Although induced pluripotent stem cells (iPSCs) are a potential source for transplantation therapy, malignant transformation (tumourigenesis) remains a major concern in their safe clinical application. iPSCs are considered more tumourigenic than embryonic stem cells (ESCs) because of genetic and epigenetic manipulations. We generated 22 human iPSC lines from normal human fibroblasts and injected three of these cell lines into SCID mice, and produced three tumours, all of which were identified as teratomas with at least two germ layers. Using cells cultured from them, RT-PCR showed that the cells expressed undifferentiated cell markers, including OCT4 and NANOG. This suggests that some undifferentiated cells remain in the teratoma during its formation. We also found emergence of cells expressing undifferentiated cell markers from teratoma-derived cells during culturing with the ESC medium. Immunocytochemical analyses showed that NANOG-, OCT4- and SSEA4-positive cells appeared and increased with time in culture. These data indicate that iPSC-like undifferentiated cells can emerge from differentiated cells under certain condition and they may present a potential risk of tumourigenesis, as do residual iPSCs.
Acute kidney injury (AKI) is a common syndrome featuring the rapid loss of the kidney's excretory function, and presents a common and important diagnostic and therapeutic challenge for clinicians. There are no effective approaches to prevent and treat AKI. During early stage, most ischaemic AKI is reversible, so further research is essential for early intervention in order to prevent and reduce AKI. Cyr61 has been shown to be expressed in renal tubular cells in hypoxic-ischaemic kidney injury, whilst there is no expression in normal tubular cells. In the present study, we have established a stably Cyr61 expressed tubular cell line Cyr61-HK-2 based on HK-2 through recombinant Cyr61-lentivirus, and found that Cyr61 expression promotes tubular epithelial cell proliferation, while the cell apoptosis is inhibited. Further study demonstrated that Cyr61 expression led to BAD phosphorylation, which protects tubular cells from apoptosis, meanwhile Cyr61 activates the Akt and ERK signalling pathways, which are essential for cell proliferation. Altogether, our data showed the Cyr61 expression promotes cell proliferation, while dampening cell apoptosis under hypoxia, potentially suggesting a novel therapeutic approach for AKI.
Oxidative stress has been progressively recognised as a key player in the development of osteoporosis. Although oxidative stress induces osteoporosis partly through inhibition of hedgehog signalling, the details of the signal interaction remain unclear. To elucidate this mechanism, we investigated osteoblast differentiation in C3H10T1/2 cells, murine embryonic mesenchymal cells, with recombinant sonic hedgehog (shh) in the presence or absence of hydrogen peroxide, and examined Gli transcriptional activity, with a focus on the interaction with mitogen-activated protein kinases (MAPKs) as possible mediators. The transcription of Gli1, Ptch1b, Alp and Bsp was facilitated by shh and strongly inhibited by oxidative stress with the hydrogen peroxide treatment; however, the other Gli isoforms, Bmp2, Runx2 and Osx, were unaffected. Oxidative stress also facilitated the phosphorylation of JNK1 and p38. Anisomycin treatment as a JNK activator or transient overexpression of the constitutive JNK1 active form inhibited hedgehog activity. Interestingly, the inhibitory effect of hydrogen peroxide on hedgehog signalling was blocked by a JNK inhibitor, but not by a p38 inhibitor. Osteoblast differentiation determined by calcium staining was, in part, rescued from oxidative stress by the JNK inhibitor. These findings suggest that oxidative stress inhibits hedgehog signalling and subsequent osteoblast differentiation through an interaction between phospho-JNK1 and GLI1.
Pluripotent mouse embryonic stem (ES) cells have the ability to generate completely ES cell-derived mice by tetraploid (TE-4N) complementation although; derivation of ES cells from TE-4N animals has not been well defined. In this study, we generated TE-4N mice from high passage number ES cells and, in turn, determined the possibility of deriving ES cells from the TE-4N mice. The results showed that adult fertile TE-4N mice could be generated by aggregation of tetraploid embryos using ES cells passaged 23 times. Furthermore, ES cells could be derived from the TE-4N male mice and as judged by molecular characterisation and a study of chimeras, these ES cells were pluripotent. The findings demonstrate that tetraploid complementation is an efficient way to produce fertile mice, which can then give rise to pluripotent ES cells.
Tissue engineering and regenerative are aiming at generating tissues to replace damaged and deteriorated organs. Recently, tissue engineering was applied to generate artificial skin for burn patients, tissue engineered trachea, cartilage for knee-replacement procedures, urinary bladder, urethra substitutes and offered cellular therapies for the treatment of urinary incontinence. The major advantage of tissue engineering approach over traditional organ transplantation is to circumvent the problem of organ shortage. Tissues reconstructed from readily available patents' stem cells induced no immunogenicity when reimplanted in the patient. However, pluripotent stem cells are major limited factors in regenerating new tissues. To overcome these problems, we developed a new technology called as ‘invivo interspecies tissue engineering’ (INVITE) and used the potential of induced pluripotent stem cells (iPSCs) to regenerate new tissues in a host organism. As a model, we used the mouse pluripotent stem cells to assess the potential of these cells to regenerate mouse tissues in rat. Three chimeric rats have been generated by mouse-induced pluripotent stem cells assessed by monitoring of green fluorescence protein (GFP) and polymerase chain reaction (PCR) assays. This study shows clearly that mouse-induced pluripotent stem cells are able to engraft in rat embryos and are capable to differentiate to multiple tissues. Beside the enormous important application of in vivo tissue engineering in human to develop new therapeutic approaches through regenerating new tissues, this model offers an accessible system for study of organ development and a valuable tool for personalised drug screening and a novel approach for personalised stem cell-based tissue regeneration.
Worldwide, the ‘Nemo’ ocellaris clownfish ( Amphiprion ocellaris , Cuvier 1830) is one of the top three most exported ornamental fishes. It also served as a subject in various fields of study except for cell culture. This first report described a simple explant method for culturing cells from the vertebra of ocellaris clownfish. The fish was first anesthetised with iced cold water and decapitated. The body trunk was disinfected in isopropanol and washed in sterile PBS. The vertebra was aseptically excised, washed two times in PBS and minced in the dissection solution (PBS containing 250 IU/mL penicillin, 250 µg/mL streptomycin, 50 µg/mL gentamycin and 2.5 µg/mL amphotericin-B). Then, the vertebral biopsies were washed three more times in PBS before being seeded in 25 cm 2 culture flasks containing 1.5 mL of RPMI-1640 supplemented with 20% FBS. A small amount of CO 2 was injected into the flask before it was tightly capped and incubated at 28°C in the regular incubator. When the monolayer reached 40–50% confluence, the vertebral biopsies were dislodged together with the medium to initiate a new primary culture. The cell monolayer was subcultured with short, cold 0.05% trypsin. The Nemo cell line was grown in the medium containing 15% FBS. The cell line at passage 4 had the population doubling time of 39.6 h and the cell line at passage 5 could be cryopreserved with 80% viability. This simple and reliable explant method has been applied successfully to culture cells of both marine and freshwater fishes for the prometaphase chromosome preparation.
Nitraria retusa leaf extracts have been investigated for their ability to induce antioxidant and antigenotoxic effects in a human chronic myelogenous leukaemia cell line. Antioxidant and antigenotoxic properties of N. retusa products were explored using antioxidant and the assays, respectively. Hex, Chl and MeOH extracts decreased oxidation induced by 2,2′-azobis (2-amidinopropane) dihydrochloride in human cells, with IC 50 concentrations of 0.6, 0.52 and 0.24 mg/mL, respectively, reflecting significant antioxidant potential. The same products inhibited the genotoxicity induced by hydroxyl radicals in the same human cell line, by 67% at 600 µg/mL, 74% at 780 µg/mL and 81.5% at 800 µg/mL, respectively.
Zebrafish keratocytes collectively migrate rapidly when established in explant cultures but little is known about the signals that initiate motility or the signal transduction pathways that result in an epithelial to mesenchymal transition. Matrix metalloproteinases (MMPs) are strong candidates for playing a role in this regulation and have previously not been analysed in this wound healing model system. Results presented here document a rapid and dramatic rise in MMP14a, MMP2, MMP9 and MMP13a mRNA levels over time. In a motility assay, a broad-spectrum MMP inhibitor and an inhibitor specific for MMP2 and MMP9 significantly decrease cell migration in a dose dependent manner but treatment with an MMP13 specific inhibitor significantly increases cell sheet area. Immunofluorescence staining with an antibody specific for the catalytic domain of MMP14 indicates that activated MMP14 protein is highly expressed on cells at the leading edge of a sheet compared with follower cells in the centre of the sheet, and is augmented further in leader cells that are stretched, thus likely in the process of detaching from the cell sheet. These data are consistent with a model in which active MMP14 at the leading edge of cell sheets in explant cultures triggers activation of MMP2 and/or MMP9, thus creating promigratory signal(s) that outweigh the inhibitory role of targets cleaved by MMP13. Taken together, these data suggest that MMPs play an important but complex role in regulating the collective cell migration of zebrafish keratocytes and provide support for the relevance of using zebrafish as a model for human disease.
We recently presented evidence showing that cementum protein 1 (CEMP1) promotes periodontal ligament (PDL) cell migration, proliferation, expression of bone, and cementum-matrix proteins and mineralisation. In other words, it induces PDL precursor cells commitment toward a cementoblast-like cells phenotype. The intracellular signalling pathways involved in cementoblast differentiation and mineralisation have not been well characterised. JNK and p38 protein kinases (MAPKs) are intracellular signalling pathways and key mediators of cellular processes such as proliferation and differentiation. Since signalling pathways involving MAPKs have been associated with osteoblastic phenotype, in this study we investigated the effect of hrCEMP1 and mineralising media containing β-glycerophosphate and ascorbic acid on the activation of p38-MAPK and JNK–MAPK in cementoblast-like cells. Our results show that mineralising media and hrCEMP1 induced phosphorylation of p38 and JNK kinases. Mineralising media containing hrCEMP1 increased the activation of p38-MAPK and its translocation to the cell nucleus; increased phosphorylation of JNK–MAPK and induced the phosphorylation of the protein C-JUN. We also demonstrate that hrCEMP1 regulates the expression of BSP, OCN, and ALP specific activity. We found that hrCEMP1 and mineralising media promote nodule formation. These findings give an insight into the signalling pathways activated by hrCEMP1 and suggest likely components of the mechanisms that regulate the formation and regeneration of cementum and surrounding connective tissues.
Spermatogenesis-associated-19 (SPATA19) is a novel spermatogenesis related gene that has important biological functions in reproduction. However, the expression aspects of SPATA19 gene are not yet well understood. We analysed SPATA19 gene expression during mouse testis development in vivo (5, 15 and 25 days old mouse testis), in parallel with mouse embryonic stem cells (ESCs) differentiation into male germ-like cells in vitro (5, 11, 19 and 27 days after differentiation), as well as Sertoli cell, mouse embryonic fibroblasts (MEF), and NIH3T3 cancerous cell line. We cultured transgenic mouse ESCs line C57BL/6J expressing Stra8-EGFP in ESC's medium and induced their differentiation by retinoic acid (RA) treatment, and meiotic cells were then sorted by FACS. RT-PCR and real-time PCR were used for analysis of SPATA19 mRNA expression. The expression of SPATA19 mRNA is increased during mouse testis development in vivo and it is highest in 25-days-old mouse testis. SPATA19 is also expressed most highly in mouse ESC-derived germ-like cells after 27 days of RA induction and NIH3T3 cell line but no expression was found in mouse ESC and Sertoli cell line. These findings demonstrate that the expression of SPATA19 increases during male germ cells development. We also suggest an additional possible role for SPATA19 in male germ cell differentiation and tumourigenic process.
While connexins (Cxs) are thought to be involved in differentiation, their expression and role has yet to be fully elucidated. We investigated the temporal expression of Cx30, Cx36 and Cx43 in two in vitro models of neuronal differentiation: human NT2/D1 and murine P19 cells, and the spatial localisation of Cx30 and Cx43 in these models. A temporal Cx43 downregulation was confirmed in both cell lines during RA-induced neuronal differentiation using RT-PCR (P < 0.05) preceding an increase in neuronal doublecortin protein. RT-PCR showed Cx36 was upregulated twofold in NT2/D1 cells (P < 0.05) and sixfold in P19 cells (P < 0.001) during neuronal differentiation. Cx30 exhibited a transient peak in expression midway through the timecourse of differentiation increasing threefold in NT2/D1 cells (P < 0.001) and eightfold in P19 cells (P < 0.01). Qualitative immunocytochemistry was used to examine spatiotemporal patterns of Cx protein distribution alongside neuronal differentiation markers. The temporal immunolabelling pattern was similar to that seen using RT-PCR. Cx43 was observed intracellularly and on cell surfaces, while Cx30 was seen as puncta. Spatially Cx43 was seen on doublecortin-negative cells, which may indicate Cx43 downregulation is requisite for differentiation in these models. Conversely, Cx30 puncta were observed on doublecortin-positive and -negative cells in NT2/D1 cells and examination of the Cx30 peak showed puncta also localized to nestin-positive cells, with few puncta on MAP2-positive cells. In P19 cells Cx30 was localized on clusters of cells surrounded by MAP2- and doublecortin-positive processes. The expression pattern of Cx30 indicates a role in neuronal differentiation; the nature of that role warrants future investigation.
Although induced pluripotent stem cells (iPSCs) are a potential source for transplantation therapy, malignant transformation (tumourigenesis) remains a major concern in their safe clinical application. iPSCs are considered more tumourigenic than embryonic stem cells (ESCs) because of genetic and epigenetic manipulations. We generated 22 human iPSC lines from normal human fibroblasts and injected three of these cell lines into SCID mice, and produced three tumours, all of which were identified as teratomas with at least two germ layers. Using cells cultured from them, RT-PCR showed that the cells expressed undifferentiated cell markers, including OCT4 and NANOG. This suggests that some undifferentiated cells remain in the teratoma during its formation. We also found emergence of cells expressing undifferentiated cell markers from teratoma-derived cells during culturing with the ESC medium. Immunocytochemical analyses showed that NANOG-, OCT4- and SSEA4-positive cells appeared and increased with time in culture. These data indicate that iPSC-like undifferentiated cells can emerge from differentiated cells under certain condition and they may present a potential risk of tumourigenesis, as do residual iPSCs.
We compared airway epithelial cell models relevant for cystic fibrosis (CF): 16HBE cells with endogenous wild-type cystic fibrosis transmembrane conductance regulator (CFTR), CFBE cells with mutated ΔF508-CFTR, corrected CFBE cells overexpressing CFTR, CFSME (CF submucosal) and Calu-3 (non-CF submucosal) cells with respect to the epithelial sodium channel (ENaC), inducible NO synthase (iNOS) and mucins (MUC) (studied by quantitative Real-Time-Polymerase Chain Reaction, qRT-PCR and Western blot), and wound healing. CFBE cells had significantly more expression of β- and γ-ENaC mRNA and of β-ENaC protein than 16HBE cells. Compared to corrected CFBE cells, CFBE cells had increased mRNA expression of all ENaC subunits and β-ENaC protein. For ENaC, the CFSME/Calu-3 mRNA ratio was very low and contradictory to the ENaC upregulation in CF cells. CFBE cells showed decreased expression of iNOS at both mRNA and protein levels compared to 16HBE cells and only at the mRNA level compared to corrected CFBE cells. CFSME cells showed expression of iNOS whereas Calu-3 cells did not. Higher expression of MUC2 and MUC5B was found in corrected CFBE cells compared to CFBE cells. Wound healing in CFBE cells was delayed compared to corrected CFBE cells, but not to 16HBE cells, and in CFSME cells compared to Calu-3 cells. Our data suggest CFSME as an inappropriate CF cell model for Calu-3 cells, and provide partial support for the theory that the differences (in ENaC, iNOS and wound healing) between these cell lines are associated to the presence of CFTR in the bronchial airway epithelial cells.
Cell Biology International ReportsVolume 20, Issue 2 p. 5-12 Research ArticleOpen Access Antigenotoxic and antioxidant activity in human chronic myelogenous leukaemia cell line K562 enhanced by Nitraria retusa leaf extracts Jihed Boubaker, Corresponding Author Jihed Boubaker Laboratory of Cellular and Molecular Biology, Faculty of Dental Medicine, University of Monastir, Rue Avicenne, Monastir, 5000 Tunisia Unit of Natural Bioactive Substances and Biotechnology, Faculty of Pharmacy, University of Monastir, Rue Avicenne, Monastir, 5000 TunisiaCorresponding author: e-mail: jihed.boubaker@yahoo.frSearch for more papers by this authorZied Ghedira, Zied Ghedira Laboratory of Cellular and Molecular Biology, Faculty of Dental Medicine, University of Monastir, Rue Avicenne, Monastir, 5000 Tunisia Unit of Natural Bioactive Substances and Biotechnology, Faculty of Pharmacy, University of Monastir, Rue Avicenne, Monastir, 5000 TunisiaSearch for more papers by this authorKamel Ghedira, Kamel Ghedira Unit of Natural Bioactive Substances and Biotechnology, Faculty of Pharmacy, University of Monastir, Rue Avicenne, Monastir, 5000 TunisiaSearch for more papers by this authorLeila Chekir-Ghedira, Leila Chekir-Ghedira Laboratory of Cellular and Molecular Biology, Faculty of Dental Medicine, University of Monastir, Rue Avicenne, Monastir, 5000 Tunisia Unit of Natural Bioactive Substances and Biotechnology, Faculty of Pharmacy, University of Monastir, Rue Avicenne, Monastir, 5000 TunisiaSearch for more papers by this author Jihed Boubaker, Corresponding Author Jihed Boubaker Laboratory of Cellular and Molecular Biology, Faculty of Dental Medicine, University of Monastir, Rue Avicenne, Monastir, 5000 Tunisia Unit of Natural Bioactive Substances and Biotechnology, Faculty of Pharmacy, University of Monastir, Rue Avicenne, Monastir, 5000 TunisiaCorresponding author: e-mail: jihed.boubaker@yahoo.frSearch for more papers by this authorZied Ghedira, Zied Ghedira Laboratory of Cellular and Molecular Biology, Faculty of Dental Medicine, University of Monastir, Rue Avicenne, Monastir, 5000 Tunisia Unit of Natural Bioactive Substances and Biotechnology, Faculty of Pharmacy, University of Monastir, Rue Avicenne, Monastir, 5000 TunisiaSearch for more papers by this authorKamel Ghedira, Kamel Ghedira Unit of Natural Bioactive Substances and Biotechnology, Faculty of Pharmacy, University of Monastir, Rue Avicenne, Monastir, 5000 TunisiaSearch for more papers by this authorLeila Chekir-Ghedira, Leila Chekir-Ghedira Laboratory of Cellular and Molecular Biology, Faculty of Dental Medicine, University of Monastir, Rue Avicenne, Monastir, 5000 Tunisia Unit of Natural Bioactive Substances and Biotechnology, Faculty of Pharmacy, University of Monastir, Rue Avicenne, Monastir, 5000 TunisiaSearch for more papers by this author First published: 25 June 2013 https://doi.org/10.1002/cbi3.10003Citations: 1AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Nitraria retusa leaf extracts have been investigated for their ability to induce antioxidant and antigenotoxic effects in a human chronic myelogenous leukaemia cell line. Antioxidant and antigenotoxic properties of N. retusa products were explored using antioxidant and the assays, respectively. Hex, Chl and MeOH extracts decreased oxidation induced by 2,2′-azobis (2-amidinopropane) dihydrochloride in human cells, with IC50 concentrations of 0.6, 0.52 and 0.24 mg/mL, respectively, reflecting significant antioxidant potential. The same products inhibited the genotoxicity induced by hydroxyl radicals in the same human cell line, by 67% at 600 µg/mL, 74% at 780 µg/mL and 81.5% at 800 µg/mL, respectively. Introduction Exposure to genotoxic chemicals present in food, the environment and medical treatment can alter the genetic material permanently, and thus may lead to cancer (De Flora and Ferguson, 2005). The genotoxic effects of toxicants damaging DNA could be minimised by modulation of the physiological detoxification systems. Many naturally occurring compounds with antioxidant activity can protect cellular components from oxidative damage, and antigenotoxic effect of plant extracts can counter or prevent the adverse effects caused by DNA-damaging chemicals. Indeed, some plant-derived polyphenolic compounds have antigenotoxic activity (Williams et al., 1989; Surh and Ferguson, 2003). These compounds scavenge free radicals and protect cell constituents against oxidative damage and therefore, limit the risk of various degenerative diseases associated with the oxidative stress. Oxidative stress is an imbalance between the production of reactive oxygen species (ROS) and antioxidant defence that may lead to oxidative damage. This can result from a deficiency in antioxidant defence mechanisms, or from an increase in ROS, due to exposure to elevated ROS levels, the presence of toxins metabolised to ROS, or excessive activation of ROS systems, such as those mediated by chronic infection and inflammation. In addition to endogenously produced antioxidants and enzymes that catalyse their metabolism, ROS can be scavenged by exogenous antioxidants, such as phenolics, carotenoids and vitamins found in fruit and vegetables (Wolfe and Liu, 2007). To explore the therapeutic potentials of Tunisian medicinal herbs for the treatment of related-diseases pathologies, a medicinal herb species traditionally used in the country was studied for its antigenotoxic and antioxidant properties on a human cell line. The objective was to use the cellular antioxidant assay (CAA) to quantify the antioxidant potential of the Hex, Chl and MeOH extracts from N. retusa leaves and their protective potential against damage provoked by free radicals. Materials and methods Chemicals All the organic solvents were obtained from Carlo ERBA (Paris, France). L-Glutamine was purchased from GIBCO BRL Life Technologies (Grand Island, NY). The N-(1-naphtyl) ethlenediaminedihydrochloride (EDTA) was purchased from Sigma–Aldrich (Steinheim, Germany). RPMI-1640, foetal bovine serum and gentamicin were bought from GIBCO BRL Life technologies Folin–Ciocalteu reagent and 2,2′-azobis (2-amidinopropane) dihydrochloride (ABAP) were purchased from Wako Chemicals USA, Inc. (Richmond, VA). 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) was purchased from Sigma–Aldrich, Inc. (St. Louis, MO). Dimethyl sulfoxide and acetic acid were obtained from Fisher Scientific (Pittsburgh, PA). Sodium carbonate, acetone, and methanol were obtained from Mallinckrodt Baker, Inc. (Phillipsburg, NJ). Plant material Nitraria retusa was collected from the saline soils at Sahline, a region situated in the centre of Tunisia, in December 2006. Identification was carried out by Prof M. Cheieb (Department of Botany, Faculty of Sciences, University of Sfax, Tunisia), according to the Flora of Tunisia (Pottier-Alaptite, 1979; Cheib and Boukhris, 1998). A voucher specimen (N.r-12.06) is kept at our laboratory for future reference. The leaves were shade-dried, powdered and stored in a tightly closed container for further use. Preparation of plant extracts A total of 350 g of powder from dried leaves was sequentially extracted in a Soxhlet apparatus (6 h) (AM Glassware, Aberdeen, UK) with hexane, chloroform, ethyl acetate and methanol solvents. Hexane (Hex), chloroform (Chl) and methanol (MeOH) extracts, with different polarities, were concentrated to dryness and the residues kept at 4°C. The extracts were resuspended in dimethyl sulfoxide solvent (DMSO). Preliminary phytochemical analysis and determination of total polyphenol, flavonoid, tannins and sterol content Plant materials were screened for the presence of tannins, flavonoids and sterols using the methods previously described by Boubaker et al. (2011). Cell culture Human chronic myelogenous leukaemia CML cell line K562 was obtained from the American Type Culture Collection (Rockville, MD). Cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) foetal calf serum, 0.1 mg/mL gentamicin and 2 mM L-glutamine as complete growth medium, and were incubated at 37°C in an air incubator with 5% CO2, and a humidified atmosphere. The cells were subcultured every 2 days. Cytotoxicity of N. retusa extract against K562 cells was estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, previously described by Boubaker et al. (2011). Cellular antioxidant activity (CAA) assay (Wolfe and Liu, 2007) K562 cells were seeded at 6 × 104/well on 96-well microplates. Twenty-four hours after seeding, the growth medium was removed and the wells washed with PBS. Triplicate wells were treated for 1 h with 100 µL of extracts plus 25 µM DCFH-DA dissolved in treatment medium. Wells were washed with 100 µL of PBS, then 600 µM ABAP solution was applied to the cells in 100 µL of PBS, and the 96-well microplate was placed into a Fluoroskan Ascent FL plate-reader (ThermoLabsystems, Franklin, MA) at 37°C. Emission at 538 nm was measured with excitation at 485 nm every 5 min for 1 h. Each plate included triplicate control and blank wells: control wells contained cells treated with DCFH-DA and oxidant ABAP; blank wells contained cells treated with PBS without oxidant (Wolfe and Liu, 2007, 2008; McDowell et al., 2011) (Figure 1). Figure 1Open in figure viewerPowerPoint Cellular antioxidant activity of plant extracts in human chronic myelogenous leukaemia cell line K562 (Wolfe and Liu, 2007). Quantification of CAA After blank subtraction from the fluorescence readings, the area under the curve of fluorescence versus time was integrated to calculate the CAA value at each concentration of sample as follows: where is the integrated area under the sample fluorescence versus time curve and is the integrated area from the control curve. The median effective dose (IC50) was determined for the pure phytochemical compounds and leaf extract from the median effect plot of log(fa/fu) versus log(dose), where fa is the fraction affected and fu is the fraction unaffected by the treatment. To quantify intraexperimental variation, the IC50 values were stated as mean ± SD for triplicate sets of data obtained from the same experiment (Wolfe and Liu, 2007, 2008; McDowell et al., 2011). Comet assay The comet assay was used to detect DNA damage. Before each experiment, frosted microscope slides were precoated with two layers (100 µL) of normal agarose (1% in milli-Q water) and left at room temperature to allow the agarose to dry. The cells were treated for 24 h with different concentrations of the samples. Treated cells were stressed with 75 µM H2O2, for 2 h. The diluted cells (5 × 105 cells in 60 µL) were mixed with an equal volume of low-melting point agarose (1.2% in PBS). This agarose cell suspension (120 µL) was spread onto each precoated slide and covered with a coverslip. After 10 min on ice, the coverslip was gently removed, and the slides were placed in lysate buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris–HCl, 1% sodium sarcosinate pH 10, 1% of Triton X-100 and 10% DMSO). They were immersed for 1 h in the buffer (4°C, in the dark). The slides were transferred into electrophoresis buffer (NaOH 10 N, EDTA 200 mmol/L, pH 13 in deionised water) for 20 min at room temperature in the dark. Electrophoresis was carried out for 15 min at 25 V, 300 mA. Finally, the slides were gently rinsed with neutralisation solution (0.4 M Tris–HCl, pH 7.5) 3 times for 5 min each. Staining of DNA was done using 50 µL ethidium bromide solution (20 µg/mL in PBS) per slide (Scassellati-Sforzolini et al., 1999). The slides were examined using an epifluorescence microscope (Zeiss Axioskop 20; Carl Zeiss, Microscope Division, Oberkochen, Germany). Quantification of the comet assay A total of 100 comets on each scored slide for each concentration of the samples were visually scored according to the relative intensity of fluorescence in the tail and classified into five classes (Figure 2). We used three slides for each concentration of extract and the experiments were repeated three times. Each comet class was given a value of 0, 1, 2, 3 or 4 (from undamaged, 0 to maximally damaged, 4) as described previously by Collins et al. (1996). The total score of DNA damage was calculated by the following equation: Figure 2Open in figure viewerPowerPoint The classification of different categories to the comet according to the relative intensity of fluorescence in the tail. The inhibition percentage of Tail DNA (%) was calculated relative to DNA damage in the control group cells treated with H2O2 only by the following formula: Statistical analysis Data were collected and expressed as the mean ± standard deviation of three independent experiments and analysed for statistical significance from the positive control. The data were examined for statistical differences by Student's t-test, with significance set at P < 0.05. Results Phytochemical study and determination of extract yield, total polyphenol, flavonoid, tannin and sterol contents of N. retusa leaf extracts The highest content of polyphenols and sterols was found in Chl extract, with the percentage of the total polyphenolic and sterols content in chloroform extract being 10.0 and 11.8%, respectively. The Hex extract contained a considerable quantity of sterols equivalent to 31% whereas the MeOH extract exhibited the highest quantities of tannins and flavonoids. As previously published, the percentage of tannins and flavonoids in MeOH extract were 18.8 and 14.8%, respectively (Boubaker et al., 2011). Cellular antioxidant activity (CAA) assay The choice of concentrations were determined by the MTT test, which were similar for the Hex, Chl and MeOH extracts in inhibiting cell growth. As previously reported, the IC50 values of Hex, Chl and MeOH were 300, 380 and >800 µg/mL, respectively (Boubaker et al., 2011). In order to follow the antioxidant effect of Hex, Chl and MeOH extracts in the intracellular environment, CAA was used, with all the extracts giving significant cellular antioxidant activity. Hex, Chl and MeOH extracts inhibited radicals resulting from ABAP intracellular oxidation concentrations of each extract inhibiting 50% of radical formation (IC50) were 0.6 mg/mL (Figure 3), 0.52 mg/mL (Figure 4) and 0.24 mg/mL (Figure 5), respectively. Figure 3Open in figure viewerPowerPoint (a) Cellular antioxidant activity (CAA) of hexane extract (Hex extract) K562 treated cells + 2′,7′-dichlorofluorescin (DCFH) + 2,2′-azobis (2-amidinopropane) dihydrochloride (ABAP). PC: Untreated cells + 2′,7′-dichlorofluorescin (DCFH) + 2,2′-azobis (2-amidinopropane) dihydrochloride (ABAP). (b) Dose–response curve for the inhibition of oxidation of the free-radical 2′,7′-dichlorofluorescin (DCFH) to DCF in K562 cells using the cellular antioxidant activity assay in the presence of different concentrations of hexane extract (Hex extract) (mean ± SD, n = 3). Figure 4Open in figure viewerPowerPoint (a) Cellular antioxidant activity (CAA) of chloroform extract (Chl extract) K562 treated cells + 2′,7′-dichlorofluorescin (DCFH) + 2,2′-azobis (2-amidinopropane) dihydrochloride (ABAP). PC: Untreated cells + 2′,7′-dichlorofluorescin (DCFH) + 2,2′-azobis (2-amidinopropane) dihydrochloride (ABAP). (b) Dose–response curve for the inhibition of oxidation of the free-radical 2′,7′-dichlorofluorescin (DCFH) to DCF in K562 cells using the cellular antioxidant activity assay in the presence of different concentrations of chloroform extract (Chl extract) (mean ± SD, n = 3). Figure 5Open in figure viewerPowerPoint (a) Cellular antioxidant activity (CAA) of methanol extract (MeOH extract) K562 treated cells + 2′,7′-dichlorofluorescin (DCFH) + 2,2′-azobis (2-amidinopropane) dihydrochloride (ABAP). PC: Untreated cells + 2′,7′-dichlorofluorescin (DCFH) + 2,2′-azobis (2-amidinopropane) dihydrochloride (ABAP). (b) Dose–response curve for the inhibition of oxidation of the free-radical 2′,7′-dichlorofluorescin (DCFH) to DCF in K562 cells using the cellular antioxidant activity assay in the presence of different concentrations of methanol extract (MeOH extract) (mean ± SD, n = 3). Comet assay DNA damage in human K562 cells after exposure to different N. retusa products was investigated using the comet assay. Regarding the direct effect of Hex, Chl and MeOH extracts (Table 1), the data of Total DNA damage (TDD) indicated that both tested samples induced no genotoxicity, as no significant difference was detected: Hex extract (TDD values were 255, 256 and 254 at, respectively, 150, 300 and 600 µg/mL), Chl extract (TDD values were 261, 252 and 255 at, respectively, 190, 380 and 760 µg/mL), and MeOH extract (TDD values 250 and 250 at, respectively, 200, 400 and 800 µg/mL), whereas the negative control had a TDD of 252 ± 6. In contrast, a significant increase of total DNA damage was observed in cells exposed to 75 µM H2O2 (TDD = 360 ± 5) compared to the vehicular treated controls. The alkaline single-cell gel electrophoresis (comet) assay to measure the antigenotoxic effect of the extracts products against 75 µM H2O2 induced DNA damage showed that Hex, Chl and MeOH extracts were all effective, each decreasing damage in a dose-dependent manner, respectively 66.7, 74.1 and 81.5% at the highest tested concentrations of each (600, 760 and 800 µg/mL, respectively). Table 1. Treatment of K562 cell DNA with N. retusa extracts in the presence and absence of H2O2 in the test of comet Extracts Comet assay on genomic DNA of K562 cells treated with N. retusa extracts Inhibitory effect of N. retusa extracts on the genotoxicity of K562 cells against H2O2 Concentrations (µg/mL) Total DNA damage (TDD) Total DNA damage (TDD) Inhibition percentage (%) T – 252 ± 6 252 ± 6 H2O2 75 µM 360 ± 5 360 ± 5 Hex extract 150 255 ± 5 302 ± 4 53.7* 300 256 ± 3 298 ± 4 57.4* 600 254 ± 4 288 ± 5 66.7* Chl extract 190 261 ± 3 301 ± 6 54.6* 380 252 ± 6 291 ± 7 63.9* 760 255 ± 4 280 ± 6 74.1* MeOH extract 200 260 ± 3 302 ± 6 53.7* 400 250 ± 3 289 ± 6 65.7* 800 250 ± 5 272 ± 5 81.5* * P < 0.05 compared to negative control without N. retusa extracts. Results are represented by the means ± SD of three experiments. Discussion The CAA capacity of MeOH extracts should be ascribed to phenolic groups of its flavonoid components, which have antioxidant properties (Manach et al., 1996). Radical chelating properties of flavonoids and tannins many contribute to their ability to inhibit oxidative damage (Morel et al., 1993). Hydroxyl groups of flavonoids participate in their antioxidant properties (Formica and Regelson, 1995; Manach et al., 1996; Yang et al., 2001). The greater the number of OH groups on A and B rings of flavonoids, the higher is their antioxidant potential. Hydroxyl groups react strongly with free radicals making them unreactive (Nijveldt et al., 2001). Cellular antioxidant properties of Hex and Chl extract may be ascribed to sterols detected in these extract and known for their antioxidant properties (Wang et al., 2002). We hypothesise that sterols inhibit free radicals and ROS produced by oxidation and redox-cycling, as reported by Argolo et al. (2004) and Ben Mansour et al. (2007). However, the polyphenolic content of Chl extract may function as potent electron and hydrogen atom donors, and therefore should terminate the radical chain reaction by converting the free radicals and the reactive oxygen species to more stable products. Similar observations about the polyphenolic constituents in terms of a dose-dependent and a reducing-power activity have been reported for several plant extracts, such as tea (Amorowicz et al., 2004). When treated with H2O2, the predominant lesions in DNA are strand breaks and base oxidation, which can increase the risk of cancer (Martins et al., 1991). Hex, Chl and MeOH extracts have significant antigenotoxic potential against stress induced by H2O2 on K562 cell DNA. This could be partly correlated with the content of polyphenols (Schnitzler et al., 2008), flavonoids (Middleton et al., 2000; Choi Cang et al., 2002), tannins (Lee et al., 2003; Bouhlel et al., 2008) and sterols (Argolo et al., 2004) detected in the different extracts, along with synergistic involvement of several of these compounds. This protective action of the tested compounds can be explained by their ability to penetrate the cell membrane and interrupt radical chain induced by H2O2, thus preventing and/or reducing free radical formation responsible for macromolecular damage, including DNA (Roginsky and Lissi, 2005). Indeed, flavonoids are effective scavengers of reactive oxygen species (ROS), and their anticancer activities heavily depend on their antioxidant and chelating properties (Afanas'ev et al., 1995; Duthie and Dobson, 1999). It is also possible that sterol compounds inhibit the free radicals and ROS produced by oxidation and redox-cycling started by H2O2 (Mahoney and Graf, 1986; Boubaker et al., 2011). Thus we suppose that antigenotoxic effect of these extracts may be ascribed to other additional mechanisms as DNA repair enzyme induction, or it may have antioxidant activity against other radical types. Acknowledgement and funding The authors acknowledge the ‘Ministère Tunisien de l'Enseignement Supérieur et de la Recherche Scientifique’ for its support. Conflict of interest The author declared no conflict of interest. References Afanas'ev IB, Ostrachovitch EA, Abramova NE, Korkina LG (1995) Different antioxidant activities of bioflavonoid rutin in normal and iron-overloaded rats. Biochem Pharmacol 50: 627– 37. Amorowicz R, Pegg RB, Rahimi-Moghaddam P, Barl B, Weil JA (2004) Free radical scavenging capacity of selected plant species from the Canadian prairies. Food Chem 84: 551– 62. 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J Agric Food Chem 56: 8404– 11. Yang B, Kotani A, Arai K, Kusu F (2001) Estimation of the antioxidant activities of flavonoids from their oxidation potentials. Anal Sci 17: 599– 604. Citing Literature Volume20, Issue2December 2013Pages 5-12 FiguresReferencesRelatedInformation
In recent years, increasing interest surrounding islet replacement therapies in human has provided the drive for advances in the methods used to isolate from humans as well as a host of animal research models. However, there has been no reports describing a technique that reliably improves the quality of RNA extracted from rat pancreatic islet. Male Sprague–Dawley rats, 10- to 12-week-old, were housed in a certified animal care facility. The rats were underwent bile duct cannulation with pancreatic inflation after clamping the distal common bile duct. The pancreas was excised and digested with ETK/Liberase TL solution at 37°C for 30 min without shaking. Cold ETK was added to stop digestion. The islets were purified by discontinuous iodixanol density gradients of 25, 23, 20 and 11% in a modified ETK/OptiPrep® solution. After a 15 min centrifugation at 1,000 g , islets were collected from the interface between the 20 and 11% layer. Immediately after purification, islets were used for RNA extraction. RNA was extracted from isolated rat pancreatic islet cells, using the commercially available kit. In the present study, we have described a technique that reliably improves the quality of RNA extracted from rat pancreatic islet using the perfusion technique in the bile duct. The islet cells that we isolated using this technique were suitable for high quality RNA extraction.
Interferon Stimulated Gene 12a, ISG12a, is a member of a family of small intracellular non-secreted proteins (10–20 kDa), mainly induced by type I IFNs and slightly induced by type II IFN. It has been shown that full length ISG12 (ISG12a) is overexpressed in breast cancer, yet the biological function of ISG12 is largely unknown. Here we show that transient transfection of ISG12a into various mammalian cell lines leads to accumulation of cells initially in the G1 phase of the cell cycle, followed by accumulation of cells in the sub G1 phase, and that cells transfected with ISG12a undergo morphological changes, such as rounding up and detachment from the plate, that are characteristic of apoptosis. Induction of apoptosis by ISG12a was confirmed by Annexin V binding assays and by TUNEL assays. Using general and specific caspase inhibitors, we also showed that ISG12a-induced apoptosis is a caspase dependent, but does not involve p53. Elevation in endogenous ISG12a levels following induction of apoptosis with reagents that induce apoptosis in the intrinsic apoptotic pathway, and reduction in ISG12a-induced apoptosis following co-transfection with Bcl-2, indicated that ISG12a induced apoptosis in the intrinsic apoptotic pathway. Our results suggest a role for the ISG12a gene as a novel pro-apoptotic gene.
In most somatic tissues, ASCs (adult stem cells) are crucial for the maintenance of tissue homoeostasis under normal physiological state and recovery from injury. LRC (label retaining cell) assay is a well-known method of identifying possible somatic stem/progenitor cells and their location both in situ and in vivo. BrdU (bromodeoxyuridine) was used here to tag the possible CSCs (cardiac stem cells)/CPCs (cardiac progenitor cells) in newborn pups, followed by a trace period of up to 24 months. In addition, we have used our newly developed 'KAL' method to rapidly Kill proliferating cells in adult heart tissues, then, Activate and Label the surviving CSCs/CPCs. LRCs that definitively exist in the heart tissues of adult mice, and some LRCs express the stem cell marker, Sca-1 or c-Kit, and are located primarily in the myocardium and vascular endothelial regions. Moreover, the number of LRCs remains nearly constant during the lifespan of the mouse. After injury induced by 5-fluorouracil, the proliferating cells were almost completely cleared on day 3, and the activated CSCs/CPCs retained their BrdU label after regeneration was complete. A small percentage of the CSCs/CPCs express Sca-1 or c-Kit. Furthermore, the LRC method together with KAL may be used to identify and locate possible CSCs/CPCs, which has potential clinical application.
Exposure to EMFs (electromagnetic fields) results in a number of important biological changes, including modification of genetic expression. We have investigated the effect of 60 Hz sinusoidal EMFs at a magnetic flux density of 80 μT on the expression of the luciferase gene contained in a plasmid labelled as pEMF (EMF plasmid). This gene construct contains the specific sequences for the induction of hsp70 (heat-shock protein 70) expression by EMFs, as well as the reporter for the luciferase gene. The pEMF vector was electrotransferred into quadriceps muscles of BALB/c mice that were later exposed to EMFs. Increased luciferase expression was observed in mice exposed to EMFs 2 h daily for 7 days compared with controls (P<0.05). These data along with other reports in the literature suggest that EMFs can have far-reaching effects on the genome.
Exposure to EMFs (electromagnetic fields) results in a number of important biological changes, including modification of genetic expression. We have investigated the effect of 60 Hz sinusoidal EMFs at a magnetic flux density of 80 μT on the expression of the luciferase gene contained in a plasmid labelled as pEMF (EMF plasmid). This gene construct contains the specific sequences for the induction of hsp70 (heat-shock protein 70) expression by EMFs, as well as the reporter for the luciferase gene. The pEMF vector was electrotransferred into quadriceps muscles of BALB/c mice that were later exposed to EMFs. Increased luciferase expression was observed in mice exposed to EMFs 2 h daily for 7 days compared with controls (P<0.05). These data along with other reports in the literature suggest that EMFs can have far-reaching effects on the genome.
A mast cell line (rat basophilic leukaemia cells, RBL-2H3) was used in vitro to study cellular responses to fluid shear stress generated by a rotating rotor in a cell dish. The [Ca2+]c (cytosolic calcium concentration) in mast cells was detected by confocal fluorescence microscopy after Fluo-3/AM (acetoxymethyl ester) staining. Cytosolic calcium fluorescence intensity oscillated, pulsed, or steadily increased after the application of a weak, moderate or strong fluid shear stress respectively. From a mathematical model, we reproduced a change in [Ca2+]c under different levels of fluid shear stress in mast cells. The model calculation confirmed another experimental observation, specifically, that the degranulation rate increases with shear stress and reaches certain steady values. Since mast cells can react clearly and quickly and obviously to mechanical stimuli, calcium signalling and degranulation dynamics could be one of the mechanisms of acupuncture and massage therapies.
