCs) is very necessary for current giant panda protection strategies. Basic fibroblast growth factor (bFGF), a member of the FGF family, is widely considered as a growth factor and differentiation inducer within the stem cell research field. However, the role of bFGF on promoting the proliferation of MSCs derived from giant panda bone marrow (BM) has not been reported. In this study, we aimed to investigate the role of bFGF on the proliferation of BM-MSCs derived from giant panda. MSCs were cultured for cell proliferation analysis at 24, 48 and 72 hrs following the addition of bFGF. With increasing concentrations of bFGF, cell numbers gradually increased. This was further demonstrated by performing 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) cell proliferation assay, 5-Bromo-2-deoxyUridine (BrdU) labeling and cell cycle testing. Furthermore, the percentage of MSCs that were OCT4 positive increased slightly following treatment with 5 ng/ml bFGF. Moreover, we demonstrated that the extracellular signal-regulated kinase (ERK) signaling pathway may play an important role in the proliferation of panda MSCs stimulated by bFGF. In conclusion, this study suggests that giant panda BM-MSCs have a high proliferative capacity with the addition of 5 ng/ml bFGF in vitro.

  3. Basic Fibroblast Growth Factor Stimulates the Proliferation of Bone Marrow Mesenchymal Stem Cells in Giant Panda (Ailuropoda melanoleuca)

    Science.gov (United States)

    Wang, Jun-Jie; Liu, Yu-Liang; Sun, Yuan-Chao; Ge, Wei; Wang, Yong-Yong; Dyce, Paul W.; Hou, Rong; Shen, Wei

    2015-01-01

    It has been widely known that the giant panda (Ailuropoda melanoleuca) is one of the most endangered species in the world. An optimized platform for maintaining the proliferation of giant panda mesenchymal stem cells (MSCs) is very necessary for current giant panda protection strategies. Basic fibroblast growth factor (bFGF), a member of the FGF family, is widely considered as a growth factor and differentiation inducer within the stem cell research field. However, the role of bFGF on promoting the proliferation of MSCs derived from giant panda bone marrow (BM) has not been reported. In this study, we aimed to investigate the role of bFGF on the proliferation of BM-MSCs derived from giant panda. MSCs were cultured for cell proliferation analysis at 24, 48 and 72 hrs following the addition of bFGF. With increasing concentrations of bFGF, cell numbers gradually increased. This was further demonstrated by performing 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) cell proliferation assay, 5-Bromo-2-deoxyUridine (BrdU) labeling and cell cycle testing. Furthermore, the percentage of MSCs that were OCT4 positive increased slightly following treatment with 5 ng/ml bFGF. Moreover, we demonstrated that the extracellular signal-regulated kinase (ERK) signaling pathway may play an important role in the proliferation of panda MSCs stimulated by bFGF. In conclusion, this study suggests that giant panda BM-MSCs have a high proliferative capacity with the addition of 5 ng/ml bFGF in vitro. PMID:26375397

  4. Basic Fibroblast Growth Factor Stimulates the Proliferation of Bone Marrow Mesenchymal Stem Cells in Giant Panda (Ailuropoda melanoleuca.

    Directory of Open Access Journals (Sweden)

    Jun-Jie Wang

    Full Text Available It has been widely known that the giant panda (Ailuropoda melanoleuca is one of the most endangered species in the world. An optimized platform for maintaining the proliferation of giant panda mesenchymal stem cells (MSCs is very necessary for current giant panda protection strategies. Basic fibroblast growth factor (bFGF, a member of the FGF family, is widely considered as a growth factor and differentiation inducer within the stem cell research field. However, the role of bFGF on promoting the proliferation of MSCs derived from giant panda bone marrow (BM has not been reported. In this study, we aimed to investigate the role of bFGF on the proliferation of BM-MSCs derived from giant panda. MSCs were cultured for cell proliferation analysis at 24, 48 and 72 hrs following the addition of bFGF. With increasing concentrations of bFGF, cell numbers gradually increased. This was further demonstrated by performing 3-(4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-H-tetrazolium bromide (MTT cell proliferation assay, 5-Bromo-2-deoxyUridine (BrdU labeling and cell cycle testing. Furthermore, the percentage of MSCs that were OCT4 positive increased slightly following treatment with 5 ng/ml bFGF. Moreover, we demonstrated that the extracellular signal-regulated kinase (ERK signaling pathway may play an important role in the proliferation of panda MSCs stimulated by bFGF. In conclusion, this study suggests that giant panda BM-MSCs have a high proliferative capacity with the addition of 5 ng/ml bFGF in vitro.

  5. The effect of gestational diabetes on proliferation capacity and viability of human umbilical cord-derived stromal cells.

    Science.gov (United States)

    Wajid, Nadia; Naseem, Rashida; Anwar, Sanam Saiqa; Awan, Sana Javaid; Ali, Muhammad; Javed, Sara; Ali, Fatima

    2015-09-01

    Stomal cells derived from Wharton's jelly of human umbilical cord (WJMSCs) are considered as the potential therapeutic agents for regeneration and are getting famous for stem cell banking. Our study aims to evaluate the effects of gestational diabetes on proliferation capacity and viability of WJMSCs. Mesenchymal stromal cells were isolated from Wharton's jelly of human umbilical cords from normal and gestational diabetic (DWJMSCs) mothers. Growth patterns of both types of cells were analyzed through MTT assay and population doubling time. Cell survival, cell death and glucose utilization were estimated through trypan blue exclusion assay, LDH assay and glucose detection assay respectively. Angiogenic ability was evaluated by immunocytochemistry and ELISA for VEGF A. Anti-cancerous potential was analyzed on HeLa cells. DWJMSCs exhibited low proliferative rate, increased population doubling time, reduced cell viability and increased cell death. Interestingly, DWJMSCs were found to have a reduced glucose utilization and anti-cancerous ability while enhanced angiogenic ability. Gestational diabetes induces adverse effects on growth, angiogenic and anti-cancerous potential of WJMSCs.

  6. Locostatin, a disrupter of Raf kinase inhibitor protein, inhibits extracellular matrix production, proliferation, and migration in human uterine leiomyoma and myometrial cells.

    Science.gov (United States)

    Janjusevic, Milijana; Greco, Stefania; Islam, Md Soriful; Castellucci, Clara; Ciavattini, Andrea; Toti, Paolo; Petraglia, Felice; Ciarmela, Pasquapina

    2016-11-01

    To investigate the presence of Raf kinase inhibitor protein (RKIP) in human myometrium and leiomyoma as well as to determine the effect of locostatin (RKIP inhibitor) on extracellular matrix (ECM) production, proliferation, and migration in human myometrial and leiomyoma cells. Laboratory study. Human myometrium and leiomyoma. Thirty premenopausal women who were admitted to the hospital for myomectomy or hysterectomy. Myometrial and leiomyoma tissues were used to investigate the localization and the expression level of RKIP through immunohistochemistry and Western blotting. Myometrial and leiomyoma cells were treated with locostatin (10 μM) to measure ECM expression by real-time polymerase chain reaction, GSK3β expression by Western blotting, cell migration by wound-healing assay, and cell proliferation by MTT assay and immunocytochemistry. The expression of RKIP in human myometrial and leiomyoma tissue; ECM components and GSK3β expression, migration, and proliferation in myometrial and leiomyoma cells. RKIP is expressed in human myometrial and leiomyoma tissue. Locostatin treatment resulted in the activation of the mitogen-activated protein kinase (MAPK) signal pathway (ERK phosphorylation), providing a powerful validation of our targeting protocol. Further, RKIP inhibition by locostatin reduces ECM components. Moreover, the inhibition of RKIP by locostatin impaired cell proliferation and migration in both leiomyoma and myometrial cells. Finally, locostatin treatment reduced GSK3β expression. Therefore, even if the activation of MAPK pathway should increase proliferation and migration, the destabilization of GSK3β leads to the reduction of proliferation and migration of myometrial and leiomyoma cells. Our results indicate that RKIP may be involved in leiomyoma pathophysiology. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  7. Biogas technology on farms 1; Biokaasuteknologiaa maatiloilla 1. Biokaasulaitoksen hankinta, kaeyttoeoenotto ja operointi - kaeytaennoen kokemuksia MTT:n maatilakohtaiselta laitokselta

    Energy Technology Data Exchange (ETDEWEB)

    Luostarinen, S. (ed.)

    2013-11-01

    Biogas technologies can be applied for several different purposes in agriculture. It is a means to utilise the energy content of manure and other organic materials, to recycle their nutrients into plant production, enhance utilisation of nitrogen and to mitigate emissions from agriculture. Of the two end-products, biogas can be utilised in the production of heat, electricity and/or vehicle fuel and digestate as fertiliser on fields. Agricultural biogas plants digest mainly animal manure in Finland. Several co-substrates are also used, including different plant biomasses and suitable by-products from especially food production. The aim of using co-substrates is usually to increase the amount of energy produced but they also affect the nutrient content and ratios in the digestate. Planning agricultural biogas plants starts from available fee materials, their amounts and characteristics. The biogas plant is designed for these materials and the technologies used are chosen to suit them. There are several options for plant design and how it can be attached into existing farm structures and it is wise to discuss these matters with an expert. In this way, correct farm-specific decisions can be made. When permitting the plant (permission for construction, environmental permit, safety issues, fertiliser legislation), it is important to make contact with the respective authority. Profitability of the biogas plant should be considered carefully. Things to consider include e.g. available financial incentives, investment cost, energy production and utilisation (own use or sale), nutrient recycling and potential avoidance of mineral fertilisers, co-substrates with gate fee, improved hygiene and less odours. Experiments at MTT Maaninka farm-scale biogas plant showed that dairy cow slurry produces 12-14 m{sup 3} of methane per ton of fresh weight. In this specific biogas plant this results potentially in methane production with an energy content of 400 MWh (3500 m{sup 3} of slurry

  8. Differential effects of platelet rich plasma and washed platelets on the proliferation of mouse MSC cells.

    Science.gov (United States)

    Duan, Jianmin; Kuang, Wei; Tan, Jiali; Li, Hongtao; Zhang, Yi; Hirotaka, Kikuchi; Tadashi, Katayama

    2011-04-01

    Multipotent mesenchymal stem cell (MSC) therapies are being tested clinically for a variety of disorders. However, despite the remarkable clinical advancements in this field, most applications still use traditional culture media containing fetal bovine serum. Platelet-rich plasma (PRP) appears as a novel application for tissue engineering and its effect on bone healing is thought to be mainly dependent on the proliferation promoting function, with the molecular mechanisms largely unknown. In this study, mouse osteogenic progenitor mesenchymal stem cells (MSCs) were cultured in PRP or washed platelet (WPLT)-treated wells or in untreated wells, and analyzed on cycloxygenase 2 (COX2) expression (qRT-PCR), cell growth (MTT assay) and cell differentiation (alkaline phosphatase activity). The results showed that PRP and WPLT stimulated cell growth similarly in the first 6 days, together with the steady induction of COX2 and PGE2. 10 μmol/l celecoxib (an inhibitor of COX2) significantly inhibited the pro-proliferation effects. Interestingly, WPLT had stronger effects than PRP in proliferation at the later time points (6-9 days). ALP activity assay and collagen 1a expression revealed PRP had a mild but statistically significant pro-differentiation effect, while no obvious effects observed in WLPT group. In summary, PRP stimulates initial growth of MSCs in a COX2 partially dependent manner and the less obvious osteogenic differentiation promoting effects of WPLT strongly indicates WPLT rather than the PRP should be the optional choice for expanding MSCs in vitro for clinical use.

  9. [Inhibitory effects of Porphyromonas endodontalis lipopolysaccharides on proliferation and differentiation of mouse osteoblast].

    Science.gov (United States)

    Li, Ren; Qiu, Li-hong; Yang, Di; Xue, Ming; Zhong, Ming

    2011-03-01

    To observe the effect of lipopolysaccharides (LPS) extracted from Porphyromonas endodontalis (Pe) on osteoblast cell proliferation and the activity of alkaline phosphatase (ALP) and interleukin (IL)-6 secretion and to investigate the role of Pe-LPS in osteoblast proliferation and differentiation. MC3T3-E1 cells were treated with different concentrations of Pe-LPS (10, 25, 50 mg/L) respectively. The relative growth rate (RGR) was detected by methyl thiazolyl tetrazolium (MTT) at different time point (12, 24, 48, 72 h). MC3T3-E1 cells were also stimulated with 10, 25 or 50 mg/L Pe-LPS for 6, 12, 24 and 48 h. The activity of ALP was detected by enzyme kinetics assay and the secretion of IL-6 was detected by enzyme-linked immunosorbent assay (ELISA). After the stimulation with 25 or 50 mg/L Pe-LPS for 72 h, the RGR of MC3T3-E1 cells descend to 87.46% and 71.12%. The ALP activities of MC3T3-E1 cells were inhibited obviously (P < 0.05) after the stimulation of different concentrations (10, 25, 50 mg/L) Pe-LPS for more than 24 hours. ELISA result showed that IL-6 increased to 32.21 ng/L treated with the 25 mg/L Pe-LPS for 6 h, 25 mg/L Pe-LPS gradually increased the expression of IL-6 from the ELISA results. Pe-LPS can induce the secretion of IL-6 in MC3T3-E1 and decrease the ALP activities of MC3T3-E1, the differentiation of osteoblasts was inhibited. with the long-time toxicity action of Pe-LPS, the proliferation rate of MC3T3-E1 also markedly decreased.

  10. [Effects of triterpenoid from Psidium guajava leaves ursolic acid on proliferation, differentiation of 3T3-L1 preadipocyte and insulin resistance].

    Science.gov (United States)

    Lin, Juan-Na; Kuang, Qiao-Ting; Ye, Kai-He; Ye, Chun-Ling; Huang, Yi; Zhang, Xiao-Qi; Ye, Wen-Cai

    2013-08-01

    To investigate the influences of triterpenoid from Psidium guajava Leaves (ursolic acid) on the proliferation, differentiation of 3T3-L1 preadipocyte, and its possible mechanism treat for insulin resistance. 3T3-L1 preadipocyte was cultured in vitro. After adding ursolic acid to the culture medium for 48h, the cell viability was tested by MTT assay. Induced for 6 days, the lipid accumulation of adipocyte was measured by Oil Red O staining. The insulin resistant cell model was established with Dexamethasone. Cellular glucose uptake was determined with GOD-POD assays and FFA concentration was determined at the time of 48h. Secreted adiponectin were measured by ELISA. The protein levels of PPARgamma and PTP1B in insulin resistant adipocyte were measured by Western Blotting. Compared with medium control group, 30, 100 micromol/L ursolic acid could increase its proliferation and differentiation significantly (P 0.05). Ursolic acid can improve the proliferation and differentiation of 3T3-L1 preadipocyte, enhance cellular glucose uptake, inhibit the production of FFA, promote the secretion of adiponectin insulin resistant adipocyte, its mechanism may be related to upregulating the expression of PPARgamma protein.

  11. Effects of recombinant human epidermal growth factor on the proliferation and radiation survival of human fibroblast cell lines in vitro

    International Nuclear Information System (INIS)

    Kim, Hyun Sook; Kang, Ki Mun; Na, Jae Boem; Chai, Gyu Young; Lee, Sang Wook

    2006-01-01

    To explore the effect of recombinant human EGF on the proliferation and survival of human fibroblast cell lines following irradiation. Fibroblast was originated human skin and primary cultured. The trypan blue stain assay and MTT assay were used to study the proliferative effects of EGF on human fibroblast cell lines in vitro. An incubation of fibroblasts with rhEGF for 24 hours immediately after irradiation was counted everyday. Cell cycle distributions were analyzed by FACS analysis. Number of fibroblast was significant more increased rhEGF (1.0 nM, 10 nM, 100 nM, 1,000 nM) treated cell than control after 8 Gy irradiation. Most effective dose of rhEGF was at 160 nM. These survival differences were maintained at 1 week later. Proportion of S phase was significantly increased on rhEGF treated cells. rhEGF cause increased fibroblast proliferation following irradiation. We expect that rhEGF was effective for radiation induced wound healing

  12. Estrogen receptor beta signaling inhibits PDGF induced human airway smooth muscle proliferation.

    Science.gov (United States)

    Ambhore, Nilesh Sudhakar; Katragadda, Rathnavali; Raju Kalidhindi, Rama Satyanarayana; Thompson, Michael A; Pabelick, Christina M; Prakash, Y S; Sathish, Venkatachalem

    2018-04-20

    Airway smooth muscle (ASM) cell hyperplasia driven by persistent inflammation is a hallmark feature of remodeling in asthma. Sex steroid signaling in the lungs is of considerable interest, given epidemiological data showing more asthma in pre-menopausal women and aging men. Our previous studies demonstrated that estrogen receptor (ER) expression increases in asthmatic human ASM; however, very limited data are available regarding differential roles of ERα vs. ERβ isoforms in human ASM cell proliferation. In this study, we evaluated the effect of selective ERα and ERβ modulators on platelet-derived growth factor (PDGF)-stimulated ASM proliferation and the mechanisms involved. Asthmatic and non-asthmatic primary human ASM cells were treated with PDGF, 17β-estradiol, ERα-agonist and/or ERβ-agonist and/or G-protein-coupled estrogen receptor 30 (GPR30/GPER) agonist and proliferation was measured using MTT and CyQuant assays followed by cell cycle analysis. Transfection of small interfering RNA (siRNA) ERα and ERβ significantly altered the human ASM proliferation. The specificity of siRNA transfection was confirmed by Western blot analysis. Gene and protein expression of cell cycle-related antigens (PCNA and Ki67) and C/EBP were measured by RT-PCR and Western analysis, along with cell signaling proteins. PDGF significantly increased ASM proliferation in non-asthmatic and asthmatic cells. Treatment with PPT showed no significant effect on PDGF-induced proliferation, whereas WAY interestingly suppressed proliferation via inhibition of ERK1/2, Akt, and p38 signaling. PDGF-induced gene expression of PCNA, Ki67 and C/EBP in human ASM was significantly lower in cells pre-treated with WAY. Furthermore, WAY also inhibited PDGF-activated PCNA, C/EBP, cyclin-D1, and cyclin-E. Overall, we demonstrate ER isoform-specific signaling in the context of ASM proliferation. Activation of ERβ can diminish remodeling in human ASM by inhibiting pro-proliferative signaling pathways

  13. The Lncrna-TUG1/EZH2 Axis Promotes Pancreatic Cancer Cell Proliferation, Migration and EMT Phenotype Formation Through Sponging Mir-382.

    Science.gov (United States)

    Zhao, Liang; Sun, Hongwei; Kong, Hongru; Chen, Zongjing; Chen, Bicheng; Zhou, Mengtao

    2017-01-01

    Pancreatic carcinoma (PC) is the one of the most common and malignant cancers worldwide. LncRNA taurine upregulated gene 1 (TUG1) was initially identified as a transcript upregulated by taurine, and the abnormal expression of TUG1 has been reported in many cancers. However, the biological role and molecular mechanism of TUG1 in PC still needs further investigation. Quantitative real-time PCR (qRT-PCR) was performed to measure the expression of TUG1 in PC cell lines and tissues. MTT and colony formation assays were used to measure the effect of TUG1 on cell proliferation. A wound healing assay, transwell assay and western blot assay were employed to determine the effect of TUG1 on cell migration and the epithelial mesenchymal transition (EMT) phenotype. RNA-binding protein immunoprecipitation (RIP) and a biotin-avidin pulldown system were performed to confirm the interaction between miR-328 and TUG1. A gene expression array analysis using clinical samples and RT-qPCR suggested that enhancer of zeste homolog 2 (EZH2) was a target of miR-382 in PC. In this study, we reported that TUG1 was overexpressed in PC tissues and cell lines, and high expression of TUG1 predicted poor prognosis. Further experiments revealed that overexpressed TUG1 promoted cell proliferation, migration and contributed to EMT formation, whereas silenced TUG1 led to opposing results. Additionally, luciferase reporter assays, an RIP assay and an RNA-pulldown assay demonstrated that TUG1 could competitively sponge miR-382 and thereby regulate EZH2. Collectively, these findings revealed that TUG1 functions as an oncogenic lncRNA that promotes tumor progression, at least partially, by functioning as an endogenous 'sponge' and competing for miR-382 binding to the miRNA target EZH2. © 2017 The Author(s). Published by S. Karger AG, Basel.

  14. The Lncrna-TUG1/EZH2 Axis Promotes Pancreatic Cancer Cell Proliferation, Migration and EMT Phenotype Formation Through Sponging Mir-382

    Directory of Open Access Journals (Sweden)

    Liang Zhao

    2017-08-01

    Full Text Available Background/Aims: Pancreatic carcinoma (PC is the one of the most common and malignant cancers worldwide. LncRNA taurine upregulated gene 1 (TUG1 was initially identified as a transcript upregulated by taurine, and the abnormal expression of TUG1 has been reported in many cancers. However, the biological role and molecular mechanism of TUG1 in PC still needs further investigation. Methods: Quantitative real-time PCR (qRT-PCR was performed to measure the expression of TUG1 in PC cell lines and tissues. MTT and colony formation assays were used to measure the effect of TUG1 on cell proliferation. A wound healing assay, transwell assay and western blot assay were employed to determine the effect of TUG1 on cell migration and the epithelial mesenchymal transition (EMT phenotype. RNA-binding protein immunoprecipitation (RIP and a biotin-avidin pulldown system were performed to confirm the interaction between miR-328 and TUG1. A gene expression array analysis using clinical samples and RT-qPCR suggested that enhancer of zeste homolog 2 (EZH2 was a target of miR-382 in PC. Results: In this study, we reported that TUG1 was overexpressed in PC tissues and cell lines, and high expression of TUG1 predicted poor prognosis. Further experiments revealed that overexpressed TUG1 promoted cell proliferation, migration and contributed to EMT formation, whereas silenced TUG1 led to opposing results. Additionally, luciferase reporter assays, an RIP assay and an RNA-pulldown assay demonstrated that TUG1 could competitively sponge miR-382 and thereby regulate EZH2. Conclusion: Collectively, these findings revealed that TUG1 functions as an oncogenic lncRNA that promotes tumor progression, at least partially, by functioning as an endogenous ‘sponge’ and competing for miR-382 binding to the miRNA target EZH2.

  15. shRNA-mediated EMMPRIN silencing inhibits human leukemic monocyte lymphoma U937 cell proliferation and increases chemosensitivity to adriamycin.

    Science.gov (United States)

    Gao, Hui; Jiang, Qixiao; Han, Yantao; Peng, Jianjun; Wang, Chunbo

    2015-03-01

    EMMPRIN is a widely distributed cell surface glycoprotein, which plays an important role in tumor progression and confers resistance to some chemotherapeutic drugs. Recent studies have shown that EMMPRIN overexpression indicates poor prognosis in acute myeloid leukemia (AML). However, little was known on the role of EMMPRIN in leukemia. Human leukemia cell line U937 was stably transfected with a EMMPRIN-targeted shRNA-containing vector to investigate the effect of EMMPRIN on cellular functions. EMMPRIN expression was monitored by qRT-PCR and Western blotting. Cell viability and proliferation were determined by trypan blue exclusion and BrdU labeling, respectively. Cell cycle and apoptosis were analyzed by flow cytometry. Cytotoxicity of chemotherapeutic agent adriamycin on cells was assessed by MTT assay. Knockdown of EMMPRIN gene significantly inhibited cell viability and decreased cell proliferation. Fluorescence-activated cell-sorting analysis revealed that the reduced EMMPRIN expression resulted in cell cycle arrest at G1 phase and induced apoptosis. Meanwhile, western blotting analysis showed that EMMPRIN knockdown was associated with downregulation of cell cycle- and apoptosis-related molecules including cyclin D1, cyclin E, as well as increase in cleavage of caspase-3 and PARP. This study also showed that silencing of EMMPRIN sensitized U937 cells to Adriamycin. EMMPRIN is involved in proliferation, growth, and chemosensitivity of human AML line U937, indicating that EMMPRIN may be a promising therapeutic target for AML.

  16. MiR-138 promotes smooth muscle cells proliferation and migration in db/db mice through down-regulation of SIRT1

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Juan [Department of Gynecology, Changzhou Maternity and Children Health Hospital, Changzhou, Jiangsu 213003 (China); Li, Li; Yun, Hui-fang [Department of Anesthesiology, Changzhou No. 2 People' s Hospital, Changzhou, Jiangsu 213003 (China); Han, Ye-shan, E-mail: yeshanhan123@163.com [Department of Anesthesiology, Changzhou No. 2 People' s Hospital, Changzhou, Jiangsu 213003 (China)

    2015-08-07

    Background: Diabetic vascular smooth muscle cells (VSMCs) exhibit significantly increased rates of proliferation and migration, which was the most common pathological change in atherosclerosis. In addition, the study about the role for miRNAs in the regulation of VSMC proliferation is just beginning to emerge and additional miRNAs involved in VSMC proliferation modulation should be identified. Methods: The expression of miR-138 and SIRT1 were examined in SMCs separated from db/db mice and in SMC lines C-12511 exposed to high glucose with qRT-PCR and western blot. The regulation of miR-138 on the expression of SMCs was detected with luciferase report assay. VSMCs proliferation and migration assays were performed to examine the effect of miR-138 inhibitor on VSMCs proliferation and migration. Results: We discovered that higher mRNA level of miR-138 and reduced expression of SIRT1 were observed in SMCs separated from db/db mice and in SMC lines C-12511. Moreover, luciferase report assay showed that the activity of SIRT1 3′-UTR was highly increased by miR-138 inhibitor and reduced by miR-138 mimic. In addition, we examined that the up-regulation of NF-κB induced by high glucose in SMCs was reversed by resveratrol and miR-138 inhibitor. MTT and migration assays showed that miR-138 inhibitor attenuated the proliferation and migration of smooth muscle cells. Conclusion: In this study, we revealed that miR-138 might promote proliferation and migration of SMC in db/db mice through suppressing the expression of SIRT1. - Highlights: • Higher mRNA level of miR-138 was observed in SMCs from db/db mice. • The mRNA and protein level of SIRT1 in SMCs from db/db mice were greatly reduced. • miR-138 could regulate the expression of SIRT1 in SMCs. • SIRT1 overexpression reversed the up-regulation of acetylized p65 and NF-κB induced by high glucose. • MiR-138 inhibitor reversed VSMCs proliferation and migration induced by high glucose.

  17. LncRNA TUG1 is upregulated and promotes cell proliferation in osteosarcoma.

    Science.gov (United States)

    Yun-Bo, Feng; Xiao-Po, Liu; Xiao-Li, Li; Guo-Long, Cao; Pei, Zhang; Fa-Ming, Tian

    2016-01-01

    To examine the expression and function of long non-coding RNA taurine up-regulated 1 ( TUG1 ) in human osteosarcoma cells. Real-time quantitive PCR was used to detect the transcription level of TUG1 in a series of osteosarcoma cell lines. Knockdown of TUG1 in U2OS cells was carried out by transient transfection of siRNAs. MTT assay was performed to access the cell growth rates. Afterwards, RNA and protein of these cells were extracted to analyze the transfection efficient as well as the expression of other molecules. Compared to the normal cell line, TUG1 exhibited a significant upregulation in osteosarcoma cells. Phenotyping analysis showed the growth-promotion activity of TUG1 , since knockdown of TUG1 resulted in declined proliferation. We also found that AKT phosphorylation was impaired after TUG1 was inhibited, suggesting that the AKT pathway was involved in the regulation of TUG1 in U2OS cells. Our data provided evidence that TUG1 was upregulated and acted as a possible oncogene via positively regulating cell proliferation in osteosarcoma cells.

  18. Effects of Bauhinia championii (Benth.) Benth. polysaccharides on the proliferation and cell cycle of chondrocytes.

    Science.gov (United States)

    Cai, Liangliang; Ye, Hongzhi; Yu, Fangrong; Li, Huiting; Chen, Jiashou; Liu, Xianxiang

    2013-05-01

    It has been recently shown that polysaccharides isolated from plants exhibit a number of beneficial therapeutic properties. Bauhinia championii (Benth.) Benth. has been widely used for the clinical treatment of knee osteoarthritis (OA) in China. However, the underlying molecular mechanisms of knee OA treatment have yet to be elucidated. In the present study, we investigated the effects of Bauhinia championii (Benth.) Benth. polysaccharides (BCBPs) on the proliferation and cell cycle of chondrocytes on 4-week-old male Sprague Dawley rats. Immunohistochemical staining was used to identify chondrocytes and an MTT assay was used to evaluate cell viability. Flow cytometry was used for cell cycle analysis. The mRNA and protein expression levels of cyclin D1, CDK4 and CDK6 in chondrocytes were detected using reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis, respectively. The data demonstrate that BCBP treatment increased the viability of chondrocytes. In addition, BCBP treatment reduced the cell population in the G0/G1 phase, whereas the cell population was increased in the S phase. Furthermore, BCBP treatment enhanced the expression of cyclin D1, CDK4 and CDK6. These results indicate that BCBP treatment promotes cell proliferation by accelerating the G1/S transition.

  19. Protease-activated receptor 2 agonist increases cell proliferation and invasion of human pancreatic cancer cells

    Science.gov (United States)

    XIE, LIQUN; DUAN, ZEXING; LIU, CAIJU; ZHENG, YANMIN; ZHOU, JING

    2015-01-01

    The aim of this study was to determine the expression of protease-activated receptor 2 (PAR-2) in the human pancreatic cancer cell line SW1990, and to evaluate its effect on cell proliferation and invasion. The expression of PAR-2 protein and mRNA in SW1990 cells was determined by immunocytochemistry and reverse transcription polymerase chain reaction (PCR), respectively. MTT and cell invasion and migration assays, as well as semi-quantitative PCR and zymography analysis, were additionally performed. PAR-2 mRNA was significantly upregulated in the cells treated with trypsin or the PAR-2 activating peptide Ser-Leu-Ile-Gly-Lys-Val (SLIGKV) (P0.05). Trypsin and SLIGKV significantly promoted SW1990 cell proliferation in a dose- and time-dependent manner (P<0.05). Compared with the control group, trypsin and SLIGKV significantly increased the mRNA expression (P<0.01) and gelatinolytic activity (P<0.01) of matrix metalloproteinase (MMP)-2. In conclusion, PAR-2 is expressed in SW1990 cells. PAR-2 activation may promote the invasion and migration of human pancreatic cancer cells by increasing MMP-2 expression. PMID:25452809

  20. LncRNA TUG1 is upregulated and promotes cell proliferation in osteosarcoma

    Directory of Open Access Journals (Sweden)

    Yun-Bo Feng

    2016-01-01

    Full Text Available Objective: To examine the expression and function of long non-coding RNA taurine up-regulated 1 (TUG1 in human osteosarcoma cells. Methods: Real-time quantitive PCR was used to detect the transcription level of TUG1 in a series of osteosarcoma cell lines. Knockdown of TUG1 in U2OS cells was carried out by transient transfection of siRNAs. MTT assay was performed to access the cell growth rates. Afterwards, RNA and protein of these cells were extracted to analyze the transfection efficient as well as the expression of other molecules. Results: Compared to the normal cell line, TUG1 exhibited a significant upregulation in osteosarcoma cells. Phenotyping analysis showed the growth-promotion activity of TUG1, since knockdown of TUG1 resulted in declined proliferation. We also found that AKT phosphorylation was impaired after TUG1 was inhibited, suggesting that the AKT pathway was involved in the regulation of TUG1 in U2OS cells. Conclusion: Our data provided evidence that TUG1 was upregulated and acted as a possible oncogene via positively regulating cell proliferation in osteosarcoma cells.

  1. Hydroxysafflor yellow A suppresses oxidized low density lipoprotein induced proliferation of vascular smooth muscle cells

    Directory of Open Access Journals (Sweden)

    Lin Sheng

    2012-06-01

    Full Text Available To investigate the relationship between the suppression of Hydroxysafflor yellow A (HSYA on the oxidized low density lipoprotein (ox-LDL induced proliferation of vascular smooth muscle cells (VSMCs and the mRNA and protein expression of extracellular signal-regulated protein kinase 1/2 (ERK1/2 and mitogen activated protein kinase phospholipase-1 (MAKP-1, VSMCs were treated with HSYA at 10 ?mol/L and/or ox-LDL at 35 mg/L for 48 h. MTT assay was done to measure cell survival rate, flow cytometry to detect cell cycle, reverse transcription PCR and Western blot to detect the expression of ERK1/2 and MAKP-1. When compared to cells treated with ox-LDL alone, the survival rate of cells treated with two reagents was reduced and the proportion of cells in G0/G1 phase significantly increased, with increased MKP-1 expression. The study suggests HSYA can inhibit VSMC proliferation via increasing MKP-1 expression, reducing p-ERK1/2 activity and suppressing cell cycle.

  2. Proliferation and mineralization ability of dental pulp cells derived from primary and permanent teeth

    Directory of Open Access Journals (Sweden)

    Suttatip Kamolmatyakul

    2011-04-01

    Full Text Available The aims of this study were to compare the proliferation and mineralization ability of CFU-F selected dental pulp cellsderived from primary and permanent teeth. Those cells were isolated by enzyme digestion and analyzed for their colonyformingcapacity. The cell proliferation was measured by the MTT assay on day 1, day 7, and day14. Alizarin Red S stainingwas used to detect mineralized nodule formation of the cells on day 7, 14, 21, and 28. Proliferation of CFU-F selected pulpcells from primary teeth was significantly higher than that of CFU-F selected pulp cells from permanent teeth in all periods ofthe experiment. Upon cultured cells in mineralization inducing media, the mineralized nodules appeared as early as day 14 inCFU-F selected pulp cells from primary teeth and MG-63, whereas those of CFU-F selected pulp cells from permanent teethcan be found at day 21. On day 21 and day 28, the mineralized nodules of the CFU-F selected pulp cells from the primaryteeth group were more than those in the CFU-F selected pulp cells from the permanent teeth group. Mineralized noduleformation in the CFU-F selected pulp cells from the permanent teeth group appeared later and were less than those ofCFU-F selected pulp cells from primary teeth. However, mineralized nodules in CFU-F selected pulp cells from the permanentteeth group increased very fast after their appearance. Those results suggest that CFU-F selected pulp cells from primaryteeth had a higher proliferation rate and mineralization rate when compared to CFU-F selected pulp cells from permanentteeth.

  3. Bio-active molecules modified surfaces enhanced mesenchymal stem cell adhesion and proliferation

    International Nuclear Information System (INIS)

    Mobasseri, Rezvan; Tian, Lingling; Soleimani, Masoud; Ramakrishna, Seeram; Naderi-Manesh, Hossein

    2017-01-01

    Surface modification of the substrate as a component of in vitro cell culture and tissue engineering, using bio-active molecules including extracellular matrix (ECM) proteins or peptides derived ECM proteins can modulate the surface properties and thereby induce the desired signaling pathways in cells. The aim of this study was to evaluate the behavior of human bone marrow mesenchymal stem cells (hBM-MSCs) on glass substrates modified with fibronectin (Fn), collagen (Coll), RGD peptides (RGD) and designed peptide (R-pept) as bio-active molecules. The glass coverslips were coated with fibronectin, collagen, RGD peptide and R-peptide. Bone marrow mesenchymal stem cells were cultured on different substrates and the adhesion behavior in early incubation times was investigated using scanning electron microscopy (SEM) and confocal microscopy. The MTT assay was performed to evaluate the effect of different bio-active molecules on MSCs proliferation rate during 24 and 72 h. Formation of filopodia and focal adhesion (FA) complexes, two steps of cell adhesion process, were observed in MSCs cultured on bio-active molecules modified coverslips, specifically in Fn coated and R-pept coated groups. SEM image showed well adhesion pattern for MSCs cultured on Fn and R-pept after 2 h incubation, while the shape of cells cultured on Coll and RGD substrates indicated that they might experience stress condition in early hours of culture. Investigation of adhesion behavior, as well as proliferation pattern, suggests R-peptide as a promising bio-active molecule to be used for surface modification of substrate in supporting and inducing cell adhesion and proliferation. - Highlights: • Bioactive molecules modified surface is a strategy to design biomimicry scaffold. • Bi-functional Tat-derived peptide (R-pept) enhanced MSCs adhesion and proliferation. • R-pept showed similar influences to fibronectin on FA formation and attachment.

  4. Fenofibrate suppressed proliferation and migration of human neuroblastoma cells via oxidative stress dependent of TXNIP upregulation

    Energy Technology Data Exchange (ETDEWEB)

    Su, Cunjin; Shi, Aiming; Cao, Guowen [Department of Pharmacy, The Second Affiliated Hospital of Soochow University, Suzhou, 215004 (China); Tao, Tao [Department of Urology, Zhongda Hospital, Medical School of Southeast University, Nanjing, 210009 (China); Chen, Ruidong [Department of Gastroenterology, The Second Affiliated Hospital of Soochow University, Suzhou, 215004 (China); Hu, Zhanhong; Shen, Zhu; Tao, Hong; Cao, Bin [Department of Pharmacy, The Second Affiliated Hospital of Soochow University, Suzhou, 215004 (China); Hu, Duanmin, E-mail: hudmsdfey@sina.com [Department of Gastroenterology, The Second Affiliated Hospital of Soochow University, Suzhou, 215004 (China); Bao, Junjie, E-mail: baojjsdfey@sina.com [Department of Pharmacy, The Second Affiliated Hospital of Soochow University, Suzhou, 215004 (China)

    2015-05-15

    There are no appropriate drugs for metastatic neuroblastoma (NB), which is the most common extra-cranial solid tumor for childhood. Thioredoxin binding protein (TXNIP), the endogenous inhibitor of ROS elimination, has been identified as a tumor suppressor in various solid tumors. It reported that fenofibrate exerts anti-tumor effects in several human cancer cell lines. However, its detail mechanisms remain unclear. The present study assessed the effects of fenofibrate on NB cells and investigated TXNIP role in its anti-tumor mechanisms. We used MTT assay to detect cells proliferation, starch wound test to investigate cells migration, H{sub 2}DCF-DA to detect intracellular ROS, siRNA to interfere TXNIP and peroxisome proliferator-androgen receptor-alpha (PPAR-α) expression, western blot to determine protein levels, flow cytometry to analyze apoptosis. Fenofibrate suppressed proliferation and migration of NB cells, remarkably increased intracellular ROS, upregulated TXNIP expression, promoted cell apoptosis. Furthermore, inhibition of TXNIP expression attenuated anti-tumor effects of fenofibrate, while inhibition of PPAR-α had no influences. Our results indicated the anti-tumor role of fenofibrate on NB cells by exacerbating oxidative stress and inducing apoptosis was dependent on the upregulation of TXNIP. - Highlights: • We found that fenofibrate suppressed proliferation and migration of NB cells. • We found that fenofibrate remarkably increased intracellular ROS, upregulated TXNIP expression, and promoted cell apoptosis. • Inhibition of TXNIP expression attenuated anti-tumor effects of fenofibrate, while inhibition of PPAR-α had no influences. • Our results indicated the anti-tumor role of fenofibrate on NB cells was dependent on the upregulation of TXNIP.

  5. Micromachined Si channel width and tortuosity on human osteoblast cell attachment and proliferation

    International Nuclear Information System (INIS)

    Leber, Christopher; Choi, Hongsoo; Bose, Susmita; Bandyopadhyay, Amit

    2010-01-01

    In this study, influence of coating chemistry, channel width and tortuosity of various two-dimensional micro-channels were explored on micromachined Si using osteoblast precursor cells line 1 (OPC1). The rationale for our study is to delineate the influence of different porosity parameters on bone cell attachment and proliferation in vitro. Channel widths of 100, 200, 300, 400, and 600 μm; channel bends of 0, 1, and 2 right angles; and gold and silicon dioxide coatings on single-crystal Si were studied. Experiments were conducted with channel tops under glass covered and uncovered conditions keeping the channel depth at 220 μm. Independent samples were evaluated using SEM imaging and MTT assay to measure bone cell morphology and quantity. Images were taken of micro-channels and exterior chambers at 50x, 500x, 1000x, and 5000x magnifications. Channel and chamber cell densities were scored as follows: bare (score = 0), scattered (1), limited (2), abundant (3), and overflowing (4). Samples were then scored and statistically analyzed for major differences. In general, OPC1 cells proliferated at least 5% or better based on cell numbers under uncovered conditions than glass covered. Channel widths of 100 μm largely prohibited cell proliferation and diffusion by narrow path inhibition with the lowest average score of 1.17. Among channel bends of 0, 1, and 2 right angles, an increase in micro-channel tortuosity from 0-2 bends amplified OPC1 cell growth upwards of ∼ 6.6%. A one-way ANOVA showed significant differences in cell quantity for alternating channel tortuosity at a significance level of p < 0.05. No preference was found for gold or silicon dioxide coatings on Si for bone cell proliferation.

  6. MiR-223 targeting MAFB suppresses proliferation and migration of nasopharyngeal carcinoma cells

    International Nuclear Information System (INIS)

    Yang, Wanyong; Lan, Xi; Li, Dongmin; Li, Tao; Lu, Shemin

    2015-01-01

    Mounting evidence suggests that miRNAs have major functions in tumor pathogenesis, and this study aimed to identify the candidate miRNA and investigate its role in nasopharyngeal carcinoma (NPC). MiRNA and mRNA expressions were screened by microarray assays. The cell proliferation, colony formation and migration ability were measured by MTT, soft agar and wound healing assays, respectively. The tumor growth suppression was evaluated by xenografting in nude mice. The plasma miR-223 levels in NPC patients were detected by TaqMan analysis. Real-time quantitative PCR and Western blotting were used to confirm miR-223 and MAFB expression levels. The targeting relationship between miR-223 and MAFB was verified using dual luciferase reporter assay. The miR-223 expression was decreased in CNE-1, CNE-2 cells as compared with NP69 cells, an immortalized human nasopharyngeal epithelial cell line, and its level also reduced in NPC patients’ plasma as compared with healthy controls. Exogenous expression of miR-223 in CNE-2 cells could inhibit cell proliferation both in vitro and in vivo. Extrogenous miR-223 in CNE-2 cells would decrease the ability of colony formation and migration. MAFB, a transcription factor of Maf family members, was identified as a target gene of miR-223. We found that migration and invasion abilities were inhibited by MAFB silencing. MiR-223 negatively regulates the growth and migration of NPC cells via reducing MAFB expression, and this finding provides a novel insight into understanding miR-223 regulation mechanism in nasopharyngeal carcinoma tumorigenesis

  7. Effect of all-trans retinoic acid on the proliferation and differentiation of brain tumor stem cells

    Directory of Open Access Journals (Sweden)

    Niu Chao

    2010-08-01

    Full Text Available Abstract Objective To investigate the effect of all-trans retinoic acid(ATRA on the proliferation and differentiation of brain tumor stem cells(BTSCs in vitro. Methods Limiting dilution and clonogenic assay were used to isolate and screen BTSCs from the fresh specimen of human brain glioblastoma. The obtained BTSCs, which were cultured in serum-free medium, were classified into four groups in accordance with the composition of the different treatments. The proliferation of the BTSCs was evaluated by MTT assay. The BTSCs were induced to differentiate in serum-containing medium, and classified into the ATRA group and control group. On the 10th day of induction, the expressions of CD133 and glial fibrillary acidic protein (GFAP in the differentiated BTSCs were detected by immunofluorescence. The differentiated BTSCs were cultured in serum-free medium, the percentage and the time required for formation of brain tumor spheres (BTS were observed. Results BTSCs obtained by limiting dilution were all identified as CD133-positive by immunofluorescence. In serum-free medium, the proliferation of BTSCs in the ATRA group was observed significantly faster than that in the control group, but slower than that in the growth factor group and ATRA/growth factor group, and the size of the BTS in the ATRA group was smaller than that in the latter two groups(P P P P Conclusion ATRA can promote the proliferation and induce the differentiation of BTSCs, but the differentiation is incomplete, terminal differentiation cannot be achieved and BTSs can be formed again.

  8. The role of integrin-α5 in the proliferation and odontogenic differentiation of human dental pulp stem cells.

    Science.gov (United States)

    Cui, Li; Xu, Shuaimei; Ma, Dandan; Gao, Jie; Liu, Ying; Yue, Jing; Wu, Buling

    2014-02-01

    It has been reported that integrin-α5 (ITGA5) activity is related to cell proliferation, differentiation, migration, and organ development. However, the involvement of ITGA5 in the biological functions of human dental pulp stem cells (hDPSCs) has not been explored. The aim of this study was to investigate the role of ITGA5 in the proliferation and odontogenic differentiation of hDPSCs. We knocked down ITGA5 in hDPSCs using lentivirus-mediated ITGA5 short hairpin RNA (shRNA). Changes in the proliferation in hDPSCs infected with lentiviruses expressing ITGA5-specific shRNA or negative control shRNA were examined using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 5-ethynyl-2'-deoxyuridine labeling. Both ITGA5 knockdown cells and shMock cells were cultured in mineralization medium for 3 weeks, and the differentiation of cells was detected with alizarin red S staining. The expression of odontogenic differentiation-related molecular markers was assessed using real-time polymerase chain reaction and Western blot assays. The knockdown of ITGA5 decreased the proliferation capacity of hDPSCs. ITGA5 shRNA promoted odontogenic differentiation of hDPSCs with the enhanced formation of mineralized nodules. It also up-regulated the messenger RNA expression of multiple markers of odontogenesis and the expression of dentin sialophosphoprotein protein. These findings suggest that ITGA5 plays an important role in maintaining hDPSCs in a proliferative state. The inhibition of ITGA5 signaling promotes the odontogenic differentiation of hDPSCs. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  9. Yi Qi Qing Re Gao-containing serum inhibits lipopolysaccharide-induced rat mesangial cell proliferation by suppressing the Wnt pathway and TGF-β1 expression.

    Science.gov (United States)

    Yang, Liping; Sun, Xueyan; Zhan, Yongli; Liu, Huijie; Wen, Yumin; Mao, Huimin; Dong, X I; Li, Ping

    2016-04-01

    The aim of the present study was to investigate the effect of Yi Qi Qing Re Gao-containing serum (YQ-S) on rat mesangial cell (MC) proliferation and to investigate the underlying mechanism. MCs were divided into the control, lipopolysaccharide (LPS)-stimulated, YQ-S and fosinopril-containing serum (For-S) groups, and cultured for 48 h. An MTT assay was used to evaluate the proliferation of MCs. In addition, reverse transcription-quantitative polymerase chain reaction and western blot analysis were conducted to detect the expression levels of Wnt4, β-catenin and transforming growth factor (TGF)-β1 in MCs. The results indicated that YQ-S inhibited LPS-induced MC proliferation. The Wnt4 and TGF-β1 mRNA expression levels were reduced in the YQ-S group (P<0.01 or P<0.05). Furthermore, the Wnt4, β-catenin and TGF-β1 protein expression levels were suppressed in the YQ-S group (P<0.01 or P<0.05). Therefore, YQ-S appears to inhibit MC proliferation, and its mechanism may involve the inhibition of the Wnt signaling pathway and downregulation of TGF-β1 expression.

  10. [Effects of 17-AAG on the proliferation and apoptosis of human lung cancer A549 and H446 cells].

    Science.gov (United States)

    Niu, Ben; Lin, Jingshuang; Feng, Tao

    2015-04-01

    To observe the effect of 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) on the apoptosis of human lung cancer cell lines A549 and H446, and to investigate the potential mechanisms. Proliferation inhibition and apoptosis assays, and the cell cycles were detected by MTT and flow cytometry respectively. Western blot was used to determine the expression level of proteins such as Hsp90, Hsp70, AKt, Her-2, Bcl-2 and Bax. After treated with 17-AAG, the proliferation of both A549 and H446 cells was inhibited significantly in a dose-dependent manner; as the concentration of 17-AAG was from 50 to 500 nmol/L, the IC₅₀ values to A549 and H446 cell lines were (222 ± 13) nmol/L and (189 ± 7) nmol/L respectively at 48 h. Cell cycle assays showed that 17-AAG was able to arrest cell cycles of A549 and H446 cell lines at the G₂/M phase. Apoptosis assay showed that 17-AAG was capable of inducing apoptosis in A549 and H446 cell lines. After treated with 17-AAG for 48 h, there were significant differences between the 400 nmol/L groups(46.3% for A549 cell line and 56.9% for H446 cell line) and the control group (11.9% for A549 cell line and 6.9% for H446 cell line, P AAG treatment: Akt and Her-2 decreased significantly while the expression of Hsp70 increased. Meanwhile, the expression of Bcl-2 decreased but that of Bax increased, indicating that 17-AAG was able to promote apoptosis mode in A549 and H446 cells. 17-AAG can regulate the expression level of apoptosis-related proteins such as Bax and Bcl-2 by Hsp90 signaling pathway in A549 and H446 cells, and ultimately inhibit cell proliferation and induce apoptosis.

  11. Selumetinib suppresses cell proliferation, migration and trigger apoptosis, G1 arrest in triple-negative breast cancer cells.

    Science.gov (United States)

    Zhou, Yan; Lin, Shuchen; Tseng, Kuo-Fu; Han, Kun; Wang, Yaling; Gan, Zhi-Hua; Min, Da-Liu; Hu, Hai-Yan

    2016-10-21

    Triple-negative breast cancer (TNBC) has aggressive progression with poor prognosis and ineffective treatments. Selumetinib is an allosteric, ATP-noncompetitive inhibitor of MEK1/2, which has benn known as effective antineoplastic drugs for several malignant tumors. We hypothesized that Selumetinib might be potential drug for TNBC and explore the mechanism. After treated with Selumetinib, the viability and mobility of HCC1937 and MDA-MB-231 were detected by MTT, tunnel, wound-healing assay, transwell assay and FCM methods. MiR array was used to analysis the change of miRs. We predicted and verified CUL1 is the target of miR-302a using Luciferase reporter assay. We also silenced the CUL1 by siRNA, to clarify whether CUL1 take part in the cell proliferation, migration and regulated its substrate TIMP1 and TRAF2. Moreover, after transfection, the antagomir of miR-302a and CUL1 over-expressed plasmid into HCC1937 and MDA-MB-231 cell accompanied with the Selumetinib treatment, we detected the proliferation and migration again. Selumetinib reduce the proliferation, migration, triggered apoptosis and G1 arrest in TNBC cell lines. In this process, the miR-302a was up-regulated and inhibited the CUL1 expression. The later negatively regulated the TIMP1 and TRAF2. As soon as we knockdown miR-302a and over-expression CUL1 in TNBC cells, the cytotoxicity of Selumetinib was reversed. MiR-302a targeted regulated the CUL1 expression and mediated the Selumetinib-induced cytotoxicity of triple-negative breast cancer.

  12. Preventing proliferation

    International Nuclear Information System (INIS)

    Rathjens, G.

    1983-01-01

    Challenging the argument that nuclear proliferation may be stabilizing, the author cites the Israeli attack on Iraq as evidence that emergent nuclear states may be moved to attack their adversaries.The larger the number of decision makers who can unleash nuclear weapons, the greater the liklihood of their use. Several reasons are cited for nations to seek nuclear capability: the accelerated spread of technology, the deterioration in US-Soviet relations and strength relative to their nations, the high cost of conventional weapons, and a loss of confidence in the international safeguards system. The imposition of constraints, such as a Comprehensive Test Ban Treaty, on nuclear trade and technology transfer are likely to have a high cost. The US position on this issue is likely to be determined by the balance of power with the Soviet Union. 5 references

  13. [Effects of cucurmosin on the cell proliferation and apoptosis in human pancreatic PANC-1 cells].

    Science.gov (United States)

    Xu, Chun-Sen; Huang, He-Guang; Chen, Ming-Huang

    2012-02-01

    To observe the effects of cucurmosin (CUS) on the cell proliferation and apoptosis in pancreatic PANC-1 cells. The inhibition of CUS on the PANC-1 cell growth was observed using MTT assay. The inhibition ratio of CUS on the pancreatic orthotopic transplantation was in vivo observed in the NOD/SCID mouse model. The changes of microstructure of the apoptosis-inducing effect of CUS on PANC-1 was observed under electron microscope. The cell cycle and apoptosis after CUS intervention was detected using flow cytometry. The Caspase-3 activity after CUS treatment was detected using enzyme linked immunospecific assay (ELISA). Treatment with CUS at the dose of 0.125, 0.25, and 0.5 mg/kg inhibited the growth of pancreatic carcinoma PANC-1 xenografs with the ratio of 45.2%, 50.0%, and 59.7%, respectively (P PANC-1 cells in a dose-dependent maner. Being exposed to 40.0 microg/mL of the CUS for 24, 48, and 72 h, the percentage of G0/ G1 phase cells was 56.60% +/- 6.65%, 67.83% +/- 6.76%, and 77.00% +/- 6.73%, respectively (P PANC-1 cells in the G0/G1 phase of the cell cycle in a time-dependent maner. CUS significantly inhibited the growth of PANC-1 cells possibly through the G0/G1 cell cycle arrest and apoptosis.

  14. [Autologous regulatory T cells can suppress the proliferation of lymphoma cell line in vitro].

    Science.gov (United States)

    Ying, Zhi-Tao; Guo, Jun; Ren, Jun; Kong, Yan; Yuan, Zhi-Hong; Liu, Xi-Juan; Zhang, Chen; Zheng, Wen; Song, Yu-Qin; Zhang, Yun-Tao; Zhu, Jun

    2009-06-01

    This study was aimed to investigate the suppressive effect of regulatory T (Treg) cells on the T cell lymphoma EL4 cell line and to explore its mechanism. C57BL/6 Mouse Treg cells were isolated by MACS (magnetic cell sorting). The purity and the expression of Foxp3 were detected by flow cytometry. The suppressive effect of sorted Treg cells on EL4 cells was detected by MTT assay. The secretion of TGF-beta1 and IL-10 was examined by enzyme-linked immunosorbent assay (ELISA). The results showed that CD4(+)CD25(+) T cells could be successfully isolated by MACS with the purity reaching 91.6% and the expression level of Foxp3 was 78.9%. The ratio of viable cells was more than 95%. Regulatory T cells could suppress the proliferation of EL4 cells effectively in the presence of antigen presenting cells (APCs). And the suppressive effect was most significant at 1:1 ratio. In addition, the suppression still existed without APCs. TGF-beta1 and IL-10 could not be detected by ELISA. It is concluded that the Treg cells can suppress T lymphoma cell in vitro. The suppressive effect of Treg cells works in dose-dependent manner, but not in cytokine-dependent manner. The mechanism of this suppression may take effect through cell-cell contact.

  15. Structure-activity relationship studies of 5,7-dihydroxyflavones as naturally occurring inhibitors of cell proliferation in human leukemia HL-60 cells.

    Science.gov (United States)

    Ninomiya, Masayuki; Nishida, Kyohei; Tanaka, Kaori; Watanabe, Kunitomo; Koketsu, Mamoru

    2013-07-01

    Flavonoids are widely occurring polyphenols that are found in plants. The aim of this study was to investigate the structure-activity relationships of 5,7-dihydroxyflavones, with a focus on the effect of B ring structure substitution on the antiproliferative effects of the compounds in human leukemia HL-60 cells. We prepared a series of 5,7-dihydroxyflavones and evaluated their ability to inhibit the proliferation of HL-60 cells by using the MTT assay. The apoptosis- and cell differentiation-inducing ability of the most potent flavones were investigated using staining and morphological analyses. This study explored the antileukemic and chemopreventive potency of 5,7-dihydroxyflavones, particularly diosmetin and chrysoeriol, which have both hydroxy and methoxy groups on the B ring.

  16. Leukemia inhibitory factor increases the proliferation of human endometrial stromal cells and expression of genes related to pluripotency

    Directory of Open Access Journals (Sweden)

    Mojdeh Salehnia

    2017-08-01

    Full Text Available Background: Concerning the low population of human endometrial mesenchymal cells within the tissue and their potential application in the clinic and tissue engineering, some researches have been focused on their in vitro expansion. Objective: The aim of this study was to evaluate the effect of leukemia inhibitory factor (LIF as a proliferative factor on the expansion and proliferation of human endometrial stromal cells. Materials and Methods: In this experimental study, the isolated and cultured human endometrial stromal cells from women at ovulatory phase aged 20-35 years, after fourth passage were divided into control and LIF-treated groups. In the experimental group, the endometrial cells were treated by 10 ng/ml LIF in culture media and the cultured cells without adding LIF considered as control group. Both groups were evaluated and compared for proliferation rate using MTT assay, for CD90 marker by flow cytometric analysis and for the expression of Oct4, Nanog, PCNA and LIFr genes using real-time RT-PCR. Results: The proliferation rate of control and LIF-treated groups were 1.17±0.17 and 1.61±0.06 respectively and there was a significant increase in endometrial stromal cell proliferation following in vitro treatment by LIF compared to control group (p=0.049. The rate of CD90 positive cells was significantly increased in LIFtreated group (98.96±0.37% compared to control group (94.26±0.08% (p=0.0498. Also, the expression ratio of all studied genes was significantly increased in the LIFtreated group compared to control group (p=0.0479. Conclusion: The present study showed that LIF has a great impact on proliferation, survival, and maintenance of pluripotency of human endometrial stromal cells and it could be applicable in cell therapies.

  17. Estrogen receptor β inhibits estradiol-induced proliferation and migration of MCF-7 cells through regulation of mitofusin 2.

    Science.gov (United States)

    Ma, Li; Liu, Yueping; Geng, Cuizhi; Qi, Xiaowei; Jiang, Jun

    2013-06-01

    In the present study, we investigated whether estrogen receptor (ER) β affected the proliferation and migration of the human breast cancer cell line MCF-7 through regulation of mitofusin 2 (mfn2). A previous study reported that mfn2 may be regulated by ER through a non-classical pathway; in this pathway, the ER modulates the activities of other transcription factors by stabilizing their binding to DNA and/or recruiting coactivators to the complex. However, the previous study, unlike the study presented here, did not directly explore the interactions between ER and mfn2. Here, RT-PCR and western blot analysis were used to test the expression of mfn2 in MCF-7 cells after exposure to different doses of estradiol (E2). The ability of cells to proliferate and migrate was determined by MTT assay and a monolayer-wounding protocol, respectively. Finally, changes in MCF-7 cell biology after transfection with ERβ or mfn2 expression vectors were investigated, and the role of ERβ in mfn2 expression was also explored. Our results showed that E2 attenuated mfn2 expression in a dose-dependent manner, concomitant with the activation of proliferation and migration of MCF-7 cells. The mfn2 expression vector effectively suppressed E2-induced upregulation of PCNA and migration in MCF-7 cells. ERβ inhibited the E2-induced mfn2 downregulation that accompanied the inhibition of proliferation and migration in MCF-7 cells. Briefly, ERβ may inhibit E2-induced proliferation and migration of MCF-7 cells through regulation of mfn2.

  18. Long Noncoding RNA Taurine-Upregulated Gene 1 Promotes Cell Proliferation and Invasion in Gastric Cancer via Negatively Modulating miRNA-145-5p.

    Science.gov (United States)

    Ren, Kewei; Li, Zhen; Li, Yahua; Zhang, Wenzhe; Han, Xinwei

    2017-05-24

    Long noncoding RNA (lncRNA) taurine-upregulated gene 1 (TUG1) is involved in the development and carcinogenesis of various tumors, suggesting the diagnostic potential of TUG1 in these cancers. However, the exact role of TUG1 and its underlying mechanism in gastric cancer (GC) remain unknown. In this study, the expression of TUG1 and miR-145-5p in GC cell lines and nonmalignant gastric epithelial cell lines was detected by qRT-PCR. BGC-823 and SGC-7901 cells were transfected with si-TUG1, pcDNA 3.1-TUG1, miR-145-5p mimics, or matched controls. The biological function of TUG1 and miR-145-5p in GC cell proliferation and invasion in vitro and tumor growth in vivo was investigated by MTT assay, Transwell invasion assay, and tumor xenograft experiments. The regulating relationship between TUG1 and miR-145-5 was confirmed by luciferase reporter assay. The results showed that TUG1 was significantly overexpressed and miR-145-5p was dramatically downregulated in GC cell lines. TUG1 knockdown strikingly inhibited cell proliferation and invasion in vitro and markedly suppressed tumor growth in vivo. Furthermore, TUG1 could directly bind to miR-145-5p and repress miR-145-5p expression. TUG1 overexpression significantly relieved the inhibition on GC cell proliferation and invasion in vitro and tumor growth in vivo, mediated by miR-145-5p overexpression. In conclusion, TUG1 promotes cell proliferation and invasion in GC via negatively modulating miRNA-145-5p, which undoubtedly contributes to understanding the mechanism of GC occurrence and development.

  19. Oxyfadichalcone C inhibits melanoma A375 cell proliferation and metastasis via suppressing PI3K/Akt and MAPK/ERK pathways.

    Science.gov (United States)

    Peng, Xiaolin; Wang, Zhengming; Liu, Yang; Peng, Xin; Liu, Yao; Zhu, Shan; Zhang, Zhe; Qiu, Yuling; Jin, Meihua; Wang, Ran; Zhang, Qingying; Kong, Dexin

    2018-08-01

    Melanoma remains to be one of the most incurable cancers. Discovery of novel antitumor agent for melanoma therapy is expected. We recently isolated Oxyfadichalcone C from Oxytropis falcate and investigated the anti-proliferative and anti-metastatic activity on human melanoma A375 cells in vitro. Cell viability was determined using MTT assay and soft agar cloning formation assay. The effect of Oxyfadichalcone C on cell cycle distribution and apoptosis were analyzed by flow cytometry. Cell metastasis was determined by wound healing assay, Transwell assay and Gelatin zymography assay. The effect of Oxyfadichalcone C on signal proteins of PI3K/Akt and MAPK/ERK pathways was examined by western blot analysis. Synergism assay was employed to determine whether combination of Oxyfadichalcone C with Vemurafenib would enhance the anti-proliferative effect. Oxyfadichalcone C potently inhibited proliferation, induced G1 phase arrest and weak apoptosis in A375 cells. Anti-migration and anti-invasion activities were also indicated. Such effects were associated with upregulation of p27, reduction of cyclin D1, p-pRb, p-Integrin β1, as well as the proteolytic activity of metalloproteinase (MMP)-2/9. Meanwhile, key molecules of PI3K/Akt and MAPK/ERK pathways were downregulated, which might be involved in the inhibition against proliferation and metastasis of A375 cells by Oxyfadichalcone C. In addition, combination of Oxyfadichalcone C with Vemurafenib at a ratio of IC50 Oxyfadichalcone C : 5 × IC 50 Vemurafenib exhibited synergistic anti-proliferative effect on A375 cells. Our findings suggest that Oxyfadichalcone C has the potential to be developed as a promising drug candidate for the treatment of melanoma. Copyright © 2018 Elsevier Inc. All rights reserved.

  20. Downregulation of long noncoding RNA TUG1 inhibits proliferation and induces apoptosis through the TUG1/miR-142/ZEB2 axis in bladder cancer cells.

    Science.gov (United States)

    Liu, Qian; Liu, Hui; Cheng, Hepeng; Li, Yang; Li, Xiaodong; Zhu, Chaoyang

    2017-01-01

    Bladder cancer is a common serious disease around the world. Long noncoding RNAs (lncRNAs) have been demonstrated to participate in the development and progression of various cancers, including bladder cancer. The aim of this study was to investigate the effects of lncRNA taurine upregulated gene 1 (TUG1) on proliferation and apoptosis in bladder cancer cell lines and the underlying mechanism. The levels of TUG1 were detected by quantitative real time polymerase chain reaction (qRT-PCR) in bladder cancer tissues and cells. The mRNA and protein levels of zinc finger E-box binding homeobox 2 (ZEB2) were measured by qRT-PCR and Western blot analysis, respectively. The functional targets of TUG1 were predicted by online softwares and confirmed by luciferase reporter assay. The effects of TUG1 on cell proliferation and apoptosis were examined by MTT and apoptosis assay, respectively. The expression levels of β-catenin, cyclinD1, and c-Myc in T24 cells were determined by Western blot analysis. The levels of TUG1 and ZEB2 were significantly increased in bladder cancer tissues and cells. Knockdown of either TUG1 or ZEB2 inhibited proliferation and induced apoptosis in bladder cancer cells. Interestingly, ZEB2 overexpression reversed the effects of TUG1 knockdown on cell proliferation and apoptosis. Moreover, ZEB2 was verified as a direct target of miR-142 and miR-142 could specially bind to TUG1. In addition, downregulation of TUG1 inhibited the Wnt/β-catenin pathway by regulating ZEB2 expression in bladder cancer cells. Downregulation of TUG1 expression inhibited proliferation and induced apoptosis in bladder cancer cells by targeting ZEB2 mediated by miR-142 through the inactivation of Wnt/β-catenin pathway.

  1. Long non-coding RNA BCAR4 promotes chondrosarcoma cell proliferation and migration through activation of mTOR signaling pathway.

    Science.gov (United States)

    Shui, Xiaolong; Zhou, Chengwei; Lin, Wei; Yu, Yang; Feng, Yongzeng; Kong, Jianzhong

    2017-05-01

    Chondrosarcoma is one of the common malignant histologic tumors, very difficult to treat, but the concrete cause and mechanism have not yet been elucidated. The present study aimed to investigate the functional involvement of BCAR4 in chondrosarcoma and its potentially underlying mechanism. QRT-PCR and western blot were used to determine the expression of BCAR4 and mTOR signaling pathway proteins both in chondrosarcoma tissues and cells. Chondrosarcoma cell proliferation and migration were assessed by MTT assay and transwell migration assay, respectively. The expression vectors were constructed and used to modulate the expression of BCAR4 and mTOR. Chondrosarcoma xenograft mouse model was established by subcutaneous injection with chondrosarcoma cell lines. The tumor volume was monitored to evaluate the effect of BCAR4 on chondrosarcoma cell tumorigenicity. The expressions of BCAR4, p-mTOR and p-P70S6K were up-regulated in chondrosarcoma tissues and cell lines. Moreover, BCAR4 overexpression had significant promoting effect on cell proliferation and migration in chondrosarcoma cells. Furthermore, mTOR signaling pathway was epigenetically activated by BCAR4-induced hyperacetylation of histone H3. We also found that mTOR overexpression abolished the decrease of chondrosarcoma cell proliferation and migration induced by BCAR4 knockdown. In vivo experiments confirmed that BCAR4 overexpression significantly accelerated tumor growth, while the knockdown of BCAR4 significantly inhibited tumor growth. BCAR4 promoted chondrosarcoma cell proliferation and migration through activation of mTOR signaling pathway, and thus contributed to chondrosarcoma progression. Impact statement LncRNA BCAR4 promoted chondrosarcoma cell proliferation and migration through activation of mTOR signaling pathway, and thus contributed to chondrosarcoma progression.

  2. The methanol seed extract of Garcinia kola attenuated angiotensin II- and lipopolyssacharide-inducedvascular smooth muscle cell proliferation and nitric oxide production

    Directory of Open Access Journals (Sweden)

    Adeolu A. Adedapo

    2016-10-01

    Full Text Available All over the world, cardiovascular diseases are a risk factor for poor health and early death with predisposing factors to include age, gender, tobacco use, physical inactivity, excessive alcohol consumption, unhealthy diet, obesity, family history of cardiovascular disease, hypertension, diabetes mellitus, hyperlipidemia, psychosocial factors, poverty and low educational status, and air pollution. It is envisaged that herbal products that can stem this trend would be of great benefit. Garcinia kola (GK, also known as bitter kola is one of such plants. Generally used as a social snack and offered to guests in some cultural settings, bitter kola has been indicated in the treatment of laryngitis, general inflammation, bronchitis, viral infections and diabetes. In this study, the effects of methanol seed extract of Garcinia kola on the proliferation of Vascular Smooth Muscle Cells (VSMCs in cell culture by Angiotensin II (Ang II and LPS-induced NO production were carried out. Confluent VSMCs were exposed to GK (25, 50 and 100 μg/ml before or after treatment with lipopolyssacharide (100μg/ml, and Angiotensin II (10-8-10-6M. Cellular proliferation was determined by MTT assay and NO production by Griess assay. Treatment with Angiotensin II (10-8, 10-6 or LPS significantly enhanced proliferation of VSM cells while LPS significantly increased nitric oxide (NO production. Treatment with GK (25, 50 & 100 μg/ml attenuated VSM cell proliferation. The results indicate that GK has potential to inhibit mitogen activated vascular cell growth and possibly inhibit inflammatory responses to LPS. Thus GK may be useful in condition that is characterized by cellular proliferation and inflammatory responses.

  3. MicroRNA-125a-5p regulates cancer cell proliferation and migration through NAIF1 in prostate carcinoma.

    Science.gov (United States)

    Fu, Yi; Cao, Fuhua

    2015-01-01

    We investigated the functional roles of microRNA-125a-5p in regulating human prostate carcinoma. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was conducted to evaluate the gene expression levels of miR-125a-5p in eight prostate cancer cell lines and nine biopsy specimens from patients with prostate cancer. miR-125a-5p was genetically knocked down in prostate cancer cell lines, DU145 and VCaP cells by lentiviral transduction. The effects of miR-125a-5p downregulation on prostate cancer cell proliferation and migration were evaluated by MTT assay and transwell assay, respectively. Direct regulation of miR-125a-5p on its downstream targets, NAIF1, and apoptotic gene caspase-3 were evaluated through dual-luciferase reporter assay, qRT-PCR, and Western blot, respectively. NAIF1 was then ectopically overexpressed in DU145 and VCaP cells to modulate prostate cancer cell proliferation and migration. Finally, the effects of miR-125a-5p downregulation or NAIF1 overexpression on the growth of in vivo prostate cancer xenograft were evaluated. miR-125a-5p was upregulated in prostate cancer cell lines and human prostate carcinomas. Lentivirus induced miR-125a-5p downregulation in DU145 and VCaP cells inhibited prostate cancer cell proliferation or migration. NAIF1 was the direct target of miR-125a-5p, as both gene and protein expression levels of NAIF1, as well as caspase-3 were upregulated by miR-125a-5p. Forced overexpression of NAIF1 had similar antitumor effects as miR-125a-5p downregulation on prostate cancer cell proliferation and migration. In vivo prostate xenograft assay confirmed the tumor-suppressive effect of miR-125a-5p downregulation or NAIF1 overexpression. miR-125a-5p regulates prostate cancer cell proliferation and migration through NAIF1.

  4. The Effect of Soy Isoflavone on the Proliferation and Differentiation of Adipose-Derived Mesenchymal Stem Cells into Chondrocytes and Expression of Collagen II and Aggrecan Genes

    Directory of Open Access Journals (Sweden)

    Fatemeh Bamdadpasand Shekarsarayi

    2017-03-01

    Full Text Available Background and Objectives: Due to the lack of blood vessels in cartilage tissue, its damage is not repairable. This study was conducted to investigate the effect of soy isoflavone on proliferation and differentiation of adipose-derived mesenchymal stem cells into chondrocytes and expression of collagen II and aggrecan genes. Methods: In this experimental study, human subcutaneous fat was obtained during liposuction and incubated with collagenase enzyme (type 1 for the breakdown of collagen, and collagenase was deactivated by DMEM medium, and was cultured in the cell sediment after centrifugation, the cells were isolated after the third passage, were placed in chondrogenic medium for differentiate into the cartilage, and were divided into three groups, including control, treatment with TGF-β1, and treatment with soy isoflavones tablets. The tablets were dissolved in distilled water, sterilized by passing through a 0.2 um filter and were added to the culture medium. After 48 hours, cell viability was determined by MTT assay, and after 14 days, collagen II and aggrecan gene expressions were assessed by real-time PCR technique. Data were statistically analyzed by one-way ANOVA and Tukey's post-hoc test using SPSS 20 and p<0.05. Results: The results of MTT assay showed a significant increase in viability in the TGF-β1 group compared to the control and soy isoflavone groups (p<0.05. The RT-PCR indicated a significant increase in the expression of collagen II and aggrecan genes in isoflavones and TGF-β1 groups compared to the control group, and also, the mean CT associated with collagen II gene had a significant increase in isoflavone and TGF-β1groups compared to the control group (p<0.05. Conclusion: Soy in culture medium increases the expression of collagen II and aggrecan genes and cell proliferation, but this increase is not high compared to the TGF-β1 group.

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

    Science.gov (United States)

    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.

  6. Effects of oridonin nanosuspension on cell proliferation and apoptosis of human prostatic carcinoma PC-3 cell line

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    Zhen Zhang

    2010-10-01

    Full Text Available Zhen Zhang, Xiumei Zhang, Wei Xue, Yuna YangYang, Derong Xu, Yunxue Zhao, Haiyan LouSchool of Medicine, Shandong University, Jinan, Republic of ChinaAbstract: This study aims to investigate the inhibitory effects of oridonin nanosuspension on human prostatic carcinoma PC-3 cell line in vitro. The PC-3 cells were incubated with increasing concentrations of oridonin solution and nanosuspensions for 12 hours, 24 hours, and 36 hours. MTT [3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide] assay was performed to measure cellular viability and investigate the effect of oridonin on cell growth of PC-3. Annexin V-FITC/PI staining method was used to determine the effect of oridonin by fluorescence microscope and flow cytometry, respectively. Nanosuspension on early apoptosis of PC-3 cells was also evaluated. Oridonin significantly inhibited the growth of PC-3 cells after 12 hours, 24 hours, and 36 hours of treatment in a dose-dependent manner (P < 0.05. Compared with the same concentration of oridonin solution, oridonin nanosuspension enhanced the inhibition ratio of proliferation. The observation of propidium iodide fluorescence staining confirmed the MTT assay results. The cell proportion of PC-3 at the G2/M phase in the nanosuspension treatment group was upregulated compared with that of the control and oridonin solution groups. Both oridonin solution and nanosuspension promoted the early apoptosis of PC-3 cells. Furthermore, while improving the ratio of early apoptosis, oridonin nanosuspensions also enhanced growth suppression, and induced apoptosis of PC-3 cells. This shows great potential in the treatment of androgen-independent carcinoma of prostate by oridonin nanosuspensions.Keywords: oridonin, nanosuspension, carcinoma of prostate, PC-3 cells, cell cycle, apoptosis

  7. The growth hormone-releasing hormone (GHRH) antagonist JV-1-36 inhibits proliferation and survival of human ectopic endometriotic stromal cells (ESCs) and the T HESC cell line.

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    Annunziata, Marta; Grande, Cristina; Scarlatti, Francesca; Deltetto, Francesco; Delpiano, Elena; Camanni, Marco; Ghigo, Ezio; Granata, Riccarda

    2010-08-01

    To determine the effect of the GHRH antagonist JV-1-36 on proliferation and survival of primary ectopic human endometriotic stromal cells (ESCs) and the T HESC cell line. Prospective laboratory study. University hospital. 22 women with endometriosis (aged 34.8+/-5.7 years) undergoing therapeutic laparoscopy. Eutopic (n=10) and ectopic (n=22) endometrial tissues were collected from women who underwent therapeutic laparoscopic surgery for endometriosis (stage III/IV). Expression of GHRH, GHRH receptor (GHRH-R) and GHRH-R splice variant (SV) 1 mRNA was determined by reverse-transcription polymerase chain reaction (RT-PCR). The ESC proliferation was assessed by 5-bromo-2-deoxyuridine incorporation, cell survival by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and Trypan blue assay. The T HESC survival was evaluated by MTT, cyclic adenosine monophosphate (cAMP) levels by ELISA, extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation by Western blot, and insulin-like growth factor (IGF)-2 mRNA by real-time PCR. The ESCs and T HESCs, but not normal endometrial tissues, expressed GHRH-R mRNA; SV1 mRNA was determined in normal endometrial tissues, ESCs, and T HESCs; GHRH mRNAwas found in T HESCs; JV-1-36 inhibited ESC proliferation and ESC and T HESC survival. In T HESCs, JV-1-36 reduced cAMP production and ERK1/2 phosphorylation but had no effect on IGF-2 mRNA expression. The GHRH antagonist JV-1-36 inhibits endometriotic cell proliferation and survival, suggesting that GHRH antagonist may represent promising tools for treatment of endometriosis. Copyright (c) 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  8. Species-specific differences in peroxisome proliferation, catalase, and SOD2 upregulation as well as toxicity in human, mouse, and rat hepatoma cells induced by the explosive and environmental pollutant 2,4,6-trinitrotoluene.

    Science.gov (United States)

    Naumenko, Ekaterina Anatolevna; Ahlemeyer, Barbara; Baumgart-Vogt, Eveline

    2017-03-01

    2,4,6-Trinitrotoluene (TNT) has been widely used as an explosive substance and its toxicity is still of interest as it persisted in polluted areas. TNT is metabolized in hepatocytes which are prone to its toxicity. Since analysis of the human liver or hepatocytes is restricted due to ethical reasons, we investigated the effects of TNT on cell viability, reactive oxygen species (ROS) production, peroxisome proliferation, and antioxidative enzymes in human (HepG2), mouse (Hepa 1-6), and rat (H4IIEC3) hepatoma cell lines. Under control conditions, hepatoma cells of all three species were highly comparable exhibiting identical proliferation rates and distribution of their cell cycle phases. However, we found strong differences in TNT toxicity with the lowest IC 50 values (highest cell death rate) for rat cells, whereas human and mouse cells were three to sevenfold less sensitive. Moreover, a strong decrease in cellular dehydrogenase activity (MTT assay) and increased ROS levels were noted. TNT caused peroxisome proliferation with rat hepatoma cells being most responsive followed by those from mouse and human. Under control conditions, rat cells contained fivefold higher peroxisomal catalase and mitochondrial SOD2 activities and a twofold higher capacity to reduce MTT than human and mouse cells. TNT treatment caused an increase in catalase and SOD2 mRNA and protein levels in human and mouse, but not in rat cells. Similarly, human and mouse cells upregulated SOD2 activity, whereas rat cells failed therein. We conclude that TNT induced oxidative stress, peroxisome proliferation and mitochondrial damage which are highest in rat cells rendering them most susceptible toward TNT. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 989-1006, 2017. © 2016 Wiley Periodicals, Inc.

  9. Galectin-3-independent Down-regulation of GABABR1 due to Treatment with Korean Herbal Extract HAD-B Reduces Proliferation of Human Colon Cancer Cells

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    Kim Kyung-Hee

    2012-09-01

    Full Text Available Objectives: Many efforts have shown multi-oncologic roles of galectin-3 for cell proliferation, angiogenesis, and apoptosis. However, the mechanisms by which galectin-3 is involved in cell proliferation are not yet fully understood, especially in human colon cancer cells. Methods: To cluster genes showing positively or negatively correlated expression with galectin-3, we employed human colon cancer cell lines, SNU-61, SNU-81, SNU-769B, SNU-C4 and SNU-C5 in high-throughput gene expression profiling. Gene and protein expression levels were determined by using real-time quantitative polymerase chain reaction (PCR and western blot analysis, respectively. The proliferation rate of human colon cancer cells was measured by using a 3-(4, 5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide (MTT assay. Results: Expression of γ-aminobutyric acid B receptor 1 (GABABR1 showed a positive correlation with galectin-3 at both the transcriptional and the translational levels. Downregulation of galectin-3 decreased not only GABABR1 expression but also the proliferation rate of human colon cancer cells. However, Korean herbal extract, HangAmDan-B (HAD-B, decreased expression of GABABR1 without any expressional change of galectin-3, and offset γ-aminobutyric acid (GABA-enhanced human colon cancer cell proliferation. Conclusions: Our present study confirmed that GABABR1 expression was regulated by galectin-3. HAD-B induced galectin-3-independent down-regulation of GABABR1, which resulted in a decreased proliferation of human colon cancer cells. The therapeutic effect of HAD-B for the treatment of human colon cancer needs to be further validated.

  10. Kalanchoe tubiflora extract inhibits cell proliferation by affecting the mitotic apparatus

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    Hsieh Yi-Jen

    2012-09-01

    Full Text Available Abstract Background Kalanchoe tubiflora (KT is a succulent plant native to Madagascar, and is commonly used as a medicinal agent in Southern Brazil. The underlying mechanisms of tumor suppression are largely unexplored. Methods Cell viability and wound-healing were analyzed by MTT assay and scratch assay respectively. Cell cycle profiles were analyzed by FACS. Mitotic defects were analyzed by indirect immunofluoresence images. Results An n-Butanol-soluble fraction of KT (KT-NB was able to inhibit cell proliferation. After a 48 h treatment with 6.75 μg/ml of KT, the cell viability was less than 50% of controls, and was further reduced to less than 10% at higher concentrations. KT-NB also induced an accumulation of cells in the G2/M phase of the cell cycle as well as an increased level of cells in the subG1 phase. Instead of disrupting the microtubule network of interphase cells, KT-NB reduced cell viability by inducing multipolar spindles and defects in chromosome alignment. KT-NB inhibits cell proliferation and reduces cell viability by two mechanisms that are exclusively involved with cell division: first by inducing multipolarity; second by disrupting chromosome alignment during metaphase. Conclusion KT-NB reduced cell viability by exclusively affecting formation of the proper structure of the mitotic apparatus. This is the main idea of the new generation of anti-mitotic agents. All together, KT-NB has sufficient potential to warrant further investigation as a potential new anticancer agent candidate.

  11. Effects of melatonin on the proliferation and differentiation of rat adipose-derived stem cells

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    Zaminy Arash

    2008-01-01

    Full Text Available Background: Osteogenesis driven by adipose-derived stem cells (ADSCs is regulated by physiological and pathological factors. Accumulating evidence from in vitro and in vivo experiments suggests that melatonin may have an influence on bone formation. However, little is known about the effects of melatonin on osteogenesis, which thus remains to be elucidated. This study was performed to determine whether melatonin at physiological concentrations (0.01-10 nM could affect the in vitro proliferation and osteogenic differentiation of rat ADSCs. Materials and Methods: ADSCs were isolated from the fat of adult rats. After cell expansion in culture media and through three passages, osteogenesis was induced in a monolayer culture using osteogenic medium with or without melatonin at physiological concentrations (0.01-10 nM. After four weeks, the cultures were examined for mineralization by Alizarin Red S and von Kossa staining and for alkaline phosphatase (ALP activity using an ALP kit. Cell viability and apoptosis were also assayed by 3-(4, 5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H-tetrazolium (MTT assay and flow cytometry, respectively. Results: The results indicated that at physiological concentrations, melatonin suppressed proliferation and differentiation of ADSCs. These data indicate that ADSCs exposed to melatonin, had a lower ALP activity in contrast to the cells exposed to osteogenic medium alone. Similarly, mineral deposition (calcium level also decreased in the presence of melatonin. Flow cytometry confirmed that cell growth had decreased and that the numbers of apoptotic cells had increased. Conclusion: These results suggest that the physiological concentration of melatonin has a negative effect on ADSC osteogenesis.

  12. Investigation of in vitro bone cell adhesion and proliferation on Ti using direct current stimulation

    International Nuclear Information System (INIS)

    Bodhak, Subhadip; Bose, Susmita; Kinsel, William C.; Bandyopadhyay, Amit

    2012-01-01

    Our objective was to establish an in vitro cell culture protocol to improve bone cell attachment and proliferation on Ti substrate using direct current stimulation. For this purpose, a custom made electrical stimulator was developed and a varying range of direct currents, from 5 to 25 μA, was used to study the current stimulation effect on bone cells cultured on conducting Ti samples in vitro. Cell–material interaction was studied for a maximum of 5 days by culturing with human fetal osteoblast cells (hFOB). The direct current was applied in every 8 h time interval and the duration of electrical stimulation was kept constant at 15 min for all cases. In vitro results showed that direct current stimulation significantly favored bone cell attachment and proliferation in comparison to nonstimulated Ti surface. Immunochemistry and confocal microscopy results confirmed that the cell adhesion was most pronounced on 25 μA direct current stimulated Ti surfaces as hFOB cells expressed higher vinculin protein with increasing amount of direct current. Furthermore, MTT assay results established that cells grew 30% higher in number under 25 μA electrical stimulation as compared to nonstimulated Ti surface after 5 days of culture period. In this work we have successfully established a simple and cost effective in vitro protocol offering easy and rapid analysis of bone cell–material interaction which can be used in promotion of bone cell attachment and growth on Ti substrate using direct current electrical stimulation in an in vitro model. - Highlights: ► D.C. stimulation was used to enhance in vitro bone cell adhesion and proliferation. ► Cells cultured on Ti were stimulated by using a custom made electrical stimulator. ► Optimization was performed by using a varying range of direct currents ∼ 5 to 25 μA. ► 25 μA stimulation was found most beneficial for promotion of cell adhesion/growth.

  13. Human Umbilical Cord Blood Serum Has Higher Potential in Inducing Proliferation of Fibroblast than Fetal Bovine Serum

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    Ferry Sandra

    2017-09-01

    Full Text Available Background: Cytokines and growth factors were reported to play an important role in stimulating fibroblast proliferation. In vitro culture, fibroblast is mostly culture in medium containing fetal bovine serum (FBS.  Human umbilical cord blood (hUCB has been reported to have low immunogenic property and potential in wound healing, so therefore hUCB serum (hUCBS could be potential and were investigated in current study. Materials and Methods: Five hUCBs were collected from healthy volunteers with normal delivering procedure. hUCB was ex utero immediately collected from umbilical vein in vacutainers and processed. NIH3T3 cells were cultured in DMEM with 10% FBS or 5-20% hUCBS for 48 hours. Cells were then quantified using MTT assay. Protein concentration of FBS and hUCBS were quantified using Bradford assay. Results: NIH3T3 cells density grown in DMEM with 10% FBS was the lowest. NIH3T3 cells densities were increased along with the increment of hUCBS concentrations. MTT results showed that average number of NIH3T3 cells grown in DMEM with 10% FBS was 6,185±1,243. Meanwhile average numbers of NIH3T3 cells grown in DMEM with 5%, 10% and 20% hUCBS were 8,126±628, 9,685±313 and 12,200±304, respectively. Average numbers of NIH3T3 cells grown in DMEM with 5% hUCBS were significantly higher than the ones with 10% FBS (p=0.000. Bradford results showed that concentration of hUCBS was significantly higher than the one of FBS (p=0.000. Conclusion: hUCBS could induce higher proliferation rate of NIH3T3 cells than FBS. Hence hUCBS could be suggested as an alternate of FBS in inducing fibroblast. Keywords: NIH3T3, fibroblast, UCB, serum, FBS, proliferation

  14. [miR-497 suppresses proliferation of human cervical carcinoma HeLa cells by targeting cyclin E1].

    Science.gov (United States)

    Han, Jiming; Huo, Manpeng; Mu, Mingtao; Liu, Junjun; Zhang, Jing

    2014-06-01

    To evaluate the effect of miR-497 on proliferation of human cervical carcinoma HeLa cells and target relationship between miR-497 and cyclin E1 (CCNE1). Pre-miR-497 sequences were synthesized and cloned into pcDNATM6.2-GW to construct recombinant plasmid pcDNATM6.2-GW-pre-miR-497 and identified by real-time quantitative PCR (qRT-PCR). In addition, sequences of the wild-type CCNE1 (WT-CCNE1) and mutant CCNE1 (MT-CCNE1) were respectively cloned into pmirGLO vectors. MTT assay was used to explore the impact of miR-497 on the proliferation of HeLa cells. Furthermore, the target effect of miR-497 on the CCNE1 was identified by dual-luciferase reporter assay system, qRT-PCR and Western blotting. The recombinant plasmids pcDNATM6.2-GW-pre-miR-497 and pmirGLO-WT-CCNE1, pmirGLO-MT-CCNE1 were successfully constructed, and the miR-497 expression level in HeLa cells transfected with pre-miR-497 was significantly higher than that in the neg-miR group (PHeLa cells (PHeLa cells with pre-miR-497 transfection (PHeLa cells transfected with pre-miR-497 (PHeLa cells could suppress cell proliferation by targeting CCNE1.

  15. Platelet-poor plasma stimulates the proliferation but inhibits the differentiation of rat osteoblastic cells in vitro.

    Science.gov (United States)

    Hamdan, Ahmad Abdel-Salam; Loty, Sabine; Isaac, Juliane; Bouchard, Philippe; Berdal, Ariane; Sautier, Jean-Michel

    2009-06-01

    Recent studies have shown that the use of platelet preparations in bone and implant surgery might stimulate bone formation. However, the biological mechanisms are not well understood. Moreover, few studies have attempted to evaluate the effect of platelet-poor plasma (PPP), which is a product of the platelet-rich plasma preparation process. Thus, this study investigated the behavior of osteoblasts isolated from fetal rat calvaria cultivated in the presence of homologous PPP. PPP was obtained by centrifugation of the rat mother's blood and used in replacement of fetal calf serum, which is classically used in primary culture procedures. Proliferation was measured by an MTT assay at 24, 48, and 72 h. Real-time PCR was performed to study the expression of Runx2, Dlx5, and osteocalcin (OC) on days 0 (4 h), 1, 3, 7, and 12. Alkaline phosphatase (ALP) biochemical activity was evaluated on days 0 (4 h), 1, 3, 7, and 12. Observations by phase-contrast microscopy showed that osteoblasts were able to differentiate until the mineralization of the matrix in the presence of PPP. PPP enhanced the proliferation significantly compared with the control group (Pexpressed by cells in the experimental group at lower levels compared with the control group. Biochemical assay of ALP showed a lower activity in the experimental group compared with the control group (P<0.001). These results suggest that, in the presence of homologous PPP, rat osteoblastic cells are able to maintain their phenotype, with a higher rate of proliferation. However, PPP seems to inhibit osteoblastic differentiation.

  16. Site-targeted complement inhibition by a complement receptor 2-conjugated inhibitor (mTT30) ameliorates post-injury neuropathology in mouse brains.

    Science.gov (United States)

    Rich, Megan C; Keene, Chesleigh N; Neher, Miriam D; Johnson, Krista; Yu, Zhao-Xue; Ganivet, Antoine; Holers, V Michael; Stahel, Philip F

    2016-03-23

    Intracerebral complement activation after severe traumatic brain injury (TBI) leads to a cascade of neuroinflammatory pathological sequelae that propagate host-mediated secondary brain injury and adverse outcomes. There are currently no specific pharmacological agents on the market to prevent or mitigate the development of secondary cerebral insults after TBI. A novel chimeric CR2-fH compound (mTT30) provides targeted inhibition of the alternative complement pathway at the site of tissue injury. This experimental study was designed to test the neuroprotective effects of mTT30 in a mouse model of closed head injury. The administration of 500 μg mTT30 i.v. at 1 h, 4 h and 24 h after head injury attenuated complement C3 deposition in injured brains, reduced the extent of neuronal cell death, and decreased post-injury microglial activation, compared to vehicle-injected placebo controls. These data imply that site-targeted alternative pathway complement inhibition may represent a new promising therapeutic avenue for the future management of severe TBI. Copyright © 2016. Published by Elsevier Ireland Ltd.

  17. Effects of composite films of silk fibroin and graphene oxide on the proliferation, cell viability and mesenchymal phenotype of periodontal ligament stem cells.

    Science.gov (United States)

    Rodríguez-Lozano, F J; García-Bernal, D; Aznar-Cervantes, S; Ros-Roca, M A; Algueró, M C; Atucha, N M; Lozano-García, A A; Moraleda, J M; Cenis, J L

    2014-12-01

    In regenerative dentistry, stem cell-based therapy often requires a scaffold to deliver cells and/or growth factors to the injured site. Graphene oxide (GO) and silk fibroin (SF) are promising biomaterials for tissue engineering as they are both non toxic and promote cell proliferation. On the other hand, periodontal ligament stem cells (PDLSCs) are mesenchymal stem cells readily accessible with a promising use in cell therapy. The purpose of this study was to investigate the effects of composite films of GO, SF and GO combined with fibroin in the mesenchymal phenotype, viability, adhesion and proliferation rate of PDLSCs. PDLSCs obtained from healthy extracted teeth were cultured on GO, SF or combination of GO and SF films up to 10 days. Adhesion level of PDSCs on the different biomaterials were evaluated after 12 h of culture, whereas proliferation rate of cells was assessed using the MTT assay. Level of apoptosis was determined using Annexin-V and 7-AAD and mesenchymal markers expression of PDLSCs were analyzed by flow cytometry. At day 7 of culture, MTT experiments showed a high rate of proliferation of PDLSCs growing on GO films compared to the other tested biomaterials, although it was slightly lower than in plastic (control). However PDLSCs growing in fibroin or GO plus fibroin films showed a discrete proliferation. Importantly, at day 10 of culture it was observed a significant increase in PDLSCs proliferation rate in GO films compared to plastic (P < 0.05), as well as in GO plus fibroin compared to fibroin alone (P < 0.001). Flow cytometry analysis showed that culture of PDLSCs in fibroin, GO or GO plus fibroin films did not significantly alter the level of expression of the mesenchymal markers CD73, CD90 or CD105 up to 168 h, being the cell viability in GO even better than obtained in plastic. Our findings suggest that the combination of human dental stem cells/fibroin/GO based-bioengineered constructs have strong potential for their therapeutic

  18. Evaluation of tetrazolium-based semiautomatic colorimetric assay for measurement of human antitumor cytotoxicity

    International Nuclear Information System (INIS)

    Heo, D.S.; Park, J.G.; Hata, K.; Day, R.; Herberman, R.B.; Whiteside, T.L.

    1990-01-01

    A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based colorimetric assay was developed and compared with 51Cr release from different adherent tumor cell targets (human squamous cell carcinoma lines of the head and neck established in our laboratory, melanoma, and colorectal carcinoma) using 5-7-day human lymphokine-activated killer cells and monocyte-depleted peripheral blood lymphocytes as effectors. With adherent tumor cell targets, MTT colorimetry was more sensitive than the 51Cr release assay in measuring the antitumor activity of effectors: median, 4385 (range, 988-8144) versus median, 1061 (range, 582-7294) lytic units (the number of effector cells required to lyse 20% of 5 x 10(3) targets)/10(7) effectors (P less than 0.01). Background effects (without effector cells) were comparable in 4-h assays (9% versus 10%) between MTT colorimetry and 51Cr release. In 24-h assays, MTT colorimetry showed higher antitumor activity (70-100% versus 40-60% lysis at 1:1 effector:target cell ratio) but lower background effects (6% versus 38%) than 51Cr release assay. Thus, MTT colorimetry was more sensitive, did not use radiolabeled targets, required fewer effector cells, and was easier, less expensive, and better adaptable to serial monitoring of effector cell function in cancer patients. This colorimetric assay is especially well suited to adherent tumor cell targets. The use of adherent tumor cell monolayers, as opposed to trypsinized single cell suspensions, provides an opportunity to measure interactions of effector cells with enzymatically unaltered solid tumor targets. Because of the greater sensitivity of the colorimetric assay, the transformation of MTT data into lytic units, as commonly used for 51Cr release assays, required an adjustment to avoid the extrapolation based on the exponential fit equation

  19. HCV core protein-induced down-regulation of microRNA-152 promoted aberrant proliferation by regulating Wnt1 in HepG2 cells.

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    Shifeng Huang

    Full Text Available Hepatitis C virus (HCV has been reported to regulate cellular microRNAs (miRNAs. The HCV core protein is considered to be a potential oncoprotein in HCV-related hepatocellular carcinoma (HCV-HCC, but HCV core-regulated miRNAs are largely unknown. Our preliminary experiments revealed significant down-regulation of microRNA-152 (miR-152 by HCV core protein in HepG2 cells. Through target gene prediction softwares, Wnt1 was predicted to be a potential target of miR-152. The present study was initiated to investigate whether miR-152 is aberrantly regulated by the HCV core protein, and involved in the regulation of the aberrant proliferation of HCV-HCC cells.MiR-152 levels were examined by stem-loop real-time RT-PCR (SLqRT-PCR. Cell proliferation was analyzed by MTT and colony formation assay. Cell cycle analysis was performed by flow cytometry. Luciferase reporter assay was conducted to confirm miRNA-target association. Wnt1 expression was determined by real-time qPCR and Western blotting.HCV core protein significantly suppressed miR-152 expression, and led to significant Wnt1 up-regulation with a concomitant aberrantly promoted proliferation. Moreover, we validated that miR-152 inhibition promoted, while miR-152 mimics inhibited cell proliferation. Using, qRT-PCR and western blot, Wnt1 was demonstrated to be regulated by miR-152. Luciferase activity assay showed that while miR-152 mimics significantly reduced the luciferase activity by 83.76% (P<0.0001, miR-152 inhibitor showed no effect on luciferase reporter. Most notably, salvage expression of miR-152 after Ad-HCV core infection for 24 h almost totally reversed the proliferation-promoting effect of the HCV core protein, and meanwhile, reduced the expression of both Wnt1 mRNA and protein to basal levels.These findings provide important evidence that the reduced miR-152 expression by HCV core protein can indirectly lose an inhibitory effect on Wnt1, which might, at least partially lead to cell

  20. Adhesion and proliferation of OCT-1 osteoblast-like cells on micro- and nano-scale topography structured poly(L-lactide).

    Science.gov (United States)

    Wan, Yuqing; Wang, Yong; Liu, Zhimin; Qu, Xue; Han, Buxing; Bei, Jianzhong; Wang, Shenguo

    2005-07-01

    The impact of the surface topography of polylactone-type polymer on cell adhesion was to be concerned because the micro-scale texture of a surface can provide a significant effect on the adhesion behavior of cells on the surface. Especially for the application of tissue engineering scaffold, the pore size could have an influence on cell in-growth and subsequent proliferation. Micro-fabrication technology was used to generate specific topography to investigate the relationship between the cells and surface. In this study the pits-patterned surfaces of polystyrene (PS) film with diameters 2.2 and 0.45 microm were prepared by phase-separation, and the corresponding scale islands-patterned PLLA surface was prepared by a molding technique using the pits-patterned PS as a template. The adhesion and proliferation behavior of OCT-1 osteoblast-like cells morphology on the pits- and islands-patterned surface were characterized by SEM observation, cell attachment efficiency measurement and MTT assay. The results showed that the cell adhesion could be enhanced on PLLA and PS surface with nano-scale and micro-scale roughness compared to the smooth surfaces of the PLLA and PS. The OCT-1 osteoblast-like cells could grow along the surface with two different size islands of PLLA and grow inside the micro-scale pits of the PS. However, the proliferation of cells on the micro- and nano-scale patterned surface has not been enhanced compared with the controlled smooth surface.

  1. Polydatin inhibits cell proliferation and induces apoptosis in laryngeal cancer and HeLa cells via suppression of the PDGF/AKT signaling pathway.

    Science.gov (United States)

    Li, Haixia; Shi, Baoyuan; Li, Yanyun; Yin, Fengfang

    2017-07-01

    Polydatin (PD), a stilbene compound extracted from Polygonum cuspidatum, is suggested to possess anti-cancer activities, including inhibition of cell proliferation, cell cycle arrest, and induction of apoptosis. The platelet-derived growth factor (PDGF)/AKT signaling pathway plays complex roles in tumor suppression. However, the effect of PD on the PDGF/AKT signaling pathway in laryngeal cancer and HeLa cells has not been explored. MTT assay and flow cytometry showed that PD inhibited cell proliferation and induced apoptosis in Hep-2 and AMC-HN-8 cells. Western blot analysis indicated that PD inhibited the expression levels of PDGF-B and phosphorylated AKT (p-AKT) in both cells. Treatment of PDGF-B siRNA or PDGFR inhibitor found that after the PDGF signaling was inactivated, p-AKT expression was significantly decreased in Hep-2 cells. Tumor xenograft experiment in nude mice indicated PD significantly inhibited the growth of Hep-2 cells in vivo. In conclusion, PD inhibited cell proliferation and induced apoptosis in laryngeal cancer and HeLa cells via inactivation of the PDGF/AKT signaling pathway. © 2017 Wiley Periodicals, Inc.

  2. Attachment and proliferation of human osteoblast-like cells (MG-63) on laser-ablated titanium implant material

    Energy Technology Data Exchange (ETDEWEB)

    Györgyey, Ágnes; Ungvári, Krisztina [Department of Oral Biology and Experimental Dental Research, Faculty of Dentistry, University of Szeged, H-6720 Szeged (Hungary); Kecskeméti, Gabriella; Kopniczky, Judit [Department of Optics and Quantum Electronics, Faculty of Science and Informatics, University of Szeged, H-6720 Szeged (Hungary); Hopp, Béla [Research Group on Laser Physics, Hungarian Academy of Sciences and University of Szeged, H-6720 Szeged (Hungary); Oszkó, Albert [Department of Physical Chemistry and Materials Science, Faculty of Science and Informatics, University of Szeged, H-6720 Szeged (Hungary); Pelsöczi, István; Rakonczay, Zoltán [Department of Oral Biology and Experimental Dental Research, Faculty of Dentistry, University of Szeged, H-6720 Szeged (Hungary); Nagy, Katalin [Department of Oral Surgery, Faculty of Dentistry, University of Szeged, H-6720 Szeged (Hungary); Turzó, Kinga, E-mail: kturzo@yahoo.com [Department of Oral Biology and Experimental Dental Research, Faculty of Dentistry, University of Szeged, H-6720 Szeged (Hungary)

    2013-10-15

    Demand is increasing for shortening the long (3–6 months) osseointegration period to rehabilitate patients' damaged chewing apparatus in as short a time as possible. For dental implants, as for biomaterials in general, the bio- and osseointegration processes can be controlled at molecular and cellular levels by modification of the implant surface. One of the most promising of such surface modifications is laser ablation, as demonstrated by our previous results [46]. Commercially pure (CP4) sand-blasted, acid-etched titanium disks (Denti® System Ltd., Hungary) were irradiated with a KrF excimer laser (248 nm, fluence 0.4 J/cm{sup 2}, FWHM 18 ns, 2000 pulses), or with a Nd:YAG laser (532 nm, 1.3 J/cm{sup 2}, 10 ns, 200 pulses) then examined by SEM, AFM, and XPS. In vitro attachment (24 h) and proliferation (72 h) of MG-63 osteoblast cells were investigated via dimethylthiazol-diphenyl tetrazolium bromide (MTT), alamarBlue (AB) assays alkaline phosphatase quantification (ALP) and SEM. SEM and AFM revealed significant changes in morphology and roughness. XPS confirmed the presence of TiO{sub 2} on each sample; after Nd:YAG treatment a reduced state of Ti (Ti{sup 3+}) was also observed. MTT, AB and ALP measurements detected an increase in the number of cells between the 24- and 72 hour observations; however, laser treatment did not affect cell attachment and proliferation significantly. - Highlights: • CP4 titanium implant surfaces were modified with Nd:YAG and KrF excimer laser. • SEM and AFM revealed significant changes in morphology and roughness. • XPS confirmed the presence of TiO{sub 2} on each sample; after Nd:YAG treatment a reduced state of Ti (Ti{sup 3+}) was found. • Cell proliferation experiments detected an increased number of MG-63 cells between the 24 h and 72 h observations. • Laser treatments neither disturbed, nor enhanced MG-63 cell attachment and proliferation significantly.

  3. Thalidomide increases human keratinocyte migration and proliferation.

    Science.gov (United States)

    Nasca, M R; O'Toole, E A; Palicharla, P; West, D P; Woodley, D T

    1999-11-01

    Thalidomide is reported to have therapeutic utility in the treatment of pyoderma gangrenosum, Behçet's disease, aphthous ulcers, and skin wounds. We investigated the effect of thalidomide on human keratinocyte proliferation and migration, two early and critical events in the re-epithelialization of skin wounds. Thalidomide at concentrations less than 1 microM did not affect keratinocyte viability. Using a thymidine incorporation assay, we found that thalidomide, at therapeutic concentrations, induced more than a 2. 5-fold increase in the proliferative potential of the cells. Keratinocyte migration was assessed by two independent motility assays: a colloidal gold assay and an in vitro scratch assay. At optimal concentrations, thalidomide increased keratinocyte migration on a collagen matrix more than 2-fold in the colloidal gold assay and more than 3-fold in the scratch assay over control. Although pro-migratory, thalidomide did not alter the level of metalloproteinase-9 secreted into culture medium. Thalidomide did, however, induce a 2-4-fold increase in keratinocyte-derived interleukin-8, a pro-migratory cellular autocrine factor. Human keratinocyte migration and proliferation are essential for re-epithelialization of skin wounds. Interleukin-8 increases human keratinocyte migration and proliferation and is chemotactic for keratinocytes. Therefore, thalidomide may modulate keratinocyte proliferation and motility by a chemokine-dependent pathway.

  4. Impact of silk fibroin-based scaffold structures on human osteoblast MG63 cell attachment and proliferation

    Directory of Open Access Journals (Sweden)

    Varkey A

    2015-10-01

    Full Text Available Aneesia Varkey,1,2 Elakkiya Venugopal,2 Ponjanani Sugumaran,2 Gopinathan Janarthanan,1 Mamatha M Pillai,2 Selvakumar Rajendran,2 Amitava Bhattacharyya1 1Advanced Textile and Polymer Research Laboratory, 2Tissue Engineering Laboratory, PSG Institute of Advanced Studies, Coimbatore, Tamil Nadu, India Abstract: The present study was carried out to investigate the impact of various types of silk fibroin (SF scaffolds on human osteoblast-like cell (MG63 attachment and proliferation. SF was isolated from Bombyx mori silk worm cocoons after degumming. Protein concentration in the degummed SF solution was estimated using Bradford method. Aqueous SF solution was used to fabricate three different types of scaffolds, viz, electrospun nanofiber mat, sponge, and porous film. The structures of the prepared scaffolds were characterized using optical micro­scopy and field emission scanning electron microscopy. The changes in the secondary structure of the proteins and the thermal behavior of the scaffolds were determined by Fourier transform infrared spectroscopy and thermo-gravimetric analysis, respectively. The biodegradation rate of scaffolds was determined by incubating the scaffolds in simulated body fluid for 4 weeks. MG63 cells were seeded on the scaffolds and their attachment and proliferation onto the scaffolds were studied. The MTT assay was carried out to deduce the toxicity of the developed scaffolds. All the scaffolds were found to be biocompatible. The amount of collagen produced by the osteoblast-like cells growing on different scaffolds was estimated. Keywords: silk fibroin scaffold, electrospun nanofiber, porous film, sponge, osteoblast

  5. Effects of homeodomain protein CDX2 expression on the proliferation and migration of lovo colon cancer cells.

    Science.gov (United States)

    Zheng, Jian-bao; Sun, Xue-jun; Qi, Jie; Li, Shou-shuai; Wang, Wei; Ren, Hai-liang; Tian, Yong; Lu, Shao-ying; Du, Jun-kai

    2011-09-01

    The homeobox gene, CDX2, plays a major role in development, especially in the gut, and also functions as a tumor suppressor in the adult colon. In the present study, we investigated the effects of CDX2 expression on the proliferation, migration, and apoptosis of the human colon cancer cell line, Lovo. Lovo cells exogenously expressing CDX2 exhibited no significant differences in the percentage of cells in G1- and S-phase or in apoptosis, as determined by flow cytometry. MTT assay also confirmed that CDX2 expression had no effect on proliferation in these cells. Interestingly, conditioned medium collected from CDX2-overexpressing Lovo cells showed a significant decrease in secretion of MMP-2 and the invasive potential of these cells was significantly inhibited. Collectively, these data suggest that CDX2 may play a critical role in the migration and metastasis of colon carcinoma and over-expression of CDX2 in colon cancer cells markedly inhibits invasion. Based on these results, exogenous expression of CDX2 might be a promising option in the treatment of colon carcinoma.

  6. BC047440 antisense eukaryotic expression vectors inhibited HepG2 cell proliferation and suppressed xenograft tumorigenicity

    International Nuclear Information System (INIS)

    Lu, Zheng; Ping, Liang; JianBo, Zhou; XiaoBing, Huang; Yu, Wen; Zheng, Wang; Jing, Li

    2012-01-01

    The biological functions of the BC047440 gene highly expressed by hepatocellular carcinoma (HCC) are unknown. The objective of this study was to reconstruct antisense eukaryotic expression vectors of the gene for inhibiting HepG 2 cell proliferation and suppressing their xenograft tumorigenicity. The full-length BC047440 cDNA was cloned from human primary HCC by RT-PCR. BC047440 gene fragments were ligated with pMD18-T simple vectors and subsequent pcDNA3.1(+) plasmids to construct the recombinant antisense eukaryotic vector pcDNA3.1(+)BC047440AS. The endogenous BC047440 mRNA abundance in target gene-transfected, vector-transfected and naive HepG 2 cells was semiquantitatively analyzed by RT-PCR and cell proliferation was measured by the MTT assay. Cell cycle distribution and apoptosis were profiled by flow cytometry. The in vivo xenograft experiment was performed on nude mice to examine the effects of antisense vector on tumorigenicity. BC047440 cDNA fragments were reversely inserted into pcDNA3.1(+) plasmids. The antisense vector significantly reduced the endogenous BC047440 mRNA abundance by 41% in HepG 2 cells and inhibited their proliferation in vitro (P < 0.01). More cells were arrested by the antisense vector at the G 1 phase in an apoptosis-independent manner (P = 0.014). Additionally, transfection with pcDNA3.1(+) BC047440AS significantly reduced the xenograft tumorigenicity in nude mice. As a novel cell cycle regulator associated with HCC, the BC047440 gene was involved in cell proliferation in vitro and xenograft tumorigenicity in vivo through apoptosis-independent mechanisms

  7. Effects of hydroxysafflor yellow A on proliferation and collagen synthesis of rat vascular adventitial fibroblasts induced by angiotensin II.

    Science.gov (United States)

    Yuan, Wendan; Yang, Dongxia; Sun, Xuhong; Liu, Wei; Wang, Liang; Li, Xiaoyan; Man, Xuejing; Fu, Qiang

    2014-01-01

    1) examine the effects of hydroxysafflor yellow A (HSYA) on the proliferation, collagen and cytokine synthesis of vascular adventitial fibroblasts as induced by angiotensin II (Ang II) in normal Sprague-Dawley (SD) rats in vitro, and 2) to assess the effects of HSYA on morphological changes and collagen accumulation of vascular adventitia in spontaneously hypertensive rats (SHR) in vivo. In vitro experiment, vascular adventitial fibroblasts from SD rats were isolated, cultured, and divided into control groups, model groups and HSYA groups. Cell morphology of adventitial fibroblasts was assessed using laser confocal microscopy, while cell proliferation with the MTT assay, and collagen synthesis was determined using hydroxyproline chromatometry. Immunocytochemistry and reverse transcription PCR were used for detecting the expression of TGF-β1, MMP-1, α-SMA and NF-κB in adventitial fibroblasts. In vivo experiment, vascular adventitia proliferation and collagen synthesis were analyzed using hematoxylin-eosin and Sirius staining. Our results showed that: 1) in vitro experiment of SD rats, HSYA inhibited proliferative activity and collagen synthesis of adventitial fibroblasts as induced by Ang II, and the inhibitory effects of HSYA on the increased expression of MMP-1, TGF-β1, α-SMA and NF-κB p65 as induced by Ang II were assessed, and 2) in vivo experiment of SHR, histological analysis displayed fewer pathological changes of vascular adventitia in HSYA treatment groups as compared with no HSYA treatment groups, and MMP-1, TGF-β1, α-SMA and NF-κB p65 expression significantly reduced after HSYA treatment (P adventitia components. This study provides experimental evidence demonstrating that HSYA has the capacity to decrease vascular adventitia proliferation and hyperplasia during vascular remodeling.

  8. Expression of Caspase-1 in breast cancer tissues and its effects on cell proliferation, apoptosis and invasion.

    Science.gov (United States)

    Sun, Yanxia; Guo, Yingzhen

    2018-05-01

    The present study aimed to detect the expression of Caspase-1 in the tumor tissues and tumor-adjacent tissues of patients with breast cancer, and to investigate the effects of Caspase-1 on the proliferation, apoptosis and invasion of breast cancer cells. Reverse transcription-quantitative polymerase chain reaction was used to detect Caspase-1 mRNA expression in breast cancer tissues and tumor-adjacent tissues from patients. Additionally, the human breast cancer MDA-MB-231 cell line was treated with the Caspase-1 small molecule inhibitor Ac-YVAD-CMK, following which the changes to Caspase-1 protein expression were detected via western blotting. The MTT method detected the changes to cell proliferation, flow cytometry detected the rate of apoptosis, and a Transwell assay was employed to assess invasion. Caspase-1 mRNA expression was significantly decreased in the breast cancer tissues of patients, compared with in the tumor-adjacent tissues, a difference that was statistically significant (P<0.05). Treatment with the Ac-YVAD-CMK markedly decreased the protein expression of Caspase-1 in MDA-MB-231 cells, and the difference was statistically significant (P<0.05). Following this treatment of Ac-YVAD-CMK cells, the proliferation and invasion abilities markedly increased, while the apoptotic levels significantly decreased (P<0.05). In conclusion, the expression of Caspase-1 is low in breast cancer tissues, which may promote the proliferation and invasion of breast cancer cells and could be closely associated with the occurrence and development of breast cancer.

  9. Andrographolide suppresses proliferation of human colon cancer SW620 cells through the TLR4/NF-κB/MMP-9 signaling pathway.

    Science.gov (United States)

    Zhang, Rui; Zhao, Jian; Xu, Jian; Jiao, De-Xin; Wang, Jian; Gong, Zhi-Qiang; Jia, Jian-Hui

    2017-10-01

    Modern pharmacological research has revealed that andrographolide has various functions, including anti-bacterial, anti-inflammatory and anti-viral effects, immunoregulation, treating cardiovascular and cerebrovascular diseases, and prevention and treatment of alcoholic liver injury. The present study investigated whether andrographolide suppresses the proliferation of human colon cancer cell through the Toll-like receptor 4 (TLR4)/nuclear factor (NF)-κB/matrix metalloproteinase-9 (MMP-9) signaling pathway. The MTT assay and lactate dehydrogenase assay were used to evaluate the anticancer effects of andrographolide on cell proliferation and cytotoxicity in human colon cancer SW620 cells. Flow cytometry was used to analyze the anticancer effects of andrographolide on apoptosis by Annexin V-fluorescein isothiocyanate/propidium iodide kit. The effects of andrographolide on the activity of caspase-3/9 were measured using ELISA. Western blot analysis was also used to analyze the protein expression of TLR4, myeloid differentiation primary response gene 88 (MyD88), NF-κB-p65 and MMP-9. In the present study, it was found that andrographolide suppressed the cell proliferation, augmented cytotoxicity, evoked cell apoptosis and activated caspase-3/9 activities in human colon cancer SW620 cells. The results revealed that the anti-proliferation effects of andrographolide on the SW620 cells was associated with the inhibition of TLR4, MyD88, NF-κB-p65 and MMP-9 signaling activation. The results suggest that andrographolide is a promising drug for treatment of human colon cancer via suppression of the TLR4/NF-κB/MMP-9 signaling pathway.

  10. miR-885-5p upregulation promotes colorectal cancer cell proliferation and migration by targeting suppressor of cytokine signaling.

    Science.gov (United States)

    Su, Meng; Qin, Baoli; Liu, Fang; Chen, Yuze; Zhang, Rui

    2018-07-01

    The aim of the present study was to investigate the role of microRNA (miR)-885-5p in colorectal cancer cell proliferation and migration, and to determine the possible underlying molecular mechanisms. The expression of miR-885-5p in colorectal cancer tissue and cells was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The expression levels of three suppressor of cytokine signaling (SOCS) factors were detected by RT-qPCR and western blotting. The effects of miR-885-5p on tumor cell proliferation and migration were studied using MTT and Transwell assays, respectively. Additionally, the expression levels of epithelial-mesenchymal transition (EMT)-related proteins (N-cadherin, E-cadherin, vimentin and Snail) were detected by RT-qPCR and western blot analysis. Furthermore, the target of miR-885-5p was predicted and confirmed using a luciferase reporter assay. miR-885-5p was demonstrated to be upregulated and SOCS was downregulated in colorectal cancer tissue, and cells. miR-885-5p suppression significantly inhibited tumor cell proliferation and migration, promoted E-cadherin expression, and inhibited the expression levels of N-cadherin, vimentin and Snail. Further studies showed that SOCS5, SOCS6 and SOCS7 were direct targets of miR-885-5p. The results suggest that miR-885-5p suppression inhibited cell proliferation and migration, and the EMT process by targeting SOCS5, SOCS6 and SOCS7 genes in colorectal cancer. miR-885-5p and SOCS may be used for the diagnosis and treatment of colorectal cancer.

  11. Trans10,cis12 conjugated linoleic acid inhibits proliferation and migration of ovarian cancer cells by inducing ER stress, autophagy, and modulation of Src.

    Directory of Open Access Journals (Sweden)

    Mian M K Shahzad

    Full Text Available The goal of this study was to investigate the anti-cancer effects of Trans10,cis12 conjugated linoleic acid (t10,c12 CLA. MTT assays and QCM™ chemotaxis 96-wells were used to test the effect of t10,c12 CLA on the proliferation and migration and invasion of cancer cells. qPCR and Western Blotting were used to determine the expression of specific factors. RNA sequencing was conducted using the Illumina platform and apoptosis was measured using a flow cytometry assay. t10,c12 CLA (IC50, 7 μM inhibited proliferation of ovarian cancer cell lines SKOV-3 and A2780. c9,t11 CLA did not attenuate the proliferation of these cells. Transcription of 165 genes was significantly repressed and 28 genes were elevated. Genes related to ER stress, ATF4, CHOP, and GADD34 were overexpressed whereas EDEM2 and Hsp90, genes required for proteasomal degradation of misfolded proteins, were downregulated upon treatment. While apoptosis was not detected, t10,c12 CLA treatment led to 9-fold increase in autophagolysosomes and higher levels of LC3-II. G1 cell cycle arrest in treated cells was correlated with phosphorylation of GSK3β and loss of β-catenin. microRNA miR184 and miR215 were upregulated. miR184 likely contributed to G1 arrest by downregulating E2F1. miR215 upregulation was correlated with increased expression of p27/Kip-1. t10,c12 CLA-mediated inhibition of invasion and migration correlated with decreased expression of PTP1b and decreased Src activation by inhibiting phosphorylation at Tyr416. Due to its ability to inhibit proliferation and migration, t10,c12 CLA should be considered for treatment of ovarian cancer.

  12. Trans10,cis12 conjugated linoleic acid inhibits proliferation and migration of ovarian cancer cells by inducing ER stress, autophagy, and modulation of Src.

    Science.gov (United States)

    Shahzad, Mian M K; Felder, Mildred; Ludwig, Kai; Van Galder, Hannah R; Anderson, Matthew L; Kim, Jong; Cook, Mark E; Kapur, Arvinder K; Patankar, Manish S

    2018-01-01

    The goal of this study was to investigate the anti-cancer effects of Trans10,cis12 conjugated linoleic acid (t10,c12 CLA). MTT assays and QCM™ chemotaxis 96-wells were used to test the effect of t10,c12 CLA on the proliferation and migration and invasion of cancer cells. qPCR and Western Blotting were used to determine the expression of specific factors. RNA sequencing was conducted using the Illumina platform and apoptosis was measured using a flow cytometry assay. t10,c12 CLA (IC50, 7 μM) inhibited proliferation of ovarian cancer cell lines SKOV-3 and A2780. c9,t11 CLA did not attenuate the proliferation of these cells. Transcription of 165 genes was significantly repressed and 28 genes were elevated. Genes related to ER stress, ATF4, CHOP, and GADD34 were overexpressed whereas EDEM2 and Hsp90, genes required for proteasomal degradation of misfolded proteins, were downregulated upon treatment. While apoptosis was not detected, t10,c12 CLA treatment led to 9-fold increase in autophagolysosomes and higher levels of LC3-II. G1 cell cycle arrest in treated cells was correlated with phosphorylation of GSK3β and loss of β-catenin. microRNA miR184 and miR215 were upregulated. miR184 likely contributed to G1 arrest by downregulating E2F1. miR215 upregulation was correlated with increased expression of p27/Kip-1. t10,c12 CLA-mediated inhibition of invasion and migration correlated with decreased expression of PTP1b and decreased Src activation by inhibiting phosphorylation at Tyr416. Due to its ability to inhibit proliferation and migration, t10,c12 CLA should be considered for treatment of ovarian cancer.

  13. Turmeric (Curcuma longa) inhibits inflammatory nuclear factor (NF)-κB and NF-κB-regulated gene products and induces death receptors leading to suppressed proliferation, induced chemosensitization, and suppressed osteoclastogenesis.

    Science.gov (United States)

    Kim, Ji H; Gupta, Subash C; Park, Byoungduck; Yadav, Vivek R; Aggarwal, Bharat B

    2012-03-01

    The incidence of cancer is significantly lower in regions where turmeric is heavily consumed. Whether lower cancer incidence is due to turmeric was investigated by examining its effects on tumor cell proliferation, on pro-inflammatory transcription factors NF-κB and STAT3, and on associated gene products. Cell proliferation and cell cytotoxicity were measured by the MTT method, NF-κB activity by EMSA, protein expression by Western blot analysis, ROS generation by FACS analysis, and osteoclastogenesis by TRAP assay. Turmeric inhibited NF-κB activation and down-regulated NF-κB-regulated gene products linked to survival (Bcl-2, cFLIP, XIAP, and cIAP1), proliferation (cyclin D1 and c-Myc), and metastasis (CXCR4) of cancer cells. The spice suppressed the activation of STAT3, and induced the death receptors (DR)4 and DR5. Turmeric enhanced the production of ROS, and suppressed the growth of tumor cell lines. Furthermore, turmeric sensitized the tumor cells to chemotherapeutic agents capecitabine and taxol. Turmeric was found to be more potent than pure curcumin for cell growth inhibition. Turmeric also inhibited NF-κB activation induced by RANKL that correlated with the suppression of osteoclastogenesis. Our results indicate that turmeric can effectively block the proliferation of tumor cells through the suppression of NF-κB and STAT3 pathways. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. NF-κB is involved in the LPS-mediated proliferation and apoptosis of MAC-T epithelial cells as part of the subacute ruminal acidosis response in cows.

    Science.gov (United States)

    Fan, Wen-Jie; Li, He-Ping; Zhu, He-Shui; Sui, Shi-Ping; Chen, Pei-Ge; Deng, Yue; Sui, Tong-Ming; Wang, Yue-Ying

    2016-11-01

    To determine the effect of NF-κB on cell proliferation and apoptosis, we investigate the expression of inflammation and apoptosis-related factors in the bovine mammary epithelial cell line, MAC-T. MAC-T cells were cultured in vitro and MTT and LDH assays used to determine the effects of lipopolysaccharide (LPS) on proliferation and cytotoxicity respectively. RT-PCR and western blotting were used to evaluate the effect of LPS and NF-κB inhibition [pyrrolidine dithiocarbamate (PDTC) treatment] on the expression of inflammation and apoptosis-related factors. LPS significantly inhibited MAC-T cell proliferation in a dose- and time-dependent manner. Furthermore, LPS promoted apoptosis while the NF-кB inhibitor PDTC attenuated this effect. After LPS treatment, the NF-кB signaling pathway was activated, and the expression of inflammation and apoptosis-related factors increased. When PDTC blocked NF-кB signaling, the expression of inflammation and apoptosis-related factors were decreased in MAC-T cells. LPS activates the TLR4/NF-κB signaling pathway, inhibits proliferation and promotes apoptosis in MAC-T cells. NF-кB inhibition attenuates MAC-T cell apoptosis and TLR4/NF-κB signaling pathway. NF-кB inhibitor alleviating MAC-T cell apoptosis is presumably modulated by NF-кB.

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

    Science.gov (United States)

    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.

  16. Effect of miR-146a-5p on proliferation and metastasis of triple-negative breast cancer via regulation of SOX5.

    Science.gov (United States)

    Si, Chengshuai; Yu, Qiao; Yao, Yufeng

    2018-05-01

    MicroRNA (miR)-146a-5p functions as a tumor suppressor in various types of cancer. However, the role of miR-146a-5p in the development of triple-negative breast cancer (TNBC) is unclear. The present study aimed to investigate the role of miR-146a-5p in TNBC. The expression level of miR-146a-5p in TNBC tissues and cell lines was initially detected using reverse transcription-quantitative polymerase chain reaction. To predict the target gene of miR-146a-5p, TargetScan software was used and a dual luciferase assay was performed to verify the prediction. Furthermore, in order to explore the role of miR-146a-5p in TNBC, miR-146a-5p was overexpressed in TNBC cells using miR-146a-5p mimics. An MTT assay was performed to detect cell proliferation, and a Transwell assay was conducted to determine cell migration and invasion. Furthermore, western blotting was performed to measure associated protein expression. It was revealed that miR-146a-5p was downregulated in TNBC tissues and cell lines. SOX5 was indicated to be a target gene of miR-146a-5p and was upregulated in TNBC cells. Additionally, miR-146a-5p could inhibit TNBC cell proliferation, migration and invasion, repress the expression of mesenchymal markers (N-cadherin, vimentin and fibronectin) and increase epithelial marker (E-cadherin) expression. Furthermore, SOX5 overexpression eliminated the effects of miR-146a-5p mimics on TNBC cells. In conclusion, the data of the present study indicated that miR-146a-5p inhibits the proliferation and metastasis of TNBC cells by regulating SOX5.

  17. Stem cell survival is severely compromised by the thymidineanalog EdU (5-ethynyl-2'-deoxyuridine), an alternative to BrdU for proliferation assays and stem cell tracing

    DEFF Research Database (Denmark)

    Andersen, Ditte C; Skovrind, Ida; Christensen, Marlene Louise

    2013-01-01

    Stem cell therapy has opened up the possibility of treating numerous degenerating diseases. However, we are still merely at the stage of identifying appropriate sources of stem cells and exploring their full differentiation potential. Thus, tracking the stem cells upon in vivo engraftment...... and during in vitro co-culture is very important and is an area of research embracing many pitfalls. 5-Ethynyl-2'-deoxyuridine (EdU), a rather new thymidine analog incorporated into DNA, has recently been suggested to be a novel highly valid alternative to other dyes for labeling of stem cells and subsequent...... tracing of their proliferation and differentiation ability. However, our results herein do not at any stage support this recommendation, since EdU severely reduces the viability of stem cells. Accordingly, we found that transplanted EdU-labeled stem cells hardly survive upon in vivo transplantation...

  18. Limb-bud and Heart Overexpression Inhibits the Proliferation and Migration of PC3M Cells.

    Science.gov (United States)

    Liu, Qicai; Li, Ermao; Huang, Long; Cheng, Minsheng; Li, Li

    2018-01-01

    Background: The limb-bud and heart gene ( LBH ) was discovered in the early 21st century and is specifically expressed in the mouse embryonic limb and heart development. Increasing evidences have indicated that LBH not only plays an important role in embryo development, it is also closely correlated with the occurance and progression of many tumors. However, its function in prostate cancer (PCa) is still not well understood. Here, we explored the effects of LBH on the proliferation and migration of the PCa cell line PC3M. Methods: LBH expression in tissues and cell lines of PCa was detected by immunohistochemistry and Western blotting. Lentivirus was used to transduct the LBH gene into the PC3M cells. Stable LBH-overexpressing PC3M-LBH cells and PC3M-NC control cells were obtained via puromycin screening. Cell proliferation was examined using the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Cell cycle distribution and apoptosis rate were investigated using flow cytometry. Cell migration was studied using the Transwell assay. Results: LBH expression level was down-regulated in 3 different PCa cell lines, especially in PC3M cells, compared with the normal prostate epithelial cells(RWPE-1). Cell lines of LBH-upregulated PC3M-LBH and PC3M-NC control were successfully constructed. Significantly increased LBH expression level and decreased cyclin D1 and cyclin E2 expression level was found in PC3M-LBH cells as compared to the PC3M-NC cells. The overexpression of LBH significantly inhibited PC3M cell proliferation in vitro and tumor growth in nude mice. LBH overexpression in PC3M cell, also induced cell cycle G0/G1 phase arrest and decreased the migration of PC3M cells. Conclusions : Our results reveal that LBH expression is down-regulated in the tissue and cell lines of PCa. LBH overexpression inhibits PC3M cell proliferation and tumor growth by inducing cell cycle arrest through down-regulating cyclin D1and cyclin E2 expression. LBH might

  19. HER2 induces cell proliferation and invasion of non-small-cell lung cancer by upregulating COX-2 expression via MEK/ERK signaling pathway

    Directory of Open Access Journals (Sweden)

    Chi F

    2016-05-01

    Full Text Available Feng Chi, Rong Wu, Xueying Jin, Min Jiang, Xike Zhu Department of Medical Oncology, Shengjing Hospital of China Medical University, Shenyang, People’s Republic of China Abstract: HER2 positivity has been well studied in various cancers, but its importance in non-small-cell lung cancer (NSCLC is still being explored. In this study, quantitative reverse transcription polymerase chain reaction (qRT-PCR was performed to detect HER2 and COX-2 expression in NSCLC tissues. Then, pcDNA3.1-HER2 was used to overexpress HER2, while HER2 siRNA and COX-2 siRNA were used to silence HER2 and COX-2 expression. MTT assay and invasion assay were used to detect the effects of HER2 on cell proliferation and invasion. Our study revealed that HER2 and COX-2 expression were upregulated in NSCLC tissues and HER2 exhibited a significant positive correlation with the levels of COX-2 expression. Overexpression of HER2 evidently elevated COX-2 expression, while silencing of HER2 evidently decreased COX-2 expression. Furthermore, overexpressed HER2 induced the ERK phosphorylation, and this was abolished by the treatment with U0126, a pharmacological inhibitor of MEK, an upstream kinase of ERK. HER2-induced expression and promoter activity of COX-2 were also suppressed by U0126, suggesting that the MEK/ERK signaling pathway regulates COX-2 expression. In addition, HER2 induced activation of AKT signaling pathway, which was reversed by pretreatment with U0126 and COX-2 siRNA. MTT and invasion assays revealed that HER2 induced cell proliferation and invasion that were reversed by pretreatment with U0126 and COX-2 siRNA. In this study, our results demonstrated for the first time that HER2 elevated COX-2 expression through the activation of MEK/ERK pathway, which subsequently induced cell proliferation and invasion via AKT pathway in NSCLC tissues. Keywords: HER2, MEK/ERK, COX-2, AKT signaling pathway, non-small-cell lung cancer

  20. PKI-587 and sorafenib targeting PI3K/AKT/mTOR and Ras/Raf/MAPK pathways synergistically inhibit HCC cell proliferation.

    Science.gov (United States)

    Gedaly, Roberto; Angulo, Paul; Hundley, Jonathan; Daily, Michael F; Chen, Changguo; Evers, B Mark

    2012-08-01

    Deregulated Ras/Raf/MAPK and PI3K/AKT/mTOR signaling pathways are found in hepatocellular carcinoma (HCC). This study aimed to test the inhibitory effects of PKI-587 and sorafenib as single agents or in combination on HCC (Huh7 cell line) proliferation. (3)H-thymidine incorporation and MTT assay were used to assess Huh7 cell proliferation. Phosphorylation of the key enzymes in the Ras/Raf/MAPK and PI3K/AKT/mTOR pathways was detected by Western blot. We found that PKI-587 is a more potent PI3K/mTOR inhibitor than PI-103. Combination of PKI-587 and sorafenib was a more effective inhibitor of Huh7 proliferation than the combination of PI-103 and sorafenib. Combination of PKI-587 and sorafenib synergistically inhibited epidermal growth factor (EGF)-stimulated Huh7 proliferation compared with monodrug therapy. EGF increased phosphorylation of Ras/Raf downstream signaling proteins MEK and ERK; EGF-stimulated activation was inhibited by sorafenib. However, sorafenib, as a single agent, increased AKT (Ser473) phosphorylation. EGF-stimulated AKT (ser473) activation was inhibited by PKI-587. PKI-587 is a potent inhibitor of AKT (Ser473), mTOR (Ser2448), and S6K (Thr389) phosphorylation; in contrast, rapamycin stimulated mTOR complex 2 substrate AKT(Ser473) phosphorylation although it inhibited mTOR complex 1 substrate S6K phosphorylation. PKI-587, as a single agent, stimulated MEK and ERK phosphorylation. However, when PKI-587 and sorafenib were used in combination, they inhibited all the tested kinases in the Ras/Raf /MAPK and PI3K/AKT/mTOR pathways. The combination of PKI-587 and sorafenib has the advantage over monodrug therapy on inhibition of HCC cell proliferation by blocking both PI3K/AKT/mTOR and Ras/Raf/MAPK signaling pathways. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. Total glucosides of paeony inhibit the proliferation of fibroblast-like synoviocytes through the regulation of G proteins in rats with collagen-induced arthritis.

    Science.gov (United States)

    Jia, Xiao-Yi; Chang, Yan; Sun, Xiao-Jing; Wu, Hua-Xun; Wang, Chun; Xu, Hong-Mei; Zhang, Lei; Zhang, Ling-Ling; Zheng, Yong-Qiu; Song, Li-Hua; Wei, Wei

    2014-01-01

    The aim of this study was to investigate the expression of G proteins in fibroblast-like synoviocytes (FLSs) from rats with collagen-induced arthritis (CIA) and to determine the effect of total glucosides of paeony (TGP). CIA rats were induced with chicken type II collagen (CCII) in Freund's complete adjuvant. The rats with experimental arthritis were randomly separated into five groups and then treated with TGP (25, 50, and 100mg/kg) from days 14 to 35 after immunization. The secondary inflammatory reactions were evaluated through the polyarthritis index and histopathological changes. The level of cyclic adenosine monophosphate (cAMP) was measured by radioimmunoassay. The FLS proliferation response was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The toxin-catalyzed ADP-ribosylation of G proteins was performed through autoradiography. The results show that TGP (25, 50, and 100mg/kg) significantly decreased the arthritis scores of CIA rats and improved the histopathological changes. TGP inhibited the proliferation of FLSs and increased the level of cAMP. Moreover, the FLS proliferation and the level of Gαi expression were significantly increased, but the level of Gαs expression was decreased after stimulation with IL-1β (10ng/ml) in vitro. TGP (12.5 and 62.5μg/ml) significantly inhibited the FLS proliferation and regulated the balance between Gαi and Gαs. These results demonstrate that TGP may exert its anti-inflammatory effects through the suppression of FLS proliferation, which may be associated with its ability to regulate the balance of G proteins. Thus, TGP may have potential as a therapeutic agent for the treatment of rheumatoid arthritis. © 2013.

  2. 2D ratiometric fluorescent pH sensor for tracking of cells proliferation and metabolism.

    Science.gov (United States)

    Ma, Jun; Ding, Changqin; Zhou, Jie; Tian, Yang

    2015-08-15

    Extracellular pH plays a vital role no matter in physiological or pathological studies. In this work, a hydrogel, CD@Nile-FITC@Gel (Gel sensor), entrapping the ratiometric fluorescent probe CD@Nile-FITC was developed. The Gel sensor was successfully used for real-time extracellular pH monitoring. In the case of CD@Nile-FITC, pH-sensitive fluorescent dye fluorescein isothiocyanate (FITC) was chosen as the response signal for H(+) and Nile blue chloride (Nile) as the reference signal. The developed fluorescent probe exhibited high selectivity for pH over other metal ions and amino acids. Meanwhile, the carbon-dots-based inorganic-organic probe demonstrated excellent photostability against long-term light illumination. In order to study the extracellular pH change in processes of cell proliferation and metabolism, CD@Nile-FITC probe was entrapped in sodium alginate gel and consequently formed CD@Nile-FITC@Gel. The MTT assay showed low cytotoxicity of the Gel and the pH titration indicated that it could monitor the pH fluctuations linearly and rapidly within the pH range of 6.0-9.0, which is valuable for physiological pH determination. As expected, the real-time bioimaging of the probe was successfully achieved. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Kefir induces apoptosis and inhibits cell proliferation in human acute erythroleukemia.

    Science.gov (United States)

    Jalali, Fatemeh; Sharifi, Mohammadreza; Salehi, Rasoul

    2016-01-01

    Acute erythroleukemia is an uncommon subtype of acute myeloid leukemia which has been considered to be a subtype of AML with a worse prognosis. Intensive chemotherapy is the first line of treatment. In recent years, the effect of kefir on some malignancies has been experimented. Kefir is a kind of beverage, which obtained by incubation of kefir grains with raw milk. Kefir grains are a symbiotic complex of different kinds of yeasts and bacteria, especially lactic acid bacteria which gather in a mostly carbohydrate matrix, named kefiran. We investigated the effect of kefir on acute erythroleukemia cell line (KG-1) and peripheral blood mononuclear cells (PBMCs). The cell line and PBMCs were treated with different doses of kefir and milk and incubated for three different times. We used Polymixin B to block the lipopolysaccharide and NaOH (1 mol/l) to neutralize the acidic media. Viability was detected by MTT assay. Apoptosis and necrosis were assessed by annexin-propidium iodide staining. Our results showed that kefir induced apoptosis and necrosis in KG-1 cell line. It was revealed that kefir decreased proliferation in erythroleukemia cell line. We did not observe a remarkable effect of kefir on PBMCs. Our study suggested that kefir may have potential to be an effective treatment for erythroleukemia.

  4. Porphyromonas gingivalis decreases osteoblast proliferation through IL-6-RANKL/OPG and MMP-9/TIMPs pathways

    Directory of Open Access Journals (Sweden)

    Le Xuan

    2009-01-01

    Full Text Available Background: Porphyromonas gingivalis, an important periodontal pathogen, is closely associated with inflammatory alveolar bone resorption. This bacterium exerts its pathogenic effect indirectly through multiple virulence factors, such as lipopolysaccharides, fimbriae, and proteases. Another possible pathogenic path may be through a direct interaction with the host′s soft and hard tissues (e.g., alveolar bone, which could lead to periodontitis. Aims and Objectives: The aim of the present study was to investigate the direct effect of live and heat-inactivated P gingivalis on bone resorption, using an in vitro osteoblast culture model. Results: Optical microscopy and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide MTT assay revealed that live P gingivalis induced osteoblast detachment and reduced their proliferation. This effect was specific to live bacteria and was dependent on their concentration. Live P gingivalis increased IL-6 mRNA expression and protein production and downregulated RANKL and OPG mRNA expression. The effect of live P gingivalis on bone resorption was strengthened by an increase in MMP-9 expression and its activity. This increase was accompanied by an increase in TIMP-1 and TIMP-2 mRNA expression and protein production by osteoblasts infected with live P gingivalis. Conclusion: Overall, the results suggest that direct contact of P gingivalis with osteoblasts induces bone resorption through an inflammatory pathway that involves IL-6, RANKL/OPG, and MMP-9/TIMPs.

  5. Enhancing proliferation and osteogenic differentiation of HMSCs on casein/chitosan multilayer films.

    Science.gov (United States)

    Li, Yan; Zheng, Zebin; Cao, Zhinan; Zhuang, Liangting; Xu, Yong; Liu, Xiaozhen; Xu, Yue; Gong, Yihong

    2016-05-01

    Creating a bioactive surface is important in tissue engineering. Inspired by the natural calcium binding property of casein (CA), multilayer films ((CA/CS)n) with chitosan (CS) as polycation were fabricated to enhance biomineralization, cell adhesion and differentiation. LBL self-assembly technique was used and the assembly process was intensively studied based on changes of UV absorbance, zeta potential and water contact angle. The increasing content of chitosan and casein with bilayers was further confirmed with XPS and TOF-SIMS analysis. To improve the biocompatibility, gelatin was surface grafted. In vitro mineralization test demonstrated that multilayer films had more hydroxyapatite crystal deposition. Human mesenchymal stem cells (HMSCs) were seeded onto these films. According to fluorescein diacetate (FDA) and cell cytoskeleton staining, MTT assay, expression of osteogenic marker genes, ALP activity, and calcium deposition quantification, it was found that these multilayer films significantly promoted HMSCs attachment, proliferation and osteogenic differentiation than TCPS control. Copyright © 2016. Published by Elsevier B.V.

  6. Urothelial carcinoma associated 1 is a hypoxia-inducible factor-1α-targeted long noncoding RNA that enhances hypoxic bladder cancer cell proliferation, migration, and invasion.

    Science.gov (United States)

    Xue, Mei; Li, Xu; Li, Zhengkun; Chen, Wei

    2014-07-01

    Urothelial carcinoma associated 1 (UCA1) has been identified as an oncogenic long noncoding RNA (lncRNA) that is involved in bladder cancer progression and acts as a diagnostic biomarker for bladder carcinoma. Here, we studied the expression and function of lncRNA-UCA1 in the hypoxic microenvironment of bladder cancer. The expression and transcriptional activity of lncRNA-UCA1 were measured by quantitative real-time polymerase chain reaction and luciferase assays. Cell proliferation and apoptosis were evaluated by MTT assays and flow cytometry. Cell migration and invasion were detected by wound healing, migration, and invasion assays. The binding of hypoxia-inducible factor-1α (HIF-1α) to hypoxia response elements (HREs) in the lncRNA-UCA1 promoter was confirmed by electrophoretic mobility shift assay and chromatin immunoprecipitation. HRE mutations were generated by using a site-directed mutagenesis kit, and HIF-1α knockdown was mediated by small interfering RNA. The effect of HIF-1α inhibition by YC-1 on lncRNA-UCA1 expression was also examined. LncRNA-UCA1 was upregulated by hypoxia in bladder cancer cells. Under hypoxic conditions, lncRNA-UCA1 upregulation increased cell proliferation, migration, and invasion and inhibited apoptosis. The underlying mechanism of hypoxia-upregulated lncRNA-UCA1 expression was that HIF-1α specifically bound to HREs in the lncRNA-UCA1 promoter. Furthermore, HIF-1α knockdown or inhibition could prevent lncRNA-UCA1 upregulation under hypoxia. These findings revealed the mechanism of lncRNA-UCA1 upregulation in hypoxic bladder cancer cells and suggested that effective blocking of lncRNA-UCA1 expression in the hypoxic microenvironment of bladder cancer could be a novel therapeutic strategy.

  7. PHA665752, a small-molecule inhibitor of c-Met, inhibits hepatocyte growth factor-stimulated migration and proliferation of c-Met-positive neuroblastoma cells

    International Nuclear Information System (INIS)

    Crosswell, Hal E; Dasgupta, Anindya; Alvarado, Carlos S; Watt, Tanya; Christensen, James G; De, Pradip; Durden, Donald L; Findley, Harry W

    2009-01-01

    c-Met is a tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF), and both c-Met and its ligand are expressed in a variety of tissues. C-Met/HGF/SF signaling is essential for normal embryogenesis, organogenesis, and tissue regeneration. Abnormal c-Met/HGF/SF signaling has been demonstrated in different tumors and linked to aggressive and metastatic tumor phenotypes. In vitro and in vivo studies have demonstrated inhibition of c-Met/HGF/SF signaling by the small-molecule inhibitor PHA665752. This study investigated c-Met and HGF expression in two neuroblastoma (NBL) cell lines and tumor tissue from patients with NBL, as well as the effects of PHA665752 on growth and motility of NBL cell lines. The effect of the tumor suppressor protein PTEN on migration and proliferation of tumor cells treated with PHA665752 was also evaluated. Expression of c-Met and HGF in NBL cell lines SH-EP and SH-SY5Y and primary tumor tissue was assessed by immunohistochemistry and quantitative RT-PCR. The effect of PHA665752 on c-Met/HGF signaling involved in NBL cell proliferation and migration was evaluated in c-Met-positive cells and c-Met-transfected cells. The transwell chemotaxis assay and the MTT assay were used to measure migration and proliferation/cell-survival of tumor cells, respectively. The PPAR-γ agonist rosiglitazone was used to assess the effect of PTEN on PHA665752-induced inhibition of NBL cell proliferation/cell-survival and migration High c-Met expression was detected in SH-EP cells and primary tumors from patients with advanced-stage disease. C-Met/HGF signaling induced both migration and proliferation of SH-EP cells. Migration and proliferation/cell-survival were inhibited by PHA665752 in a dose-dependent manner. We also found that induced overexpression of PTEN following treatment with rosiglitazone significantly enhanced the inhibitory effect of PHA665752 on NBL-cell migration and proliferation. c-Met is highly expressed in most tumors from

  8. Assessments of proliferation capacity and viability of New Zealand rabbit peripheral blood endothelial progenitor cells labeled with superparamagnetic particles.

    Science.gov (United States)

    Mai, Xiao-Li; Ma, Zhan-Long; Sun, Jun-Hui; Ju, Sheng-Hong; Ma, Ming; Teng, Gao-Jun

    2009-01-01

    Magnetic resonance imaging (MRI) has proven to be effective in tracking the distribution of transplanted stem cells to target organs by way of labeling cells with superparamagnetic iron oxide particles (SPIO). However, the effect of SPIO upon labeled cells is still unclear on a cellular level. With this study, the proliferation and viability of New Zealand rabbit peripheral blood endothelial progenitor cells (EPCs) labeled with SPIO were evaluated and in vitro images were obtained using a 1.5 T MR scanner. Mononuclear cells (MNCs) were isolated from peripheral blood of the adult New Zealand rabbit and cultured in fibronectin-coated culture flasks, in which EPCs were identified from cell morphology, outgrowth characteristics, and internalization of DiI-Ac-LDL and binding to FITC-UEA I. EPCs were incubated with the self-synthesized poly-L-lysine-conjugated SPIO (PLL-SPIO) particles in a range of concentrations. The prevalence of iron-containing vesicles or endosomes in the cytoplasm of labeled cells was confirmed with Prussian blue staining and transmission electron microscopy. Tetrazolium salt (MTT) assay, cell apoptosis, and cycle detection were assessed to evaluate proliferation and function of various concentrations, magnetically labeled EPCs. The quantity of iron per cell was determined by atomic absorption spectrometry. The cells underwent MRI with different sequences. The result showed that rabbit EPCs were efficiently labeled with the home synthesized PLL-SPIO. There was found to be no statistically significant difference in the MTT values of light absorption measured on the third and fifth days. Between labeled and unlabeled cells, there were also no aberrations found in the cell cycles, apoptosis, or growth curves. The atomic absorption spectrophotometer showed that the intracellular content of Fe decreased as more time elapsed after labeling. The labeled EPCs demonstrated a loss of MRI signal intensity (SI) when compared with the SI of unlabeled cells

  9. Hormone assay

    International Nuclear Information System (INIS)

    Eisentraut, A.M.

    1977-01-01

    An improved radioimmunoassay is described for measuring total triiodothyronine or total thyroxine levels in a sample of serum containing free endogenous thyroid hormone and endogenous thyroid hormone bound to thyroid hormone binding protein. The thyroid hormone is released from the protein by adding hydrochloric acid to the serum. The pH of the separated thyroid hormone and thyroid hormone binding protein is raised in the absence of a blocking agent without interference from the endogenous protein. 125 I-labelled thyroid hormone and thyroid hormone antibodies are added to the mixture, allowing the labelled and unlabelled thyroid hormone and the thyroid hormone antibody to bind competitively. This results in free thyroid hormone being separated from antibody bound thyroid hormone and thus the unknown quantity of thyroid hormone may be determined. A thyroid hormone test assay kit is described for this radioimmunoassay. It provides a 'single tube' assay which does not require blocking agents for endogenous protein interference nor an external solid phase sorption step for the separation of bound and free hormone after the competitive binding step; it also requires a minimum number of manipulative steps. Examples of the assay are given to illustrate the reproducibility, linearity and specificity of the assay. (UK)

  10. Assay system

    International Nuclear Information System (INIS)

    Patzke, J.B.; Rosenberg, B.J.

    1984-01-01

    The accuracy of assays for monitoring concentrations of basic drugs in biological fluids containing a 1 -acid glycoproteins, such as blood (serum or plasma), is improved by the addition of certain organic phosphate compounds to minimize the ''protein effect.'' Kits containing the elements of the invention are also disclosed

  11. Crosstalk between EGFR and integrin affects invasion and proliferation of gastric cancer cell line, SGC7901

    Directory of Open Access Journals (Sweden)

    Dan L

    2012-10-01

    Full Text Available Li Dan,1,* Ding Jian,2,* Lin Na,1 Wang Xiaozhong,1 1Digestive Department, the Union Hospital of Fujian Medical University, Fujian, People’s Republic of China; 2Digestive Department, the First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian, People’s Republic of China*These authors contributed equally to this workBackground/objective: To investigate the crosstalk between epidermal growth factor receptor (EGFR and integrin-mediated signal transduction pathways in human gastric adenocarcinoma cells.Methods: EGF was used as a ligand of EGFR to stimulate the gastric adenocarcinoma cell, SGC7901. Signal molecules downstream of the integrin, FAK(Y397 and p130cas(Y410 phosphorylation, were measured by immunoprecipitation and western blot. Fibronectin (Fn was used as a ligand of integrin to stimulate the same cell line. Signal molecules downstream of EGFR and extracellular signal-regulated kinase (ERK general phosphorylation were also measured. Focal adhesion kinase (FAK small-interfering RNA was designed and transfected into SGC7901 cells to decrease the expression of FAK. Modified Boyden chambers and MTT assay were used to examine the effect of FAK inhibition on the invasiveness and proliferation of SGC7901.Results: EGF activated FAK(Y397 and p130cas(Y410 phosphorylation, while Fn activated ERK general phosphorylation. Inhibition of FAK expression decreased p130cas(Y410 phosphorylation activated by EGF and ERK general phosphorylation activated by Fn, also decreased the invasiveness and proliferation of SGC7901 cells activated by EGF or Fn.Conclusion: There is crosstalk between EGFR and integrin signal transduction. FAK may be a key cross point of the two signal pathways and acts as a potential target for human gastric cancer therapy.Keywords: gastric adenocarcinoma, epidermal growth factor receptor, integrin, focal adhesion kinase, crosstalk

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

    Science.gov (United States)

    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.

  13. [Effect of M007 mediated photodynamic therapy on proliferation of human osteosarcoma MG63 cells in vitro].

    Science.gov (United States)

    Zhou, Yu-Kai; Wu, Wen-Zhi; Zhang, Lan; Yang, Chun-Hui; Wang, Yan-Ping

    2012-01-01

    To investigate the effect of a new photosensitizer, M007 mediated photodynamic therapy on proliferation of human osteosarcoma MG63 cells in vitro. Human osteosarcoma MG63 cells were prepared as 1 x 10(6) /mL single-cell suspension, and 1 mL cells were transferred into 60 mL culture dish, then treated with 5 different gradient dosages (0, 2, 4, 8, 16 micromol/L) of M007 followed by photodynamic therapy or dark reaction for 10 min. The survival rate of the cells and the mode of cell death were detected by flow cytometry with the stain of Annexin V-FITC/PI. The effect on proliferation of survival cells was observed by MTT assay and colony-forming assay. M007 mediated photodynamic therapy induced the inactivation of MG63 human osteosarcoma cells in the way of late apoptosis/necrosis or becoming naked nucleus predominately. More than 90% MG63 cells in M007-PDT group were dead under the treatment of 2-16 micromol/L M007. The survival rates of 4-16 micromol/L M007-PDT group were steadily less than 1%. The optical densities did not increase with extension of culture time in 2-8 micromol/L M007-PDT group (P > 0.05). There were 16 survival alive cells found occasionally in 2 micromol/L M007-PDT group, but no colonies found in other groups. M007 mediated photodynamic therapy totally inactivated human osteosarcoma MG63 cells in vitro with the dosage more than 4 micromol/L.

  14. Epigenetic activation of SIN1 promotes NSCLC cell proliferation and metastasis by affecting the epithelial–mesenchymal transition

    International Nuclear Information System (INIS)

    Hu, Zhongwu; Wang, Yaqin; Wang, Yuemei; Zang, Bao; Hui, Hongxia; You, Zhenbing; Wang, Xiaowei

    2017-01-01

    Stress-activated protein kinase (SAPK) interacting protein 1 (SIN1) is an essential component of mTORC2. Previous studies have shown that SIN1 is a key regulator of Akt pathway which plays an important role in various pathological conditions including cancer. While its effects and mechanisms on the progression of NSCLC remain unknown. In this study, we report that SIN1 is able to promote the growth and migration of NSCLC cells both in vitro and in vivo. Overexpression of SIN1 promoted A549 and H1299 cells proliferation by both MTT and colony formation assays. Consistently, knockdown of SIN1 inhibited the proliferation of these cells. In transwell assay, overexpression of SIN1 increased the migration of A549 and H1299 cells, while SIN1 knockdown reduced their migration. In a tumor xenograft model, overexpression of SIN1 promoted tumor growth of A549 cells in vivo, while SIN1 knockdown suppresses the tumor growth. We also found a mechanistic link between SIN1 and H3K4me3, H3K4me3 is involved in SIN1 upregulation. Moreover, SIN1 can significantly promote the in vitro migration and invasion of NSCLC cells via induction epithelial mesenchymal transition (EMT) process, which subsequently leads to transcriptional downregulation of epithelial marker E-cadherin and upregulation of mesenchymal markers N-cadherin and Vimentin expression. Together, our results reveal that SIN1 plays an important role in NSCLC and SIN1 is a potential biomarker and a promising target in the treatment of NSCLC.

  15. VEGF induces proliferation of human hair follicle dermal papilla cells through VEGFR-2-mediated activation of ERK

    International Nuclear Information System (INIS)

    Li, Wei; Man, Xiao-Yong; Li, Chun-Ming; Chen, Jia-Qi; Zhou, Jiong; Cai, Sui-Qing; Lu, Zhong-Fa; Zheng, Min

    2012-01-01

    Vascular endothelial growth factor (VEGF) is one of the strongest regulators of physiological and pathological angiogenesis. VEGF receptor 2 (VEGFR-2), the primary receptor for VEGF, is thought to mediate major functional effects of VEGF. Previously, we have localized both VEGF and VEGFR-2 in human hair follicles. In this study, we further defined the expression and roles of VEGFR-2 on human hair follicle dermal papilla (DP) cells. The expression of VEGFR-2 on DP cells was examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis separately, and localization of VEGFR-2 was defined by immunofluorescence. The effect of VEGF on DP cells was analyzed by MTT assays and specific inhibitors. Finally, the role of VEGF involved in the signaling pathways was investigated by Western blot. RT-PCR and Western blot analysis demonstrated the expression of VEGFR-2 on DP cells. Immunostaining for VEGFR-2 showed strong signal on cultured human DP cells in vitro. Exogenous VEGF 165 stimulated proliferation of DP cells in a dose-dependent manner. Furthermore, this stimulation was blocked by a VEGFR-2 neutralizing antibody (MAB3571) and an ERK inhibitor (PD98059). VEGF 165 -induced phosphorylation of ERK1/2 was abolished by MAB3571 and PD98059, while the phosphorylation of p38, JNK and AKT were not changed by VEGF 165 . Taken together, VEGFR-2 is expressed on primary human hair follicle DP cells and VEGF induces proliferation of DP cells through VEGFR-2/ERK pathway, but not p38, JNK or AKT signaling. -- Highlights: ► We examine the expression of VEGFR-2 on cultured human dermal papilla (DP) cells. ► VEGF 165 stimulated proliferation of human DP cells in a dose-dependent manner. ► This stimulation was through VEGFR-2-mediated activation of ERK.

  16. FOXA1 promotes tumor cell proliferation through AR involving the Notch pathway in endometrial cancer

    International Nuclear Information System (INIS)

    Qiu, Meiting; Bao, Wei; Wang, Jingyun; Yang, Tingting; He, Xiaoying; Liao, Yun; Wan, Xiaoping

    2014-01-01

    Increasing evidence suggests that forkhead box A1 (FOXA1) is frequently dysregulated in many types of human cancers. However, the exact function and mechanism of FOXA1 in human endometrial cancer (EC) remains unclear. FOXA1 expression, androgen receptor (AR) expression, and the relationships of these two markers with clinicopathological factors were determined by immunohistochemistry analysis. FOXA1 and AR were up-regulated by transient transfection with plasmids, and were down-regulated by transfection with siRNA or short hairpin RNA (shRNA). The effects of FOXA1 depletion and FOXA1 overexpression on AR-mediated transcription as well as Notch pathway and their impact on EC cell proliferation were examined by qRT-PCR, western blotting, co-immunoprecipitation, ChIP-PCR, MTT, colony-formation, and xenograft tumor–formation assays. We found that the expression of FOXA1 and AR in ECs was significantly higher than that in a typical hyperplasia and normal tissues. FOXA1 expression was significantly correlated with AR expression in clinical tissues. High FOXA1 levels positively correlated with pathological grade and depth of myometrial invasion in EC. High AR levels also positively correlated with pathological grade in EC. Moreover, the expression of XBP1, MYC, ZBTB16, and UHRF1, which are downstream targets of AR, was promoted by FOXA1 up-regulation or inhibited by FOXA1 down-regulation. Co-immunoprecipitation showed that FOXA1 interacted with AR in EC cells. ChIP-PCR assays showed that FOXA1 and AR could directly bind to the promoter and enhancer regions upstream of MYC. Mechanistic investigation revealed that over-expression of Notch1 and Hes1 proteins by FOXA1 could be reversed by AR depletion. In addition, we showed that down-regulation of AR attenuated FOXA1-up-regulated cell proliferation. However, AR didn’t influence the promotion effect of FOXA1 on cell migration and invasion. In vivo xenograft model, FOXA1 knockdown reduced the rate of tumor growth. These

  17. Effects of PARP-1 inhibitors AG-014699 and AZD2281 on proliferation and apoptosis of human hepatoma cell line HepG2

    Directory of Open Access Journals (Sweden)

    DU Senrong

    2015-06-01

    Full Text Available ObjectiveTo observe the inhibitory and pro-apoptotic effects of two poly(ADP-ribose polymerase (PARP-1 inhibitors, AG-014699 and AZD2281, on human hepatoma HepG2 cells and preliminarily explore the mechanism by which AG-014699 induces HepG2 cell apoptosis, and to provide a new therapeutic target for hepatoma. MethodsThe effects of different concentrations of AG-014699 and AZD2281 on HepG2 cell proliferation were determined by MTT assay. The cell apoptosis rate was measured by flow cytometry. The expression levels of caspase-3 and caspase-8 were measured by Western Blot. Inter-group comparison was made by t test. ResultsBoth AG-014699 and AZD2281 suppressed HepG2 cell proliferation in a time- and dose-dependent manner. However, the sensitivity of HepG2 cells to the two PARP-1 inhibitors was different. The half-maximal inhibitory concentrations of AG-014699 and AZD2281 at 48 h determined by MTT assay were about 20 μmol/L and 400 μmol/L, respectively. Flow cytometry and Western blot were not used to evaluate the apoptosis of HepG2 cells exposed to AZD2281 to which these cells were not sensitive. HepG2 cell apoptosis could be induced by 10, 30, and 50 μmol/L AG-014699, and the highest apoptosis rate at 48 h was significantly higher than that of the control group (3100%±2.13% vs 09%±0013%, P<0.01. Compared with those in the control group, the protein levels of caspase-3 and caspase-8 in HepG2 cells after 48-h exposure to 30, and 50 μmol/L AG-014699 increased. ConclusionThe two PARP-1 inhibitors AG-014699 and AZD2281 can inhibit the proliferation of HepG2 cells, which showed different sensitivities to the two inhibitors. AG-014699 can induce HepG2 cell apoptosis by up-regulating the protein expression of caspase-3 and caspase-8.

  18. The threat of proliferation

    International Nuclear Information System (INIS)

    Palme, Olof.

    1986-01-01

    The paper on the threat of proliferation, is a keynote speech delivered to the Colloquium on Nuclear War, Nuclear Proliferation and their Consequences, Geneva, 1985. Topics discussed in the address include: nuclear weapons, nuclear war, terrorists, Non-Proliferation Treaty, nuclear disarmament, and leadership in world affairs. (UK)

  19. Fibrin promotes proliferation and matrix production of intervertebral disc cells cultured in three-dimensional poly(lactic-co-glycolic acid) scaffold.

    Science.gov (United States)

    Sha'ban, Munirah; Yoon, Sun Jung; Ko, Youn Kyung; Ha, Hyun Jung; Kim, Soon Hee; So, Jung Won; Idrus, Ruszymah Bt Hj; Khang, Gilson

    2008-01-01

    Previously, we have proven that fibrin and poly(lactic-co-glycolic acid) (PLGA) scaffolds facilitate cell proliferation, matrix production and early chondrogenesis of rabbit articular chondrocytes in in vitro and in vivo experiments. In this study, we evaluated the potential of fibrin/PLGA scaffold for intervertebral disc (IVD) tissue engineering using annulus fibrosus (AF) and nucleus pulposus (NP) cells in relation to potential clinical application. PLGA scaffolds were soaked in cells-fibrin suspension and polymerized by dropping thrombin-sodium chloride (CaCl(2)) solution. A PLGA-cell complex without fibrin was used as control. Higher cellular proliferation activity was observed in fibrin/PLGA-seeded AF and NP cells at each time point of 3, 7, 14 and 7 days using the MTT assay. After 3 weeks in vitro incubation, fibrin/PLGA exhibited a firmer gross morphology than PLGA groups. A significant cartilaginous tissue formation was observed in fibrin/PLGA, as proven by the development of cells cluster of various sizes and three-dimensional (3D) cartilaginous histoarchitecture and the presence of proteoglycan-rich matrix and glycosaminoglycan (GAG). The sGAG production measured by 1,9-dimethylmethylene blue (DMMB) assay revealed greater sGAG production in fibrin/PLGA than PLGA group. Immunohistochemical analyses showed expressions of collagen type II, aggrecan core protein and collagen type I genes throughout in vitro culture in both fibrin/PLGA and PLGA. In conclusion, fibrin promotes cell proliferation, stable in vitro tissue morphology, superior cartilaginous tissue formation and sGAG production of AF and NP cells cultured in PLGA scaffold. The 3D porous PLGA scaffold-cell complexes using fibrin can provide a vehicle for delivery of cells to regenerate tissue-engineered IVD tissue.

  20. Gambogic acid-loaded magnetic Fe(3)O(4) nanoparticles inhibit Panc-1 pancreatic cancer cell proliferation and migration by inactivating transcription factor ETS1.

    Science.gov (United States)

    Wang, Cailian; Zhang, Haijun; Chen, Yan; Shi, Fangfang; Chen, Baoan

    2012-01-01

    E26 transformation-specific sequence-1 (ETS1) transcription factor plays important roles in both carcinogenesis and the progression of a wide range of malignancies. Aberrant ETS1 expression correlates with aggressive tumor behavior and a poorer prognosis in patients with various malignancies. The aim of the current study was to evaluate the efficacy of a drug delivery system utilizing gambogic acid-loaded magnetic Fe(3)O(4) nanoparticles (GA-MNP-Fe(3)O(4)) on the suppression of ETS1-mediated cell proliferation and migration in Panc-1 pancreatic cancer cells. The effects caused by GA-MNP-Fe(3)O(4) on the proliferation of Panc-1 pancreatic cancer cells were evaluated using a MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay while inhibition of tumor cell migration was investigated in a scratch assay. The expressions of ETS1, cyclin D1, urokinase-type plasminogen activator (u-PA), and VEGF (vascular endothelial growth factor) were examined by Western blot to elucidate the possible mechanisms involved. In Panc-1 pancreatic cancer cells, we observed that application of GA-MNP-Fe(3)O(4) was able to suppress cancer cell proliferation and prevent cells from migrating effectively. After treatment, Panc-1 pancreatic cancer cells showed significantly decreased expression of ETS1, as well as its downstream target genes for cyclin D1, u-PA, and VEGF. Our novel finding reaffirmed the significance of ETS1 in the treatment of pancreatic cancer, and application of GA-MNP-Fe(3)O(4) nanoparticles targeting ETS1 should be considered as a promising contribution for better pancreatic cancer care.

  1. A Novel Natural Product, KL-21, Inhibits Proliferation and Induces Apoptosis in Chronic Lymphocytic Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Aysun Adan Gökbulut

    2015-06-01

    Full Text Available INTRODUCTION: The aims of this study were to examine the cytotoxic and apoptotic effects of KL-21, a novel plant product (produced by Naturin Natural Products, İzmir, Turkey, on 232B4 chronic lymphocytic leukemia (CLL cells and to determine the cytotoxic effects on healthy BEAS-2B human bronchial epithelial cells. METHODS: The cytotoxic effect of KL-21 was determined by MTT cell proliferation assay. Changes in caspase-3 enzyme activity were measured using the caspase-3 colorimetric assay. Changes in mitochondrial membrane potential were determined using the JC-1 dye-based method. Annexin V-FITC/PI double staining was performed to measure the apoptotic cell population. Effects of KL-21 on cell cycle profiles of CLL cells were investigated by flow cytometry. RESULTS: We detected time- and concentration-dependent increases in the cytotoxic effect of KL-21 on 232B4 CLL cells. However, we also showed that, especially at higher concentrations, KL-21 was less cytotoxic towards BEAS-2B healthy cells than towards CLL cells. Annexin-V/PI double staining results showed that the apoptotic cell population increased in 232B4 cells. Increasing concentrations of KL-21 increased caspase-3 enzyme activity and induced loss of mitochondrial membrane potential. KL-21 administration resulted in small increases in the percentage of the cells in the G0/G1 phase while it decreased the S phase cell population up to 1 mg/mL. At the highest concentration, most of the cells accumulated in the G0/G1 phase. DISCUSSION AND CONCLUSION: KL-21 has a growth-inhibitory effect on 232B4 CLL cells. KL-21 causes apoptosis and cell cycle arrest at G0/G1.

  2. TRIM8 downregulation in glioma affects cell proliferation and it is associated with patients survival

    International Nuclear Information System (INIS)

    Micale, Lucia; Fusco, Carmela; Fontana, Andrea; Barbano, Raffaela; Augello, Bartolomeo; De Nittis, Pasquelena; Copetti, Massimiliano; Pellico, Maria Teresa; Mandriani, Barbara; Cocciadiferro, Dario; Parrella, Paola; Fazio, Vito Michele; Dimitri, Lucia Maria Cecilia; D’Angelo, Vincenzo; Novielli, Chiara; Larizza, Lidia; Daga, Antonio; Merla, Giuseppe

    2015-01-01

    Human gliomas are a heterogeneous group of primary malignant brain tumors whose molecular pathogenesis is not yet solved. In this regard, a major research effort has been directed at identifying novel specific glioma-associated genes. Here, we investigated the effect of TRIM8 gene in glioma. TRIM8 transcriptional level was profiled in our own glioma cases collection by qPCR and confirmed in the independent TCGA glioma cohort. The association between TRIM8 expression and Overall Survival and Progression-free Survival in TCGA cohort was determined by using uni-multivariable Cox regression analysis. The effect of TRIM8 on patient glioma cell proliferation was evaluated by performing MTT and clonogenic assays. The mechanisms causing the reduction of TRIM8 expression were explored by using qPCR and in vitro assays. We showed that TRIM8 expression correlates with unfavorable clinical outcome in glioma patients. We found that a restored TRIM8 expression induced a significant reduction of clonogenic potential in U87MG and patient’s glioblastoma cells. Finally we provide experimental evidences showing that miR-17 directly targets the 3′ UTR of TRIM8 and post-transcriptionally represses the expression of TRIM8. Our study provides evidences that TRIM8 may participate in the carcinogenesis and progression of glioma and that the transcriptional repression of TRIM8 might have potential value for predicting poor prognosis in glioma patients. The online version of this article (doi:10.1186/s12885-015-1449-9) contains supplementary material, which is available to authorized users

  3. In Vivo and In Vitro Effects of ATM/ATR Signaling Pathway on Proliferation, Apoptosis, and Radiosensitivity of Nasopharyngeal Carcinoma Cells.

    Science.gov (United States)

    Wang, Ming; Liu, Gang; Shan, Guo-Ping; Wang, Bing-Bing

    2017-08-01

    The study investigated the ability of ataxia-telangiectasia mutated (ATM)/Rad3-related (ATR) signaling pathway to influence the proliferation, apoptosis, and radiosensitivity of nasopharyngeal carcinoma (NPC) cells. NPC tissues and corresponding adjacent normal tissues were collected from 143 NPC patients. The NPC CNE2 cells were assigned into a control group, X-ray group, CGK-733 group, and X-ray+CGK-733 group. The mRNA levels of ATM and ATR were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR) and the protein levels of ATM and ATR using western blotting. The positive expression of ATM and ATR in tissues and nude mouse tumor tissues was determined by immunohistochemistry. Cell proliferation, migration, invasion, and apoptosis rates were analyzed by the 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) assay, scratch test, transwell assay, and flow cytometry, respectively. A nude mouse model of NPC was established to observe tumor volume and growth. The mRNA levels of ATR and ATM and the expression of ATR and ATM protein in NPC tissues were significantly higher than those in adjacent normal tissues. The colony formation assay showed that the colony-forming rate decreased, showing radiation dose-dependent and CGK-733 concentration-dependent manners. Expression of ATM, ATR, Chk1, and Chk2 was evidently increased in the X-ray, CGK-733, and X-ray+CGK-733groups compared with the control group, and the aforementioned expression was highest in the X-ray+CGK-733 group among the four groups. The cell proliferation, invasion, and migration were decreased, tumor volume decreased and cell apoptosis increased in the X-ray, CGK-733, and X-ray+CGK-733 groups compared with the control group; the X-ray+CGK-733 group exhibited lowest cell proliferation, invasion and migration, smallest tumor volume, and highest cell apoptosis among the four groups. Inhibition of ATM/ATR signaling pathway reduces proliferation and enhances apoptosis and

  4. Expression of MiR-9 promotes proliferation, migration and ...

    African Journals Online (AJOL)

    Purpose: To investigate the effect of miR-9 on the proliferation, differentiation and migration of human neural stem cells (NSCs). Methods: The expression of miR-9 was investigated by quantitative real-time polymerase chain reaction (RT-PCR). Cell proliferation was assessed by cell counting kit-8 (CCK8) assay, while cell ...

  5. MiR-371-5p facilitates pancreatic cancer cell proliferation and decreases patient survival.

    Directory of Open Access Journals (Sweden)

    De He

    Full Text Available microRNAs (miRNAs play a critical role in tumorigenesis, either as a tumor suppressor or as an oncogenic miRNA, depending on different tumor types. To date, scientists have obtained a substantial amount of knowledge with regard to miRNAs in pancreatic cancer. However, the expression and function of miR-371-5p in pancreatic cancer has not been clearly elucidated. The aim of this study was to investigate the roles of miR-371-5p in pancreatic cancer and its association with the survival of patients with pancreatic cancer.The expression of miR-371-5p was examined in pancreatic duct adenocarcinoma (PDAC and their adjacent normal pancreatic tissues (ANPT or in pancreatic cancer cell lines by qRT-PCR. The association of miR-371-5p expression with overall survival was determined. The proliferation and apoptosis of SW-1990 and Panc-1 cells, transfected with miR-371-5p mimics or inhibitor, were assessed using MTT assay and flow cytometry, respectively. The tumorigenicity was evaluated via mice xenograft experiments. miR-371-5p promoter interactions were analyzed by chromatin immunoprecipitation assays (ChIP. Protein expression was analyzed by Western blot.The expression level of miR-371-5p was dramatically upregulated in clinical PDAC tissues compared with ANPT. Patients with high miR-371-5p expression had a significantly shorter survival than those with low miR-371-5p expression. The in vitro and in vivo assays showed that overexpression of miR-371-5p resulted in cell proliferation and increased tumor growth, which was associated with inhibitor of growth 1 (ING1 downregulation. Interestingly, we also found that ING1, in turn, inhibited expression of miR-371-5p in the promoter region.our study demonstrates a novel ING1-miR-371-5p regulatory feedback loop, which may have a critical role in PDAC. Thus miR-371-5p can prove to be a novel prognostic factor and therapeutic target for pancreatic cancer treatment.

  6. MC3T3-E1 proliferation and differentiation on biphasic mixtures of Mg substituted β-tricalcium phosphate and amorphous calcium phosphate

    International Nuclear Information System (INIS)

    Singh, Satish S.; Roy, Abhijit; Lee, Boeun E.; Banerjee, Ipsita; Kumta, Prashant N.

    2014-01-01

    A low temperature aqueous approach was used to synthesize nanocrystalline, high surface area Mg 2+ substituted β-tricalcium phosphate (β-TCMP) to assess its potential use as a synthetic bone graft substitute. X-ray diffraction indicated that β-TCMP was the predominant crystalline phase formed. However, thermal analysis revealed the presence of a secondary amorphous phase which increased with increasing Mg 2+ concentration. Further analysis by Rietveld refinement indicated that the level of ionic substitution of Ca 2+ by Mg 2+ was significantly lower than the amount of Mg 2+ measured using elemental analysis, confirming the formation of a Mg 2+ rich secondary amorphous phase. MC3T3-E1 proliferation on substrates prepared using β-TCMP was assessed using the MTT assay. In comparison to commercially available β-TCP, increased proliferation was observed on samples prepared with 50% Mg, despite elevated Mg 2+ and PO 4 3− concentrations in culture media. Alkaline phosphatase (ALP) activity and qRT-PCR were used to study the effect of varying Mg 2+ substitution on osteogenic differentiation. Cells cultured on β-TCMP substrates prepared with increased Mg 2+ concentrations expressed significantly increased levels of ALP activity and osteogenic genes such as, osteocalcin, collagen-1, and Runx2, in comparison to those cultured on commercially available β-TCP

  7. LncRNA TUG1 influences papillary thyroid cancer cell proliferation, migration and EMT formation through targeting miR-145.

    Science.gov (United States)

    Lei, Hongwei; Gao, Yan; Xu, Xiaoying

    2017-07-01

    LncRNA TUG1, a tumor oncogene associated with various human cancers, has been reported to be involved in regulating various cellular processes, such as proliferation, apoptosis and invasion through targeting multiple genes. However, its biological function in thyroid cancer cells has not been elucidated. The aim of this study is to measure TUG1 expression level and evaluate its function in thyroid cancer cells. LncRNA TUG1 expression levels in thyroid cancer tissues and three thyroid cancer cell lines (the ATC cell lines SW1736 and KAT18 and the FTC cell line FTC133) were assessed by qRT-PCR and compared with that of the human normal breast epithelial cell HGC-27. MTT assay, colony formation assay, transwell assay and western blot analysis were performed to assess the effects of TUG1 on proliferation, metastasis and EMT formation in thyroid cancer cells in vitro. Rescue assay was performed to further confirm that TUG1 contributes to the progression of thyroid cancer cells through regulating miR-145/ZEB1 signal pathway. LncRNA TUG1 was found to be up-regulated in thyroid cancer tissues and thyroid cancer cells compared with that in the human normal breast epithelial cell HGC-27. Increased lncRNA TUG1 expression was found to significantly promote tumor cell proliferation, and facilitate cell invasion, while down-regulated TUG1 could obviously inhibit cell proliferation, migration/invasion and reverse EMT to MET. These results indicated that TUG1 may contribute to the progression of thyroid cancer cells by function as a ceRNA competitive sponging miR-145, and that lncRNA TUG1 is associated with tumor progression in thyroid cancer cells. © The Author 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. [Over-expression of miR-151a-3p inhibits proliferation and migration of PC-3 prostate cancer cells].

    Science.gov (United States)

    Zhang, Yi; Hao, Tongtong; Zhang, Han; Wei, Pengtao; Li, Xiaohui

    2018-03-01

    Objective To observe the effect of microRNA-151a-3p (miR-151a-3p) up-regulation on the proliferation and migration of prostate cancer cells and explore the possible molecular mechanism. Methods The expression of miR-151a-3p in PC-3M, C4-2B, 22RV1, DU-145, PC-3, LNCap human prostate cancer cells and RWPE-1 human normal prostate epithelial cells was detected by real-time fluorescence quantitative PCR. PC-3 cells with the lowest expression of miR-151a-3p were used for subsequent experiments. Bioinformatics and dual-luciferase reporter assay were performed to predict and test potential target genes of miR-151a-3p. The miR-151a-3p mimics or negative control microRNAs (miR-NCs) were transfected into PC-3 cells. Real-time fluorescence quantitative PCR was used to detect the expression of miR-151a-3p and potential target gene mRNA. The protein expressions of target genes and downstream signaling pathway proteins were analyzed by Western blotting. The proliferation of PC-3 cells was examined by MTT assay, and the migration of PC-3 cells was detected by Transwell TM assay. Results The expression level of miR-151a-3p in the prostate cancer cells was significantly lower than that in RWPE-1 normal human prostate epithelial cells. PC-3 cells had the lowest expression level of miR-151a-3p. The bioinformatics and dual-luciferase reporter assay showed that NEK2 was the potential target gene for miR-151a-3p. After transfection with miR-151a-3p mimics, the expression of miR-151a-3p in PC-3 cells significantly increased and the expression of NEK2 mRNA significantly decreased. The protein expressions of PI3K-AKT-mTOR signaling pathway were also reduced. Up-regulation of miR-151a-3p significantly inhibited the proliferation and migration of PC-3 cells. Conclusion The expression of miR-151a-3p is reduced in prostate cancer cells. Up-regulation of miR-151a-3p can inhibit the proliferation and migration of P-3 in prostate cancer by decreasing the expression of NEK2 and PI3K

  9. Fissile material proliferation risk

    International Nuclear Information System (INIS)

    Dreicer, J.S.; Rutherford, D.A.

    1996-01-01

    The proliferation risk of a facility depends on the material attractiveness, level of safeguards, and physical protection applied to the material in conjunction with an assessment of the impact of the socioeconomic circumstances and threat environment. Proliferation risk is a complementary extension of proliferation resistance. The authors believe a better determination of nuclear proliferation can be achieved by establishing the proliferation risk for facilities that contain nuclear material. Developing a method that incorporates the socioeconomic circumstances and threat environment inherent to each country enables a global proliferation assessment. To effectively reduce the nuclear danger, a broadly based set of criteria is needed that provides the capability to relatively assess a wide range of nuclear related sites and facilities in different countries and still ensure a global decrease in proliferation risk for fissile material (plutonium and highly enriched uranium)

  10. The p-ERK–p-c-Jun–cyclinD1 pathway is involved in proliferation of smooth muscle cells after exposure to cigarette smoke extract

    Energy Technology Data Exchange (ETDEWEB)

    Li, Tianjia [Department of Vascular surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005 (China); Song, Ting [Nursing Department of Orthopedics 3rd Ward, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005 (China); Ni, Leng; Yang, Genhuan; Song, Xitao; Wu, Lifei [Department of Vascular surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005 (China); Liu, Bao, E-mail: liubao72@yahoo.com.cn [Department of Vascular surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005 (China); Liu, Changwei, E-mail: liucw@vip.sina.com [Department of Vascular surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005 (China)

    2014-10-24

    Highlights: • Smooth muscle cells proliferated after exposure to cigarette smoke extract. • The p-ERK, p-c-Jun, and cyclinD1 expressions increased in the process. • The p-ERK inhibitor, U0126, can reverse these effects. • The p-ERK → p-c-Jun → cyclinD1 pathway is involved in the process. - Abstract: An epidemiological survey has shown that smoking is closely related to atherosclerosis, in which excessive proliferation of vascular smooth muscle cells (SMCs) plays a key role. To investigate the mechanism underlying this unusual smoking-induced proliferation, cigarette smoke extract (CSE), prepared as smoke-bubbled phosphate-buffered saline (PBS), was used to induce effects mimicking those exerted by smoking on SMCs. As assessed by Cell Counting Kit-8 detection (an improved MTT assay), SMC viability increased significantly after exposure to CSE. Western blot analysis demonstrated that p-ERK, p-c-Jun, and cyclinD1 expression increased. When p-ERK was inhibited using U0126 (inhibitor of p-ERK), cell viability decreased and the expression of p-c-Jun and cyclinD1 was reduced accordingly, suggesting that p-ERK functions upstream of p-c-Jun and cyclinD1. When a c-Jun over-expression plasmid was transfected into SMCs, the level of cyclinD1 in these cells increased. Moreover, when c-Jun was knocked down by siRNA, cyclinD1 levels decreased. In conclusion, our findings indicate that the p-ERK–p-c-Jun–cyclinD1 pathway is involved in the excessive proliferation of SMCs exposed to CSE.

  11. Effects of exosomes derived from MDA-MB-231 on proliferation of endothelial cells and the role of MAPK/ERK and PI3K/Akt pathways

    Directory of Open Access Journals (Sweden)

    Shuang LONG

    2012-11-01

    Full Text Available Objective  To investigate the effects of exosomes derived from breast cancer cell line MDA-MB-231 on proliferation of human umbilical cord vein endothelial cells (HUVECs, and evaluate the role of MAPK/ERK and PI3K/Akt signal transduction pathway during the process. Methods  Exosomes were derived and purified from MDA-MB-231 by cryogenic ultracentrifugation and density gradient centrifugation. MTT assay was carried out for measurement of cell proliferation in HUVECs with exosome of 50, 100, 200 and 400μg/ml. The states of cell cycle of HUVECs co-cultured with 200μg/ml exosomes were detected by flow cytometry. The effects of 200μg/ml exosomes on the expression of ERK, Akt and phosphorylated ERK, Akt in HUVECs were detected with Western blotting. Results  Exosomes derived from MDA-MB-231 significantly promoted HUVECs proliferation in a classical time-and dose-dependent manner. Flow cytometry revealed that, co-cultured with 200μg/ml exosomes for 24h, S-phase cells in HUVECs increased, while G1/S phase cells in HUVECs decreased. Western blotting showed that, cocultured with 200μg/ml exosomes for 24h, 48h and 72h, the expressions of phosphorylated ERK and Akt were up-regulated in a time-dependent manner. Conclusion  Exosomes derived from breast cancer cell line MDA-MB-231 may promote HUVECs proliferation, the changes in cell cycle and the continuous activation of the MAPK/ERK and PI3K/Akt signal transduction pathways may be the underlying mechanism.

  12. The p-ERK–p-c-Jun–cyclinD1 pathway is involved in proliferation of smooth muscle cells after exposure to cigarette smoke extract

    International Nuclear Information System (INIS)

    Li, Tianjia; Song, Ting; Ni, Leng; Yang, Genhuan; Song, Xitao; Wu, Lifei; Liu, Bao; Liu, Changwei

    2014-01-01

    Highlights: • Smooth muscle cells proliferated after exposure to cigarette smoke extract. • The p-ERK, p-c-Jun, and cyclinD1 expressions increased in the process. • The p-ERK inhibitor, U0126, can reverse these effects. • The p-ERK → p-c-Jun → cyclinD1 pathway is involved in the process. - Abstract: An epidemiological survey has shown that smoking is closely related to atherosclerosis, in which excessive proliferation of vascular smooth muscle cells (SMCs) plays a key role. To investigate the mechanism underlying this unusual smoking-induced proliferation, cigarette smoke extract (CSE), prepared as smoke-bubbled phosphate-buffered saline (PBS), was used to induce effects mimicking those exerted by smoking on SMCs. As assessed by Cell Counting Kit-8 detection (an improved MTT assay), SMC viability increased significantly after exposure to CSE. Western blot analysis demonstrated that p-ERK, p-c-Jun, and cyclinD1 expression increased. When p-ERK was inhibited using U0126 (inhibitor of p-ERK), cell viability decreased and the expression of p-c-Jun and cyclinD1 was reduced accordingly, suggesting that p-ERK functions upstream of p-c-Jun and cyclinD1. When a c-Jun over-expression plasmid was transfected into SMCs, the level of cyclinD1 in these cells increased. Moreover, when c-Jun was knocked down by siRNA, cyclinD1 levels decreased. In conclusion, our findings indicate that the p-ERK–p-c-Jun–cyclinD1 pathway is involved in the excessive proliferation of SMCs exposed to CSE

  13. Inhibition of SK4 Potassium Channels Suppresses Cell Proliferation, Migration and the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells.

    Directory of Open Access Journals (Sweden)

    Panshi Zhang

    Full Text Available Treatments for triple-negative breast cancer (TNBC are limited; intermediate-conductance calcium-activated potassium (SK4 channels are closely involved in tumor progression, but little is known about these channels in TNBC. We aimed to investigate whether SK4 channels affect TNBC. First, by immunohistochemistry (IHC and western blotting (WB, increased SK4 protein expression in breast tumor tissues was detected relative to that in non-tumor breast tissues, but there was no apparent expression difference between various subtypes of breast cancer (p>0.05. Next, functional SK4 channels were detected in the TNBC cell line MDA-MB-231 using WB, real-time PCR, immunofluorescence and patch-clamp recording. By employing SK4 specific siRNAs and blockers, including TRAM-34 and clotrimazole, in combination with an MTT assay, a colony-formation assay, flow cytometry and a cell motility assay, we found that the suppression of SK4 channels significantly inhibited cell proliferation and migration and promoted apoptosis in MDA-MB-231 cells (p<0.05. Further investigation revealed that treatment with epidermal growth factor (EGF/basic fibroblast growth factor (bFGF caused MDA-MB-231 cells to undergo the epithelial-mesenchymal transition (EMT and to show increased SK4 mRNA expression. In addition, the down-regulation of SK4 expression inhibited the EMT markers Vimentin and Snail1. Collectively, our findings suggest that SK4 channels are expressed in TNBC and are involved in the proliferation, apoptosis, migration and EMT processes of TNBC cells.

  14. RNAi-mediated knockdown of pituitary tumor-transforming gene-1 (PTTG1) suppresses the proliferation and invasive potential of PC3 human prostate cancer cells

    International Nuclear Information System (INIS)

    Huang, S.Q.; Liao, Q.J.; Wang, X.W.; Xin, D.Q.; Chen, S.X.; Wu, Q.J.; Ye, G.

    2012-01-01

    Pituitary tumor-transforming gene-1 (PTTG1) is a proto-oncogene that promotes tumorigenesis and metastasis in numerous cell types and is overexpressed in a variety of human tumors. We have demonstrated that PTTG1 expression was up-regulated in both human prostate cancer specimens and prostate cancer cell lines. For a more direct assessment of the function of PTTG1 in prostate tumorigenesis, RNAi-mediated knockdown was used to selectively decrease PTTG1 expression in PC3 human prostate tumor cells. After three weeks of selection, colonies stably transfected with PTTG1-targeted RNAi (the knockdown PC3 cell line) or empty vector (the control PC3 cell line) were selected and expanded to investigate the role of PTTG1 expression in PC3 cell growth and invasion. Cell proliferation rate was significantly slower (28%) in the PTTG1 knockdown line after 6 days of growth as indicated by an MTT cell viability assay (P < 0.05). Similarly, a soft agar colony formation assay revealed significantly fewer (66.7%) PTTG1 knockdown PC3 cell colonies than control colonies after three weeks of growth. In addition, PTTG1 knockdown resulted in cell cycle arrest at G1 as indicated by fluorescence-activated cell sorting. The PTTG1 knockdown PC3 cell line also exhibited significantly reduced migration through Matrigel in a transwell assay of invasive potential, and down-regulation of PTTG1 could lead to increased sensitivity of these prostate cancer cells to a commonly used anticancer drug, taxol. Thus, PTTG1 expression is crucial for PC3 cell proliferation and invasion, and could be a promising new target for prostate cancer therapy

  15. The Long Non-Coding RNA XIST Controls Non-Small Cell Lung Cancer Proliferation and Invasion by Modulating miR-186-5p

    Directory of Open Access Journals (Sweden)

    Haoyou Wang

    2017-04-01

    Full Text Available Background/Aims: Long non-coding RNAs (lncRNAs are key players in the development and progression of human cancers. The lncRNA XIST (X-inactive specific transcript has been shown to be upregulated in human non-small cell lung cancer (NSCLC; however, its role and molecular mechanisms in NSCLC cell progression remain unclear. Methods: qRT-PCR was conducted to assess the expression of XIST and miR-186. Cell proliferation was detected using MTT assay. Cell invasion and migration were evaluated using transwell assay. Cell cycle distribution and apoptosis rates were analyzed by flow cytometry. Luciferase reporter assay was used to identify the direct regulation of XIST and miR-186. A RNA immunoprecipitation was used to analyze whether XIST was associated with the RNA-induced silencing complex (RISC. Results: We confirmed that XIST was upregulated in NSCLC cell lines and tissues. Functionally, XIST knockdown inhibited cancer cell proliferation and invasion, and induced apoptosis in vitro, and suppressed subcutaneous tumor growth in vivo. Mechanistic investigations revealed a reciprocal repressive interaction between XIST and miR-186-5p. Furthermore, we showed that miR-186-5p has a binding site for XIST. Our data also indicated that XIST and miR-186-5p are likely in the same RNA induced silencing complex. Conclusion: Together, our data revealed that XIST knockdown confers suppressive function in NSCLC and XIST may be a novel therapeutic marker in this disease.

  16. miR-130b targets NKD2 and regulates the Wnt signaling to promote proliferation and inhibit apoptosis in osteosarcoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Li, Zhi [Department of Human Anatomy and Histoembryology, College of Basic Medical Sciences, Jilin University (China); Li, Youjun, E-mail: liyoujunn@126.com [Department of Human Anatomy and Histoembryology, College of Basic Medical Sciences, Jilin University (China); Wang, Nan; Yang, Lifeng; Zhao, Wei; Zeng, Xiandong [Central Hospital Affiliated to Shenyang Medical College (China)

    2016-03-18

    miR-130b was significantly up-regulated in osteosarcoma (OS) cells. Naked cuticle homolog 2 (NKD2) inhibited tumor growth and metastasis in OS by suppressing Wnt signaling. We used three miRNA target analysis tools to identify potential targets of miR-130b, and found that NKD2 is a potential target of miR-130b. Based on these findings, we hypothesize that miR-130b might target NKD2 and regulate the Wnt signaling to promote OS growth. We detected the expression of miR-130b and NKD2 mRNA and protein by quantitative Real-Time PCR (qRT-PCR) and western blot assays, respectively, and found up-regulation of miR-130b and down-regulation of NKD2 mRNA and protein exist in OS cell lines. MTT and flow cytometry assays showed that miR-130b inhibitors inhibit proliferation and promote apoptosis in OS cells. Furthermore, we showed that NKD2 is a direct target of miR-130b, and miR-130b regulated proliferation and apoptosis of OS cells by targeting NKD2. We further investigated whether miR-130b and NKD2 regulate OS cell proliferation and apoptosis by inhibiting Wnt signaling, and the results confirmed our speculation that miR-130b targets NKD2 and regulates the Wnt signaling to promote proliferation and inhibit apoptosis of OS cells. These findings will offer new clues for OS development and progression, and novel potential therapeutic targets for OS. - Highlights: • miR-130b is up-regulated and NKD2 is down-regulated in osteosarcoma cell lines. • Down-regulation of miR-130b inhibits proliferation of osteosarcoma cells. • Down-regulation of miR-130b promotes apoptosis of osteosarcoma cells. • miR-130b directly targets NKD2. • NKD2 regulates OS cell proliferation and apoptosis by inhibiting the Wnt signaling.

  17. miR-130b targets NKD2 and regulates the Wnt signaling to promote proliferation and inhibit apoptosis in osteosarcoma cells

    International Nuclear Information System (INIS)

    Li, Zhi; Li, Youjun; Wang, Nan; Yang, Lifeng; Zhao, Wei; Zeng, Xiandong

    2016-01-01

    miR-130b was significantly up-regulated in osteosarcoma (OS) cells. Naked cuticle homolog 2 (NKD2) inhibited tumor growth and metastasis in OS by suppressing Wnt signaling. We used three miRNA target analysis tools to identify potential targets of miR-130b, and found that NKD2 is a potential target of miR-130b. Based on these findings, we hypothesize that miR-130b might target NKD2 and regulate the Wnt signaling to promote OS growth. We detected the expression of miR-130b and NKD2 mRNA and protein by quantitative Real-Time PCR (qRT-PCR) and western blot assays, respectively, and found up-regulation of miR-130b and down-regulation of NKD2 mRNA and protein exist in OS cell lines. MTT and flow cytometry assays showed that miR-130b inhibitors inhibit proliferation and promote apoptosis in OS cells. Furthermore, we showed that NKD2 is a direct target of miR-130b, and miR-130b regulated proliferation and apoptosis of OS cells by targeting NKD2. We further investigated whether miR-130b and NKD2 regulate OS cell proliferation and apoptosis by inhibiting Wnt signaling, and the results confirmed our speculation that miR-130b targets NKD2 and regulates the Wnt signaling to promote proliferation and inhibit apoptosis of OS cells. These findings will offer new clues for OS development and progression, and novel potential therapeutic targets for OS. - Highlights: • miR-130b is up-regulated and NKD2 is down-regulated in osteosarcoma cell lines. • Down-regulation of miR-130b inhibits proliferation of osteosarcoma cells. • Down-regulation of miR-130b promotes apoptosis of osteosarcoma cells. • miR-130b directly targets NKD2. • NKD2 regulates OS cell proliferation and apoptosis by inhibiting the Wnt signaling.

  18. Efficacy Study of Broken Rice Maltodextrin in In Vitro Wound Healing Assay

    Directory of Open Access Journals (Sweden)

    Zahiah Mohamed Amin

    2015-01-01

    Full Text Available Maltodextrins that contain both simple sugars and polymers of saccharides have been widely used as ingredients in food products and pharmaceutical delivery systems. To date, no much work has been reported on the applications of maltodextrin from broken rice (RB sources. Therefore, the objective of this work was to investigate the in vitro wound healing efficacy of RB maltodextrin at different conditions. Wounds treated with lower dextrose equivalent (DE range (DE 10–14 of maltodextrins at a concentration of 10% obtained from RB were found to be able to heal the wounds significantly faster (p<0.01 than maltodextrin with higher DE ranges (DE 15–19 and DE 20–24 and concentrations of 5% and 20%. The findings from both BrdU and MTT assay further confirmed its wound healing properties as the NIH 3T3 fibroblast wounded cells were able to proliferate without causing cytotoxic effect when wounded cell was treated with maltodextrin. All these findings indicated that the RB maltodextrin could perform better than the commercial maltodextrin at the same DE range. This study showed that RB maltodextrins had better functionality properties than other maltodextrin sources and played a beneficial role in wound healing application.

  19. Efficacy Study of Broken Rice Maltodextrin in In Vitro Wound Healing Assay

    Science.gov (United States)

    Mohamed Amin, Zahiah; Koh, Soo Peng; Abdul Hamid, Nur Syazwani

    2015-01-01

    Maltodextrins that contain both simple sugars and polymers of saccharides have been widely used as ingredients in food products and pharmaceutical delivery systems. To date, no much work has been reported on the applications of maltodextrin from broken rice (RB) sources. Therefore, the objective of this work was to investigate the in vitro wound healing efficacy of RB maltodextrin at different conditions. Wounds treated with lower dextrose equivalent (DE) range (DE 10–14) of maltodextrins at a concentration of 10% obtained from RB were found to be able to heal the wounds significantly faster (p < 0.01) than maltodextrin with higher DE ranges (DE 15–19 and DE 20–24) and concentrations of 5% and 20%. The findings from both BrdU and MTT assay further confirmed its wound healing properties as the NIH 3T3 fibroblast wounded cells were able to proliferate without causing cytotoxic effect when wounded cell was treated with maltodextrin. All these findings indicated that the RB maltodextrin could perform better than the commercial maltodextrin at the same DE range. This study showed that RB maltodextrins had better functionality properties than other maltodextrin sources and played a beneficial role in wound healing application. PMID:26436094

  20. Effects of microRNA-129 and its target gene c-Fos on proliferation and apoptosis of hippocampal neurons in rats with epilepsy via the MAPK signaling pathway.

    Science.gov (United States)

    Wu, Dong-Mei; Zhang, Yu-Tong; Lu, Jun; Zheng, Yuan-Lin

    2018-09-01

    This study aims to investigate the effect of microRNA-129 (miR-129) on proliferation and apoptosis of hippocampal neurons in epilepsy rats by targeting c-Fos via the MAPK signaling pathway. Thirty rats were equally classified into a model group (successfully established as chronic epilepsy models) and a normal group. Expression of miR-129, c-Fos, bax, and MAPK was detected by RT-qPCR and Western blotting. Hippocampal neurons were assigned into normal, blank, negative control (NC), miR-129 mimic, miR-129 inhibitor, siRNA-c-Fos, miR-129 inhibitor+siRNA-c-Fos groups. The targeting relationship between miR-129 and c-Fos was predicted and verified by bioinformatics websites and dual-luciferase reporter gene assay. Cell proliferation after transfection was measured by MTT assay, and cell cycle and apoptosis by flow cytometry. c-Fos is a potential target gene of miR-129. Compared with the normal group, the other six groups showed a decreased miR-129 expression; increased expression of expression of c-Fos, Bax, and MAPK; decreased proliferation; accelerated apoptosis; more cells arrested in the G1 phase; and fewer cells arrested in the S phase. Compared with the blank and NC groups, the miR-129 mimic group and the siRNA-c-Fos group showed decreased expression of c-Fos, Bax, and MAPK, increased cells proliferation, and decreased cell apoptosis, fewer cells arrested in the G1 phase and more cells arrested in the S phase. However, the miR-129 inhibitor groups showed reverse consequences. This study suggests that miR-129 could inhibit the occurrence and development of epilepsy by repressing c-Fos expression through inhibiting the MAPK signaling pathway. © 2017 Wiley Periodicals, Inc.

  1. Combination of NRP1-mediated iRGD with 5-fluorouracil suppresses proliferation, migration and invasion of gastric cancer cells.

    Science.gov (United States)

    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.

  2. Ethyl pyruvate inhibits proliferation and induces apoptosis of hepatocellular carcinoma via regulation of the HMGB1–RAGE and AKT pathways

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Ping; Dai, Weiqi; Wang, Fan; Lu, Jie; Shen, Miao; Chen, Kan; Li, Jingjing; Zhang, Yan; Wang, Chengfen; Yang, Jing; Zhu, Rong; Zhang, Huawei; Zheng, Yuanyuan; Guo, Chuan-Yong, E-mail: guochuanyong@hotmail.com; Xu, Ling, E-mail: xuling606@sina.com

    2014-01-24

    Highlights: • Ethyl pyruvate inhibits liver cancer. • Promotes apoptosis. • Decreased the expression of HMGB1, p-Akt. - Abstract: Ethyl pyruvate (EP) was recently identified as a stable lipophilic derivative of pyruvic acid with significant antineoplastic activities. The high mobility group box-B1 (HMGB1)–receptor for advanced glycation end-products (RAGE) and the protein kinase B (Akt) pathways play a crucial role in tumorigenesis and development of many malignant tumors. We tried to observe the effects of ethyl pyruvate on liver cancer growth and explored its effects in hepatocellular carcinoma model. In this study, three hepatocellular carcinoma cell lines were treated with ethyl pyruvate. An MTT colorimetric assay was used to assess the effects of EP on cell proliferation. Flow cytometry and TUNEL assays were used to analyze apoptosis. Real-time PCR, Western blotting and immunofluorescence demonstrated ethyl pyruvate reduced the HMGB1–RAGE and AKT pathways. The results of hepatoma orthotopic tumor model verified the antitumor effects of ethyl pyruvate in vivo. EP could induce apoptosis and slow the growth of liver cancer. Moreover, EP decreased the expression of HMGB1, RAGE, p-AKT and matrix metallopeptidase-9 (MMP9) and increased the Bax/Bcl-2 ratio. In conclusion, this study demonstrates that ethyl pyruvate induces apoptosis and cell-cycle arrest in G phase in hepatocellular carcinoma cells, plays a critical role in the treatment of cancer.

  3. Proliferation: myth or reality?

    International Nuclear Information System (INIS)

    2005-01-01

    This article analyzes the proliferation approach, its technical condition and political motivation, and the share between the myth (political deception, assumptions and extrapolations) and the reality of proliferation. Its appreciation is complicated by the irrational behaviour of some political actors and by the significant loss of the non-use taboo. The control of technologies is an important element for proliferation slowing down but an efficient and autonomous intelligence system remains indispensable. (J.S.)

  4. Augmenter of liver regeneration causes different kinetics of ERK1/2 and Akt/PKB phosphorylation than EGF and induces hepatocyte proliferation in an EGF receptor independent and liver specific manner

    Energy Technology Data Exchange (ETDEWEB)

    Ilowski, Maren; Putz, Christine [Department of Surgery, Ludwig-Maximilians-University of Munich Hospital Grosshadern, Munich (Germany); Weiss, Thomas S. [Department of Surgery, University of Regensburg Hospital, Regensburg (Germany); Brand, Stephan [Department of Internal Medicine II, Ludwig-Maximilians-University of Munich Hospital Grosshadern, Munich (Germany); Jauch, Karl-Walter [Department of Surgery, Ludwig-Maximilians-University of Munich Hospital Grosshadern, Munich (Germany); Hengstler, Jan G. [Leibniz Research Centre for Working Environment and Human Factors, TU Dortmund University, Dortmund (Germany); Thasler, Wolfgang Erwin, E-mail: wolfgang.thasler@med.uni-muenchen.de [Department of Surgery, Ludwig-Maximilians-University of Munich Hospital Grosshadern, Munich (Germany)

    2010-04-16

    Background/Aim: Augmenter of liver regeneration (ALR) is a potent growth factor which supports liver regeneration in experimental animals. The aim of this study was to compare proliferation as well as the kinetics of ERK1/2 and Akt/PKB phosphorylation by recombinant human ALR (rhALR) and EGF in human hepatocytes and extrahepatic cells. Methods: Kinetics of ERK1/2 and Akt/PKB phosphorylation were determined in primary human hepatocytes (phh) after stimulation with rhALR and EGF. Induction of proliferation was analyzed in phh and several cell lines of hepatic and extrahepatic origin by the MTT and [{sup 3}H]-thymidine assay. Results: The kinetics of ERK phosphorylation showed clear differences, whereby rhALR caused a transient and EGF a permanent increase during the observation period of 60 min. For both, Akt and ERK phosphorylation, EGF caused a faster effect with maximal levels observed already after 2 min, whereas rhALR caused maximal phosphorylation between 10 and 15 min. Using the EGF receptor inhibitor AG1478 we provide evidence of an EGF receptor independent induction of proliferation by rhALR. Furthermore, rhALR induced proliferation only in phh and the human liver derived cell lines HepG2 and Chang. In contrast, EGF enhanced proliferation in all analyzed cell types including cell lines of colon, bronchial, pancreatic and gastric origin (SW480, BC1, L36PL and GC1). Conclusion: rhALR and EGF induce different kinetics of ERK and Akt phosphorylation in human hepatocytes. The mitogenic effect of rhALR is liver specific and seems to be at least partially independent from EGF receptor mediated signaling.

  5. Effect of microRNA-135a on Cell Proliferation, Migration, Invasion, Apoptosis and Tumor Angiogenesis Through the IGF-1/PI3K/Akt Signaling Pathway in Non-Small Cell Lung Cancer.

    Science.gov (United States)

    Zhou, Yufei; Li, Shaoxia; Li, Jiangtao; Wang, Dongfeng; Li, Quanxing

    2017-01-01

    This study explored the ability of microRNA-135a (miR-135a) to influence cell proliferation, migration, invasion, apoptosis and tumor angiogenesis through the IGF-1/PI3K/Akt signaling pathway in non-small cell lung cancer (NSCLC). NSCLC tissues and adjacent normal tissues were collected from 138 NSCLC patients. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression levels of miR-135a and IGF-1, PI3K, Akt, VEGF, bFGF and IL-8 mRNA; western blotting was used to determine the expression levels of IGF-1, PI3K and Akt protein; and enzyme-linked immunosorbent assay (ELISA) was used to analyze the expression levels of VEGF, bFGF and IL-8 protein. Human NSCLC cell lines (A549, H460, and H1299) and the human bronchial epithelial cell line (HBE) were selected. A549 cells were assigned to blank, negative control (NC), miR-135a mimics, miR-135a inhibitors, IGF-1 siRNA and miR-135a inhibitors + IGF-1 siRNA groups. The following were performed: an MTT assay to assess cell proliferation, a scratch test to detect cell migration, a Transwell assay to measure cell invasion, and a flow cytometry to analyze cell apoptosis. The expression level of miR-135a was lower while those of IGF-1, PI3K and Akt mRNA were higher in NSCLC tissues than in the adjacent normal tissues. Dual-luciferase reporter assay indicated IGF-1 as a target of miR-135a. The in vitro results showed that compared with the blank group, cell proliferation, migration and invasion were suppressed, mRNA and protein levels of IGF-1, PI3K, Akt, VEGF, bFGF and IL-8 were reduced, and cell apoptosis was enhanced in the miR-135a mimics and IGF-1 siRNA groups. Compared with the IGF-1 siRNA group, cells in the miR-135a inhibitors + IGF-1 siRNA group demonstrated increased cell proliferation, migration and invasion, elevated mRNA and protein levels of IGF-1, PI3K, Akt, VEGF, bFGF and IL-8 and reduced cell apoptosis. These findings indicated that miR-135a promotes cell apoptosis and inhibits

  6. Piper betle shows antioxidant activities, inhibits MCF-7 cell proliferation and increases activities of catalase and superoxide dismutase

    Directory of Open Access Journals (Sweden)

    Abrahim Noor

    2012-11-01

    Full Text Available Abstract Background Breast cancer is the most common form of cancer and the focus on finding chemotherapeutic agents have recently shifted to natural products. Piper betle is a medicinal plant with various biological activities. However, not much data is available on the anti-cancer effects of P. betle on breast cancer. Due to the current interest in the potential effects of antioxidants from natural products in breast cancer treatment, we investigated the antioxidant activities of the leaves of P. betle and its inhibitory effect on the proliferation of the breast cancer cell line, MCF-7. Methods The leaves of P. betle were extracted with solvents of varying polarities (water, methanol, ethyl acetate and hexane and their phenolic and flavonoid content were determined using colorimetric assays. Phenolic composition was characterized using HPLC. Antioxidant activities were measured using FRAP, DPPH, superoxide anion, nitric oxide and hyroxyl radical scavenging assays. Biological activities of the extracts were analysed using MTT assay and antioxidant enzyme (catalase, superoxide dismutase, glutathione peroxidase assays in MCF-7 cells. Results Overall, the ethyl acetate extract showed the highest ferric reducing activity and radical scavenging activities against DPPH, superoxide anion and nitric oxide radicals. This extract also contained the highest phenolic content implying the potential contribution of phenolics towards the antioxidant activities. HPLC analyses revealed the presence of catechin, morin and quercetin in the leaves. The ethyl acetate extract also showed the highest inhibitory effect against the proliferation of MCF-7 cells (IC50=65 μg/ml. Treatment of MCF-7 cells with the plant extract increased activities of catalase and superoxide dismutase. Conclusions Ethyl acetate is the optimal solvent for the extraction of compounds with antioxidant and anti-proliferative activities. The increased activities of catalase and superoxide

  7. Piper betle shows antioxidant activities, inhibits MCF-7 cell proliferation and increases activities of catalase and superoxide dismutase.

    Science.gov (United States)

    Abrahim, Noor Nazirahanie; Kanthimathi, M S; Abdul-Aziz, Azlina

    2012-11-15

    Breast cancer is the most common form of cancer and the focus on finding chemotherapeutic agents have recently shifted to natural products. Piper betle is a medicinal plant with various biological activities. However, not much data is available on the anti-cancer effects of P. betle on breast cancer. Due to the current interest in the potential effects of antioxidants from natural products in breast cancer treatment, we investigated the antioxidant activities of the leaves of P. betle and its inhibitory effect on the proliferation of the breast cancer cell line, MCF-7. The leaves of P. betle were extracted with solvents of varying polarities (water, methanol, ethyl acetate and hexane) and their phenolic and flavonoid content were determined using colorimetric assays. Phenolic composition was characterized using HPLC. Antioxidant activities were measured using FRAP, DPPH, superoxide anion, nitric oxide and hyroxyl radical scavenging assays. Biological activities of the extracts were analysed using MTT assay and antioxidant enzyme (catalase, superoxide dismutase, glutathione peroxidase) assays in MCF-7 cells. Overall, the ethyl acetate extract showed the highest ferric reducing activity and radical scavenging activities against DPPH, superoxide anion and nitric oxide radicals. This extract also contained the highest phenolic content implying the potential contribution of phenolics towards the antioxidant activities. HPLC analyses revealed the presence of catechin, morin and quercetin in the leaves. The ethyl acetate extract also showed the highest inhibitory effect against the proliferation of MCF-7 cells (IC50=65 μg/ml). Treatment of MCF-7 cells with the plant extract increased activities of catalase and superoxide dismutase. Ethyl acetate is the optimal solvent for the extraction of compounds with antioxidant and anti-proliferative activities. The increased activities of catalase and superoxide dismutase in the treated cells could alter the antioxidant defense

  8. Director's series on proliferation

    International Nuclear Information System (INIS)

    Bailey, K.C.; Price, M.E.

    1995-01-01

    This is an occasional publication of essays on the topics of nuclear, chemical, biological, and missile proliferation. The views represented are those of the author's. Essay topics include: Nuclear Proliferation: Myth and Reality; Problems of Enforcing Compliance with Arms Control Agreements; The Unreliability of the Russian Officer Corps: Reluctant Domestic Warriors; and Russia's Nuclear Legacy

  9. Proliferation Networks and Financing

    International Nuclear Information System (INIS)

    Gruselle, Bruno

    2007-01-01

    The objective of this study is to propose practical solutions aimed at completing and strengthening the existing arrangement for the control of nuclear proliferation through a control of financial as well as material or immaterial flows. In a first part, the author proposes a systemic analysis of networks of suppliers and demanders. He notably evokes the Khan's network and the Iraqi acquisition network during the 1993-2001 period. He also proposes a modelling of proliferation networks (supplier networks and acquisition networks) and of their interactions. In a second part, the author examines possible means and policies aimed at neutralising proliferation networks: organisation, adaptation and improvement of intelligence tools in front of proliferation networks, and means, limitations and perspectives of network neutralisation. He also briefly addresses the possibility of military action to contain proliferation flows

  10. Downregulation of long noncoding RNA TUG1 inhibits proliferation and induces apoptosis through the TUG1/miR-142/ZEB2 axis in bladder cancer cells

    Directory of Open Access Journals (Sweden)

    Liu Q

    2017-05-01

    Full Text Available Qian Liu,* Hui Liu,* Hepeng Cheng, Yang Li, Xiaodong Li, Chaoyang Zhu Department of Urology Surgery, Huaihe Hospital of Henan University, Kaifeng, People’s Republic of China *These authors contributed equally to this work Background: Bladder cancer is a common serious disease around the world. Long noncoding RNAs (lncRNAs have been demonstrated to participate in the development and progression of various cancers, including bladder cancer. The aim of this study was to investigate the effects of lncRNA taurine upregulated gene 1 (TUG1 on proliferation and apoptosis in bladder cancer cell lines and the underlying mechanism.Methods: The levels of TUG1 were detected by quantitative real time polymerase chain reaction (qRT-PCR in bladder cancer tissues and cells. The mRNA and protein levels of zinc finger E-box binding homeobox 2 (ZEB2 were measured by qRT-PCR and Western blot analysis, respectively. The functional targets of TUG1 were predicted by online softwares and confirmed by luciferase reporter assay. The effects of TUG1 on cell proliferation and apoptosis were examined by MTT and apoptosis assay, respectively. The expression levels of β-catenin, cyclinD1, and c-Myc in T24 cells were determined by Western blot analysis.Results: The levels of TUG1 and ZEB2 were significantly increased in bladder cancer tissues and cells. Knockdown of either TUG1 or ZEB2 inhibited proliferation and induced apoptosis in bladder cancer cells. Interestingly, ZEB2 overexpression reversed the effects of TUG1 knockdown on cell proliferation and apoptosis. Moreover, ZEB2 was verified as a direct target of miR-142 and miR-142 could specially bind to TUG1. In addition, downregulation of TUG1 inhibited the Wnt/β-catenin pathway by regulating ZEB2 expression in bladder cancer cells.Conclusion: Downregulation of TUG1 expression inhibited proliferation and induced apoptosis in bladder cancer cells by targeting ZEB2 mediated by miR-142 through the inactivation of Wnt

  11. Evaluation of the attachment, proliferation, and differentiation of osteoblast on a calcium carbonate coating on titanium surface

    Energy Technology Data Exchange (ETDEWEB)

    Liu Yi; Jiang Tao; Zhou Yi; Zhang Zhen; Wang Zhejun [Key Laboratory for Oral Biomedical Engineering, Ministry of Education, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China); Tong Hua; Shen Xinyu [College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072 (China); Wang Yining, E-mail: wang.yn@whu.edu.cn [Key Laboratory for Oral Biomedical Engineering, Ministry of Education, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China)

    2011-07-20

    Titanium has been reported to have some limitations in dental and orthopaedic clinical application. This study described a coating process using a simple chemical method to prepare calcium carbonate coatings on smooth titanium (STi) and sandblasted and acid-etched titanium (SATi), and evaluated the biological response of the materials in vitro. The surfaces of STi, SATi, calcium carbonate coated STi (CC-STi) and calcium carbonate coated SATi (CC-SATi) were characterized for surface roughness, contact angles, surface morphology and surface chemistry. The morphology of MG63 cells cultured on the surfaces was observed by SEM and Immuno-fluorescence staining. Cell attachment/proliferation was assessed by MTT assay, and cell differentiation was evaluated by alkaline phosphatase (ALP) activity. MG63 was found to attach favorably to calcium carbonate crystals with longer cytoplasmic extensions on CC-STi and CC-SATi, resulting in lower cell proliferation but higher ALP activity when compared to STi and SATi respectively. Moreover, CC-SATi is more favorable than CC-STi in terms of biological response. In conclusion, the calcium carbonate coatings on titanium were supposed to improve the osteointegration process and stimulate osteoblast differentiation, especially in early stage. And this method could possibly be a feasible alternative option for future clinical application. Highlights: {yields} Calcium carbonate coatings were prepared on titanium substrates. {yields} The coating process is simple and cost-effective. {yields} Calcium carbonate coating could induce differentiation toward an osteoblastic phenotype. {yields} Calcium carbonate coating could enhance the osteointegration process especially in early stage.

  12. [Inhibitory effect of RNA interference targeting GFI-1 on the proliferation of atypical chronic myelogenous leukemia NT1 cells].

    Science.gov (United States)

    Yang, X; Liu, H; Lin, Z H; Qian, J; Xu, X R

    2016-08-01

    To investigate the inhibitory effects of RNA interference targeting GFI-1 on growth and proliferation of atypical chronic myelogenous leukemia (aCML) NT1 cells. NT1 cells were transfected with PBS and liposome complex (vehicle group), scrambled siRNA and liposome complex (negative control, NC group), and GFI-1 siRNA and liposome complex (GFI-1 siRNA group), respectively. Real-time quantitative RT-PCR (qRT-PCR) and Western blot were performed to examine the expression levels of GFI-1 mRNA and protein, respectively. The proliferation abilities of NT1 cells of the three groups were evaluated by MTT assay. The cell cycle in cells of the three groups was analyzed by flow cytometry. Moreover, nude mouse xenograft model was used to detect the tumor formation ability in the three group cells. Quantitative real-time PCR data showed that the expression level of GFI-1 mRNA in GFI-1 siRNA group was significantly lower than those of NC group and vehicle group [(0.367±0.017) vs. (0.918±0.006) and (1.010±0.005), respectively, (PNT1 cells in the GFI-1 siRNA group (0.667±0.059) was significantly lower than those of the NC group (1.096±0.049) and vehicle group (1.193±0.064, P=0.023). Flow cytometry data showed that sub-G1 and G0/G1 phase proportions of the GFI-1 siRNA group were significantly higher than those of the NC and vehicle groups [sub-G1: (8.2±2.5)% vs. (1.9±1.3)% and (2.0±3.6)%, respectively, (PNT1 cells, which may provide a new therapeutic target for atypical chronic myelogenous leukemia.

  13. Prostaglandin receptor EP3 regulates cell proliferation and migration with impact on survival of endometrial cancer patients.

    Science.gov (United States)

    Zhu, Junyan; Trillsch, Fabian; Mayr, Doris; Kuhn, Christina; Rahmeh, Martina; Hofmann, Simone; Vogel, Marianne; Mahner, Sven; Jeschke, Udo; von Schönfeldt, Viktoria

    2018-01-02

    Prostaglandin E2 (PGE2) receptor 3 (EP3) regulates tumor cell proliferation, migration, and invasion in numerous cancers. The role of EP3 as a prognostic biomarker in endometrial cancer remains unclear. The primary aim of this study was to analyze the prognostic significance of EP3 expression in endometrial cancer. We analyzed the EP3 expression of 140 endometrial carcinoma patients by immunohistochemistry. RL95-2 endometrial cancer cell line was chosen from four endometrial cancer cell lines (RL95-2, Ishikawa, HEC-1-A, and HEC-1-B) according to EP3 expression level. Treated with PGE2 and EP3 antagonist, RL95-2 cells were investigated by MTT, BrdU, and wound healing assay for functional assessment of EP3. EP3 staining differed significantly according to WHO tumor grading in both whole cohort (p = 0.01) and the subgroup of endometrioid carcinoma (p = 0.01). Patients with high EP3 expression in their respective tumors had impaired progression-free survival as well as overall survival in both cohorts above. EP3 expression in the overall cohort was identified as an independent prognostic marker for progression-free survival (HR 1.014, 95%CI 1.003-1.024, p = 0.01) when adjusted for age, stage, grading, and recurrence. Treatment with EP3 antagonists induced upregulation of estrogen receptor β and decreased activity of Ras and led to attenuated proliferation and migration of RL95-2 cells. EP3 seems to play a crucial role in endometrial cancer progression. In the context of limited systemic treatment options for endometrial cancer, this explorative analysis identifies EP3 as a potential target for diagnostic workup and therapy.

  14. Hesperidin inhibits HeLa cell proliferation through apoptosis mediated by endoplasmic reticulum stress pathways and cell cycle arrest

    International Nuclear Information System (INIS)

    Wang, Yaoxian; Yu, Hui; Zhang, Jin; Gao, Jing; Ge, Xin; Lou, Ge

    2015-01-01

    Hesperidin (30, 5, 9-dihydroxy-40-methoxy-7-orutinosyl flavanone) is a flavanone that is found mainly in citrus fruits and has been shown to have some anti-neoplastic effects. The aim of the present study was to investigate the effect of hesperidin on apoptosis in human cervical cancer HeLa cells and to identify the mechanism involved. Cells were treated with hesperidin (0, 20, 40, 60, 80, and 100 μM) for 24, 48, or 72 h and relative cell viability was assessed using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Hesperidin inhibited the proliferation of HeLa cells in a concentration- and time-dependent manner. Hesperidin-induced apoptosis in HeLa cells was characterized by increased nuclear condensation and DNA fragmentation. Furthermore, increased levels of GADD153/CHOP and GRP78 indicated hesperidin-induced apoptosis in HeLa cells involved a caspase-dependent pathway, presumably downstream of the endoplasmic reticulum stress pathway. Both of these proteins are hallmarks of endoplasmic reticulum stress. Hesperidin also promoted the formation of reactive oxygen species, mobilization of intracellular Ca 2+ , loss of mitochondrial membrane potential (ΔΨm), increased release of cytochrome c and apoptosis-inducing factor from mitochondria, and promoted capase-3 activation. It also arrested HeLa cells in the G0/G1 phase in the cell cycle by downregulating the expression of cyclinD1, cyclinE1, and cyclin-dependent kinase 2 at the protein level. The effect of hesperidin was also verified on the human colon cancer cell HT-29 cells. We concluded that hesperidin inhibited HeLa cell proliferation through apoptosis involving endoplasmic reticulum stress pathways and cell cycle arrest

  15. Cytotoxicity of Thirdhand Smoke and Identification of Acrolein as a Volatile Thirdhand Smoke Chemical That Inhibits Cell Proliferation.

    Science.gov (United States)

    Bahl, Vasundhra; Weng, Nikki J-H; Schick, Suzaynn F; Sleiman, Mohamad; Whitehead, Jacklyn; Ibarra, Allison; Talbot, Prue

    2016-03-01

    Thirdhand smoke (THS) is a mixture of chemicals that remain on indoor surfaces after smoking has ceased. These chemicals can be inhaled, ingested, or absorbed dermally, and thus could impact human health. We evaluated the cytotoxicity and mode of action of fresh and aged THS, the toxicity of volatile organic chemicals (VOCs) in THS, and the molecular targets of acrolein, a VOC in THS. Experiments were done using mouse neural stem cells (mNSC), human pulmonary fibroblasts (hPF), and lung A549 epithelial cells. THS-exposed cotton cloth was extracted in Dulbecco's Eagle Medium and caused cytotoxicity in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. THS extracts induced blebbing, immotility, vacuolization, cell fragmentation, severing of microfilaments and depolymerization of microtubules in mNSC. Cytotoxicity was inversely related to headspace volume in the extraction container and was lost upon aging, suggesting that VOCs in THS were cytotoxic. Phenol, 2',5'-dimethyl furan and acrolein were identified as the most cytotoxic VOCs in THS, and in combination, their cytotoxicity increased. Acrolein inhibited proliferation of mNSC and hPF and altered expression of cell cycle regulatory genes. Twenty-four hours of treatment with acrolein decreased expression of transcription factor Dp-1, a factor needed for the G1 to S transition in the cell cycle. At 48 h, WEE1 expression increased, while ANACP1 expression decreased consistent with blocking entry into and completion of the M phase of the cell cycle. This study identified acrolein as a highly cytotoxic VOC in THS which killed cells at high doses and inhibited cell proliferation at low doses. © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  16. NLS-RARα promotes proliferation and inhibits differentiation in HL-60 cells.

    Science.gov (United States)

    Hu, Xiu-Xiu; Zhong, Liang; Zhang, Xi; Gao, Yuan-Mei; Liu, Bei-Zhong

    2014-01-01

    A unique mRNA produced in leukemic cells from a t(15;17) acute promyelocytic leukemia (APL) patient encodes a fusion protein between the retinoic acid receptor α (RARα) and a myeloid gene product called PML. Studies have reported that neutrophil elastase (NE) cleaves bcr-1-derived PML-RARα in early myeloid cells, leaving only the nuclear localization signal (NLS) of PML attached to RARα. The resultant NLS-RARα fusion protein mainly localizes to, and functions within, the cell nucleus. It is speculated that NLS-RARα may act in different ways from the wild-type RARα, but its biological characteristics have not been reported. This study takes two approaches. Firstly, the NLS-RARα was silenced with pNLS-RARα-shRNA. The mRNA and protein expression of NLS-RARα were detected by RT-PCR and Western blot respectively. Cell proliferation in vitro was assessed by MTT assay. Flow cytometry (FCM) was used to detect the differentiation of cells. Secondly, the NLS-RARα was over-expressed by preparation of recombinant adenovirus HL-60/pAd-NLS-RARα. The assays of mRNA and protein expression of NLS-RARα, and cell proliferation, were as above. By contrast, cell differentiation was stimulated by all trans retinoic acid (ATRA) (2.5µmol/L) at 24h after virus infection of pAd-NLS-RARα, and then detected by CD11b labeling two days later. The transcription and translation of C-MYC was detected in HL-60/pAd-NLS-RARα cells which treated by ATRA. Our results showed that compared to the control groups, the expression of NLS-RARα was significantly reduced in the HL-60/pNLS-RARα-shRNA cells, and increased dramatically in the HL-60/pAd-NLS-RARα cells. The proliferation was remarkably inhibited in the HL-60/pNLS-RARα-shRNA cells in a time-dependent manner, but markedly promoted in the HL-60/pAd-NLS-RARα cells. FCM outcome revealed the differentiation increased in HL-60/pNLS-RARα-shRNA cells, and decreased in the HL-60/pAd-NLS-RARα cells treated with 2.5µmol/L ATRA. The

  17. Knockdown of hypoxia-inducible factor-1 alpha reduces proliferation, induces apoptosis and attenuates the aggressive phenotype of retinoblastoma WERI-Rb-1 cells under hypoxic conditions.

    Science.gov (United States)

    Xia, Tian; Cheng, Hao; Zhu, Yu

    2014-01-01

    Hypoxia-inducible factor-1 alpha (HIF-1α) plays a critical role in tumor cell adaption to hypoxia by inducing the transcription of numerous genes. The role of HIF-1α in malignant retinoblastoma remains unclear. We analyzed the role of HIF-1α in WERI-Rb-1 retinoblastoma cells under hypoxic conditions. CoCl2 (125 mmol/L) was added to the culture media to mimic hypoxia. HIF-1α was silenced using siRNA. Gene and protein expression were measured by semi-quantitative RT-PCR and Western blotting. Cell cycle and apoptosis were analyzed by flow cytometry. Cell proliferation, adhesion and invasion were assayed using MTT, Transwell invasion, and cell adhesion assays respectively. Hypoxia significantly upregulated HIF-1α protein expression and the HIF-1α target genes VEGF, GLUT1, and Survivin mRNA. HIF-1α mRNA expression was not affected by hypoxia. Transfection of the siRNA expression plasmid pRNAT-CMV3.2/Neo-HIF-1α silenced HIF-1α by approximately 80% in hypoxic WERI-Rb-1 cells. The knockdown of HIF-1α under hypoxic conditions downregulated VEGF, GLUT1, and Survivin mRNA. It also inhibited proliferation, promoted apoptosis, induced the G0/G1 phase cell cycle arrest, and reduced the adhesion and invasion of WERI-Rb-1 cells. HIF-1α plays a major role in the survival and aggressive phenotype of retinoblastoma cells under hypoxic conditions. Targeting HIF-1α may be a promising therapeutic strategy for human malignant retinoblastoma.

  18. Non-proliferation

    International Nuclear Information System (INIS)

    Manley, I.T.

    1981-01-01

    Proliferation is a problem that can only be solved when the political problems which lead countries to contemplate, the possession of nuclear weapons are solved; in the meantime it can only be managed. Non-proliferation policy has to deal both with the political and the technical aspects of proliferation. It must seek to buy time by addressing the reasons why nations feel the political need to construct nuclear weapons, as well as delaying the moment when such nations feel capable of doing so. The subject is examined and proposals made. (author)

  19. Getting serious about proliferation

    International Nuclear Information System (INIS)

    Leventhal, P.

    1984-01-01

    The US needs to give a higher priority to nuclear non-proliferation, but Reagan's policies assume that proliferation is inevitable and that it is more important to be a reliable supplier than to cause trade frictions by trading only with those nations which sign the non-proliferation treaty (NPT). This undercuts US leadership and the intent of the agreement. Several bills now before Congress could help to restore US leadership by tightening export restrictions and the use of plutonium from the US

  20. Androgen receptor or estrogen receptor-beta blockade alters DHEA-, DHT-, and E(2)-induced proliferation and PSA production in human prostate cancer cells.

    Science.gov (United States)

    Arnold, Julia T; Liu, Xunxian; Allen, Jeffrey D; Le, Hanh; McFann, Kimberly K; Blackman, Marc R

    2007-08-01

    Dehydroepiandrosterone (DHEA) is an endogenous steroid that is metabolized to androgens and/or estrogens in the human prostate. DHEA levels decline with age, and use of DHEA supplements to retard the aging process is of unproved effectiveness and safety. LNCaP and LAPC-4 prostate cancer cells were used to determine whether DHEA-modulated proliferation and prostate specific antigen (PSA) production were mediated via the androgen receptor (AR) and/or ERbeta. Cells were treated with DHEA, DHT, or E(2) and antagonists to AR (Casodex-bicalutamide) or ER (ICI 182,780) or siRNA to the respective receptors. Proliferation was assessed by MTT assay and PSA mRNA and protein secretion were measured by quantitative real-time PCR and ELISA. Associations of AR and ERbeta were analyzed by co-immunoprecipitation studies and fluorescent confocal microscopy. DHEA-, T-, and E(2)-induced proliferation of LNCaP cells was blunted by Casodex but not by ICI treatment. In LNCaP cells, Casodex and ICI suppressed hormone-induced PSA production. In LAPC-4 cells, DHT-stimulated PSA mRNA was inhibited by Casodex and ICI, and the minimal stimulation by DHEA was inhibited by ICI. Use of siRNAs confirmed involvement of AR and ERbeta in hormone-induced PSA production while AR-ERbeta co-association was suggested by immunoprecipitation and nuclear co-localization. These findings support involvement of both AR and ERbeta in mediating DHEA-, DHT-, and E(2)-induced PSA expression in prostate cancer cells. (c) 2007 Wiley-Liss, Inc.

  1. Effect of tamoxifen, methoxyprogesterone acetate and combined treatment on cellular proliferation and apoptosis in SKOV3/DDP cells via the regulation of vascular endothelial growth factor.

    Science.gov (United States)

    Wen, Lv; Hong, Ding; Yanyin, Wu; Mingyue, Zhang; Baohua, Li

    2013-05-01

    The aim of this study was to investigate the effect of tamoxifen (TAM), methoxyprogesterone acetate (MPA) and their combined treatment on cisplatin-resistant ovarian cancer SKOV3/DDP cells, as well as the potential mechanisms. MTT assay was used to investigate the effect of different concentrations (0.01, 0.1, 1, 10 and 100 μM) of TAM, MPA and their combined treatment on the proliferation of cisplatin-resistant ovarian cancer SKOV3/DDP cells. Flow cytometry was employed to analyze the cell cycle and apoptosis rate of SKOV3/DDP cells treated with medium concentration (10 μM) of TAM, MPA and their combined treatment. Change in the protein level of vascular endothelial growth factor (VEGF) in response to drug treatments was measured using Western-blot. The proliferation of SKOV3/DDP cells was inhibited by 1, 10 and 100 μM of TAM or MPA in a dose-dependent manner. Compared to the control group, 10 μM TAM could significantly arrest SKOV3/DDP cells in the G0/G1 stage and induce apoptosis (p < 0.01). However, 10 μM MPA only promoted cell apoptosis, while exhibited little effect on the cell cycle. We further found that 10 μM TAM could remarkably reduce the protein expression of VEGF, while 10 μM MPA only induce a slight reduction. Strikingly, the combined treatment of TAM and MPA exhibited additive effect on the proliferation, cell cycle, apoptosis rate and VEGF expression of SKOV3/DDP cells. We found that TAM, MPA and their combined treatment exhibited significant inhibitory effect on the cisplatin-resistant ovarian cancer SKOV3/DDP cells. Hence, TAM and MPA could be potential cytotoxic drugs to treat cisplatin-resistant patients with advanced ovarian cancer.

  2. Effects of RF-EMF Exposure from GSM Mobile Phones on Proliferation Rate of Human Adipose-derived Stem Cells: An In-vitro Study

    Directory of Open Access Journals (Sweden)

    Shahbazi-Gahrouei D

    2016-12-01

    Full Text Available Background: As the use of mobile phones is increasing, public concern about the harmful effects of radiation emitted by these devices is also growing. In addition, protection questions and biological effects are among growing concerns which have remained largely unanswered. Stem cells are useful models to assess the effects of radiofrequency electromagnetic fields (RF-EMF on other cell lines. Stem cells are undifferentiated biological cells that can differentiate into specialized cells. Adipose tissue represents an abundant and accessible source of adult stem cells. The aim of this study is to investigate the effects of GSM 900 MHz on growth and proliferation of mesenchymal stem cells derived from adipose tissue within the specific distance and intensity. Materials and Methods: ADSCs were exposed to GSM mobile phones 900 MHz with intensity of 354.6 µW/cm2 square waves (217 Hz pulse frequency, 50% duty cycle, during different exposure times ranging from 6 to 21 min/day for 5 days at 20 cm distance from the antenna. MTT assay was used to determine the growth and metabolism of cells and trypan blue test was also done for cell viability. Statistical analyses were carried out using analysis of one way ANOVA. P<0.05 was considered to be statistically significant. Results: The proliferation rates of human ADSCs in all exposure groups were significantly lower than control groups (P<0.05 except in the group of 6 minutes/day which did not show any significant difference with control groups. Conclusion: The results show that 900 MHz RF signal radiation from antenna can reduce cell viability and proliferation rates of human ADSCs regarding the duration of exposure.

  3. Bioinformatics prediction of miR-30a targets and its inhibition of cell proliferation of osteosarcoma by up-regulating the expression of PTEN

    Directory of Open Access Journals (Sweden)

    Biao Zhong

    2017-11-01

    Full Text Available Abstract Background MiRNAs are frequently abnormally expressed in the progression of human osteosarcoma. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN is one of the tumor suppressors in various types of human cancer. In the present study, we detected how hsa-miR-30a-3p regulated PTEN and further tested the role of hsa-miR-30a-3p in the cell proliferation of osteosarcoma cells. Methods The levels of miR-30a were determined by real time PCR. The expression of PTEN was tested by western blotting analysis. Cell distribution of PTEN was observed with confocal laser scanning microscope. Cell viability was determined by MTT assay. Results The expression of miR-30a and PTEN was obviously decreased in MG-63, 143B and Saos-2 cells compared with primary osteoblasts. TargetScan analysis data showed miR-30a might bind with position 30-57 of 3’UTR of PTEN. Transfection with miR-30a-3p increased the level of PTEN in MG-63 cells, while transfection with miR-30a-3p inhibitor significantly decreased the expression of PTEN in osteosarcoma cells. Transfection with miR-30a-3p significantly inhibited cell proliferation of osteosarcoma cells, while miR-30a inhibitor obviously promoted cell viability of MG63 cells and Saos-2 cells. Inhibition of PTEN eliminated the proliferation inhibitory effect of miR-30a-3p. Conclusion Thus, all these findings revealed the anti-tumor effects of miR-30a in human osteosarcoma cells, which could be mediated by regulating the level of PTEN.

  4. Synthesis and Evaluation of the Antioxidant Activity of Lipophilic Phenethyl Trifluoroacetate Esters by In Vitro ABTS, DPPH and in Cell-Culture DCF Assays

    Directory of Open Access Journals (Sweden)

    Roberta Bernini

    2018-01-01

    Full Text Available Polyphenols are natural compounds showing a variety of health-promoting effects. Unfortunately, due to low lipid solubility, their applications in the pharmaceutical, food, and cosmetic industries are limited. With the aim of obtaining novel lipophilic derivatives, the present study reports the synthesis of a series of phenethyl trifluoroacetate esters containing up to two hydroxyl groups in the aromatic ring. Experimental logP values confirmed a greater lipophilicity of the novel compounds compared to the parent compounds. The radical scavenging capacity of all phenethyl trifluoroacetate esters was evaluated by in vitro assays (ABTS, DPPH and in cultured cells (L6 myoblasts and THP-1 leukemic monocytes using 2′,7′-dichlorodihydrofluorescein diacetate. These data revealed that the esters showed a good antioxidant effect that was strictly dependent on the grade of hydroxylation of the phenyl ring. The lack of toxicity, evaluated by the MTT assay and proliferation curves, makes these trifluoroacetates attractive derivatives for pharmaceutical, food, and cosmetic applications.

  5. A flow cytometric assay for simultaneously measuring the ...

    African Journals Online (AJOL)

    This research objective was to exploit a novel method for measuring the proliferation, cytotoxicity of cytokine-induced killer (CIK) cells using carboxyfluorescein succinimidyl ester/proliferation index (CFSE/PI) and flow cytometric assay. As cells divide, CFSE is apportioned equally between the two daughter cells, leading to a ...

  6. MeCP2 silencing of LncRNA H19 controls hepatic stellate cell proliferation by targeting IGF1R

    International Nuclear Information System (INIS)

    Yang, Jing-Jing; Liu, Li-Ping; Tao, Hui; Hu, Wei; Shi, Peng; Deng, Zi-Yu; Li, Jun

    2016-01-01

    Highlights: • H19 plays a key role in HSCs proliferation and fibrosis. • MeCP2/H19 axis involvement in HSCs activation and fibrosis. • MeCP2 negative controls H19 expression in activated HSCs. • Identification of IGF1R as new target of H19 in HSC. - Abstract: Methyl-CpG-binding protein 2 (MeCP2) plays a key role in liver fibrosis. However, the potential mechanism of MeCP2 in liver fibrosis remains unclear. Early reports suggest that LncRNA H19 is important epigenetic regulator with critical roles in cell proliferation, but its role in hepatic fibrosis remains elusive. Sprague-Dawley rats liver fibrosis was generated by 12-weeks treatment with CCl 4 intraperitoneal injection. HSC-T6 cells were used in vitro study. The expression levels of MeCP2, H19, IGF1R, α-SMA, and Col1A1 were estimated by Western blotting, qRT-PCR and Immunohistochemistry. HSC-T6 cells were transfected with MeCP2-siRNA, pEGF-C1-MeCP2, pEX-3-H19, and H19-siRNA. Finally, cell proliferation ability was assessed by the MTT assay. Here, we found that H19 was significantly down-regulated in HSCs and fibrosis tissues, and an opposite pattern is observed for MeCP2 and IGF1R. Silencing of MeCP2 blocked HSCs proliferation. Knockdown of MeCP2 elevated H19 expression in activated HSCs, and over-expression of MeCP2 inhibited H19 expression in activated HSCs. Moreover, we investigated the effect of H19 on IGF1R expression. Overexpression of H19 in HSCs repressed the expression of IGF1R, and an opposite pattern is observed for H19 silenced. In addition, we reported that overexpression of H19 inhibited the TGF-β1-induced proliferation of HSCs. Furthermore, MeCP2 negative regulation of H19 by targeting the protein IGF1R. Taken together, these results demonstrated that MeCP2 silencing of H19 can alter the IGF1R overexpression, thus contributing to HSCs proliferation. These data could suggest the development of combination therapies that target the MeCP2.

  7. G Protein-Coupled Receptor 87 (GPR87 Promotes Cell Proliferation in Human Bladder Cancer Cells

    Directory of Open Access Journals (Sweden)

    Xia Zhang

    2015-10-01

    Full Text Available G protein-coupled receptor 87 (GPR87 is a newly deorphanized member of the cell surface molecule G protein-coupled receptor family. GPR signaling was shown to play a role in promotion of cell growth and survival, metastasis, and drug resistance. The overexpression of GPR87 has also been reported in many malignant tumors including bladder cancer. The aim of the present study is to examine the effect of silencing GPR87 expression with a replication-deficient recombinant adenoviral vector expressing short hairpin RNA targeting GPR87 (Ad-shGPR87 and to explore the underlying molecular mechanisms in bladder cancer cells. Six GPR87-expressing human bladder cancer cells, HT1197, HT1376, J82, RT112, TCCSUP and UMUC3, were used. Infection with Ad-shGPR87 effectively downregulated the GPR87 expression, and significantly reduced the percentage of viable cells in 4 of 6 cell lines as detected by an MTT assay. Significant inhibition on cell proliferation with Ad-shGPR87 was observed in the wild-type p53 bladder cancer cell lines (HT1197, RT112, TCCSUP and UMUC3, but not in the mutant p53 cells (HT1376 and J82. As represented by a wild-type p53 RT112 cell, Ad-shGPR87 infection significantly enhanced p53 and p21 expression and caused caspase-dependent apoptosis. Furthermore, the treatment with Ad-shGPR87 exerted a significant antitumor effect against the GPR87-expressing RT112 xenografts. GPR87 appeared to be a promising target for gene therapy, and Ad-shGPR87 had strong antitumor effects, specifically anti-proliferative and pro-apoptotic effects, against GPR87-expressing human bladder cancer cells.

  8. Nuclear proliferation and terrorism

    International Nuclear Information System (INIS)

    Anon.

    1977-01-01

    This section of the book, Part III, has two chapters (9 and 10). Chapter 9, Nuclear Power and Proliferation of Nuclear Weapons, is disucssed under these subjects: nuclear nonproliferation: origins and status; requirements for nuclear weapons manufacture; current nuclear programs and proliferation capabilities; encouraging decisions to forego weapons; arms control; safeguards; attitudes and expectations. Chapter 10, Nuclear Terrorism, discusses these areas: theft of nuclear materials; attacks on nuclear reactors; responding to nuclear terrorism; security and civil liberties

  9. Paris polyphylla Suppresses Proliferation and Vasculogenic Mimicry of Human Osteosarcoma Cells and Inhibits Tumor Growth In Vivo.

    Science.gov (United States)

    Yao, Nan; Ren, Ke; Wang, Yimin; Jin, Qiaomei; Lu, Xiao; Lu, Yan; Jiang, Cuihua; Zhang, Dongjian; Lu, Jun; Wang, Chen; Huo, Jiege; Chen, Yong; Zhang, Jian

    2017-01-01

    Paris polyphylla, a traditional antipyretic-detoxicate chinese medicinal herb, has been applied extensively in cancer treatments for nearly 2000 years. The purpose of the present study is to evaluate the potential anti-osteosarcoma effects of Paris polyphylla ethanol extract (PPEE) and to investigate its underlying mechanisms. The antiproliferation activity of PPEE was tested on 143B, MG-63, U-2 OS and hFOB1.19 cells using MTT assay. The pro-apoptotic and cell cycle arrest effects of PPEE were confirmed by Hoechst 33342 staining and flow cytometry. The antimigratory, anti-invasive and antivasculogenic mimicry (VM) effects of PPEE were investigated by wound healing, Transwell and 3D culture assays. Mouse xenograft model was used to examine its anti-osteosarcoma efficacy in vivo. Hematologic profiles and hepatorenal functions were evaluated to assess the toxicity of PPEE. PPEE evidently suppressed cell proliferation of 143B, MG-63 and U-2 OS with IC50 values of 10-60[Formula: see text][Formula: see text]g/mL, but showed little cytotoxicity against normal osteoblastic cell. PPEE promoted apoptosis in 143B cell via caspase activation, increased Bax/Bcl-2 ratio and PARP cleavage. It also induced G2/M phase arrest associated with elevated phosphorylation of CDK1, Cdc25C, Chk2 and down-regulation of cyclin B1, CDK1, Cdc25C expression. Additionally, PPEE inhibited 143B cell migration, invasion and VM formation at noncytotoxic concentrations through decreasing the expression of FAK, Mig-7, MMP2 and MMP9. Finally, daily oral administration of PPEE for four weeks exhibits potent antitumor and anti-VM activity in 143B xenograft model with low toxicity. Taken together, these findings demonstrated PPEE possesses anti-osteosarcoma and anti-VM activity in vitro and in vivo, and therefore is a potential candidate for osteosarcoma treatment.

  10. Lymphocyte Proliferation Response in Patients with Acute and Chronic Brucellosis

    Directory of Open Access Journals (Sweden)

    Khadijeh Khosravi

    2016-05-01

    Full Text Available Abstract Background: Brucella is an intracellular bacterium that causes chronic infection in humans and domestic animals. The underlying mechanisms that cause prolonged illness are complex and not fully understood. Immune responses may have an important role in the chronicity of infection. Here, we evaluated the lymphocyte proliferation responses in patients with chronic and acute brucellosis. Materials and Methods: This descriptive - analytical study was performed on 22 patients with acute brucellosis, 21 patients with chronic brucellosis and 21 healthy people with the similar age, sex and genetic background as control group. Peripheral lymphocytes were isolated using Ficoll and the cellular proliferation was quantified in presence of antigen and phytohemaglutinin-A by MTT method. Results: The brucella antigen-specific stimulation index in patients with chronic brucellosis was significantly lower than the acute brucellosis patients (p=0.001. Also, stimulating the lymphocytes with phytohemaglutinin-A has shown that proliferative response in patients with chronic brucellosis was lower than the other groups (p=0.04. Conclusion: The results indicated that chronic brucellosis inhibits lymphocyte proliferation. This inhibition of lymphocyte proliferation may be due to the induction of anergy.

  11. miR-124 suppresses proliferation and invasion of nasopharyngeal carcinoma cells through the Wnt/β-catenin signaling pathway by targeting Capn4

    Directory of Open Access Journals (Sweden)

    Hu H

    2017-05-01

    Full Text Available Haili Hu,1,* Guanghui Wang,1,* Congying Li2 1Department of Otorhinolaryngology, Huaihe Hospital of Henan University, 2Department of Otorhinolaryngology, School of Medicine, Kaifeng University, Kaifeng, People’s Republic of China *These authors contributed equally to this work Background: Recent studies have demonstrated that microRNA 124 (miR-124 acts as a tumor suppressor in nasopharyngeal carcinoma (NPC; however, the exact molecular mechanism by which miR-124 exerts tumor suppression has not been well elucidated.Materials and methods: We performed quantitative real-time PCR (qRT-PCR to measure the expression of metastasis associated lung adenocarcinoma transcript 1, miR-124, and calpain small subunit 1 (Capn4 mRNAs in NPC cell lines. We also performed western blot analysis to detect the levels of Capn4. Furthermore, we performed MTT assay and transwell invasion assay to determine the proliferation and invasion ability of two NPC cell lines, namely, HONE1 and CNE2 cells, respectively. The verification of targets of miR-124 was performed using prediction softwares and luciferase reporter analysis.Results: According to our results, the expression of Capn4 was found to be elevated, whereas the expression of miR-124 was lowered in NPC cell lines compared with normal nasopharyngeal cells. When we preformed overexpression of miR-124, it suppressed the proliferation and invasion of NPC cells. Moreover, miR-124 suppressed the expression of Capn4 by targeting Capn4 in HONE1 and CNE2 cells. When we preformed overexpression of Capn4, it reversed the inhibitory effect of miR-124 on the proliferation and invasion of NPC cells. Furthermore, miR-124–Capn4 axis decreased the levels of β-catenin, cyclin D1, and c-Myc, the components of the Wnt/β-catenin signaling pathway.Conclusion: The suppression of proliferation and invasion of NPC cells by miR-124 were achieved by the regulation of Wnt/β-catenin signaling pathway by targeting Capn4. The results of

  12. [X-linked inhibitor of apoptosis protein (XIAP) and Survivin suppression on human pancreatic cancer cells Panc-1 proliferation and chemosensitivety].

    Science.gov (United States)

    Zai, Hong-yan; Yi, Xiao-ping; Li, Yi-xiong; You, Xue-ying; Cao, Li-ping; Liu, Hui

    2013-04-18

    To investigate the effect on cell proliferation and chemosensitivity of human pancreatic cancer cells Panc-1 after X-linked inhibitor of apoptosis protein (XIAP) and Survivin are inhibited simultaneously, and to compare it with the separate gene suppression strategy by which expression of XIAP or Survivin is inhibited respectively. Panc-1 (Panc-1-X, Panc-1-S and Panc-1-XS) in which expression of XIAP and/or Survivin was inhibited, was established by using XIAP-shRNA lentiviral and Survivin-shRNA lentiviral we had built. The expressions of XIAP and Survivin mRNA and protein were evaluated by Real-time PCR and Semi-quantitatively Western blot analysis; cell proliferation was investigated by cell counting and colony formation assay; cell apoptosis was investigated by Caspase-3/7 activity assay kit and flow cytometry; gemcitabine (Gem) chemosensitivity was investigated by MTT assay. The pancreatic cell line Panc-1 in which the expression of XIAP and/or Survivin was stablely inhibited was successfully established. The cell proliferation of Panc-1-XS cells decreased significantly. The colony formation rate of Panc-1-XS cells (10.12%± 1.33%), was significantly lower than that of Panc-1-XncSnc cells (96.61% ± 7.89%) and Panc-1 cells (100.28% ± 8.97%) respectively (PPanc-1-XS cells (15.02 ± 0.57) was significantly higher than that of Panc-1 cells and Panc-1-XncSnc cells (8.87 ± 0.19 and 9.05 ± 0.23, respectively; PPanc-1-XS cells (24.09% ± 2.75%) was significantly higher than that of Panc-1-XncSnc cells and Panc-1 cells (12.09% ± 1.97% and 12.06% ± 1.22%, respectively; PPanc-1-XS cells [(0.47 ± 0.04) mg/L] was significantly lower than that of Panc-1-XncSnc cells [(2.18 ± 0.13) mg/L] and Panc-1 cells [(2.13 ± 0.18) mg/L, PPanc-1-XS cells [(0.47 ± 0.04) mg/L] was significantly lower than that of Panc-1-X cells [(0.76 ± 0.07) mg/L] and Panc-1-S cells [(0.87 ± 0.09) mg/L, PPanc-1 cells was significantly suppressed and the Gem chemosensitivity was significantly

  13. Targeting of GIT1 by miR-149* in breast cancer suppresses cell proliferation and metastasis in vitro and tumor growth in vivo

    Directory of Open Access Journals (Sweden)

    Dong Y

    2017-12-01

    Full Text Available Yan Dong,1,* Cai Chang,2,* Jingtian Liu,3 Jinwei Qiang4 1Department of Ultrasonography, Jinshan Hospital, 2Department of Ultrasonography, Cancer Center, 3Department of General Surgery, 4Department of Radiology, Jinshan Hospital, Fudan University, Shanghai, China *These authors contributed equally to this work Abstract: Breast cancer remains a major cause of cancer-related death in women worldwide. Dysregulation of microRNAs (miRNAs is involved in the initiation and progression of breast cancer. Moreover, it was found that GIT1 was widely involved in the development of many human cancers. Herein, we aimed to investigate the expression changes of miR-149* and GIT1 and the functional effects of miR-149*/GIT1 link in breast cancer. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR and Western blot (WB were used to examine the expression levels of miR-149* and GIT1. Dual luciferase reporter assay was utilized to confirm the target interaction between miR-149* and GIT1. The biological functions, including cell proliferation, invasion, and migration, of miR-149* and GIT1 were determined by MTT assay and Transwell assays, respectively. Eventually, the tumor xenograft model in nude mice injected with stable transfected MDA-MB-231 cells was established to verify the effects of miR-149* and GIT1 on tumor growth. Our results showed that miR-149* expression was decreased, whereas GIT1 expression was increased in clinical samples of breast cancer. Based on the inverse expression trend between miR-149* and GIT1, we further demonstrated that miR-149* indeed directly targets GIT1. Subsequently, it was observed that inhibition of miR-149* significantly promoted cell proliferation, invasion, and migration, but the ability of cell proliferation, invasion, and migration was obviously declined after silencing of GIT1 in MDA-MB-231 cells transfected with miR-149* mimic and/or si-GIT1. Finally, it was also found that elevated miR-149* decelerated

  14. [Overexpression of miR-519d-3p inhibits the proliferation of DU-145 prostate cancer cells by reducing TRAF4].

    Science.gov (United States)

    Li, Xiaohui; Han, Xingtao; Yang, Jinhui; Sun, Jiantao; Wei, Pengtao

    2018-01-01

    Objective To observe the effect of microRNA-519d-3p (miR-519d-3p) on the proliferation of prostate cancer cells and explore the possible molecular mechanism. Methods The expression level of miR-519d-3p in PC-3, DU-145, 22RV1, PC-3M, LNCaP human prostate cancer cells and RWPE-1 human normal prostate epithelial cells was detected by real-time quantitative PCR. miR-519d-3p mimics or negative control microRNAs (miR-NC) was transfected into the prostate cancer cells with the lowest level of miR-519d-3p expression. Transfection efficiency was examined. The effect of miR-519d-3p on the cell cycle of prostate cancer was detected by flow cytometry. MTT assay and plate clone formation assay were used to detect its effect on the proliferation of prostate cancer cells. Bioinformatics software was used to predict and dual luciferase reporter assay was used to validate the target gene of miR-519d-3p. Real-time quantitative PCR was used to detect the expression of miR-519d-3p target gene. Western blot analysis was used to detect the expression of target gene protein and downstream protein. Results The expression of miR-519d-3p in normal prostate epithelial cells was significantly higher than that in prostate cancer cells, and the lowest was found in DU-145 cells. After transfected with miR-519d-3p mimics, the expression level of miR-519d-3p in DU-145 cells increased significantly. Bioinformatics prediction and dual luciferase reporter gene confirmed that tumor necrosis factor receptor associated factor 4 (TRAF4) was the target gene of miR-519d-3p. Overexpression of miR-519d-3p significantly reduced the expression of TRAF4 gene and its downstream TGF-β signaling pathway proteins in the prostate cancer cells. Conclusion The expression of miR-519d-3p is down-regulated in prostate cancer cells. Overexpression of miR-519d-3p can inhibit the proliferation of prostate cancer cells. The possible mechanism is that miR-519d-3p inhibits the expression of TRAF4.

  15. Molecular mechanisms of radiation-induced cell proliferation in human carcinoma cells

    International Nuclear Information System (INIS)

    Schmidt-Ullrich, R.K.; Mikkelsen, R.; Valerie, K.; Todd, D.; Kavanagh, B.; Contessa, J.; Rorrer, K.; Chen, P.

    1996-01-01

    Purpose: At therapeutically applied ionizing radiation (IR) doses of 0.5 to 5 Gy, a certain proportion of cells will undergoes radiation-induced death while a varied proportion of cells will survive and be able of furnishing adaptive responses. One of these adaptive responses has been experimentally and clinically described as repopulation. Despite description of this phenomenon more than 20 years ago, the mechanisms of this response have remained relatively unknown until modern experimental techniques have been applied to studies on cellular radiation responses. materials and Methods: Human mammary, MCF-7 and MDA-MB-231, and squamous, A431, carcinoma cells (MCC and SCC), expressing epidermal growth factor-receptor (EGF-R) at widely varied levels, have been exposed under defined culture conditions to single and repeated IR at doses between 0.5 and 5 Gy. Cellular IR responses of activation and expression changes of growth regulatory genes and activation of signal transduction pathways were linked to IR-induced proliferation responses. Specifically, EGF-R activation and expression were assessed by levels of Tyr phosphorylation (Y p ) of the receptor protein and mRNA, respectively. Phospholipase (PL-C) activation was quantified by Y p levels and production of inositol-triphosphate (IP 3 ), elevation of cytoplasmic Ca 2+ by video-intensified florescence microscopy after Fura-2 loading. Mitogen-activated protein (MAP) kinase activation was measured by a MBP receptor assay. The EGF-R and signal transduction activation events were correlated with a proliferation response of irradiated cells as quantified by MTT assay. Results: The cell lines tested showed an about 3-fold stimulation of EGF-R Y p levels within 5 min of IR which was associated with a 2.5-fold upregulation of EGF-R after 24 hr. Repeated daily 2 Gy exposures of MCF-7 and MDA-cells resulted in up to 9-fold increases in EGF-R mRNA. EGF-R downstream signal transduction was evidenced by activation of the

  16. Nuclear non-proliferation

    International Nuclear Information System (INIS)

    Anon.

    1984-01-01

    DOE's nuclear non-proliferation responsibilities are defined by the provisions of the Atomic Energy Act of 1954, as amended, and of the Nuclear Non-Proliferation Act of 1978 (NNPA). The Department's major responsibilities in this area are to: (1) provide technical assistance to the Department of State in negotiating agreements for civil cooperation in the peaceful uses of nuclear energy with other countries and international organizations; (2) join with other agencies to reach executive branch judgments with respect to the issuance of export licenses by the Nuclear Regulatory Commission; (3) be responsible for processing subsequent arrangements with other agencies as required by the Nuclear Non-Proliferation Act; (4) control the distribution of special nuclear materials, components, equipment, and nuclear technology exports; (5) participate in bilateral and multilateral cooperation with foreign governments and organizations to promote the peaceful uses of nuclear energy; and (6) act as a primary technical resource with respect to US participation in the International Atomic Energy Agency

  17. Dynamics of nuclear proliferation

    International Nuclear Information System (INIS)

    Meyer, S.M.

    1984-01-01

    This book looks beyond policy disputes to make a systematic examination of the assumptions and contending hypotheses that constitute contemporary thinking on nuclear proliferation. Rather than determine who is right or wrong, the intent is to develop a better picture by using the various schools of thought as analytic windows. A better understanding of how the process operates should offer better guidance for predicting future nuclear proliferation and, ultimately, for controlling it. Separate chapters deal with the contending views, the technological and motivational bases of nuclear proliferation, the presence of a technological imperative, testing the motivational hypothesis, the dynamics of the process, and forecasting. Four appendices present historical decisions, the technical model, cost-estimating procedures, and procedures for estimating nuclear propensities. 288 references, 17 figures, 26 tables

  18. Estradiol agonists inhibit human LoVo colorectal-cancer cell proliferation and migration through p53.

    Science.gov (United States)

    Hsu, Hsi-Hsien; Kuo, Wei-Wen; Ju, Da-Tong; Yeh, Yu-Lan; Tu, Chuan-Chou; Tsai, Ying-Lan; Shen, Chia-Yao; Chang, Sheng-Huang; Chung, Li-Chin; Huang, Chih-Yang

    2014-11-28

    To investigate the effects of 17β-estradiol via estrogen receptors (ER) or direct administration of ER agonists on human colorectal cancer. LoVo cells were established from the Bioresource Collection and Research Center and cultured in phenol red-free DMEM (Sigma, United States). To investigate the effects of E2 and/or ER selective agonists on cellular proliferation, LoVo colorectal cells were treated with E2 or ER-selective agonists for 24 h and 48 h and subjected to the MTT (Sigma) assay to find the concentration. And investigate the effects of E2 and/or ER selective agonists on cell used western immunoblotting to find out the diversification of signaling pathways. In order to observe motility and migration the wound healing assay and a transwell chamber (Neuro Probe) plate were tased. For a quantitative measure, we counted the number of migrating cells to the wound area post-wounding for 24 h. We further examined the cellular migration-regulating factors urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA) and matrix metalloproteinase (MMP)-9 in human LoVo cells so gelatin zymography that we used and gelatinolytic activity was visualized by Coomassie blue staining. And these results are presented as means ± SE, and statistical comparisons were made using Student's t-test. The structure was first compared with E2 and ER agonists. We then treated the LoVo cells with E2 and ER agonists (10(-8) mol/L) for 24 h and 48 h and subsequently measured the cell viability using MTT assay. Our results showed that treatment with 17β-estradiol and/or ER agonists in human LoVo colorectal cancer cells activated p53 and then up-regulated p21 and p27 protein levels, subsequently inhibiting the downstream target gene, cyclin D1, which regulates cell proliferation. Taken together, our findings demonstrate the anti-tumorigenesis effects of 17β-estradiol and/or ER agonists and suggest that these compounds may prove to be a potential alternative

  19. Anti-CTGF single-chain variable fragment dimers inhibit human airway smooth muscle (ASM) cell proliferation by down-regulating p-Akt and p-mTOR levels.

    Science.gov (United States)

    Gao, Wei; Cai, Liting; Xu, Xudong; Fan, Juxiang; Xue, Xiulei; Yan, Xuejiao; Qu, Qinrong; Wang, Xihua; Zhang, Chen; Wu, Guoqiu

    2014-01-01

    Connective tissue growth factor (CTGF) contributes to airway smooth muscle (ASM) cell hyperplasia in asthma. Humanized single-chain variable fragment antibody (scFv) was well characterized as a CTGF antagonist in the differentiation of fibroblast into myofibroblast and pulmonary fibrosis in our previous studies. To further improve the bioactivity of scFv, we constructed a plasmid to express scFv-linker-matrilin-6×His fusion proteins that could self-assemble into the scFv dimers by disulfide bonds in matrilin under non-reducing conditions. An immunoreactivity assay demonstrated that the scFv dimer could highly bind to CTGF in a concentration-dependent manner. The MTT and EdU assay results revealed that CTGF (≥10 ng/mL) promoted the proliferation of ASM cells, and this effect was inhibited when the cells were treated with anti-CTGF scFv dimer. The western blot analysis results showed that increased phosphorylation of Akt and mTOR induced by CTGF could be suppressed by this scFv dimer. Based on these findings, anti-CTGF scFv dimer may be a potential agent for the prevention of airway remodeling in asthma.

  20. Proliferation resistance modeling

    International Nuclear Information System (INIS)

    Bari, R.; Peterson, P.; Roglans, J.; Mladineo, S.; Nuclear Engineering Division; BNL; Univ. of California at Berkely; PNNL

    2004-01-01

    The National Nuclear Security Administration is developing methods for nonproliferation assessments. A working group on Nonproliferation Assessment Methodology (NPAM) assembled a toolbox of methods for various applications in the nonproliferation arena. One application of this methodology is to the evaluation of the proliferation resistance of Generation IV nuclear energy systems. This paper first summarizes the key results of the NPAM program and then provides results obtained thus far in the ongoing application, which is co-sponsored by the DOE Office of Nuclear Energy Science and Technology. In NPAM, a top-level measure of proliferation resistance for a fuel cycle system is developed from a hierarchy of metrics. The problem is decomposed into: metrics to be computed, barriers to proliferation, and a finite set of threats. The analyst models the process undertaken by the proliferant to overcome barriers to proliferation and evaluates the outcomes. In addition to proliferation resistance (PR) evaluation, the application also addresses physical protection (PP) evaluation against sabotage and theft. The Generation IV goal for future nuclear energy systems is to assure that they are very unattractive and the least desirable route for diversion or theft of weapons-usable materials, and provide increased physical protection against terrorism. An Expert Group, addressing this application, has identified six high-level measures for the PR goals (six measures have also been identified for the PP goals). Combined together, the complete set of measures provides information for program policy makers and system designers to compare specific system design features and integral system characteristics and to make choices among alternative options. The Group has developed a framework for a phased evaluation approach to analyzing PR and PP of system characteristics and to quantifying metrics and measures. This approach allows evaluations to become more detailed and representative

  1. Inhibition of CD147 expression by RNA interference reduces proliferation, invasion and increases chemosensitivity in cancer stem cell-like HT-29 cells.

    Science.gov (United States)

    Chen, Jie; Pan, Yuqin; He, Bangshun; Ying, Houqun; Wang, Feng; Sun, Huiling; Deng, Qiwen; Liu, Xian; Lin, Kang; Peng, Hongxin; Cho, William C; Wang, Shukui

    2015-10-01

    The association between CD147 and cancer stem cells (CSCs) provides a new angle for cancer treatments. The aim of this study was to investigate the biological roles of CD147 in colorectal CSCs. The Oct4-green fluorescent protein (GFP) vector was used to isolate CSCs and pYr-mir30-shRNA was used to generate short hairpin RNA (shRNA) specifically for CD147. After RNA interference (RNAi), CD147 was evaluated by reverse transcription‑quantitative PCR and western blot analysis, and its biological functions were assessed by MTT and invasion assays. The results showed that the differentiation of isolated CSC-like HT-29 cells was blocked and these cells were highly positive for CD44 and CD147. RNAi-mediated CD147 silencing reduced the expression of CD147 at both mRNA and protein levels. Moreover, the activities of proliferation and invasion were decreased obviously in CSCs. Knockdown of CD147 increased the chemosensitivity of CSC-like cells to gemcitabine, cisplatin, docetaxel at 0.1, 1 and 10 µM respectively, however, there was no significant difference among the three groups to paclitaxel at 10 µM. In conclusion, these results suggest that CD147 plays an important role in colorectal CSCs and might be regarded as a novel CSC-specific targeted strategy against colorectal cancer.

  2. [Inhibitory effect of exogenous insulin-like growth factor binding protein 7 on proliferation of human breast cancer cell line MDA-MB-453 and its mechanism].

    Science.gov (United States)

    Yuan, Lei; Fan, Wen-Juan; Yang, Xu-Guang; Rao, Shu-Mei; Song, Jin-Ling; Song, Guo-Hua

    2013-10-25

    The present study was to investigate the effects of exogenous insulin-like growth factor binding protein 7 (IGFBP7) on the proliferation of human breast cancer cell line MDA-MB-453 and its possible mechanism. By means of MTT method in vitro, the results showed exogenous IGFBP7 inhibited the growth of MDA-MB-453 cells (IC50 of IGFBP7 = 8.49 μg/mL) in time- and concentration-dependent manner. SB203580, p38(MAPK) inhibitor, blocked the anti-proliferative effect of exogenous IGFBP7. The flow cytometry assay showed that exogenous IGFBP7 remarkably induced G0/G1 arrest in MDA-MB-453 cells. The Western blot showed that exogenous IGFBP7 promoted phosphorylation of p38(MAPK), up-regulated expression of p21(CIP1/WAF1), and inhibited phosphorylation of Rb. SB203580 restrained exogenous IGFBP7-induced regulation of p21(CIP1/WAF1) and p-Rb in MDA-MB-453 cells. In conclusion, the present study suggests that exogenous IGFBP7 could activate the p38(MAPK) signaling pathway, upregulate p21(CIP1/WAF1) expression, inhibit phosphorylation of Rb, and finally induce G0/G1 arrest in MDA-MB-453 cells.

  3. Effects of Germinated Brown Rice and Its Bioactive Compounds on the Expression of the Peroxisome Proliferator-Activated Receptor Gamma Gene

    Directory of Open Access Journals (Sweden)

    Zaki Tubesha

    2013-02-01

    Full Text Available Dysregulated metabolism is implicated in obesity and other disease conditions like type 2 diabetes mellitus and cardiovascular diseases, which are linked to abnormalities of peroxisome proliferator-activated receptor gamma (PPARγ. PPARγ has been the focus of much research aimed at managing these diseases. Also, germinated brown rice (GBR is known to possess antidiabetic, antiobesity and hypocholesterolemic effects. We hypothesized that GBR bioactive compounds may mediate some of the improvements in metabolic indices through PPARγ modulation. Cultured HEP-G2 cells were treated with 50 ppm and 100 ppm of extracts from GBR (GABA, ASG and oryzanol after determination of cell viabilities using MTT assays. Results showed that all extracts upregulated the expression of the PPARγ. However, combination of all three extracts showed downregulation of the gene, suggesting that, in combination, the effects of these bioactives differ from their individual effects likely mediated through competitive inhibition of the gene. Upregulation of the gene may have therapeutic potential in diabetes mellitus and cardiovascular diseases, while its downregulation likely contributes to GBR’s antiobesity effects. These potentials are worth studying further.

  4. The mechanism of kaempferol induced apoptosis and inhibited proliferation in human cervical cancer SiHa cell: From macro to nano.

    Science.gov (United States)

    Tu, Lv-Ying; Bai, Hai-Hua; Cai, Ji-Ye; Deng, Sui-Ping

    2016-11-01

    Kaempferol has been identified as a potential cancer therapeutic agent by an increasing amount of evidences. However, the changes in the topography of cell membrane induced by kaempferol at subcellular- or nanometer-level were still unclear. In this work, the topographical changes of cytomembrane in human cervical cancer cell (SiHa) induced by kaempferol, as well as the role of kaempferol in apoptosis induction and its possible mechanisms, were investigated. At the macro level, MTT assays showed that kaempferol inhibited the proliferation of SiHa cells in a time- and dose-dependent manner. Flow cytometry analysis demonstrated that kaempferol could induce SiHa cell apoptosis, mitochondrial membrane potential disruption, and intracellular free calcium elevation. At the micro level, fluorescence imaging by laser scanning confocal microscopy (LSCM) indicated that kaempferol could also destroy the networks of microtubules. Using high resolution atomic force microscopy (AFM), we determined the precise changes of cellular membrane induced by kaempferol at subcellular or nanometer level. The spindle-shaped SiHa cells shrank after kaempferol treatment, with significantly increased cell surface roughness. These data showed structural characterizations of cellular topography in kaempferol-induced SiHa cell apoptosis and might provide novel integrated information from macro to nano level to assess the impact of kaempferol on cancer cells, which might be important for the understanding of the anti-cancer mechanisms of drugs. SCANNING 38:644-653, 2016. © 2016 Wiley Periodicals, Inc. © Wiley Periodicals, Inc.

  5. Peroxisome proliferator-activated receptor γ is expressed in hippocampal neurons and its activation prevents β-amyloid neurodegeneration: role of Wnt signaling

    International Nuclear Information System (INIS)

    Inestrosa, Nibaldo C.; Godoy, Juan A.; Quintanilla, Rodrigo A.; Koenig, Cecilia S.; Bronfman, Miguel

    2005-01-01

    The molecular pathogenesis of Alzheimer's disease (AD) involves the participation of the amyloid-β-peptide (Aβ), which plays a critical role in the neurodegeneration that triggers the disease. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors, which are members of the nuclear receptor family. We report here that (1) PPARγ is present in rat hippocampal neurons in culture. (2) Activation of PPARγ by troglitazone and rosiglitazone protects rat hippocampal neurons against Aβ-induced neurodegeneration, as shown by the 3-[4,5 -2yl]-2,5-diphenyltetrazolium bromide (MTT) reduction assay, immunofluorescence using an anti-heavy neurofilament antibody, and quantitative electron microscopy. (3) Hippocampal neurons treated with several PPARγ agonists, including troglitazone, rosiglitazone, and ciglitazone, prevent the excitotoxic Aβ-induced rise in bulk-free Ca 2+ . (4) PPARγ activation results in the modulation of Wnt signaling components, including the inhibition of glycogen synthase kinase-3β (GSK-3β) and an increase of the cytoplasmic and nuclear β-catenin levels. We conclude that the activation of PPARγ prevents Aβ-induced neurodegeneration by a mechanism that may involve a cross talk between neuronal PPARγ and the Wnt signaling pathway. More important, the fact that the activation of PPARγ attenuated Aβ-dependent neurodegeneration opens the possibility to fight AD from a new therapeutic perspective

  6. Serum from Chronic Hepatitis B Patients Promotes Growth and Proliferation via the IGF-II/IGF-IR/MEK/ERK Signaling Pathway in Hepatocellular Carcinoma Cells.

    Science.gov (United States)

    Ji, Yuanyuan; Wang, Zhidong; Chen, Haiyan; Zhang, Lei; Zhuo, Fei; Yang, Qingqing

    2018-05-09

    Chronic hepatitis B virus (HBV) infection (CHB) plays a central role in the etiology of hepatocellular carcinoma (HCC). Emerging evidence implicates insulin-like growth factor (IGF)-II as a major risk factor for the growth and development of HCC. However, the relationship between HBV infection and IGF-II functions remains to be elucidated. Levels of circulating IGF-II and IGF-I receptor (IGF-IR) in healthy donors (HDs) and CHB patients were tested by ELISA. Human HCC cell lines (HepG-2, SMMC-7721, MHCC97-H) were incubated with serum from HDs and CHB patients at various concentrations for 24, 48, and 72 h. MTT and plate colony formation assays, BrdU ELISA, ELISA, small-interfering RNA (siRNA) transfection, quantitative real-time PCR, and western blot were applied to assess the functional and molecular mechanisms in HCC cell lines. Serum levels of IGF-II and IGF-IR were significantly higher in CHB patients than in HDs. Additionally, serum from CHB patients directly induced cell growth, proliferation, IGF-II secretion, and HDGF-related protein-2 (HRP-2) and nuclear protein 1 (NUPR1) mRNA and protein expression in HCC cells. Moreover, serum from CHB patients increased IGF-II-induced cell growth, proliferation, and HRP-2 and NUPR1 mRNA and protein expression in HCC cells. Blockade of IGF-IR clearly inhibited the above effects. Most importantly, interference with IGF-II function markedly repressed the cell proliferation and HRP-2 and NUPR1 mRNA and protein expression induced by serum from CHB patients. Furthermore, serum from CHB patients induced ERK phosphorylation via IGF-IR, with the MEK inhibitor PD98059 significantly decreasing CHB patient serum-induced IGF-II secretion, cell proliferation, and HRP-2 and NUPR1 mRNA and protein expression. Serum from CHB patients increases cell growth and proliferation and enhances HRP-2 and NUPR1 expression in HCC cells via the IGF-II/IGF-IR/MEK/ERK signaling pathway. These findings help to explain the molecular mechanisms

  7. Effects of Antioxidants and Vitamins on the Proliferation of Human Diploid Cells

    Directory of Open Access Journals (Sweden)

    Gaziza Dаnlybaeva

    2014-01-01

    Full Text Available Introduction: Microelements, essential nutrients that are needed in small amounts including minerals such as calcium, zinc, iron and other vitamins (A, B, C, and etc., are macronutrients necessary for a healthy life. The role of micronutrients in vivo is well known, and there are several publications that have examined the effects of micronutrients on genomic stability. Furthermore, a number of vitamins and microelements are substrates and/or cofactors in metabolic pathways, which regulate DNA synthesis and/or repair and gene expression. A deficiency in such nutrients may result in disruption of genomic integrity and alterations in DNA methylation patterns, linking cellular nutrition with change in gene expression. For example, lack of vitamin C is known to cause increased DNA oxidation and chromosomal damage. Vitamin A, as well as other micronutrients, have a protective effect, whereas higher concentrations are associated with increased DNA damage. Ubiquinone (coenzyme Q10 and dihydroquercetin are used in therapy as antioxidant compounds and electron carriers, which reduce lipid peroxidation of cell membranes. However, previous studies indicate that various ubiquinone analogs may cause a divergent effect on oxidative stress and oxidative phosphorylation. The aim of our study was to investigate the effect of vitamins A and C, coenzyme Q10, and dihydroquercetin on the proliferative potential of cultured human embryonic diploid fibroblasts (M-22. Methods: In the first series of experiments, nontoxic concentrations of vitamins for the cells were identified using MTT assay. Results: Vitamins A and C, dihydroquercetin of 1µM, and coenzyme Q10 of 5µM were nontoxic for human skin fibroblasts. In the second series of experiments, cell cultivation was carried out with nontoxic concentrations. A vitamin C concentration of 1µM for 7 consecutive passages increased the proliferation index (PI compared to the control. Thus, the average PI in the

  8. microRNA-10b Is Overexpressed and Critical for Cell Survival and Proliferation in Medulloblastoma.

    Directory of Open Access Journals (Sweden)

    Rekha Pal

    Full Text Available This study demonstrates the effects of miRNA-10b on medulloblastoma proliferation through transcriptional induction of the anti-apoptotic protein BCL2. Using a cancer specific miRNA-array, high expression of miRNA-10b in medulloblastoma cell lines compared to a normal cerebellar control was shown, and this was confirmed with real time PCR (RT-PCR. Two medulloblastoma cell lines (DAOY and UW228 were transiently transfected with control miRNA, miRNA-10b inhibitor or miRNA-10b mimic and subjected to RT-PCR, MTT, apoptosis, clonogenic assay and western blot analysis. Transfection of miRNA-10b inhibitor induced a significant down-regulation of miRNA-10b expression, inhibited proliferation, and induced apoptosis, while miRNA-10b mimic exerted an opposite effect. Inhibition of miRNA-10b abrogated the colony-forming capability of medulloblastoma cells, and markedly down-regulated the expression of BCL2. Down-regulation of BCL2 by antisense oligonucleotides or siRNA also significantly down-regulated miRNA-10b, suggesting that BCL2 is a major mediator of the effects of miRNA-10b. ABT-737 and ABT-199, potent inhibitors of BCL2, downregulated the expression of miRNA-10b and increased apoptosis. Analysis of miRNA-10b levels in 13 primary medulloblastoma samples revealed that the 2 patients with the highest levels of miRNA-10b had multiple recurrences (4.5 and died within 8 years of diagnosis, compared with the 11 patients with low levels of miRNA-10b who had a mean of 1.2 recurrences and nearly 40% long-term survival. The data presented here indicate that miRNA-10b may act as an oncomir in medulloblastoma tumorigenesis, and reveal a previously unreported mechanism with Bcl-2 as a mediator of the effects of miRNA-10b upon medulloblastoma cell survival.

  9. Cell Proliferation in Neuroblastoma

    Science.gov (United States)

    Stafman, Laura L.; Beierle, Elizabeth A.

    2016-01-01

    Neuroblastoma, the most common extracranial solid tumor of childhood, continues to carry a dismal prognosis for children diagnosed with advanced stage or relapsed disease. This review focuses upon factors responsible for cell proliferation in neuroblastoma including transcription factors, kinases, and regulators of the cell cycle. Novel therapeutic strategies directed toward these targets in neuroblastoma are discussed. PMID:26771642

  10. Controlling nuclear proliferation

    International Nuclear Information System (INIS)

    Sweet, W.

    1981-01-01

    Nuclear non-proliferation policy depends on the 1968 Non-Proliferation Treaty, in which countries promise not to acquire nuclear weapons in exchange for open access to peaceful nuclear technology, and a system of international safeguards that are imposed on exported nuclear equipment and facilities operated by parties to the treaty. Critics have feared all along that non-nuclear countries might circumvent or exploit the system to obtain nuclear weapons and that the Atoms for Peace plan would spread the very technology it sought to control. The nuclear weapons states would like everyone else to believe that atomic bombs are undesirable, but they continue to rely on the bombs for their own defense. Israel's raid on Iraq's nuclear reactor focused world attention on the proliferation problem and helped to broaden and sterengthen its prospects. It also highlighted the weakness that there are no effective sanctions against violators. Until the international community can ageee on enforcement measures powerful enough to prevent nuclear proliferation, individual countries may be tempted to follow Israel's example, 19 references

  11. Interference with PSMB4 Expression Exerts an Anti-Tumor Effect by Decreasing the Invasion and Proliferation of Human Glioblastoma Cells

    Directory of Open Access Journals (Sweden)

    Yu-Chen Cheng

    2018-01-01

    Full Text Available Background/Aims: Glioblastoma (GBM is a malignant brain tumor with a poor prognosis. Proteasome subunit beta type-4 (PSMB4 is an essential subunit that contributes to the assembly of the 20S proteasome complex. However, the role of PSMB4 in glioblastomas remains to be clarified. The aim of this study was to investigate the role of PSMB4 in GBM tumor progression. Methods: We first analyzed the PSMB4 protein and mRNA expression in 80 clinical brain specimens and 77 datasets from the National Center for Biotechnology Information (NCBI Gene Expression Omnibus (GEO database. Next, we inhibited the PSMB4 expression by siRNA in cellular and animal models to explore PSMB4’s underlying mechanisms. The cell survival after siPSMB4 transfection was assayed by MTT assay. Annexin V and propidium iodide staining was used to monitor the apoptosis by flow cytometric analysis. Moreover, the migration and invasion were evaluated by wound healing and Transwell assays. The expression of migration-related and invasion-related proteins after PSMB4 inhibition was detected by Western blotting. In addition, an orthotropic xenograft mouse model was used to assay the effect of PSMB4 knockdown in the in vivo study. Results: Basis on the results of bioinformatics study, glioma patients with higher PSMB4 expression had a shorter survival time than those with lower PSMB4 expression. The staining of clinical brain tissues showed elevated PSMB4 expression in GBM tissues compared with normal brain tissues. The PSMB4 inhibition decreased proliferation, migration and invasion abilities in human GBM cells. Downregulated PSMB4 resulted in cell cycle arrest and apoptosis in vitro. In an orthotropic xenograft mouse model, the glioma tumors progression was reduced when PSMB4 was down-regulated. The decreased PSMB4 enhanced the anti-tumor effect of temozolomide (TMZ on tumor growth. In addition, the absence of PSMB4 decreased the expression of phosphorylated focal adhesion kinase and

  12. Engineered electrospun poly(caprolactone)/polycaprolactone-g-hydroxyapatite nano-fibrous scaffold promotes human fibroblasts adhesion and proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Keivani, F. [Biology Department, Science and Research Branch, Islamic Azad University, Tehran (Iran, Islamic Republic of); Shokrollahi, P., E-mail: p.shokrolahi@ippi.ac.ir [Department of Biomaterials, Faculty of Science, Iran Polymer and Petrochemical Institute, Tehran (Iran, Islamic Republic of); Zandi, M. [Department of Biomaterials, Faculty of Science, Iran Polymer and Petrochemical Institute, Tehran (Iran, Islamic Republic of); Irani, S. [Biology Department, Science and Research Branch, Islamic Azad University, Tehran (Iran, Islamic Republic of); Shokrolahi, F. [Department of Biomaterials, Faculty of Science, Iran Polymer and Petrochemical Institute, Tehran (Iran, Islamic Republic of); Khorasani, S.C. [Biology Department, Science and Research Branch, Islamic Azad University, Tehran (Iran, Islamic Republic of)

    2016-11-01

    Polycaprolactone (PCL)/hydroxyapatite nano-composites are among the best candidates for tissue engineering. However, interactions between nHAp and PCL are difficult to control leading to inhomogeneous dispersion of the bio-ceramic particles. Grafting of polymer chains at high density/chain length while promotes the phase compatibility may result in reduced HAp exposed surface area and therefore, bioactivity is compromised. This issue is addressed here by grafting PCL chains onto HAp nano-particles through ring opening polymerization of ε-caprolactone (PCL-g-HAp). FTIR and TGA analysis showed that PCL (6.9 wt%), was successfully grafted on the HAp. PCL/PCL-g-HAp nano-fibrous scaffold showed up to 10 and 33% enhancement in tensile strength and modulus, respectively, compared to those of PCL/HAp. The effects of HAp on the in vitro HAp formation were investigated for both the PCL/HAp and PCL/PCL-g-HAp scaffolds. Precipitation of HAp on the nano-composite scaffolds observed after 15 days incubation in simulated body fluid (SBF), as confirmed by scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDX). Human fibroblasts were seeded on PCL, PCL/HAp and PCL/PCL-g-HAp scaffolds. According to MTT assay, the highest cell proliferation was recorded for PCL/PCL-g-HAp nano-composite, at all time intervals (1–21 days, P < 0.001). Fluorescent microscopy (of DAPI stained samples) and electron microscopy images showed that all nano-fibrous scaffolds (PCL, PCL/HAp, and PCL/PCL-g-HAp), were non-toxic against cells, while more cell adhesion, and the most uniform cell distribution observed on the PCL/PCL-g-HAp. Overall, grafting of relatively short chains of PCL on the surface of HAp nano-particles stimulates fibroblasts adhesion and proliferation on the PCL/PCL-g-HAp nano-composite. - Highlights: • PCL chains were grafted on HAp nano-particles at relatively low density, through ROP of ε-caprolactone (PCL-g-HAp) • PCL-g-HAp featured a relatively high

  13. Hypoxic stress up-regulates Kir2.1 expression and facilitates cell proliferation in brain capillary endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Yamamura, Hideto; Suzuki, Yoshiaki; Yamamura, Hisao [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan); Asai, Kiyofumi [Department of Molecular Neurobiology, Graduate School of Medical Sciences, Nagoya City University, Nagoya (Japan); Imaizumi, Yuji, E-mail: yimaizum@phar.nagoya-cu.ac.jp [Department of Molecular & Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya (Japan)

    2016-08-05

    The blood-brain barrier (BBB) is mainly composed of brain capillary endothelial cells (BCECs), astrocytes and pericytes. Brain ischemia causes hypoxic encephalopathy and damages BBB. However, it remains still unclear how hypoxia affects BCECs. In the present study, t-BBEC117 cells, an immortalized bovine brain endothelial cell line, were cultured under hypoxic conditions at 4–5% oxygen for 72 h. This hypoxic stress caused hyperpolarization of resting membrane potential. Patch-clamp recordings revealed a marked increase in Ba{sup 2+}-sensitive inward rectifier K{sup +} current in t-BBEC117 cells after hypoxic culture. Western blot and real-time PCR analyses showed that Kir2.1 expression was significantly up-regulated at protein level but not at mRNA level after the hypoxic culture. Ca{sup 2+} imaging study revealed that the hypoxic stress enhanced store-operated Ca{sup 2+} (SOC) entry, which was significantly reduced in the presence of 100 μM Ba{sup 2+}. On the other hand, the expression of SOC channels such as Orai1, Orai2, and transient receptor potential channels was not affected by hypoxic stress. MTT assay showed that the hypoxic stress significantly enhanced t-BBEC117 cell proliferation, which was inhibited by approximately 60% in the presence of 100 μM Ba{sup 2+}. We first show here that moderate cellular stress by cultivation under hypoxic conditions hyperpolarizes membrane potential via the up-regulation of functional Kir2.1 expression and presumably enhances Ca{sup 2+} entry, resulting in the facilitation of BCEC proliferation. These findings suggest potential roles of Kir2.1 expression in functional changes of BCECs in BBB following ischemia. -- Highlights: •Hypoxic culture of brain endothelial cells (BEC) caused membrane hyperpolarization. •This hyperpolarization was due to the increased expression of Kir2.1 channels. •Hypoxia enhanced store-operated Ca{sup 2+} (SOC) entry via Kir2.1 up-regulation. •Expression levels of putative SOC

  14. Hypoxic stress up-regulates Kir2.1 expression and facilitates cell proliferation in brain capillary endothelial cells

    International Nuclear Information System (INIS)

    Yamamura, Hideto; Suzuki, Yoshiaki; Yamamura, Hisao; Asai, Kiyofumi; Imaizumi, Yuji

    2016-01-01

    The blood-brain barrier (BBB) is mainly composed of brain capillary endothelial cells (BCECs), astrocytes and pericytes. Brain ischemia causes hypoxic encephalopathy and damages BBB. However, it remains still unclear how hypoxia affects BCECs. In the present study, t-BBEC117 cells, an immortalized bovine brain endothelial cell line, were cultured under hypoxic conditions at 4–5% oxygen for 72 h. This hypoxic stress caused hyperpolarization of resting membrane potential. Patch-clamp recordings revealed a marked increase in Ba 2+ -sensitive inward rectifier K + current in t-BBEC117 cells after hypoxic culture. Western blot and real-time PCR analyses showed that Kir2.1 expression was significantly up-regulated at protein level but not at mRNA level after the hypoxic culture. Ca 2+ imaging study revealed that the hypoxic stress enhanced store-operated Ca 2+ (SOC) entry, which was significantly reduced in the presence of 100 μM Ba 2+ . On the other hand, the expression of SOC channels such as Orai1, Orai2, and transient receptor potential channels was not affected by hypoxic stress. MTT assay showed that the hypoxic stress significantly enhanced t-BBEC117 cell proliferation, which was inhibited by approximately 60% in the presence of 100 μM Ba 2+ . We first show here that moderate cellular stress by cultivation under hypoxic conditions hyperpolarizes membrane potential via the up-regulation of functional Kir2.1 expression and presumably enhances Ca 2+ entry, resulting in the facilitation of BCEC proliferation. These findings suggest potential roles of Kir2.1 expression in functional changes of BCECs in BBB following ischemia. -- Highlights: •Hypoxic culture of brain endothelial cells (BEC) caused membrane hyperpolarization. •This hyperpolarization was due to the increased expression of Kir2.1 channels. •Hypoxia enhanced store-operated Ca 2+ (SOC) entry via Kir2.1 up-regulation. •Expression levels of putative SOC channels were not affected by hypoxia.

  15. DNA binding, antioxidant, cytotoxicity (MTT, lactate dehydrogenase, NO), and cellular uptake studies of structurally different nickel(II) thiosemicarbazone complexes: synthesis, spectroscopy, electrochemistry, and X-ray crystallography.

    Science.gov (United States)

    Prabhakaran, R; Kalaivani, P; Huang, R; Poornima, P; Vijaya Padma, V; Dallemer, F; Natarajan, K

    2013-02-01

    Three new nickel(II) thiosemicarbazone complexes have been synthesized and characterized by analytical, spectral, and single-crystal X-ray diffraction studies. In complex 1, the ligand 2-hydroxy-1-naphthaldehydethiosemicarbazone coordinated as a monobasic tridentate donor, whereas in complexes 2 and 3, the ligands salicylaldehyde-4(N)-ethylthiosemicarbazone and 2-hydroxy-1-naphthaldehyde-4(N)-ethylthiosemicarbazone coordinated as a dibasic tridentate donor. The DNA binding ability of the complexes in calf thymus DNA was explored by absorption and emission titration experiments. The antioxidant property of the new complexes was evaluated to test their free-radical scavenging ability. In vitro cytotoxicity assays were performed for the new complexes in A549 and HepG2 cell lines. The new compounds overcome cisplatin resistance in the A549 cell line and they were also active in the HepG2 cell line. The cellular uptake study showed the accumulation of the complexes in tumor cells depended on the nature of the ligand attached to the nickel ion.

  16. Nuclear non-proliferation

    International Nuclear Information System (INIS)

    Anon.

    1990-01-01

    This patent describes the treaty on the non-proliferation of nuclear weapons which is the corner-stone of an international non-proliferation regime which has grown to embrace the overwhelming majority of countries in the world in the period since the Treaty. The other elements of the regime include, first of all, the safeguards system of IAEA-which operates to prevent the diversion of nuclear materials to military or other prohibited activities and must be accepted by all non-nuclear-weapon parties to the Treaty and, secondly, the Antarctic Treaty, the Treaty for the Prohibition of Nuclear Weapons in Latin America (Treaty of Tlatelolco) and the south Pacific Nuclear Free zone Treaty (Treaty of Rarotonga)-which serve to extend the regime geographically. The last two Treaties require safeguards agreements with IAEA. In addition, the Treaty of Tlatelolco contains provisions establishing the agency for the Prohibition of Nuclear Weapons in Latin America and the Caribbean to ensure compliance

  17. Proliferation in cycle

    Energy Technology Data Exchange (ETDEWEB)

    Piao Yunsong [College of Physical Sciences, Graduate School of Chinese Academy of Sciences, Beijing 100049 (China)], E-mail: yspiao@gucas.ac.cn

    2009-06-15

    In the contracting phase with w{approx_equal}0, the scale invariant spectrum of curvature perturbation is given by the increasing mode of metric perturbation. In this Letter, it is found that if the contracting phase with w{approx_equal}0 is included in each cycle of a cycle universe, since the metric perturbation is amplified on super horizon scale cycle by cycle, after each cycle the universe will be inevitably separated into many parts independent of one another, each of which corresponds to a new universe and evolves up to next cycle, and then is separated again. In this sense, a cyclic multiverse scenario is actually presented, in which the universe proliferates cycle by cycle. We estimate the number of new universes proliferated in each cycle, and discuss the implications of this result.

  18. Proliferation in cycle

    International Nuclear Information System (INIS)

    Piao Yunsong

    2009-01-01

    In the contracting phase with w≅0, the scale invariant spectrum of curvature perturbation is given by the increasing mode of metric perturbation. In this Letter, it is found that if the contracting phase with w≅0 is included in each cycle of a cycle universe, since the metric perturbation is amplified on super horizon scale cycle by cycle, after each cycle the universe will be inevitably separated into many parts independent of one another, each of which corresponds to a new universe and evolves up to next cycle, and then is separated again. In this sense, a cyclic multiverse scenario is actually presented, in which the universe proliferates cycle by cycle. We estimate the number of new universes proliferated in each cycle, and discuss the implications of this result.

  19. LncRNA UCA1 promotes proliferation and cisplatin resistance of oral squamous cell carcinoma by sunppressing miR-184 expression.

    Science.gov (United States)

    Fang, Zheng; Zhao, Junfang; Xie, Weihong; Sun, Qiang; Wang, Haibin; Qiao, Bin

    2017-12-01

    Chemotherapy resistance has become the main obstacle for the effective treatment of human cancers. Long non-coding RNA urothelial cancer associated 1 (UCA1) is generally regarded as an oncogene in some cancers. However, the function and molecular mechanism of UCA1 implicated in cisplatin (CDDP) chemoresistance of oral squamous cell carcinoma (OSCC) is still not fully established. UCA1 expression in tumor tissues and cells was tested by qRT-PCR. MTT, flow cytometry and caspase-3 activity analysis were explored to evaluate the CDDP sensitivity in OSCC cells. Western blot analysis was used to measure BCL2, Bax and SF1 protein expression. Luciferase reporter assay was conducted to investigate the molecular relationship between UCA1, miR-184, and SF1. Nude mice model was used to confirm the functional role of UCA1 in CDDP resistance in vivo. UCA1 expression was upregulated in OSCC tissues, cell lines, and CDDP resistant OSCC cells. Function analysis revealed that UCA1 facilitated proliferation, enhanced CDDP chemoresistance, and suppressed apoptosis in OSCC cells. Mechanisms investigation indicated that UCA1 could interact with miR-184 to repress its expression. Rescue experiments suggested that downregulation of miR-184 partly reversed the tumor suppression effect and CDDP chemosensitivity of UCA1 knockdown in CDDP-resistant OSCC cells. Moreover, UCA1 could perform as a miR-184 sponge to modulate SF1 expression. The OSCC nude mice model experiments demonstrated that depletion of UCA1 further boosted CDDP-mediated repression effect on tumor growth. UCA1 accelerated proliferation, increased CDDP chemoresistance and restrained apoptosis partly through modulating SF1 via sponging miR-184 in OSCC cells, suggesting that targeting UCA1 may be a potential therapeutic strategy for OSCC patients. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  20. Deficiency of Functional Iron-Sulfur Domains in ABCE1 Inhibits the Proliferation and Migration of Lung Adenocarcinomas By Regulating the Biogenesis of Beta-Actin In Vitro

    Directory of Open Access Journals (Sweden)

    Qian Yu

    2017-11-01

    Full Text Available Background/Aims: ATP-binding cassette transporter E1 (ABCE1, a unique ABC superfamily member that bears two Fe-S clusters, is essential for metastatic progression in lung cancer. Fe-S clusters within ABCE1 are crucial for ribosome dissociation and translation reinitiation; however, whether these clusters promote tumor proliferation and migration is unclear. Methods: The interaction between ABCE1 and β-actin was confirmed using GST pull-down. The lung adenocarcinoma (LUAD cell line A549 was transduced with lentiviral packaging vectors overexpressing either wild-type ABCE1 or ABCE1 with Fe-S cluster deletions (ΔABCE1. The role of Fe-S clusters in the viability and migration of cancer cells was evaluated using clonogenic, MTT, Transwell and wound healing assays. Cytoskeletal rearrangement was determined using immunofluorescent techniques. Results: Fe-S clusters were the key domains in ABCE1 involved in binding to β-actin. The proliferative and migratory capacity increased in cells overexpressing ABCE1. However, the absence of Fe-S clusters reversed these effects. A549 cells overexpressing ABCE1 exhibited irregular morphology and increased levels of cytoskeletal polymerization as indicated by the immunofluorescence images. In contrast, cells expressing the Fe-S cluster deletion mutant presented opposing effects. Conclusion: These results demonstrate the indispensable role of Fe-S clusters when ABCE1 participates in the proliferation and migration of LUADs by interacting with β-actin. The Fe-S clusters of ABCE1 may be potential targets for the prevention of lung cancer metastasis.

  1. microRNA-494 is a potential prognostic marker and inhibits cellular proliferation, migration and invasion by targeting SIRT1 in epithelial ovarian cancer.

    Science.gov (United States)

    Yang, Aijun; Wang, Xuenan; Yu, Chunna; Jin, Zhenzhen; Wei, Lingxia; Cao, Jinghe; Wang, Qin; Zhang, Min; Zhang, Lin; Zhang, Lei; Hao, Cuifang

    2017-09-01

    Ovarian cancer is one of the most common types of gynecological malignancy worldwide, and is the fourth leading cause of cancer-associated mortality among women. Despite improvements in therapeutic treatments, the prognosis for epithelial ovarian cancer (EOC) remains poor, mainly due to the rapid growth and metastasis of ovarian cancer tumors. An increasing number of studies have indicated that microRNAs (miRNAs) are involved in the carcinogenesis and progression of human cancer, suggesting that miRNAs may be used in clinical prognosis and as a therapeutic target in EOC. The aim of the present study was to investigate the expression levels of miRNA-494 in EOC tissues and cell lines. The clinical significance of miRNA-494 in patients with EOC was also evaluated. The results demonstrated that miRNA-494 was significantly downregulated in EOC tissues and cell lines. Low expression levels of miRNA-494 were associated with poor prognostic features, including International Federation of Gynecology and Obstetrics stage, tumor size and lymph node metastasis. In vitro functional studies demonstrated that overexpression of miRNA-494 inhibited proliferation, migration and invasion in EOC cells. By contrast, knockdown of miRNA-494 enhanced cell growth, migration and invasion in EOC cells. Notably, sirtuin 1 (SIRT1) was identified as a direct target of miRNA-494 in EOC. Furthermore, MTT, cell migration and invasion assays verified that EOC cell proliferation, migration and invasion were completely restored with forced miRNA-494 expression and SIRT1 restoration. Together, these findings suggest that miRNA-494 is a potential prognostic marker, and may provide novel therapeutic regimens of targeted therapy for EOC.

  2. MicroRNA-2400 promotes bovine preadipocyte proliferation

    International Nuclear Information System (INIS)

    Wei, Yao; Cui, Ya Feng; Tong, Hui Li; Zhang, Wei Wei; Yan, Yun Qin

    2016-01-01

    MicroRNAs (miRNAs) play critical roles in the proliferation of bovine preadipocytes. miR-2400 is a novel and unique miRNA from bovines. In the present study, we separated and identified preadipocytes from bovine samples. miR-2400 overexpression increased the rate of preadipocyte proliferation, which was analyzed with a combination of EdU and flow cytometry. Simultaneously, functional genes related to proliferation (PCNA, CCND2, CCNB1) were also increased, which was detected by real-time PCR. Furthermore, luciferase reporter assays showed that miR-2400 bound directly to the 3'untranslated regions (3′UTRs) of PRDM11 mRNA. These data suggested that miR-2400 could promote preadipocyte proliferation by targeting PRDM11. - Highlights: • miRNAs are important in bovine preadipocyte proliferation. • miR-2400 is a novel miRNA from bovines. • miR-2400 overexpression increased preadipocyte proliferation. • Functional genes related to preadipocyte proliferation were upregulated. • Preadipocyte proliferation was promoted by targeting PRDM11.

  3. Global proliferation concerns

    International Nuclear Information System (INIS)

    Simpkins, R.

    1978-01-01

    The Non-Proliferation Treaty and the IAGA Safeguards System are discussed. President Carter's program to defer commercial reprocessing and recycle, to restructure the breeder program, to develop alternative fuel cycles, to increase US uranium enrichment capability, to provide fuel assurance for consumer nations, to continue the embargo of sensitive technology and equipment and to develop the International Nuclear Fuel Cycle Evaluation Program is outlined

  4. Intermittent pressure decreases human keratinocyte proliferation in vitro.

    Science.gov (United States)

    Nasca, Maria R; Shih, Alan T; West, Dennis P; Martinez, Wanda M; Micali, Giuseppe; Landsman, Adam S

    2007-01-01

    The aim of this study was to investigate the correlation between pressure changes and keratinocyte proliferation by determining whether keratinocytes exposed to altered mechanical pressures would proliferate at different rates compared to control cells not subjected to pressure changes. Tissue culture flasks of human keratinocytes plated at an approximate density of 15,000 cells/cm(2) undergoing an intermittent cyclic pressure of 362 mm Hg at a frequency of 2.28 or 5.16 cycles/min (0.038 or 0.086 Hz) for 8 h were compared to control flasks grown at ambient room pressure. An in-line pressure transducer was used to monitor and adjust pressure within the cell chambers, using a solenoid valve. A thymidine incorporation assay assessed the amount of cell proliferation in each set of experiments. Differences in proliferation between keratinocytes subjected to cyclic pressure changes and control cells were found to be statistically significant (p < 0.05) in 4 out of 5 proliferation assays. Also, a higher frequency of pressure changes consistently generated a reduced proliferation rate compared to that seen in cells exposed to a lower frequency of pressure changes. These data indicate that keratinocytes undergoing intermittent pressure changes exhibit decreased proliferation rates compared to controls. Furthermore, an increased frequency rate seems to have a greater effect on proliferation than low-frequency rate pressure changes, suggesting that the stress caused by frequently changed pressure may play a greater role in reducing keratinocyte proliferation than the actual magnitude of load applied to the cells. Our results support the current treatment protocol of reducing speed and duration of walking on the site of the wound to promote healing of foot ulcers. (c) 2007 S. Karger AG, Basel.

  5. Chloroquinone Inhibits Cell Proliferation and Induces Apoptosis in ...

    African Journals Online (AJOL)

    Purpose: To demonstrate the role of chloroquinone (CQ) in inducing apoptosis in HONE-1 and HNE-1, nasopharyngeal carcinoma (NPC) cell lines. Methods: Water-soluble tetrazolium salt (WST)-1 assay was used for the determination of cell proliferation while an inverted microscope was employed for the analysis of ...

  6. Bryostatin I inhibits growth and proliferation of pancreatic cancer ...

    African Journals Online (AJOL)

    Purpose: To evaluate the effect of bryostatin I on proliferation of pancreatic cancer cells as well as tumor growth in mice tumor xenograft model. Methods: Activation of NF-κB was evaluated by preparing nuclear material extract using nuclear extract kit (Carlsbad, CA, USA) followed by enzyme-linked immunosorbent assay ...

  7. Proliferation after the Iraq war

    International Nuclear Information System (INIS)

    Daguzan, J.F.

    2004-09-01

    This article uses the Iraq war major event to analyze the approach used by the US to fight against proliferation. It questions the decision and analysis process which has led to the US-British intervention and analyzes the consequences of the war on the proliferation of other countries and on the expected perspectives. Finally, the future of proliferation itself is questioned: do we have to fear more threat or is the virtuous circle of non-proliferation well started? (J.S.)

  8. [Overexpression of N-myc downstream regulated gene 2 (NDRG2) inhibits proliferation, migration and promotes apoptosis in SW480 rectal cancer cells].

    Science.gov (United States)

    Li, Zhiqiang; Sun, Yang; Wan, Hongxing; Chai, Fang

    2017-01-01

    Objective To investigate the role of N-myc downstream regulated gene 2 (NDRG2) gene in the proliferation, migration and apoptosis of rectal cancer cells. Methods Human rectal cancer SW480 cells were cultured and transfected with pCDNA3.1-NDRG2 and empty vector (SW480-Ve). SW480 cells were set as a control group. Cell proliferation was detected in SW480 cells, SW480-Ve cells and SW480-NDRG2 cells by MTT assay; cell migration distance in the three groups at 24, 48, 72 hours was tested by wound healing assay; apoptosis rate was determined in the three groups at 48 hours by flow cytometry; the expressions of Bax, caspase-3, Bcl-2 proteins in the three groups were examined by Western blotting. Results After the cells were cultured for 7 days, cell survival rate in SW480-NDRG2 group was significantly lower than that in SW480 cells and SW480-Ve cells; the cell survival rate decreased gradually with the prolongation of the culture time; and it had no significant difference between SW480-Ve group and SW480 group. Cell migration distance in SW480-NDRG2 group was significantly lower than that in SW480-Ve cells and SW480 cells, and it had also no significant difference between SW480-Ve cells and SW480 cells. The apoptosis rate in SW480-NDRG2 group was significantly higher than that in SW480 group and SW480-Ve group, and SW480 cells and SW480-Ve cells had no significant difference in the rate. The expressions of Bax and caspase-3 proteins in SW480-NDRG2 group were significantly higher than those in SW480 cells and SW480-Ve cells; Bcl-2 protein expression was significantly lower in SW480-NDRG2 group than in SW480 cells and SW480-Ve cells; and the expressions of Bax, caspase-3 and Bcl-2 proteins were not significantly different between SW480 cells and SW480-Ve cells. Conclusion Overexpression of NDRG2 can inhibit the proliferation, reduce cell migration, and promote cell apoptosis by regulating the expressions of Bcl-2, Bax and caspase-3 proteins in SW480 cells.

  9. Can we predict nuclear proliferation

    International Nuclear Information System (INIS)

    Tertrais, Bruno

    2011-01-01

    The author aims at improving nuclear proliferation prediction capacities, i.e. the capacities to identify countries susceptible to acquire nuclear weapons, to interpret sensitive activities, and to assess nuclear program modalities. He first proposes a retrospective assessment of counter-proliferation actions since 1945. Then, based on academic studies, he analyzes what causes and motivates proliferation, with notably the possibility of existence of a chain phenomenon (mechanisms driving from one program to another). He makes recommendations for a global approach to proliferation prediction, and proposes proliferation indices and indicators

  10. MicroRNA-101 inhibits cell proliferation, promotes cell apoptosis and increases sensitivity of breast cancer MDA-MB-231 cells to paclitaxel

    Directory of Open Access Journals (Sweden)

    Qiu-Lin Ke

    2016-02-01

    Full Text Available Objective: To explore the effect that miR-101 inhibits breast cancer MDA-MB-231 cell proliferation and increases the chemosensitivity of paclitaxel to breast cancer MDA-MB-231 cells and its influence on protein expression level of target gene Bcl2. Methods: miR-101 was artificially synthesized, it used liposome 3000 to transfect MDA-MB-231 cells, and experiment was divided into three groups: blank control group, negative control group and miR-101 group. MTT assay was used to detect the effect of miR-101 on MDA-MB-231 cell proliferation and chemosensitivity of paclitaxel-mediated MDA-MB-231 cells; flow cytometer was used to detect cell apoptosis. Real-time PCR and Western bloting were used to detect the changes of mRNA and protein expression levels of Bcl2. Results: After miR-101 transfected MDA-MB- 231 cells, cell proliferation ability significantly decreased compared with negative control group, and differences had statistical significance (P<0.01; after paclitaxel was used to process cells, IC50 of miR-101-processing group decreased by 2.45 times compared with blank control group, differences had statistical significance (P<0.05 and differences between blank control group and negative control group had no statistical significance; detection results by flow cytometer showed that both early-stage and late-stage apoptosis rates of MDA-MB-231 cells of miR-101 group were significantly higher than those of negative control group (P<0.05, and early-stage apoptosis rate was more significant (P<0.01; after transfection of miR-101, mRNA and protein levels of Bcl2 of MDA-MB-231 cells significantly decreased, and differences had statistical significance (P<0.05. Conclusion: miR-101 can inhibit breast cancer MDAMB- 231 cell proliferation through targeting and downregulating Bcl2, thereby increasing the chemosensitivity of breast cancer cells to paclitaxel and promoting cell apoptosis.

  11. miR-139 is up-regulated in osteoarthritis and inhibits chondrocyte proliferation and migration possibly via suppressing EIF4G2 and IGF1R

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Weihua; Zhang, Weikai; Li, Feng; Guo, Fengjing; Chen, Anmin, E-mail: chenanmin6072@126.com

    2016-05-27

    Osteoarthritis (OA) is one of the most progressive articular cartilage erosions. microRNAs (miRNAs) play pivotal roles in OA modulation, but the role of miR-139 in OA remains elusive. This study aims to reveal the effects and possible mechanism of miR-139 in OA and chondrocytes. The levels of miR-139 and its possible targets eukaryotic translation initiation factor 4 gamma 2 (EIF4G2) and insulin-like growth factor 1 receptor (IGF1R) were detected by qRT-PCR in the articular cartilages of 20 OA patients and 20 non-OA patients. Human chondrocyte CHON-001 cells were transfected with miR-139 mimic or inhibitor, as well as the siRNAs of EIF4G2 and IGF1R. Cell viability by MTT assay, proliferation by colony formation assay and migration by Transwell assay were performed. Results showed that miR-139 was up-regulated, while EIF4G2 and IGF1R mRNAs down-regulated in OA cartilages (P < 0.001), and negative correlations existed between the level of miR-139 and EIF4G2 or IGF1R. Overexpression of miR-139 in CHON-001 cells suppressed both mRNA and protein levels of EIF4G2 and IGF1R, and inhibited cell viability, colony formation number and cell migration, while miR-139 inhibitor induced the opposite effects. Knockdown of EIF4G2 or IGF1R in CHON-001 cells reversed the effects of miR-139 inhibitor on cell viability, colony formation and cell migration. These results indicate that miR-139 is capable of inhibiting chondrocyte proliferation and migration, thus being a possible therapeutic target for OA. The mechanism of miR-139 in chondrocytes may be related to its regulation on EIF4G2 and IGF1R.

  12. Inhibition of Zoledronic Acid on Cell Proliferation and Invasion of Lung Cancer Cell Line 95D

    Directory of Open Access Journals (Sweden)

    Mingming LI

    2009-03-01

    Full Text Available Background and objective Abnormal proliferation and metastasis is the basic characteristic of malignant tumors. The aim of this work is to explore the effects of zoledronic acid on cell proliferation and invasion in lung cancer cell line 95D. Methods The effect of zoledrnic acid (ZOL on proliferation of lung cancer cell line 95D was detected by MTT. The expression of proliferation and invasion-relation genes and proteins were detected by Western blot, RT-PCR and immunofluorescence. Changes of invasion of lung cancer cell numbers were measured by polycarbonates coated with Matrigel. Results ZOL could inhibit the proliferation of lung cancer cell line 95D in vitro in a time-dependant and a dose-dependant manner. With time extending after ZOL treated, the mRNA expresion of VEGF, MMP9, MMP2 and protein expression of VEGF, MMP9, ERK1/ ERK2 were decreased. The results of Tanswell invasion showed the numbers of invasive cells were significantly reduced in 95D cells treated with ZOL 4 d and 6 d later. Conclusion ZOL could inhibit cell proliferation and invasion of lung cancer cell line 95D.

  13. Non-proliferation

    International Nuclear Information System (INIS)

    Deiseroth, D.; Gustafsson, S.

    1993-01-01

    The issue of Nuclear Non Proliferation has been moved to a leading place on the contemporary international security agenda. What about the situation of nuclear weapons and nuclear technology in Russia, Kazakhstan, Ukraine and Belorussia? Why did the IAEA-inspectors totally failed to discover any sign of Iraq's clandestine nuclear-weapon programme before the Gulf War? Do the NATO and their nuclear power states violate Art. VI of the Non-Proliferation-Treaty (NPT), because they are - despite the end of the cold war - not willing to renounce of the ''option of the first use of nuclear weapons''? Does the NPT establish a form of nuclear apartheid? What will be the situation if the NPT-Extension-Conference in 1995 will be unable to obtain a majority of the parties for any one extension proposal? Do we need a new international nuclear control agency with severe powers, a sort of nuclear Interpol? The Colloquium ''Saving NPT and abolishing Nuclear Weapons'', held in Stockholm in September 1992, organized by the Swedish and the German Sections of IALANA, tried to analyse some of the raised issues. (orig.) [de

  14. Non Proliferation of Nuclear

    International Nuclear Information System (INIS)

    Bambang S Irawan

    2004-01-01

    Non-Proliferation Treaty of Nuclear Weapons is the international community's efforts to maintain the security of the world, in order to prevent the spread of nuclear technology and the use of nuclear weapons, promoting cooperation for the use of nuclear peaceful purposes, build mutual trust (Confidence Building Measures) as well as to achieve the ultimate goal of disarmament overall (General and Complete Disarmament). Addressing the post-WTC tragedy, 11 September 2001, the Indonesian government should set up a National Measures (National Action Plan), among others formed the National Security Council and NBC Counter Proliferation Unit, or the National Authority for Nuclear Treaty, preparing national legislation, to prevent the abuse nuclear materials for terrorist acts, prevent Illicit Trafficking of Nuclear materials, developed a National Preparedness and Emergency Response Management in the event of a nuclear accident or attack by the use of nuclear terrorism. Importance of a National Action Plan meant the existence of a national commitment in the context of compliance with treaties and conventions which have been ratified relating to safety, security, safeguards towards a general and complete disarmament, to safeguard national security and maintain peace (safeguards) international

  15. Initiatives for proliferation prevention

    International Nuclear Information System (INIS)

    1997-04-01

    Preventing the proliferation of weapons of mass destruction is a central part of US national security policy. A principal instrument of the Department of Energy's (DOE's) program for securing weapons of mass destruction technology and expertise and removing incentives for scientists, engineers and technicians in the newly independent states (NIS) of the former Soviet Union to go to rogue countries or assist terrorist groups is the Initiatives for Proliferation Prevention (IPP). IPP was initiated pursuant to the 1994 Foreign Operations Appropriations Act. IPP is a nonproliferation program with a commercialization strategy. IPP seeks to enhance US national security and to achieve nonproliferation objectives by engaging scientists, engineers and technicians from former NIS weapons institutes; redirecting their activities in cooperatively-developed, commercially viable non-weapons related projects. These projects lead to commercial and economic benefits for both the NIS and the US IPP projects are funded in Russian, Ukraine, Kazakhstan and Belarus. This booklet offers an overview of the IPP program as well as a sampling of some of the projects which are currently underway

  16. Uncertainties in Nuclear Proliferation Modeling

    International Nuclear Information System (INIS)

    Kim, Chul Min; Yim, Man-Sung; Park, Hyeon Seok

    2015-01-01

    There have been various efforts in the research community to understand the determinants of nuclear proliferation and develop quantitative tools to predict nuclear proliferation events. Such systematic approaches have shown the possibility to provide warning for the international community to prevent nuclear proliferation activities. However, there are still large debates for the robustness of the actual effect of determinants and projection results. Some studies have shown that several factors can cause uncertainties in previous quantitative nuclear proliferation modeling works. This paper analyzes the uncertainties in the past approaches and suggests future works in the view of proliferation history, analysis methods, and variable selection. The research community still lacks the knowledge for the source of uncertainty in current models. Fundamental problems in modeling will remain even other advanced modeling method is developed. Before starting to develop fancy model based on the time dependent proliferation determinants' hypothesis, using graph theory, etc., it is important to analyze the uncertainty of current model to solve the fundamental problems of nuclear proliferation modeling. The uncertainty from different proliferation history coding is small. Serious problems are from limited analysis methods and correlation among the variables. Problems in regression analysis and survival analysis cause huge uncertainties when using the same dataset, which decreases the robustness of the result. Inaccurate variables for nuclear proliferation also increase the uncertainty. To overcome these problems, further quantitative research should focus on analyzing the knowledge suggested on the qualitative nuclear proliferation studies

  17. Proliferation: myth or reality?; La proliferation: mythe ou realite?

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2005-07-01

    This article analyzes the proliferation approach, its technical condition and political motivation, and the share between the myth (political deception, assumptions and extrapolations) and the reality of proliferation. Its appreciation is complicated by the irrational behaviour of some political actors and by the significant loss of the non-use taboo. The control of technologies is an important element for proliferation slowing down but an efficient and autonomous intelligence system remains indispensable. (J.S.)

  18. The nuclear proliferation

    International Nuclear Information System (INIS)

    Gere, F.

    1995-04-01

    In this book is detailed the beginning of nuclear military power, with the first bomb of Hiroshima, the different ways of getting uranium 235 and plutonium 239, and how the first countries (Usa, Ussr, China, United kingdom, France) got nuclear weapons. Then the most important part is reviewed with the details of non-proliferation treaty and the creation of IAEA to promote civilian nuclear power in the world and to control the use of plutonium and uranium in nuclear power plants. The cases of countries who reached the atom mastery, such Israel, South Africa, Pakistan, Iraq, North Korea, Argentina, Brazil, Iran, Algeria, Taiwan and the reasons which they wanted nuclear weapon for or why they gave up, are exposed

  19. Microbead agglutination based assays

    KAUST Repository

    Kodzius, Rimantas

    2013-01-21

    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling the macroscopic observation. Such tests are most often used to explore antibody-antigen reactions. Agglutination has been used for protein assays using a biotin/streptavidin system as well as a hybridization based assay. The agglutination systems are prone to selftermination of the linking analyte, prone to active site saturation and loss of agglomeration at high analyte concentrations. We investigated the molecular target/ligand interaction, explaining the common agglutination problems related to analyte self-termination, linkage of the analyte to the same bead instead of different microbeads. We classified the agglutination process into three kinds of assays: a two- component assay, a three-component assay and a stepped three- component assay. Although we compared these three kinds of assays for recognizing DNA and protein molecules, the assay can be used for virtually any molecule, including ions and metabolites. In total, the optimized assay permits detecting analytes with high sensitivity in a short time, 5 min, at room temperature. Such a system is appropriate for POC testing.

  20. SOX15 regulates proliferation and migration of endometrial cancer cells.

    Science.gov (United States)

    Rui, Xiaohui; Xu, Yun; Jiang, Xiping; Guo, Caixia; Jiang, Jingting

    2017-10-31

    The study aimed to investigate the effects of Sry-like high mobility group box 15 ( SOX15 ) on proliferation and migration of endometrial cancer (EC) cells. Immunohistochemistry (IHC) was applied to determine the expression of SOX15 in EC tissues and adjacent tissues. We used cell transfection method to construct the HEC-1-A and Ishikawa cell lines with stable overexpression and low expression SOX15 Reverse-transcription quantitative real-time PCR (RT-qPCR) and Western blot were performed to examine expression of SOX15 mRNA and SOX15 protein, respectively. By conducting a series of cell proliferation assay and migration assay, we analyzed the influence of SOX15 overexpression or low expression on EC cell proliferation and migration. The expression of SOX15 mRNA and protein in EC tissues was significantly lower than that in adjacent tissues. After lentivirus-transfecting SOX15 , the expression level of SOX15 mRNA and protein was significantly increased in cells of SOX15 group, and decreased in sh- SOX15 group. Overexpression of SOX15 could suppress cell proliferation, while down-regulation of SOX15 increased cell proliferation. Flow cytometry results indicated that overexpression of SOX15 induced the ratio of cell-cycle arrest in G 1 stage. In addition, Transwell migration assay results showed that SOX15 overexpression significantly inhibited cell migration, and also down-regulation of SOX15 promoted the migration. As a whole, SOX15 could regulate the proliferation and migration of EC cells and up- regulation of SOX15 could be valuable for EC treatment. © 2017 The Author(s).

  1. Radioreceptor opioid assay

    International Nuclear Information System (INIS)

    Miller, R.J.; Chang, K.-J.

    1981-01-01

    A radioreceptor assay is described for assaying opioid drugs in biological fluids. The method enables the assay of total opioid activity, being specific for opioids as a class but lacking specificity within the class. A radio-iodinated opioid and the liquid test sample are incubated with an opiate receptor material. The percentage inhibition of the binding of the radio-iodinated compound to the opiate receptor is calculated and the opioid activity of the test liquid determined from a standard curve. Examples of preparing radio-iodinated opioids and assaying opioid activity are given. A test kit for the assay is described. Compared to other methods, this assay is cheap, easy and rapid. (U.K.)

  2. Absolute nuclear material assay

    Science.gov (United States)

    Prasad, Manoj K [Pleasanton, CA; Snyderman, Neal J [Berkeley, CA; Rowland, Mark S [Alamo, CA

    2010-07-13

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  3. Nuclear proliferation: linkages and solutions

    International Nuclear Information System (INIS)

    Quester, G.H.

    1979-01-01

    Nuclear proliferation must be periodically re-examined as a moral as well as a practical foreign policy dilemma. The question is asked whether proliferation precludes a safe and peaceful world, or if a halt to proliferation is adequate without other arms control. The moral dilemma in foreign policy arises over the need to make practical choices which often serve one goal while sacrificing another. The ramifications of nuclear proliferation are examined and the conclusions reached that it is not an acceptable option. It is also decided that, because general disarmament steps will be more difficult to achieve, the world may have to accept a small number of nuclear arsenals as the price of state sovereignties. A high priority for making the effort to prevent proliferation is advised. 8 references

  4. Effects of plant lectins on in vitro fibroblast proliferation

    Directory of Open Access Journals (Sweden)

    Ana Maria Sell

    2003-06-01

    Full Text Available Lectins are carbohydrate-binding proteins that have been isolated from various sources and presented a wide spectrum of biological activities. The effects of four lectins, namely, Phaseolus vulgaris phytohemagglutinin, PHA, wheat germ agglutinin, WGA, Artocarpus integrifolia seed lectins, jacalin and artocarpin, on in vitro fibroblasts proliferation were investigated. The lectins did not influence the initial cell adhesion to the plate. PHA and WGA at 10-20 µg/mL concentrations significantly decreased fibroblasts proliferation. At these concentrations, they caused morphological alterations on cells and over 80 µg/mL, promoted cell death. Neither jacalin nor artocarpin significantly affected cell proliferation.O objetivo deste trabalho foi avaliar a influência das lectinas PHA, WGA, jacalina e artocarpina sobre a proliferação de fibroblastos in vitro. Para tanto, fibroblastos gengivais de voluntários saudáveis foram cultivados, por cinco dias, em DMEM suplementado com soro bovino fetal (10% v/v e na presença das lectinas nas concentrações finais de 0.1 a 300 µg/mL. A adesão, o crescimento e a morfologia celular foram acompanhados por microscopia de inversão e contraste de fase. O índice de proliferação foi avaliado pelo método calorimétrico usando MTT. As lectinas não alteraram a adesão inicial dos fibroblastos à placa de poliestireno. PHA e WGA, nas concentrações de 10 a 20 µg/mL, diminuíram significativamente a proliferação celular. Nestas concentrações a morfologia celular é alterada e acima de 80 µg/mL, houve100% de morte celular. As lectinas jacalina e artocarpina não influenciaram a proliferação celular.

  5. [miR-143 inhibits cell proliferation through targeted regulating the expression of K-ras gene in HeLa cells].

    Science.gov (United States)

    Qin, H X; Cui, H K; Pan, Y; Hu, R L; Zhu, L H; Wang, S J

    2016-12-23

    Objective: To explore the effect of microRNA miR-143 on the proliferation of cervical cancer HeLa cells through targeted regulating the expression of K-ras gene. Methods: The luciferase report carrier containing wild type 3'-UTR of K-ras gene (K-ras-wt) or mutated 3'-UTR of the K-ras (K-ras-mut) were co-transfected with iR-143 mimic into the HeLa cells respectively, and the targeting effect of miR-143 in the transfectants was verified by the dual luciferase report system. HeLa cells were also transfected with miR-143 mimic (miR-143 mimic group), mimic control (negative control group), and miR-143 mimic plus K-ras gene (miR-143 mimic+ K-ras group), respectively. The expression of miR-143 in the transfected HeLa cells was detected by real-time PCR (RT-PCR), and the expression of K-ras protein was detected by Western blot. The cell proliferation activity of each group was examined by MTT assay. In addition, human cervical cancer tissue samples ( n =5) and cervical intraepithelial neoplasia tissue samples ( n =5) were also examined for the expression of miR-143 and K-ras protein by RT-PCR and Western blot, respectively. Results: The luciferase report assay showed that co-transfection with miR-143 mimic decreased the luciferase activity of the K-ras-wt significantly, but did not inhibit the luciferase activity of the K-ras-mut. The expression of miR-143 in the HeLa cells transfected with miR-143 mimic was significantly higher than that in the HeLa cells transfected with the mimic control (3.31±0.45 vs 0.97±0.22, P cell proliferative activity of the miR-143 mimic group was significantly lower than that of the negative control group ( P cell proliferative activity of the miR-143 mimic+ K-ras group was also significantly lower than the control group ( P HeLa cells through targeted regulating the expression of K-ras gene. In human cervical cancer tissues of a small sample set, the expression of miR-143 is downregulated, and the expression of K-ras is upregulated.

  6. Cucurbitacin B inhibits human breast cancer cell proliferation through disruption of microtubule polymerization and nucleophosmin/B23 translocation

    Directory of Open Access Journals (Sweden)

    Duangmano Suwit

    2012-10-01

    Full Text Available Abstract Background Cucurbitacin B, an oxygenated tetracyclic triterpenoid compound extracted from the Thai medicinal plant Trichosanthes cucumerina L., has been reported to have several biological activities including anti-inflammatory, antimicrobial and anticancer. Cucurbitacin B is great of interest because of its biological activity. This agent inhibits growth of various types of human cancer cells lines. Methods In this study, we explored the novel molecular response of cucurbitacin B in human breast cancer cells, MCF-7 and MDA-MB-231. The growth inhibitory effect of cucurbitacin B on breast cancer cells was assessed by MTT assay. The effects of cucurbitacin B on microtubules morphological structure and tubulin polymerization were analyzed using immunofluorescence technique and tubulin polymerization assay kit, respectively. Proteomic analysis was used to identify the target-specific proteins that involved in cucurbitacin B treatment. Some of the differentially expressed genes and protein products were validated by real-time RT-PCR and western blot analysis. Cell cycle distributions and apoptosis were investigated using flow cytometry. Results Cucurbitacin B exhibited strong antiproliferative effects against breast cancer cells in a dose-dependent manner. We show that cucurbitacin B prominently alters the cytoskeletal network of breast cancer cells, inducing rapid morphologic changes and improper polymerization of the microtubule network. Moreover, the results of 2D-PAGE, real-time RT-PCR, and western blot analysis revealed that the expression of nucleophosmin/B23 and c-Myc decreased markedly after cucurbitacin B treatment. Immunofluorescence microscopy showed that cucurbitacin B induced translocation of nucleophosmin/B23 from the nucleolus to nucleoplasm. Treatment with cucurbitacin B resulted in cell cycle arrest at G2/M phase and the enhancement of apoptosis. Conclusions Our findings suggest that cucurbitacin B may inhibit the

  7. Regulator of G-protein signaling 5 (RGS5) inhibits cell proliferation and enhances radiosensitivity of human lung cancer cells

    International Nuclear Information System (INIS)

    Xu Zumin; Wang Jin; Zuo Yufang; Yu Zhonghua; Peng Fang; Hu Xiao; Zhou Qichao; Ma Honglian; Bao Yong; Chen Ming

    2014-01-01

    Objective: To investigate the effects of regulator and the underlying molecular mechanisms of G-protein signaling 5 (RGS5) on radiation response in human lung cancer cells. Methods: The effects of RGS5 on viability were determined by MTT assay, and apoptosis rate was detected by flow cytometry, in human lung cancer cells. The combined effect of ionizing radiation and RGS5 on tumor cells was detected by colony formation assay. The protein expression was detected by Western blot. Results: RGS5 overexpression remarkably inhibited the survival of human lung cancer cells, and the growth inhibition rate of RGS5 overexpression on A549 and Calu-3 cells were 44.4% (F = 29.18, P < 0.05) and 39.27% (F = 23.04, P < 0.05) at 48 h, and 54.3%(F = 103.45, P < 0.05), 44.7%(F = 108.02, P < 0.05) at 72 h post-irradiation, respectively. RGS5 might exert its inhibitory effects on human lung cancer cells by inducing tumor cell apoptosis, while the apoptotic cells rate in A549 and Calu-3 cells in control group, pTRiEX group and pTRiEX-RGS5 group were (1.3±0.2)%, (3.4±0.6)%, (19.6±2.3)% (F = 86.62, P < 0.05), and (3.2±0.8)%, (3.0±0.9)%, (12.8±1.8)% (F = 28.80, P < 0.05) at 36 h post-irradiation, respectively. Furthermore, RGS5 could sensitize the lung cancer cells to radiation. Conclusions: RGS5 might play an inhibitory role in human lung cancer cell proliferation, which may explain the pathoclinical observation thet high expression of RGSS is a favorable prognostic factor in NSCLC patients. In addition, RGS5 also enhance the anti-tumor effects of radiation in human lung cancer cells. (authors)

  8. Endogenous Locus Reporter Assays.

    Science.gov (United States)

    Liu, Yaping; Hermes, Jeffrey; Li, Jing; Tudor, Matthew

    2018-01-01

    Reporter gene assays are widely used in high-throughput screening (HTS) to identify compounds that modulate gene expression. Traditionally a reporter gene assay is built by cloning an endogenous promoter sequence or synthetic response elements in the regulatory region of a reporter gene to monitor transcriptional activity of a specific biological process (exogenous reporter assay). In contrast, an endogenous locus reporter has a reporter gene inserted in the endogenous gene locus that allows the reporter gene to be expressed under the control of the same regulatory elements as the endogenous gene, thus more accurately reflecting the changes seen in the regulation of the actual gene. In this chapter, we introduce some of the considerations behind building a reporter gene assay for high-throughput compound screening and describe the methods we have utilized to establish 1536-well format endogenous locus reporter and exogenous reporter assays for the screening of compounds that modulate Myc pathway activity.

  9. L-type calcium channels play a crucial role in the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Wen, Li [Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi' an 710032 (China); Wang, Yu [Department of Oncology, Xijing Hospital, Fourth Military Medical University, Xi' an 710032 (China); Wang, Huan [Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi' an 710032 (China); Kong, Lingmin [Department of Fundamental Medicine, Cell Engineering Research Centre, Fourth Military Medical University, Xi' an 710032 (China); Zhang, Liang [Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi' an 710032 (China); Chen, Xin [Department of General Dentistry, The 174th Hospital of Chinese PLA, Xiamen 361003 (China); Ding, Yin, E-mail: dingyin@fmmu.edu.cn [Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi' an 710032 (China)

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer We detect the functional Ca{sup 2+} currents and mRNA expression of VDCC{sub L} in rMSCs. Black-Right-Pointing-Pointer Blockage of VDCC{sub L} exert antiproliferative and apoptosis-inducing effects on rMSCs. Black-Right-Pointing-Pointer Inhibiting VDCC{sub L} can suppress the ability of rMSCs to differentiate into osteoblasts. Black-Right-Pointing-Pointer {alpha}1C of VDCC{sub L} may be a primary functional subunit in VDCC{sub L}-regulating rMSCs. -- Abstract: L-type voltage-dependent Ca{sup 2+} channels (VDCC{sub L}) play an important role in the maintenance of intracellular calcium homeostasis, and influence multiple cellular processes. They have been confirmed to contribute to the functional activities of osteoblasts. Recently, VDCC{sub L} expression was reported in mesenchymal stem cells (MSCs), but the role of VDCC{sub L} in MSCs is still undetermined. The aim of this study was to determine whether VDCC{sub L} may be regarded as a new regulator in the proliferation and osteogenic differentiation of rat MSC (rMSCs). In this study, we examined functional Ca{sup 2+} currents (I{sub Ca}) and mRNA expression of VDCC{sub L} in rMSCs, and then suppressed VDCC{sub L} using nifedipine (Nif), a VDCC{sub L} blocker, to investigate its role in rMSCs. The proliferation and osteogenic differentiation of MSCs were analyzed by MTT, flow cytometry, alkaline phosphatase (ALP), Alizarin Red S staining, RT-PCR, and real-time PCR assays. We found that Nif exerts antiproliferative and apoptosis-inducing effects on rMSCs. ALP activity and mineralized nodules were significantly decreased after Nif treatment. Moreover, the mRNA levels of the osteogenic markers, osteocalcin (OCN), bone sialoprotein (BSP), and runt-related transcription factor 2 (Runx2), were also down-regulated. In addition, we transfected {alpha}1C-siRNA into the cells to further confirm the role of VDCC{sub L} in rMSCs, and a similar effect on osteogenesis was found. These

  10. Dendrobium candidum inhibits MCF-7 cells proliferation by inducing cell cycle arrest at G2/M phase and regulating key biomarkers

    Directory of Open Access Journals (Sweden)

    Sun J

    2015-12-01

    Full Text Available Jing Sun,1 Yidi Guo,1 Xueqi Fu,1–3 Yongsen Wang,1 Ye Liu,1 Bo Huo,1 Jun Sheng,4 Xin Hu1–3 1School of Life Sciences, 2Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, 3National Engineering Laboratory of AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, 4Yunnan Research Centre for Advance Tea Processing, Yunnan Agricultural University, Kunming, People’s Republic of China Background: Breast cancer is one of the most frequently occurring cancers in women. In recent years, Dendrobium candidum has played a part in antihyperthyroidism and anticancer drugs. This study aims to examine the antitumor effect of D. candidum on breast cancer. Methods: Human breast cancer cell line MCF-7 and normal breast epithelial cell line MCF10A were used to observe the effects of D. candidum treatment on human breast cancer. 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay was employed to examine the cell proliferation of the MCF-7 and MCF10A cells. Western blot analysis and reverse transcription polymerase chain reaction were used to detect the key molecules and biomarkers in breast cancer pathology. Cell cycle was analyzed by using Becton Dickinson FACScan cytofluorometer. Results: The results indicated that D. candidum significantly decreased cell viability at different concentrations compared to the control group (P<0.05. D. candidum-treated MCF-7 cells in the G2/M phase was significantly increased compared to the control group (P<0.05. The messenger RNA levels of estrogen receptor alpha, IGFBP2, IGFBP4, and GATA3 were significantly decreased, and the messenger RNA and protein levels of ELF5, p53, p21, p18, CDH1, CDH2, and p12 were significantly increased, compared to the control group (P<0.05. The protein levels of estrogen receptor alpha, PGR, GATA3, and Ki67 were significantly decreased and the protein levels of p53 and ELF5 were significantly increased compared to the control group (P

  11. LncRNA TUG1 is upregulated and promotes cell proliferation in osteosarcoma

    OpenAIRE

    Yun-Bo, Feng; Xiao-Po, Liu; Xiao-Li, Li; Guo-Long, Cao; Pei, Zhang; Fa-Ming, Tian

    2016-01-01

    Abstract Objective: To examine the expression and function of long non-coding RNA taurine up-regulated 1 (TUG1) in human osteosarcoma cells. Methods: Real-time quantitive PCR was used to detect the transcription level of TUG1 in a series of osteosarcoma cell lines. Knockdown of TUG1 in U2OS cells was carried out by transient transfection of siRNAs. MTT assay was performed to access the cell growth rates. Afterwards, RNA and protein of these cells were extracted to analyze the transfection eff...

  12. Thorium cycles and proliferation

    International Nuclear Information System (INIS)

    Lovins, A.B.

    1979-01-01

    This paper analyzes several prevalent misconceptions about nuclear fuel cycles that breed fissile uranium-233 from thorium. Its main conclusions are: U-233, despite the gamma radioactivity of associated isotopes, is a rather attractive material for making fission bombs, and is a credible material for subnational as well as national groups to use for this purpose; (2) pure thorium cycles, which in effect merely substitute U-233 for Pu, would take many decades and much U to establish, and offer no significant safeguards advantage over Pu, cycles; (3) denatured Th-U cycles, which dilute the U-233 with inert U-238 to a level not directly usable in bombs, are not an effective safeguard even against subnational bomb-making; (4) several other features of mixed Th-U cycles are rather unattractive from a safeguards point of view; (5) thus, Th cycles of any kind are not a technical fix for proliferation (national or subnational) and, though probably more safeguardable than Pu cycles, are less so than once-through U cycles that entail no reprocessing; (6) while thorium cycles have some potential technical advantages, including flexibility, they cannot provide major savings in nuclear fuel resources compared to simpler ways of saving neutrons and U; and (7) while advocates of nuclear power may find Th cycles worth exploring, such cycles do not differ fundamentally from U cycles in any of the respects--including safeguards and fuel resources--that are relevant to the broader nuclear debate, and should not be euphorically embraced as if they did

  13. Theoretical Approaches to Nuclear Proliferation

    Directory of Open Access Journals (Sweden)

    Konstantin S. Tarasov

    2015-01-01

    Full Text Available This article analyses discussions between representatives of three schools in the theory of international relations - realism, liberalism and constructivism - on the driving factors of nuclear proliferation. The paper examines major theoretical approaches, outlined in the studies of Russian and foreign scientists, to the causes of nuclear weapons development, while unveiling their advantages and limitations. Much of the article has been devoted to alternative approaches, particularly, the role of mathematical modeling in assessing proliferation risks. The analysis also reveals a variety of different approaches to nuclear weapons acquisition, as well as the absence of a comprehensive proliferation theory. Based on the research results the study uncovers major factors both favoring and impeding nuclear proliferation. The author shows that the lack of consensus between realists, liberals and constructivists on the nature of proliferation led a number of scientists to an attempt to explain nuclear rationale by drawing from the insights of more than one school in the theory of IR. Detailed study of the proliferation puzzle contributes to a greater understating of contemporary international realities, helps to identify mechanisms that are most likely to deter states from obtaining nuclear weapons and is of the outmost importance in predicting short- and long-term security environment. Furthermore, analysis of the existing scientific literature on nuclear proliferation helps to determine future research agenda of the subject at hand.

  14. Microculture tetrazolium assays: a comparison between two new tetrazolium salts, XTT and MTS.

    Science.gov (United States)

    Goodwin, C J; Holt, S J; Downes, S; Marshall, N J

    1995-02-13

    Microculture tetrazolium assays are being widely exploited to investigate the mechanisms of both cell activation and cell damage. They are colorimetric assays which are based upon the bioreduction of a tetrazolium salt to an intensely coloured formazan. We contrast the responses obtainable with two new tetrazolium salts, MTS and XTT, when used on the rat lymphoma cell line (Nb2 cells), which has been activated by human growth hormone. These tetrazolium salts, unlike the more commonly used MTT, form soluble formazans upon bioreduction by the activated cells. This has the advantage that it eliminates the error-prone solubilisation step which is required for the microculture tetrazolium assays which employ MTT. Bioreduction of XTT and MTS usually requires addition of an intermediate electron acceptor, phenazine methosulphate (PMS). We found that the XTT/PMS, but not the MTS/PMS, reagent mixture was unstable. Nucleation and crystal formation in the XTT/PMS reagent mixture, prepared in DPBS, could occur within 1-3 min. This resulted in a decline in XTT-formazan production and manifested itself in the microculture tetrazolium assay as both poor within-assay precision and serious assay drift. Several features of the system suggested that the formation of charge-transfer complexes between XTT and PMS accounted for this instability. No such instability was encountered when MTS and PMS were mixed. We demonstrate that MTS/PMS provides microculture tetrazolium assays for hGH which are free from these serious artefacts and which are uniquely precise. In conclusion we therefore advocate the use of MTS in preference to XTT for the new generation of microculture tetrazolium assays.

  15. Nuclear non proliferation and disarmament

    International Nuclear Information System (INIS)

    2000-01-01

    In the framework of the publication of a document on the ''weapons mastership, disarmament and non proliferation: the french action'', by the ministry of Foreign Affairs and the ministry of Defense, the French Documentation organization presents a whole document. This document describes and details the following topics: the conference on the treaty of non proliferation of nuclear weapons, the France, Usa and Non Governmental Organizations position, the threats of the proliferation, the french actions towards the disarmament, the disarmament in the world, a chronology and some bibliographic resources. (A.L.B.)

  16. International proliferation on nuclear weapons

    International Nuclear Information System (INIS)

    Hill, J.

    1977-01-01

    The subject is dealt with under the following headings: introduction; routes to proliferation (preparation of U 235 , Pu 239 , U 233 ); nuclear power fuel cycles and proliferation; the fast reactor fuel cycle; security aspects of the existing fuel cycle; the IAEA and the nuclear non-proliferation treaty. It is concluded that 'the basis for sound international control exists, and taken together with the further technical steps which will be taken to make the existing fuel cycles more robust against the diversion of materials by terrorists and the abuse of civil nuclear power programmes by governments, we have good reason to proceed now with the orderly exploitation of ...nuclear energy...'. (U.K.)

  17. Solid phase assays

    International Nuclear Information System (INIS)

    Reese, M.G.; Johnson, L.R.; Ransom, D.K.

    1980-01-01

    In a solid phase assay for quantitative determination of biological and other analytes, a sample such as serum is contacted with a receptor for the analyte being assayed, the receptor being supported on a solid support. No tracer for the analyte is added to the sample before contacting with the receptor; instead the tracer is contacted with the receptor after unbound analyte has been removed from the receptor. The assay can be otherwise performed in a conventional manner but can give greater sensitivity. (author)

  18. Alkyl-fluorinated thymidine derivatives for imaging cell proliferation

    International Nuclear Information System (INIS)

    Toyohara, Jun; Hayashi, Akio; Gogami, Akie; Hamada, Masahiro; Hamashima, Yoshio; Katoh, Takahiro; Node, Manabu; Fujibayashi, Yasuhisa

    2006-01-01

    Derivatives of 2'-deoxyuridine that contain fluoroalkyl groups at the C5 position and derivatives of thymidine that contain fluoroalkyl groups at the N3 position were synthesized and examined in three in vitro assays designed to evaluate their potential as radiopharmaceuticals for imaging cellular proliferation. Three of the former nucleosides and five of the latter were synthesized. The three assays were as follows: (a) phosphoryl transfer assay, which showed that all three of the former nucleosides and four of the latter ones were phosphorylated by recombinant human thymidine kinase 1 (TK1) and that N 3 -(2-fluoroethyl)-thymidine (NFT202) was the most potent substrate of the eight nucleosides studied; (b) transport assay, which indicated that all eight nucleosides had good affinity for an 6-[(4-nitrobenzyl)thio]-9-β-D-ribofuranosylpurine-sensitive mouse erythrocyte nucleoside transporter, with inhibition constants in the range of 0.02-0.55 mM; and (c) degradation assay, which showed that all but one of the former nucleosides and none of the latter were degraded by recombinant Escherichia coli thymidine phosphorylase (an enzyme that catalyzes the glycosidic bond of thymidine and 2'-deoxyuridine derivatives). From these in vitro screening assays, we selected NFT202 as a candidate for subsequent in vivo evaluation because this compound met the three minimum requirements of the in vitro screening assays and had the most potent phosphorylation activity as a substrate for recombinant human TK1

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

    Science.gov (United States)

    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.

  20. Factor IX assay

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003679.htm Factor IX assay To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  1. Factor VIII assay

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003678.htm Factor VIII assay To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  2. Factor II assay

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003674.htm Factor II assay To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  3. Factor VII assay

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003676.htm Factor VII assay To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

  4. Microbead agglutination based assays

    KAUST Repository

    Kodzius, Rimantas; Castro, David; Foulds, Ian G.; Parameswaran, Ash M.; Sumanpreet, K. Chhina

    2013-01-01

    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling

  5. Telmisartan Exerts Anti-Tumor Effects by Activating Peroxisome Proliferator-Activated Receptor-γ in Human Lung Adenocarcinoma A549 Cells

    Directory of Open Access Journals (Sweden)

    Juan Li

    2014-03-01

    Full Text Available Telmisartan, a member of the angiotensin II type 1 receptor blockers, is usually used for cardiovascular diseases. Recent studies have showed that telmisartan has the property of PPARγ activation. Meanwhile, PPARγ is essential for tumor proliferation, invasion and metastasis. In this work we explore whether telmisartan could exert anti-tumor effects through PPARγ activation in A549 cells. MTT and trypan blue exclusion assays were included to determine the survival rates and cell viabilities. RT-PCR and western blotting were used to analyze the expression of ICAM-1, MMP-9 and PPARγ. DNA binding activity of PPARγ was evaluated by EMSA. Our data showed that the survival rates and cell viabilities of A549 cells were all reduced by telmisartan in a time- and concentration-dependent manner. Meanwhile, our results also demonstrated that telmisartan dose-dependently inhibited the expression of ICAM-1 and MMP-9. Moreover, the cytotoxic and anti-proliferative effects, ICAM-1 and MMP-9 inhibitive properties of telmisartan were totally blunted by the PPARγ antagonist GW9662. Our findings also showed that the expression of PPARγ was up-regulated by telmisartan in a dose dependent manner. And, the EMSA results also figured out that DNA binding activity of PPARγ was dose-dependently increased by telmisartan. Additionally, our data also revealed that telmisartan-induced PPARγ activation was abrogated by GW9662. Taken together, our results indicated that telmisartan inhibited the expression of ICAM-1 and MMP-9 in A549 cells, very likely through the up-regulation of PPARγ synthesis.

  6. Dithiolethione modified valproate and diclofenac increase E-cadherin expression and decrease proliferation of non-small cell lung cancer cells.

    Science.gov (United States)

    Moody, Terry W; Switzer, Christopher; Santana-Flores, Wilmarie; Ridnour, Lisa A; Berna, Marc; Thill, Michelle; Jensen, Robert T; Sparatore, Anna; Del Soldato, Piero; Yeh, Grace C; Roberts, David D; Giaccone, Giuseppe; Wink, David A

    2010-05-01

    The effects of dithiolethione modified valproate, diclofenac and sulindac on non-small cell lung cancer (NSCLC) cells were investigated. Sulfur(S)-valproate and S-diclofenac at 1 microg/ml concentrations significantly reduced prostaglandin (PG)E(2) levels in NSCLC cell lines A549 and NCI-H1299 as did the COX-2 inhibitor DuP-697. In vitro, S-valproate, S-diclofenac and S-sulindac half-maximally inhibited the clonal growth of NCI-H1299 cells at 6, 6 and 15 microg/ml, respectively. Using the MTT assay, 10 microg/ml S-valproate, NO-aspirin and Cay10404, a selective COX-2 inhibitor, but not SC-560, a selective COX-1 inhibitor, inhibited the growth of A549 cells. In vivo, 18mg/kg i.p. of S-valproate and S-diclofenac, but not S-sulindac, significantly inhibited A549 or NCI-H1299 xenograft proliferation in nude mice, but had no effect on the nude mouse body weight. The mechanism by which S-valproate and S-diclofenac inhibited the growth of NSCLC cells was investigated. Nitric oxide-aspirin but not S-valproate caused apoptosis of NSCLC cells. By Western blot, S-valproate and S-diclofenac increased E-cadherin but reduced vimentin and ZEB1 (a transcriptional suppressor of E-cadherin) protein expression in NSCLC cells. Because S-valproate and S-diclofenac inhibit the growth of NSCLC cells and reduce PGE(2) levels, they may prove beneficial in the chemoprevention and/or therapy of NSCLC. Published by Elsevier Ireland Ltd.

  7. Effects of valproic acid and pioglitazone on cell cycle progression and proliferation of T-cell acute lymphoblastic leukemia Jurkat cells

    Directory of Open Access Journals (Sweden)

    Marie Saghaeian Jazi

    2016-07-01

    Full Text Available Objective(s: T-cell acute lymphoblastic leukemia (T-ALL is an aggressive hematologic malignant tumor. Administration of chemical compounds influencing apoptosis and T cell development has been discussed as promising novel therapeutic strategies. Valproic acid (VPA as a recently emerged anti-neoplastic histone deacetylase (HDAC inhibitor and pioglitazone (PGZ as a high-affinity peroxisome proliferator-activated receptor-gamma (PPARγ agonist have been shown to induce apoptosis and cell cycle arrest in different studies. Here, we aimed to investigate the underlying molecular mechanisms involved in anti-proliferative effects of these compounds on human Jurkat cells. Materials and Methods: Treated cells were evaluated for cell cycle progression and apoptosis using flowcytometry and MTT viability assay. Real-time RT-PCR was carried out to measure the alterations in key genes associated with cell death and cell cycle arrest. Results: Our findings illustrated that both VPA and PGZ can inhibit Jurkat E6.1 cells in vitro after   24 hr; however, PGZ 400 μM presents the most anti-proliferative effect. Interestingly, treated cells have been arrested in G2/M with deregulated cell division cycle 25A (Cdc25A phosphatase and cyclin-dependent kinase inhibitor 1B (CDKN1B or p27 expression. Expression of cyclin D1 gene was inhibited when DNA synthesis entry was declined. Cell cycle deregulation in PGZ and VPA-exposed cells generated an increase in the proportion of aneuploid cell population, which has not reported before. Conclusion: These findings define that anti-proliferative effects of PGZ and VPA on Jurkat cell line are mediated by cell cycle deregulation. Thus, we suggest PGZ and VPA may relieve potential therapeutic application against apoptosis-resistant malignancies.

  8. Non-proliferation and nuclear data

    International Nuclear Information System (INIS)

    Sowerby, M.G.

    1978-01-01

    A review is made of the problem of the proliferation of nuclear weapons with particular emphasis on proliferation and nuclear power. Some indications of the nuclear data requirements associated with methods of reducing proliferation risks are presented

  9. [miR-503-5p inhibits the proliferation of T24 and EJ bladder cancer cells by interfering with the Rb/E2F signaling pathway].

    Science.gov (United States)

    Li, Xiaohui; Han, Xingtao; Yang, Jinhui; Sun, Jiantao; Wei, Pengtao

    2017-10-01

    Objective To observe the effect of microRNA-503-5p (miR-503-5p) on the growth of T24 and EJ bladder cancer cells, and explore the possible molecular mechanism. Methods The miR-504-5p mimics or miR-NC was transfected into T24 and EJ cells. The target gene of miR-503-5p was predicted by bioinformatics. The expressions of E2F transcription factor 3 (E2F3) mRNA and Rb/E2F signaling pathway mRNA were detected by the real-time quantitative PCR (qPCR). The expressions of Rb/E2F signal pathway proteins E2F3, cyclin E, CDK2, Rb and p-Rb were detected by Western blotting. The cell cycle of bladder cancer cell lines was determined by flow cytometry. MTT assay and plate cloning assay were performed to observe the proliferation ability of bladder cancer cells. Results After miR-503-5p mimics transfection, the expression of miR-503-5p in bladder cancer cells significantly increased. The increased expression of miR-503-5p significantly reduced the expressions of E2F3 mRNA and Rb/E2F signaling pathway mRNA in bladder cancer cells. What's more, the expressions of Rb/E2F signal pathway proteins were down-regulated. The bladder cancer cells were arrested in G0/G1 phase, and their growth was significantly inhibited by miR-503-5p. Conclusion The miR-503-5p over-expression can inhibit the growth of bladder cancer cell lines T24 and EJ by down-regulating the expression of the Rb/E2F signaling pathway.

  10. MicroRNA-148b is frequently down-regulated in gastric cancer and acts as a tumor suppressor by inhibiting cell proliferation

    Directory of Open Access Journals (Sweden)

    Jiang Li

    2011-01-01

    Full Text Available Abstract Background MicroRNAs (miRNAs are involved in cancer development and progression, acting as tumor suppressors or oncogenes. Our previous studies have revealed that miR-148a and miR-152 are significantly down-regulated in gastrointestinal cancers. Interestingly, miR-148b has the same "seed sequences" as miR-148a and miR-152. Although aberrant expression of miR-148b has been observed in several types of cancer, its pathophysiologic role and relevance to tumorigenesis are still largely unknown. The purpose of this study was to elucidate the molecular mechanisms by which miR-148b acts as a tumor suppressor in gastric cancer. Results We showed significant down-regulation of miR-148b in 106 gastric cancer tissues and four gastric cancer cell lines, compared with their non-tumor counterparts by real-time RT-PCR. In situ hybridization of ten cases confirmed an overt decrease in the level of miR-148b in gastric cancer tissues. Moreover, the expression of miR-148b was demonstrated to be associated with tumor size (P = 0.027 by a Mann-Whitney U test. We also found that miR-148b could inhibit cell proliferation in vitro by MTT assay, growth curves and an anchorage-independent growth assay in MGC-803, SGC-7901, BGC-823 and AGS cells. An experiment in nude mice revealed that miR-148b could suppress tumorigenicity in vivo. Using a luciferase activity assay and western blot, CCKBR was identified as a target of miR-148b in cells. Moreover, an obvious inverse correlation was observed between the expression of CCKBR protein and miR-148b in 49 pairs of tissues (P = 0.002, Spearman's correlation. Conclusions These findings provide important evidence that miR-148b targets CCKBR and is significant in suppressing gastric cancer cell growth. Maybe miR-148b would become a potential biomarker and therapeutic target against gastric cancer.

  11. Fisetin regulates astrocyte migration and proliferation in vitro

    Science.gov (United States)

    Wang, Nan; Yao, Fang; Li, Ke; Zhang, Lanlan; Yin, Guo; Du, Mingjun; Wu, Bingyi

    2017-01-01

    Fisetin (3,3′,4′,7-tetrahydroxyflavone) is a plant flavonol found in fruits and vegetables that has been reported to inhibit migration and proliferation in several types of cancer. Reactive astrogliosis involves astrocyte migration and proliferation, and contributes to the formation of glial scars in central nervous system (CNS) disorders. However, the effect of fisetin on the migration and proliferation of astrocytes remains unclear. In this study, we found that fisetin inhibited astrocyte migration in a scratch-wound assay and diminished the phosphorylation of focal adhesion kinase (FAK; Tyr576/577 and paxillin (Tyr118). It also suppressed cell proliferation, as indicated by the decreased number of 5-ethynyl-2′-deoxyuridine (EdU)-positive cells, induced cell cycle arrest in the G1 phase, reduced the percentage of cells in the G2 and S phase (as measured by flow cytometry), and decreased cyclin D1 expression, but had no effect on apoptosis. Fisetin also decreased the phosphorylation levels of Akt and extracellular signal-regulated kinase (Erk)1/2, but had no effect on the phosphorylation of p38 mitogen-activated protein kinase (MAPK). These results indicate that fisetin inhibits aggressive cell phenotypes by suppressing cell migration and proliferation via the Akt/Erk signaling pathway. Fisetin may thus have potential for use as a therapeutic strategy targeting reactive astrocytes, which may lead to the inhibition of glial scar formation in vitro. PMID:28204814

  12. Fibroblast response to initial attachment and proliferation on titanium and zirconium surfaces.

    Directory of Open Access Journals (Sweden)

    Araceli Meza-Rodríguez

    2016-08-01

    Full Text Available Introduction: In recent decades, dental implants have become one of the best options for comprehensive dental restoration; their placement is a multidisciplinary task that requires a solid understanding of biological, periodontal, surgical and prosthetic principles. Objective: The aim of this study was to quantify in vitro the adhesion and proliferation of human gingival fibroblasts (HGF response on titanium (Ti and zirconia (Zr surfaces. Methodology: Samples of Ti and Zr were observed under atomic force microscopy (AFM. HGFs were inoculated in each sample to determine adhesion and cell proliferation. The reagent MTT was mixed with medium DMEM and inoculated in each plate; formazan was dissolved with dimethyl sulfoxide and analyzed at 540nm in a microplate spectrophotometer. The test was performed with three independent experiments. Data were analyzed with Kolmogorov-Smirnov tests (Lilliefors, Kruskal-Wallis tests and Mann-Whitney test comparisons. Results: Topography of the Zr plates showed greater roughness (Ra=0.39μm than Ti (Ra=0.049μm. Quantification of HGF adhesion was significantly higher (p<0.05 in Ti, while proliferation showed no statistically significant differences between the groups. Conclusion: It is noteworthy that, even though Ti initially showed increased cell adhesion on the surface, after 24h Zr samples showed similar proliferation; this demonstrates that both surfaces have a comparable biological response.

  13. Monitoring in real time the effect of TLX overexpression on proliferation and migration of C6 cells.

    Science.gov (United States)

    Li, G L; Fang, S H; Xu, B

    2017-01-01

    Orphan nuclear receptor TLX has been shown to play an essential role in regulating the self-renewal and proliferation of neural stem cells (NSCs). However, TLX overexpression in NSCs induces long-term NSC expansion and further leads to glioma initiation in mouse when combined with p53 mutations. Whether overexpression of TLX plays a role in glioma stem cell (GSC) proliferation and migration still remains largely unknown. In this study, we infected C6 cells, a special glioma cell line which is mainly composed of cancer stem cells(CSCs), with lentiviruses expressing GFP(LV-GFP) or GFP-T2A-TLX(LV-TLX) and then monitored cell proliferation and migration using the real-time analyzer system (RTCA, xCELLigence, Roche). We found that the cell index (CI) observed for the TLX overexpressing C6 cells showed a lower value than that of the LV-GFP transduced cells. And the MTT results correlated highly with the RTCA proliferation assessments. Furthermore, the expression of p21 was decreased while other downstream genes PTEN and p53 were not significantly changed in TLX overexpressing C6 cells . These findings strongly indicate that TLX overexpression has the ability to decrease the proliferating and migratory properties of C6 cells by targeting p21. Further, our results suggest that TLX overexpression may also have a similar inhibitory effect on GSC proliferation and migration.

  14. Effects of currently used pesticides in assays for estrogenicity, androgenicity, and aromatase activity in vitro

    DEFF Research Database (Denmark)

    Andersen, Helle Raun; Vinggaard, Anne; Rasmussen, Thomas Høj

    2002-01-01

    , chlorpyrifos, deltamethrin, tolclofos-methyl, and tribenuron-methyl induced weak responses in one or both estrogenicity assays. Upon cotreatment with 17beta-estradiol, the response was potentiated by endosulfan in the proliferation assay and by pirimicarb, propamocarb, and daminozid in the ER transactivation...

  15. Assay method and compositions

    International Nuclear Information System (INIS)

    1977-01-01

    Methods are described for measuring catecholamine levels in human and animal body fluids and tissues using the catechol-O-methyl-transferase (COMT) radioassay. The assay involves incubating the biological sample with COMT and the tritiated methyl donor, S-adenosyl-L-methionine( 3 H)-methyl. The O-methylated ( 3 H) epinephrine and/or norepinephrine are extracted and oxidised to vanillin- 3 H which in turn is extracted and its radioactivity counted. When analysing dopamine levels the assay is extended by vanillin- 3 H and raising the pH of the aqueous periodate phase from which O-methylated ( 3 H) dopamine is extracted and counted. The assay may be modified depending on whether measurements of undifferentiated total endogenous catecholamine levels or differential analyses of the catecholamine levels are being performed. The sensitivity of the assay can be as low as 5 picograms for norepinephrine and epinephrine and 12 picograms for dopamine. The assemblance of the essential components of the assay into a kit for use in laboratories is also described. (U.K.)

  16. Future non-proliferation challenges

    International Nuclear Information System (INIS)

    Yelchenko, Volodymyr

    2008-01-01

    Having chaired the Second Session of the Preparatory Committee Mr. Volodymyr Yelchenko noted that the NPT States parties reaffirmed the important role of the Treaty as the cornerstone of the global non-proliferation regime. They stressed that non-compliance with the Treaty provisions by States parties undermined non-proliferation and placed emphasis on the mutually reinforcing nature of disarmament and non-proliferation, and due respect for the right of States parties to the peaceful use of nuclear energy in conformity with the treaty. They reaffirmed the importance of promoting the peaceful uses of nuclear energy and international nuclear cooperation for peaceful purposes in ways consistent with the non-proliferation goal of the Treaty. The universality aspect was brought to the front with the lack of progress in this area. States parties called upon India, Israel and Pakistan to accede to the Treaty as non-nuclear-weapons states, promptly and without conditions and to bring into force comprehensive safeguards agreements, together with Additional Protocols, for ensuring non-proliferation. There is concern that non-States actors could gain access to weapons of mass destruction. One of the underlying themes at the Second Prepcom was the total elimination of nuclear weapons as the only absolute guarantee against their proliferation. Negative consequences to nuclear non-proliferation were also mentioned in the context of the abrogation of the Anti-Ballistic Missile Treaty and the development of missile defense systems, with the risk of a new arms race on Earth and in outer space. The importance of the immediate commencement of negotiations in the Conference of Disarmament on a treaty concerning fissile material for nuclear weapons or other nuclear explosive devices and the urgent conclusion of such a treaty as a beneficial step towards non-proliferation was stressed. The NPT states parties reaffirmed the role of the IAEA as the sole competent authority responsible for

  17. H2A/K pseudogene mutation may promote cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Jisheng; Jing, Ruirui; Lv, Xin; Wang, Xiaoyue; Li, Junqiang; Li, Lin; Li, Cuiling; Wang, Daoguang; Bi, Baibing; Chen, Xinjun [Cancer Research Center, Shandong University School of Medicine, Jinan 250012 (China); Yang, Jing-Hua, E-mail: sdu_crc_group1@126.com [Cancer Research Center, Shandong University School of Medicine, Jinan 250012 (China); Department of Surgery, VA Boston Healthcare System, Boston University School of Medicine, Boston 510660, MA (United States)

    2016-05-15

    Highlights: • The mutant H2A/K pseudogene is active. • The mutant H2A/K pseudogene can promote cell proliferation. - Abstract: Little attention has been paid to the histone H2A/K pseudogene. Results from our laboratory showed that 7 of 10 kidney cancer patients carried a mutant H2A/K pseudogene; therefore, we were interested in determining the relationship between mutant H2A/K and cell proliferation. We used shotgun and label-free proteomics methods to study whether mutant H2A/K lncRNAs affected cell proliferation. Quantitative proteomic analysis indicated that the expression of mutant H2A/K lncRNAs resulted in the upregulation of many oncogenes, which promoted cell proliferation. Further interaction analyses revealed that a proliferating cell nuclear antigen (PCNA)-protein interaction network, with PCNA in the center, contributes to cell proliferation in cells expressing the mutant H2A/K lncRNAs. Western blotting confirmed the critical upregulation of PCNA by mutant H2A/K lncRNA expression. Finally, the promotion of cell proliferation by mutant H2A/K lncRNAs (C290T, C228A and A45G) was confirmed using cell proliferation assays. Although we did not determine the exact mechanism by which the oncogenes were upregulated by the mutant H2A/K lncRNAs, we confirmed that the mutant H2A/K lncRNAs promoted cell proliferation by upregulating PCNA and other oncogenes. The hypothesis that cell proliferation is promoted by the mutant H2A/K lncRNAs was supported by the protein expression and cell proliferation assay results. Therefore, mutant H2A/K lncRNAs may be a new factor in renal carcinogenesis.

  18. Novel T lymphocyte proliferation assessment using whole mouse cryo-imaging

    Science.gov (United States)

    Wuttisarnwattana, Patiwet; Raza, Syed A.; Eid, Saada; Cooke, Kenneth R.; Wilson, David L.

    2014-03-01

    New imaging technologies enable one to assess T-cell proliferation, an important feature of the immunological response. However, none of the traditional imaging modalities allow one to examine quantiatively T-cell function with microscopic resolution and single cell sensitivity over an entire mouse. To address this need, we established T-cells proliferation assays using 3D microscopic cryo-imaging. Assays include: (1) biodistribution of T-cells, (2) secondary lymphoid organ (SLO) volume measurement, (3) carboxyfluorescein succinimidyl ester (CFSE) dilution per cell as cells divide. To demonstrate the application, a graft-versus-host-disease (GVHD) model was used. 3D visualization show that T-cells specifically homed to the SLOs (spleen and lymph nodes) as well as GVHD target organs (such as GI-tract, liver, skin and thymus).The spleen was chosen as representative of the SLOs. For spleen size analysis, volumes of red and white pulp were measured. Spleen volumes of the allogeneic mice (with GVHD) were significantly larger than those of the syngeneic mice (without GVHD) at 72 to 120 hours post-transplant. For CFSE dilution approach, we employed color-coded volume rendering and probability density function (PDF) of single cell intensity to assess T-cell proliferation in the spleen. As compared to syngeneic T-cells, the allogeneic T-cells quickly aggregated in the spleen as indicated by increasing of CFSE signal over the first 48 hours. Then they rapidly proliferated as evidenced by reduced CFSE intensity (at 48-96 hours). Results suggest that assays can be used to study GVHD treatments using T-cell proliferation and biodistibution as assays. In summary, this is the first time that we are able to track and visualize T-cells in whole mouse with single cell sensitivity. We believe that our technique can be an alternative choice to traditional in vitro immunological proliferation assays by providing assessment of proliferation in an in vivo model.

  19. Rover waste assay system

    Energy Technology Data Exchange (ETDEWEB)

    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J. [Idaho National Engineering Lab., Idaho Falls, ID (United States)

    1997-11-01

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched {sup 235}U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for {sup 137}Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs.

  20. Rover waste assay system

    International Nuclear Information System (INIS)

    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J.

    1997-01-01

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched 235 U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for 137 Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs

  1. Radioreceptor assay for insulin

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Kazuo [Tokyo Univ. (Japan). Faculty of Medicine

    1975-04-01

    Radioreceptor assay of insulin was discussed from the aspects of the measuring method, its merits and problems to be solved, and its clinical application. Rat liver 10 x g pellet was used as receptor site, and enzymatic degradation of insulin by the system contained in this fraction was inhibited by adding 1 mM p-CMB. /sup 125/I-labelled porcine insulin was made by lactoperoxidase method under overnight incubation at 4/sup 0/C and later purification by Sephadex G-25 column and Whatman CF-11 cellulose powder. Dog pancreatic vein serum insulin during and after the glucose load was determined by radioreceptor assay and radioimmunoassay resulting that both measurements accorded considerably. Radioreceptor assay would clarify the pathology of disorders of glucose metabolism including diabetes.

  2. Clonogenic assay: adherent cells.

    Science.gov (United States)

    Rafehi, Haloom; Orlowski, Christian; Georgiadis, George T; Ververis, Katherine; El-Osta, Assam; Karagiannis, Tom C

    2011-03-13

    The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 1956. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811). Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant

  3. Scintillation proximity assay

    International Nuclear Information System (INIS)

    Hart, H.

    1980-01-01

    In a method of immunological assay two different classes of particles which interact at short distances to produce characteristic detectable signals are employed in a modification of the usual latex fixation test. In one embodiment an aqueous suspension of antigen coated tritiated latex particles (LH) and antigen coated polystyrene scintillant particles (L*) is employed to assay antibody in the aqueous medium. The amount of (LH) (L*) dimer formation and higher order aggregation induced and therefore the concentration of antibody (or antigen) present which caused the aggregation can be determined by using standard liquid scintillation counting equipment. (author)

  4. Assays for calcitonin receptors

    International Nuclear Information System (INIS)

    Teitelbaum, A.P.; Nissenson, R.A.; Arnaud, C.D.

    1985-01-01

    The assays for calcitonin receptors described focus on their use in the study of the well-established target organs for calcitonin, bone and kidney. The radioligand used in virtually all calcitonin binding studies is 125 I-labelled salmon calcitonin. The lack of methionine residues in this peptide permits the use of chloramine-T for the iodination reaction. Binding assays are described for intact bone, skeletal plasma membranes, renal plasma membranes, and primary kidney cell cultures of rats. Studies on calcitonin metabolism in laboratory animals and regulation of calcitonin receptors are reviewed

  5. Nuclear proliferation and safeguards. Summary

    International Nuclear Information System (INIS)

    1982-03-01

    This comprehensive analysis of the technological, economic, and political factors affecting the potential spread of nuclear weapons proved useful in the congressional debate which culminated in the Nuclear Non-Proliferation Act of 1978. The report was subsequently published commercially and has been a frequently cited reference in the literature on proliferation and nuclear power. Despite developments since 1977, the information in the OTA report is still useful to those wishing to obtain an indepth understanding of the issues. Included is an analysis of why a nation might want nuclear weapons development program and the various sources of nuclear material are discussed. The control of proliferation is considered as well as its relation to the nuclear industry

  6. Domestic Politics and Nuclear Proliferation

    International Nuclear Information System (INIS)

    Kim, Chul Min; Yim, Man Sung

    2016-01-01

    The external security threat is known as the most important factor of nuclear weapons program, the domestic politics situation can also affect the nuclear proliferation decision of a country. For example, when a leader wants nuclear weapons as an ultimate weapon, the domestic politics situation can determine the effectiveness of the weapons program of a country. This study analyzes the current knowledge of the relationship between domestic politics and nuclear proliferation and suggests the main challenges of the quantitative models trying to calculate nuclear proliferation risk of countries. The domestic politics status is one of the most important indicators of nuclear program. However, some variables have never been used in quantitative analyses; for example, number of veto players and the public opinion on nuclear weapons; despite they are considered to be important in various qualitative studies. Future studies should focus on how should they be coded and how can they be linked with existing domestic politics variables

  7. Domestic Politics and Nuclear Proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Chul Min; Yim, Man Sung [KAIST, Daejeon (Korea, Republic of)

    2016-05-15

    The external security threat is known as the most important factor of nuclear weapons program, the domestic politics situation can also affect the nuclear proliferation decision of a country. For example, when a leader wants nuclear weapons as an ultimate weapon, the domestic politics situation can determine the effectiveness of the weapons program of a country. This study analyzes the current knowledge of the relationship between domestic politics and nuclear proliferation and suggests the main challenges of the quantitative models trying to calculate nuclear proliferation risk of countries. The domestic politics status is one of the most important indicators of nuclear program. However, some variables have never been used in quantitative analyses; for example, number of veto players and the public opinion on nuclear weapons; despite they are considered to be important in various qualitative studies. Future studies should focus on how should they be coded and how can they be linked with existing domestic politics variables.

  8. Ultracentrifuge and non-proliferation

    International Nuclear Information System (INIS)

    Voortman, A.J.

    1977-01-01

    The author states that there is no meaningful difference, from the point of view of proliferation between peaceful, civil, scientific application of nuclear fission, and the use of it in nuclear weapons. The proliferation of the nuclear technology for weapons appeared and appears to be closely connected with the spread of peaceful applications of nuclear technology. In connection with this, he discusses the Ultracentrifuge plant at Almelo (Netherlands) and the supply of nuclear technology by West-Germany especially to Brazil. Further the changed American policy and the possibility of an American/Russian deal to prevent the spread of the nuclear enrichment technology is discussed

  9. Lateral flow assays

    NARCIS (Netherlands)

    Posthuma-Trumpie, G.A.; Amerongen, van A.

    2012-01-01

    A simple version of immunochemical-based methods is the Lateral Flow Assay (LFA). It is a dry chemistry technique (reagents are included); the fluid from the sample runs through a porous membrane (often nitrocellulose) by capillary force. Typically the membrane is cut as a strip of 0.5*5 cm. In most

  10. Microchemiluminescent assay system

    Energy Technology Data Exchange (ETDEWEB)

    Kiel, J.L.

    1986-04-09

    The patent concerns a microchemiluminescent assay system, which can be used to detect ionizing radiation, heat or specific substances. The method involves the use of a complex formed from serum albumin and a luminescer which, in the presence of ionizing radiation (heat, or a specific analyte), will emit light in an amount proportional to the amount of radiation, etc. (U.K.).

  11. Hyaluronic Acid Assays

    DEFF Research Database (Denmark)

    Itenov, Theis S; Kirkby, Nikolai S; Bestle, Morten H

    2015-01-01

    BACKGROUD: Hyaluronic acid (HA) is proposed as a marker of functional liver capacity. The aim of the present study was to compare a new turbidimetric assay for measuring HA with the current standard method. METHODS: HA was measured by a particle-enhanced turbidimetric immunoassay (PETIA) and enzyme...

  12. FLUIDICS DEVICE FOR ASSAY

    DEFF Research Database (Denmark)

    2007-01-01

    The present invention relates to a device for use in performing assays on standard laboratory solid supports whereon chemical entities are attached. The invention furthermore relates to the use of such a device and a kit comprising such a device. The device according to the present invention is a...

  13. Heme-induced Trypanosoma cruzi proliferation is mediated by CaM kinase II

    International Nuclear Information System (INIS)

    Souza, C.F.; Carneiro, A.B.; Silveira, A.B.; Laranja, G.A.T.; Silva-Neto, M.A.C.; Costa, S.C. Goncalves da; Paes, M.C.

    2009-01-01

    Trypanosoma cruzi, the etiologic agent of Chagas disease, is transmitted through triatomine vectors during their blood-meal on vertebrate hosts. These hematophagous insects usually ingest approximately 10 mM of heme bound to hemoglobin in a single meal. Blood forms of the parasite are transformed into epimastigotes in the crop which initiates a few hours after parasite ingestion. In a previous work, we investigated the role of heme in parasite cell proliferation and showed that the addition of heme significantly increased parasite proliferation in a dose-dependent manner . To investigate whether the heme effect is mediated by protein kinase signalling pathways, parasite proliferation was evaluated in the presence of several protein kinase (PK) inhibitors. We found that only KN-93, a classical inhibitor of calcium-calmodulin-dependent kinases (CaMKs), blocked heme-induced cell proliferation. KN-92, an inactive analogue of KN-93, was not able to block this effect. A T. cruzi CaMKII homologue is most likely the main enzyme involved in this process since parasite proliferation was also blocked when Myr-AIP, an inhibitory peptide for mammalian CaMKII, was included in the cell proliferation assay. Moreover, CaMK activity increased in parasite cells with the addition of heme as shown by immunological and biochemical assays. In conclusion, the present results are the first strong indications that CaMKII is involved in the heme-induced cell signalling pathway that mediates parasite proliferation.

  14. Heme-induced Trypanosoma cruzi proliferation is mediated by CaM kinase II

    Energy Technology Data Exchange (ETDEWEB)

    Souza, C.F. [Laboratorio de Imunomodulacao e Protozoologia, Instituto Oswaldo Cruz, Fiocruz (Brazil); Carneiro, A.B.; Silveira, A.B. [Laboratorio de Sinalizacao Celular, Instituto de Bioquimica Medica, UFRJ (Brazil); Laranja, G.A.T. [Laboratorio de Interacao Tripanosomatideos e Vetores, Departamento de Bioquimica, IBRAG, UERJ, 20551-030 Rio de Janeiro (Brazil); Silva-Neto, M.A.C. [Laboratorio de Sinalizacao Celular, Instituto de Bioquimica Medica, UFRJ (Brazil); INCT, Entomologia Molecular (Brazil); Costa, S.C. Goncalves da [Laboratorio de Imunomodulacao e Protozoologia, Instituto Oswaldo Cruz, Fiocruz (Brazil); Paes, M.C., E-mail: mcpaes@uerj.br [Laboratorio de Interacao Tripanosomatideos e Vetores, Departamento de Bioquimica, IBRAG, UERJ, 20551-030 Rio de Janeiro (Brazil); INCT, Entomologia Molecular (Brazil)

    2009-12-18

    Trypanosoma cruzi, the etiologic agent of Chagas disease, is transmitted through triatomine vectors during their blood-meal on vertebrate hosts. These hematophagous insects usually ingest approximately 10 mM of heme bound to hemoglobin in a single meal. Blood forms of the parasite are transformed into epimastigotes in the crop which initiates a few hours after parasite ingestion. In a previous work, we investigated the role of heme in parasite cell proliferation and showed that the addition of heme significantly increased parasite proliferation in a dose-dependent manner . To investigate whether the heme effect is mediated by protein kinase signalling pathways, parasite proliferation was evaluated in the presence of several protein kinase (PK) inhibitors. We found that only KN-93, a classical inhibitor of calcium-calmodulin-dependent kinases (CaMKs), blocked heme-induced cell proliferation. KN-92, an inactive analogue of KN-93, was not able to block this effect. A T. cruzi CaMKII homologue is most likely the main enzyme involved in this process since parasite proliferation was also blocked when Myr-AIP, an inhibitory peptide for mammalian CaMKII, was included in the cell proliferation assay. Moreover, CaMK activity increased in parasite cells with the addition of heme as shown by immunological and biochemical assays. In conclusion, the present results are the first strong indications that CaMKII is involved in the heme-induced cell signalling pathway that mediates parasite proliferation.

  15. Initial attachment, subsequent cell proliferation/viability and gene expression of epithelial cells related to attachment and wound healing in response to different titanium surfaces.

    Science.gov (United States)

    An, Na; Rausch-fan, Xiaohui; Wieland, Marco; Matejka, Michael; Andrukhov, Oleh; Schedle, Andreas

    2012-12-01

    A tight seal between the epithelium and the dental implant surface is required to prevent bacterial inflammation and soft tissue recession and therefore to demonstrate a long-term success. Surface hydrophilicity was recently shown to promote osseointegration. The aim of this study was to investigate the influence of surface hydrophilicity in combination with surface topography of Ti implant surfaces on the behavior and activation/differentiation of epithelial cells using a set of in vitro experiments mimicking the implant-soft tissue contact. Hydrophobic acid-etched (A) and coarse-grit-blasted, acid-etched (SLA) surfaces and hydrophilic acid-etched (modA) and modSLA surfaces were produced. The behavior of an oral squamous cell carcinoma cell line (HSC-2) grown on all surfaces was compared through determination of cell attachment and proliferation/viability (CCK-8 and MTT assay), time-lapse microscopy of fluorescence labeled cells and determination of gene expression by real time polymerase chain reaction. Within the surfaces with similar wettability cell spreading and cell movements observed by time-lapse microscopy after one day of incubation were most pronounced on smoother (A and modA) surfaces compared to rougher (SLA and modSLA) surfaces. Within the surfaces with similar roughness the hydrophilic surfaces (modA and modSLA) showed more cell spreading and cell activity compared to the hydrophobic surfaces (A and SLA). The relative gene expressions of cytokeratin14, integrin α6, integrin β4, vinculin, transforming growth factor (TGF)-β, TGF-β1, and TGF-β3 were decreased in HSC-2 on all four types of Ti surfaces compared to control surfaces (tissue culture polystyrene; pmodA). These results suggest that surface hydrophilicity might positively influence the epithelial seal around dental implants. All tested titanium surfaces downregulate cell attachment, cell proliferation, expression of adhesion promoters, and cytokines involved in wound healing in HSC-2

  16. α-Ketoglutarate Promotes Pancreatic Progenitor-Like Cell Proliferation

    Directory of Open Access Journals (Sweden)

    Jing Song

    2018-03-01

    Full Text Available A major source of β cell generation is pancreatic progenitor-like cell differentiation. Multiple studies have confirmed that stem cell metabolism plays important roles in self-renewal and proliferation. In the absence of glucose, glutamine provides the energy for cell division and growth. Furthermore, α-ketoglutarate (αKG, a precursor for glutamine synthesis, is sufficient for enabling glutamine-independent cell proliferation. We have demonstrated that αKG contributes to the large-scale proliferation of pancreatic progenitor-like cells that can provide an ample amount of clinically relevant β cells. We compared the mRNA expression of a subset of genes, the abundance of ATP, reactive oxide species, mitochondrial number, and the colony-forming frequency between mouse pancreatic CD133+ and CD133− cells. We employed Real-Time PCR, immunostaining and passage assays to investigate self-renewal and proliferation of pancreatic progenitor-like cells in a 3D culture system in the presence and absence of αKG. The energy metabolism of CD133+ cells was more prone to oxidative phosphorylation. However, in the 3D culture system, when αKG was supplemented to the culture medium, the proliferation of the pancreatic progenitor-like cells was significantly elevated. We confirmed that the presence of αKG correlated with the up-regulation of Ten-Eleven Translocation (Tet. αKG can promote the proliferation of pancreatic progenitor-like cells via the up-regulation of Tet.

  17. α-Ketoglutarate Promotes Pancreatic Progenitor-Like Cell Proliferation.

    Science.gov (United States)

    Song, Jing; Ma, Dongshen; Xing, Yun; Tang, Shanshan; Alahdal, Murad; Guo, Jiamin; Pan, Yi; Zhang, Yanfeng; Shen, Yumeng; Wu, Qiong; Lu, Zhou; Jin, Liang

    2018-03-22

    A major source of β cell generation is pancreatic progenitor-like cell differentiation. Multiple studies have confirmed that stem cell metabolism plays important roles in self-renewal and proliferation. In the absence of glucose, glutamine provides the energy for cell division and growth. Furthermore, α-ketoglutarate (αKG), a precursor for glutamine synthesis, is sufficient for enabling glutamine-independent cell proliferation. We have demonstrated that αKG contributes to the large-scale proliferation of pancreatic progenitor-like cells that can provide an ample amount of clinically relevant β cells. We compared the mRNA expression of a subset of genes, the abundance of ATP, reactive oxide species, mitochondrial number, and the colony-forming frequency between mouse pancreatic CD133⁺ and CD133 - cells. We employed Real-Time PCR, immunostaining and passage assays to investigate self-renewal and proliferation of pancreatic progenitor-like cells in a 3D culture system in the presence and absence of αKG. The energy metabolism of CD133⁺ cells was more prone to oxidative phosphorylation. However, in the 3D culture system, when αKG was supplemented to the culture medium, the proliferation of the pancreatic progenitor-like cells was significantly elevated. We confirmed that the presence of αKG correlated with the up-regulation of Ten-Eleven Translocation (Tet). αKG can promote the proliferation of pancreatic progenitor-like cells via the up-regulation of Tet.

  18. Lipid nanocarriers based on natural oils with high activity against oxygen free radicals and tumor cell proliferation

    International Nuclear Information System (INIS)

    Lacatusu, I.; Badea, N.; Badea, G.; Oprea, O.; Mihaila, M.A.; Kaya, D.A.; Stan, R.; Meghea, A.

    2015-01-01

    The development of nano-dosage forms of phytochemicals represents a significant progress of the scientific approach in the biomedical research. The aim of this study was to assess the effectiveness of lipid nanocarriers based on natural oils (grape seed oil, fish oil and laurel leaf oil) in counteracting free radicals and combating certain tumor cells. No drug was encapsulated in the nanocarriers. The cytotoxic effect exerted by bioactive nanocarriers against two tumor cells, MDA-MB 231 and HeLa cell lines, and two normal cells, L929 and B16 cell lines, was measured using the MTT assay, while oxidative damage was assessed by measuring the total antioxidant activity using chemiluminescence analysis. The best performance was obtained for nanocarriers based on an association of grape seed and laurel leaf oils, with a capacity to scavenge about 98% oxygen free radicals. A dose of nanocarriers of 5 mg·mL −1 has led to a drastic decrease in tumor cell proliferation even in the absence of an antitumor drug (e.g. about 50% viability for MDA-MB 231 cell line and 60% viability for HeLa cell line). A comparative survival profile of normal and tumor cells, which were exposed to an effective dose of 2.5 mg·mL −1 lipid nanocarriers, has revealed a death rate of 20% for normal B16 cells and of 40% death rate for MDA-MB 231 and HeLa tumor cells. The results in this study imply that lipid nanocarriers based on grape seed oil in association with laurel leaf oil could be a candidate to reduce the delivery system toxicity and may significantly improve the therapeutic efficacy of antitumor drugs in clinical applications. - Highlights: • Functional lipid nanocarriers with unique features and broad spectrum effectiveness • Lipid nanocarriers based on laureal leaf oil (LLO) and grape seed oil (GSO) • Antioxidant activity has reached 98% for nanocarriers containing 25% GSO and 2% LLO. • LLO exerts a significant cytotoxic effect against HeLa and MDA-MB 231 tumor cells. • 50

  19. Polymeric nanoparticle-based delivery of microRNA-199a-3p inhibits proliferation and growth of osteosarcoma cells

    Directory of Open Access Journals (Sweden)

    Zhang L

    2015-04-01

    Full Text Available Linlin Zhang,1,2,* Arun K lyer,3,4,* Xiaoqian Yang,1 Eisuke Kobayashi,1 Yuqi Guo,1,2 Henry Mankin,1 Francis J Hornicek,1 Mansoor M Amiji,3 Zhenfeng Duan1 1Sarcoma Biology Laboratory, Center for Sarcoma and Connective Tissue Oncology, Massachusetts General Hospital, Boston, Massachusetts, USA; 2Department of Pathology, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, People’s Republic of China; 3Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University, Boston, Massachusetts, USA; 4Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI, USA *These authors contributed equally to this work Abstract: Our prior screening of microRNAs (miRs identified that miR-199a-3p expression is reduced in osteosarcoma cells, one of the most common types of bone tumor. miR-199a-3p exhibited functions of tumor cell growth inhibition, suggesting the potential application of miR-199a-3p as an anticancer agent. In the study reported here, we designed and developed a lipid-modified dextran-based polymeric nanoparticle platform for encapsulation of miRs, and determined the efficiency and efficacy of delivering miR-199a-3p into osteosarcoma cells. In addition, another potent miR, let-7a, which also displayed tumor suppressive ability, was selected as a candidate miR for evaluation. Fluorescence microscopy studies and real-time polymerase chain reaction results showed that dextran nanoparticles could deliver both miR-199a-3p and let-7a into osteosarcoma cell lines (KHOS and U-2OS successfully. Western blotting analysis and 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assays demonstrated that dextran nanoparticles loaded with miRs could efficiently downregulate the expression of target proteins and effectively inhibit the growth and proliferation of osteosarcoma cells. These results demonstrate that a lipid-modified dextran

  20. Lipid nanocarriers based on natural oils with high activity against oxygen free radicals and tumor cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Lacatusu, I.; Badea, N.; Badea, G.; Oprea, O. [University Politehnica of Bucharest, Faculty of Applied Chemistry and Materials Science, Polizu Street No 1, 011061 Bucharest (Romania); Mihaila, M.A. [Institute of Virusology “Stefan S. Nicolau”, Center of Immunology, Bravu Road, No. 285, 030304 Bucharest (Romania); Kaya, D.A. [Department of Field Crops, Faculty of Agriculture, Mustafa Kemal University, 31030 Antakya, Hatay (Turkey); Stan, R., E-mail: rl_stan2000@yahoo.com [University Politehnica of Bucharest, Faculty of Applied Chemistry and Materials Science, Polizu Street No 1, 011061 Bucharest (Romania); Meghea, A. [University Politehnica of Bucharest, Faculty of Applied Chemistry and Materials Science, Polizu Street No 1, 011061 Bucharest (Romania)

    2015-11-01

    The development of nano-dosage forms of phytochemicals represents a significant progress of the scientific approach in the biomedical research. The aim of this study was to assess the effectiveness of lipid nanocarriers based on natural oils (grape seed oil, fish oil and laurel leaf oil) in counteracting free radicals and combating certain tumor cells. No drug was encapsulated in the nanocarriers. The cytotoxic effect exerted by bioactive nanocarriers against two tumor cells, MDA-MB 231 and HeLa cell lines, and two normal cells, L929 and B16 cell lines, was measured using the MTT assay, while oxidative damage was assessed by measuring the total antioxidant activity using chemiluminescence analysis. The best performance was obtained for nanocarriers based on an association of grape seed and laurel leaf oils, with a capacity to scavenge about 98% oxygen free radicals. A dose of nanocarriers of 5 mg·mL{sup −1} has led to a drastic decrease in tumor cell proliferation even in the absence of an antitumor drug (e.g. about 50% viability for MDA-MB 231 cell line and 60% viability for HeLa cell line). A comparative survival profile of normal and tumor cells, which were exposed to an effective dose of 2.5 mg·mL{sup −1} lipid nanocarriers, has revealed a death rate of 20% for normal B16 cells and of 40% death rate for MDA-MB 231 and HeLa tumor cells. The results in this study imply that lipid nanocarriers based on grape seed oil in association with laurel leaf oil could be a candidate to reduce the delivery system toxicity and may significantly improve the therapeutic efficacy of antitumor drugs in clinical applications. - Highlights: • Functional lipid nanocarriers with unique features and broad spectrum effectiveness • Lipid nanocarriers based on laureal leaf oil (LLO) and grape seed oil (GSO) • Antioxidant activity has reached 98% for nanocarriers containing 25% GSO and 2% LLO. • LLO exerts a significant cytotoxic effect against HeLa and MDA-MB 231 tumor

  1. Pravastatin and simvastatin inhibit the adhesion, replication and proliferation of Toxoplasma gondii (RH strain) in HeLa cells.

    Science.gov (United States)

    Sanfelice, Raquel Arruda; da Silva, Suelen Santos; Bosqui, Larissa Rodrigues; Miranda-Sapla, Milena Menegazzo; Barbosa, Bellisa Freitas; Silva, Rafaela José; Ferro, Eloísa A Vieira; Panagio, Luciano Aparecido; Navarro, Italmar Teodorico; Bordignon, Juliano; Conchon-Costa, Ivete; Pavanelli, Wander Rogerio; Almeida, Ricardo Sergio; Costa, Idessania Nazareth

    2017-03-01

    The conventional treatment for toxoplasmosis with pyrimethamine and sulfadiazine shows toxic effects to the host, and it is therefore necessary to search for new drugs. Some studies suggest the use of statins, which inhibit cholesterol synthesis in humans and also the initial processes of isoprenoid biosynthesis in the parasite. Thus, the objective of this study was to evaluate the activity of the statins pravastatin and simvastatin in HeLa cells infected in vitro with the RH strain of T. gondii. HeLa cells (1×10 5 ) were infected with T. gondii tachyzoites (5×10 5 ) following two different treatment protocols. In the first protocol, T. gondii tachyzoites were pretreated with pravastatin (50 and 100μg/mL) and simvastatin (1.56 and 3.125μg/mL) for 30min prior to infection. In the second, HeLa cells were first infected (5×10 5 ) with tachyzoites and subsequently treated with pravastatin and simvastatin for 24h at the concentrations noted above. Initially, we evaluated the cytotoxicity of drugs by the MTT assay, number of tachyzoites adhered to cells, number of infected cells, and viability of tachyzoites by trypan blue exclusion. The supernatant of the cell cultures was collected post-treatment for determination of the pattern of Th1/Th2/Th17 cytokines by cytometric bead array. There was no cytotoxicity to HeLa cells with 50 and 100μg/mL pravastatin and 1.56 and 3.125μg/mL simvastatin. There was no change in the viability of tachyzoites that received pretreatment. Regarding the pre- and post-treatment of the cells with pravastatin and simvastatin alone, there was a reduction in adhesion, invasion and proliferation of cells to T. gondii. As for the production of cytokines, we found that IL-6 and IL-17 were significantly reduced in cells infected with T. gondii and treated with pravastatin and simvastatin, when compared to control. Based on these results, we can infer that pravastatin and simvastatin alone possess antiproliferative effects on tachyzoites forms

  2. Effects of L-carnitine against oxidative stress in human hepatocytes: involvement of peroxisome proliferator-activated receptor alpha

    Directory of Open Access Journals (Sweden)

    Li Jin-Lian

    2012-03-01

    Full Text Available Abstract Background Excessive oxidative stress and lipid peroxidation have been demonstrated to play important roles in the production of liver damage. L-carnitine is a natural substance and acts as a carrier for fatty acids across the inner mitochondrial membrane for subsequent beta-oxidation. It is also an antioxidant that reduces metabolic stress in the cells. Recent years L-carnitine has been proposed for treatment of various kinds of disease, including liver injury. This study was conducted to evaluate the protective effect of L-carnitine against hydrogen peroxide (H2O2-induced cytotoxicity in a normal human hepatocyte cell line, HL7702. Methods We analyzed cytotoxicity using MTT assay and lactate dehydrogenase (LDH release. Antioxidant activity and lipid peroxidation were estimated by reactive oxygen species (ROS levels, activities and protein expressions of superoxide dismutase (SOD and catalase (CAT, and malondialdehyde (MDA formation. Expressions of peroxisome proliferator-activated receptor (PPAR-alpha and its target genes were evaluated by RT-PCR or western blotting. The role of PPAR-alpha in L-carnitine-enhanced expression of SOD and CAT was also explored. Statistical analysis was performed by a one-way analysis of variance, and its significance was assessed by Dennett's post-hoc test. Results The results showed that L-carnitine protected HL7702 cells against cytotoxity induced by H2O2. This protection was related to the scavenging of ROS, the promotion of SOD and CAT activity and expression, and the prevention of lipid peroxidation in cultured HL7702 cells. The decreased expressions of PPAR-alpha, carnitine palmitoyl transferase 1 (CPT1 and acyl-CoA oxidase (ACOX induced by H2O2 can be attenuated by L-carnitine. Besides, we also found that the promotion of SOD and CAT protein expression induced by L-carnitine was blocked by PPAR-alpha inhibitor MK886. Conclusions Taken together, our findings suggest that L-carnitine could protect HL

  3. Development of fluorescent methods for DNA methyltransferase assay

    Science.gov (United States)

    Li, Yueying; Zou, Xiaoran; Ma, Fei; Tang, Bo; Zhang, Chun-yang

    2017-03-01

    DNA methylation modified by DNA methyltransferase (MTase) plays an important role in regulating gene transcription, cell growth and proliferation. The aberrant DNA MTase activity may lead to a variety of human diseases including cancers. Therefore, accurate and sensitive detection of DNA MTase activity is crucial to biomedical research, clinical diagnostics and therapy. However, conventional DNA MTase assays often suffer from labor-intensive operations and time-consuming procedures. Alternatively, fluorescent methods have significant advantages of simplicity and high sensitivity, and have been widely applied for DNA MTase assay. In this review, we summarize the recent advances in the development of fluorescent methods for DNA MTase assay. These emerging methods include amplification-free and the amplification-assisted assays. Moreover, we discuss the challenges and future directions of this area.

  4. Proliferating macrophages prevail in atherosclerosis.

    Science.gov (United States)

    Randolph, Gwendalyn J

    2013-09-01

    Macrophages accumulate in atherosclerotic lesions during the inflammation that is part of atherosclerosis development and progression. A new study in mice indicates that the accumulation of macrophages in atherosclerotic plaques depends on local macrophage proliferation rather than the recruitment of circulating monocytes.

  5. Nuclear Proliferation Technology Trends Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Zentner, Michael D.; Coles, Garill A.; Talbert, Robert J.

    2005-10-04

    A process is underway to develop mature, integrated methodologies to address nonproliferation issues. A variety of methodologies (both qualitative and quantitative) are being considered. All have one thing in common, a need for a consistent set of proliferation related data that can be used as a basis for application. One approach to providing a basis for predicting and evaluating future proliferation events is to understand past proliferation events, that is, the different paths that have actually been taken to acquire or attempt to acquire special nuclear material. In order to provide this information, this report describing previous material acquisition activities (obtained from open source material) has been prepared. This report describes how, based on an evaluation of historical trends in nuclear technology development, conclusions can be reached concerning: (1) The length of time it takes to acquire a technology; (2) The length of time it takes for production of special nuclear material to begin; and (3) The type of approaches taken for acquiring the technology. In addition to examining time constants, the report is intended to provide information that could be used to support the use of the different non-proliferation analysis methodologies. Accordingly, each section includes: (1) Technology description; (2) Technology origin; (3) Basic theory; (4) Important components/materials; (5) Technology development; (6) Technological difficulties involved in use; (7) Changes/improvements in technology; (8) Countries that have used/attempted to use the technology; (9) Technology Information; (10) Acquisition approaches; (11) Time constants for technology development; and (12) Required Concurrent Technologies.

  6. [HSP90 Inhibitor 17-AAG Inhibits Multiple Myeloma Cell Proliferation by Down-regulating Wnt/β-Catenin Signaling Pathway].

    Science.gov (United States)

    Chen, Kan-Kan; He, Zheng-Mei; Ding, Bang-He; Chen, Yue; Zhang, Li-Juan; Yu, Liang; Gao, Jian

    2016-02-01

    To investigate the inhibitory effect of HSP90 inhibitory 17-AAG on proliferation of multiple myeloma cells and its main mechanism. The multiple myeloma cells U266 were treated with 17-AAG of different concentrations (200, 400, 600 and 800 nmol/L) for 24, 48, and 72 hours respectively, then the proliferation rate, expression levels of β-catenin and C-MYC protein, as well as cell cycle of U266 cells were treated with 17-AAG and were detected by MTT method, Western blot and flow cytometry, respectively. The 17-AAG showed inhibitory effect on the proliferation of U266 cells in dose- and time-depetent manners (r = -0.518, P AAG displayed no inhibitory effect on proliferation of U266 cells (P > 0.05). The result of culturing U266 cells for 72 hours by 17-AAG of different concentrations showed that the more high of 17-AAG concentration, the more low level of β-catenin and C-MYC proteins (P AAG concentration, the more high of cell ratio in G1 phase (P AAG, the more long time of culture, the more high of cell ratio in G1 phase (P AAG can inhibit the proliferation of multiple myeloma cells, the down-regulation of Wnt/β-catenin signaling pathway and inhibition of HSP90 expression may be the main mechnisms of 17-AAG effect.

  7. Estimation of Cell Proliferation Dynamics Using CFSE Data

    Science.gov (United States)

    Banks, H.T.; Sutton, Karyn L.; Thompson, W. Clayton; Bocharov, Gennady; Roose, Dirk; Schenkel, Tim; Meyerhans, Andreas

    2010-01-01

    Advances in fluorescent labeling of cells as measured by flow cytometry have allowed for quantitative studies of proliferating populations of cells. The investigations (Luzyanina et al. in J. Math. Biol. 54:57–89, 2007; J. Math. Biol., 2009; Theor. Biol. Med. Model. 4:1–26, 2007) contain a mathematical model with fluorescence intensity as a structure variable to describe the evolution in time of proliferating cells labeled by carboxyfluorescein succinimidyl ester (CFSE). Here, this model and several extensions/modifications are discussed. Suggestions for improvements are presented and analyzed with respect to statistical significance for better agreement between model solutions and experimental data. These investigations suggest that the new decay/label loss and time dependent effective proliferation and death rates do indeed provide improved fits of the model to data. Statistical models for the observed variability/noise in the data are discussed with implications for uncertainty quantification. The resulting new cell dynamics model should prove useful in proliferation assay tracking and modeling, with numerous applications in the biomedical sciences. PMID:20195910

  8. Radioreceptor assay for oxyphenonium

    International Nuclear Information System (INIS)

    Ensing, K.; Zeeuw, R.A. de

    1984-01-01

    The development of a radioreceptor assay for the quaternary anticholinergic drug, oxyphenonium, in plasma is reported. It is based on competition between this drug and 3 H-dexetimide for binding to muscarinic receptors. After ion pair extraction and reextraction, the drug can be determined in plasma at concentrations down to a value of 100 pg/ml. This permits pharmacokinetic studies to be made after inhalation of oxyphenonium. (author)

  9. Dual isotope assays

    International Nuclear Information System (INIS)

    Smith, G.F.W.; Stevens, R.A.J.; Jacoby, B.

    1980-01-01

    Dual isotope assays for thyroid function are performed by carrying out a radio-immunoassay for two of thyroxine (T4), tri-iodothyronine (T3), thyroid stimulating hormone (TSH), and thyroxine binding globulin (TBG), by a method wherein a version of one of the thyroid components, preferably T4 or T3 is labelled with Selenium-75 and the version of the other thyroid component is labelled with a different radionuclide, preferably Iodine-125. (author)

  10. In vitro screening of six anthelmintic plant products against larval Haemonchus contortus with a modified methyl-thiazolyl-tetrazolium reduction assay.

    Science.gov (United States)

    Hördegen, P; Cabaret, J; Hertzberg, H; Langhans, W; Maurer, V

    2006-11-03

    Because of the increasing anthelmintic resistance and the impact of conventional anthelmintics on the environment, it is important to look for alternative strategies against gastrointestinal nematodes. Phytotherapy could be one of the major options to control these pathologies. Extracts or ingredients of six different plant species were tested against exsheathed infective larvae of Haemonchus contortus using a modified methyl-thiazolyl-tetrazolium (MTT) reduction assay. Pyrantel tartrate was used as reference anthelmintic. Bromelain, the enzyme complex of the stem of Ananas comosus (Bromeliaceae), the ethanolic extracts of seeds of Azadirachta indica (Meliaceae), Caesalpinia crista (Caesalpiniaceae) and Vernonia anthelmintica (Asteraceae), and the ethanolic extracts of the whole plant of Fumaria parviflora (Papaveraceae) and of the fruit of Embelia ribes (Myrsinaceae) showed an anthelmintic efficacy of up to 93%, relative to pyrantel tartrate. Based on these results obtained with larval Haemonchus contortus, the modified MTT reduction assay could be a possible method for testing plant products with anthelmintic properties.

  11. The novel protein C9orf116 promotes rat liver cell line BRL-3A proliferation.

    Directory of Open Access Journals (Sweden)

    Chunyan Zhang

    Full Text Available Our previous study has proved that the chromosome 9 open reading frame 116 (C9orf116 (NM_001106564.1 was significantly up-regulated in the proliferation phase of liver regeneration. To study its possible physiological function, we analyzed the effect of C9orf116 on BRL-3A cells via over-expression and interference technique. MTT results showed that the cell viability of the interference group was significantly lower than the control group at 48h after transfection (P<0.05, whereas it was significantly higher in the over-expression group (P<0.05. The flow cytometry results showed that C9orf116 knockdown or over-expression had little effect on BRL-3A cell apoptosis. However, the number of cells in division phase (G2/M was significantly reduced in the interference group (P<0.05, but significantly increased in the over-expression group (P<0.01. Furthermore, the expressions of cell proliferation-related genes CCNA2, CCND1 and MYC both at mRNA and protein levels were down-regulated in the interference group and up-regulated in the over-expression group. Therefore, we concluded that C9orf116 may promote cell proliferation by modulating cell cycle transition and the expression of key genes CCNA2, CCND1 and MYC in BRL-3A cells.

  12. Regorafenib delays the proliferation of hepatocellular carcinoma by inducing autophagy.

    Science.gov (United States)

    Han, Rui; Li, Shixin

    2018-04-02

    The aim of the present study was to investigate the effects of regorafenib on hepatocellular carcinoma autophagy, thereby supressing the malignancy of HCC. First, HepG2 and Hep3B cell autophagy was investigated using GFP-LC3 transfection after the treatment of regorafenib. Then, the activation of Akt/mTOR signaling was analyzed using western blot. Our data showed that liver cancer cell autophagy was significantly induced by 20 μM regorafenib using GFP-LC3 transfection. Meanwhile, regorafenib-induced cell death could largely be abolished by 3-MA or CQ treatment, suggesting that regorafenib-induced HepG2 cell death was partially dependent on autophagy. Moreover, the activation of Akt/mTOR signaling was inhibited by regorafenib pre-incubation. MTT assay showed the combination use of regorafenib and CDDP led to a stronger growth inhibitory effect on HepG2 and Hep3B cells. In summary, regorafenib may acts an adjunctive therapy for liver cancer patients via modulating autophagy-dependent cell death even when apoptosis resistance is induced in cancer cells.

  13. In vitro screening of six anthelmintic plant products against larval Haemonchus contortus with a modified methyl-thiazolyl-tetrazolium reduction assay

    OpenAIRE

    Hördegen, P.; Cabaret, J.; Hertzberg, H.; Langhans, W.; Maurer, V.

    2006-01-01

    Because of the increasing anthelmintic resistance and the impact of conventional anthelmintics on the environment, it is important to look for alternative strategies against gastrointestinal nematodes. Phytotherapy could be one of the major options to control these pathologies. Extracts or ingredients of six different plant species were tested against exsheathed infective larvae of Haemonchus contortus using a modified methyl-thiazolyltetrazolium (MTT) reduction assay. Pyrantel tartrate was u...

  14. The local lymph node assay being too sensitive?

    Science.gov (United States)

    Hans-Werner, Vohr; Jürgen, Ahr Hans

    2005-12-01

    The local lymph node assay (LLNA) and modifications thereof were recently recognized by the OECD as stand-alone methods for the detection of skin-sensitizing potential. However, although the validity of the LLNA was acknowledged by the ICCVAM, attention was drawn to one major problem, i.e., the possibility of false positive results caused by non-specific cell activation as a result of inflammatory processes in the skin (irritation). This is based on the fact that inflammatory processes in the skin may lead to non-specific activation of dendritic cells, cell migration and non-specific proliferation of lymph node cells. Measuring cell proliferation by radioactive or non-radioactive methods, without taking the irritating properties of test items into account, leads thus to false positive reactions. In this paper, we have compared both endpoints: (1) cell proliferation alone and (2) cell proliferation in combination with inflammatory (irritating) processes. It turned out that a considerable number of tests were "false positive" to the definition mentioned above. By excluding such false positive results the LLNA seems not to be more sensitive than relevant guinea pig assays. These various methods and results are described here.

  15. Non proliferation of nuclear weapons?

    International Nuclear Information System (INIS)

    Le Guelte, Georges

    2015-10-01

    After having evoked the behaviour of nuclear countries regarding the development of nuclear weapons and uranium procurement, or nuclear programmes after the Second World War until nowadays, the author presents the non proliferation Treaty (NPT) as a construction at the service of super-powers. He comments and discusses the role of the IAEA control system and its evolutions: a control limited to declared installations, an export control with the spectre of plutonium, a control system thwarted by some technological innovations, information systems coming in, and an additional protocol related to the application of guarantees. He comments the evolution of the context from a bipolar world to a world without pole which raises the issue of how to have commitments respected: description of the role and practice of non proliferation during the Cold War, after the Cold War, and in a world without governance

  16. Proliferation Vulnerability Red Team report

    Energy Technology Data Exchange (ETDEWEB)

    Hinton, J.P.; Barnard, R.W.; Bennett, D.E. [and others

    1996-10-01

    This report is the product of a four-month independent technical assessment of potential proliferation vulnerabilities associated with the plutonium disposition alternatives currently under review by DOE/MD. The scope of this MD-chartered/Sandia-led study was limited to technical considerations that could reduce proliferation resistance during various stages of the disposition processes below the Stored Weapon/Spent Fuel standards. Both overt and covert threats from host nation and unauthorized parties were considered. The results of this study will be integrated with complementary work by others into an overall Nonproliferation and Arms Control Assessment in support of a Secretarial Record of Decision later this year for disposition of surplus U.S. weapons plutonium.

  17. N,N'-di-(m-methylphenyi)-3,6-dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide (ZGDHu-1) suppresses the proliferation of PANC-1 pancreatic cancer cells via apoptosis and G2/M cell cycle arrest.

    Science.gov (United States)

    Chen, Su-Feng; Xia, Jun; Lv, Ya-Ping; Liu, Jin-Lin; Li, Wan-Xiang; Yu, Xi-Ping; Hu, Wei-Xiao; Zhou, Yong-Lie

    2015-04-01

    Pancreatic cancer is one of the human gastrointestinal malignancies with a high mortality and poor prognosis. Approximately eighty percent of patients are diagnosed with unresectable or metastatic disease. Thus, development of novel chemicals in the treatment of pancreatic cancer is imperative. This study aimed to investigate the anticancer effects of N,N'-di-(m-methylphenyi)-3,6-dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide (ZGDHu-1), a new tetrazine derivative, on the PANC-1 pancreatic cancer cell line and clarify the underlying molecular mechanism. Using an MTT assay, we found that ZGDHu-1 significantly suppressed the proliferation of PANC-1 cells in a time- and dose-dependent manner. Moreover, according to the morphological and flow cytometric analysis, the results indicated that ZGDHu-1 induced PANC-1 cell apoptosis and G2/M cell cycle arrest in a dose-dependent manner. In the western blot analysis, expression of the pro-apoptotic Bax gene was upregulated while the anti-apoptotic Bcl-2 gene was downregulated following treatment with ZGDHu-1. ZGDHu-1 also activated pro-caspase-3 and PARP and increased the expression of NF-κB inhibitor IκB. Furthermore, the expression levels of G2/M regulatory molecules such as cyclin B1 and cdc2 were decreased while that of Chk1 was increased. These results suggested that ZGDHu-1 suppressed the proliferation of pancreatic cancer cells, rendering it a potential therapeutic drug for the treatment of pancreatic cancer.

  18. Black cohosh inhibits 17β-estradiol-induced cell proliferation of endometrial adenocarcinoma cells.

    Science.gov (United States)

    Park, So Yun; Kim, Hee Ja; Lee, Sa Ra; Choi, Youn-Hee; Jeong, Kyungah; Chung, Hyewon

    2016-10-01

    This study was conducted to investigate the effect of black cohosh (BC) extract on the proliferation and apoptosis of Ishikawa cells. Ishikawa human endometrial adenocarcinoma cells were treated with or without BC (1, 5, 10 and 25 μM) and cell proliferation and cytotoxicity were measured by CCK-8 assays and flow cytometry analysis. Additionally, Ishikawa cells were treated with 17β-estradiol (E2), E2 + progesterone and E2 + BC (5 and 10 μM) and the effect of BC and progesterone on E2-induced cell proliferation was analyzed. BC decreased the proliferation of Ishikawa cells at a dose-dependent rate compared with the control group (p < 0.05). The proliferation of Ishikawa cells increased in the presence of E2, whereas the subsequent addition of progesterone or BC decreased proliferation to the level of the control group (p < 0.05). The inhibitory effect of BC on E2-induced cell proliferation was greater than the inhibitory effect of progesterone. In conclusion, BC induces apoptosis in Ishikawa cells and suppresses E2-induced cell proliferation in Ishikawa cells. BC could be considered a candidate co-treatment agent of estrogen-dependent tumors, especially those involving endometrial cells.

  19. Nuclear power and weapons proliferation

    International Nuclear Information System (INIS)

    Greenwood, T.; Rathjens, C.W.; Ruina, J.

    1977-01-01

    The relationship between nuclear weapons development and nuclear electric power is examined. A brief description of nuclear weapons design is first given. This is then followed by a discussion of various aspects of nuclear power technology and of how they affect a nuclear weapon programme. These include fuel cycles, chemical reprocessing of spent fuel, uranium enrichment, and the control of dissemination of nuclear technology. In conclusion there is a discussion of possible political and institutional controls for limiting nuclear proliferation. (U.K.)

  20. CBRN and proliferation in Asia

    International Nuclear Information System (INIS)

    Brisset, Jean-Vincent

    2015-01-01

    The author proposes a brief overview of the history, evolution and status of military nuclear weapons and programmes as well as bacteriologic and chemical weapons (nuclear weapons, ballistic missile, and position with respect with the Conventions on chemical and bacteriologic weapons) in Asian countries (China, Japan, India, Pakistan, North Korea). In a second part, he discusses issues related to exports and possible proliferation from these countries

  1. CD147-induced cell proliferation is associated with Smad4 signal inhibition.

    Science.gov (United States)

    Qin, Hui; Rasul, Azhar; Li, Xin; Masood, Muqaddas; Yang, Guang; Wang, Na; Wei, Wei; He, Xi; Watanabe, Nobumoto; Li, Jiang; Li, Xiaomeng

    2017-09-15

    CD147 is a multifunctional trans-membrane glycoprotein, which is highly expressed in many cancers. However, the mechanism by which CD147 modulates cell proliferation is not fully understood. The aim of this study is to investigate the role of CD147 in cell proliferation associated with the TGF-β/Smad4 signaling pathway. Here, we used cell viability and clone formation assays in LNCaP prostate cancer cells to demonstrate that CD147 promotes cell proliferation. The luciferase assay and western blotting show that silencing CD147 using shRNA enhances transcription and expression of p21 WAF1 . Using immunofluorescence and nuclear-cytoplasmic separation, we show that this is primarily attributed to transport of Smad4 from the cytoplasm to nucleus. Other assays (GST pull-down, co-immunoprecipitation and immunofluorescence) demonstrate that Smad4 is a new interaction partner of CD147, with the Smad4 MH2 domain and CD147 intracellular domain (CD147-ICD) being involved in the interaction. Furthermore, we report that a phosphoserine (pSer) in CD147 (pSer252) is responsible for this interaction and inhibition of the Smad4/p21 WAF1 signal that promotes cell proliferation. Our results provide a novel molecular mechanism for CD147-induced cell proliferation associated with Smad4 signal inhibition. Copyright © 2017. Published by Elsevier Inc.

  2. miR-613 inhibits proliferation and invasion of breast cancer cell via VEGFA

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Junzhao; Yuan, Peng; Mao, Qixin [Breast Surgery, The Affiliated Cancer Hospital of Zhengzhou University, Henan (China); Lu, Peng [Gastrointestinal Surgery Department, People' s Hospital of Zhengzhou, Henan (China); Xie, Tian; Yang, Hanzhao [Breast Surgery, The Affiliated Cancer Hospital of Zhengzhou University, Henan (China); Wang, Chengzheng, E-mail: wangchengzheng@126.com [Breast Surgery, The Affiliated Cancer Hospital of Zhengzhou University, Henan (China)

    2016-09-09

    MicroRNAs (miRNAs) play important roles in the pathogenesis of many types of cancers by negatively regulating gene expression at posttranscriptional level. However, the role of microRNAs in breast cancer, has remained elusive. Her